Effects of dietary nanoliposome-coated astaxanthin on haematological parameters, immune responses and the antioxidant status of rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Astaxanthin is the most prevalent carotenoid in the marine environment and is widely used as an additive in formulated aquafeeds. OBJECTIVES: A 60-day feeding trial was conducted to consider the effect of dietary nanoliposome-coated astaxanthin (NA) on haematological parameters, serum antioxidant activities and immune responses of rainbow trout, Oncorhynchus mykiss. METHODS: A total of 450 healthy fish weighing 31.00 ± 2.09 g were randomly assigned in triplicate (30 fish per replicate) to 5 dietary treatments: 0 (control), 25.00, 50.00, 75.00, and 100.00 mg kg-1 NA. RESULTS: Fish fed the diet supplemented with 50.00 mg kg-1 NA exhibited the highest values of red blood cells, white blood cells, haemoglobin and haematocrit of 1.64 ± 0.01 × 106 mm-3, 5.54 ± 0.21 × 103 mm-3, 8.73 ± 0.24 g dL-1 and 46.67% ± 0.88%, respectively, which were significantly higher than those fed the basal diet (p < 0.05). The lowest and highest percentages of lymphocytes (67.67% ± 0.33%) and neutrophils (27.33% ± 1.20%) were also obtained in fish fed 50.00 mg kg-1 NA compared to those fed the basal diet (p < 0.05). Fish receiving diet supplemented with 50.00 mg kg-1 NA revealed the highest serum activity in superoxide dismutase, catalase, glutathione peroxidase, lysozyme and alternative complement and the lowest level of total cholesterol, cortisol, aspartate aminotransferase and alanine aminotransferase than fish receiving the basal diet (p < 0.05). Serum immunoglobulin (Ig) and ACH50 contents significantly increased with increasing dietary NA supplementation to the highest values of 43.17 ± 1.46 and 293.33 ± 2.03 U mL-1, respectively, in fish fed diet supplemented with 50 mg kg-1 NA (p < 0.05). CONCLUSIONS: Supplementation of NA in rainbow trout diet at 50 mg kg-1 exhibited a positive effect on haematological parameters, antioxidant capacity and immune responses. Administration of such dosage can enhance rainbow trout immune responses against unfavourable or stressful conditions, for example disease outbreaks, hypoxic condition, thermal stress and sudden osmotic fluctuations, which usually happen in an intensive culture system.

Assessment of dietary Selenium and its role in Mercury fate in cultured fish rainbow trout with two sustainable aquafeeds.

This study enhances the current limited understanding of the interaction between mercury (Hg) and selenium (Se) species in fish. Rainbow trout (Oncorhynchus mykiss), a model aquaculture fish, was exposed to Hg and Se species through controlled dietary conditions. Over a 6-month feeding trial, the impact of dietary Se on Hg bioaccumulation in fish, including flesh, brain, and liver, was tracked. Twelve dietary conditions were tested, including plant-based diets (0.25 µgSe g-1) and tuna byproduct diets (0.25 µgHg g-1, 8.0 µgSe g-1) enriched with methylmercury and/or Se as selenite or selenomethionine. The tuna byproduct diet resulted in lower Hg levels than the plant-based diets, with muscle Hg content below the European Commission's safe threshold. This study highlights the significant impact of specific Se compounds in the diet, particularly from tuna-based aquafeed, on Hg bioaccumulation. These promising results provide a strong recommendation for future use of fisheries byproducts in sustainable aquafeeds.

Study of the influence of tributyrin-supplemented diets on the gut bacterial communities of rainbow trout (Oncorhynchus mykiss).

Dietary supplementation with triglyceride tributyrin (TBT), a butyrate precursor, has been associated with beneficial effects on fish health and improvements in the ability of carnivorous fish to tolerate higher levels of plant-based protein. In this study, we aimed to investigate the effects of a plant-based diet supplemented with TBT on the structural diversity and putative function of the digesta-associated bacterial communities of rainbow trout (Oncorhynchus mykiss). In addition to this, we also assessed the response of fish gut digestive enzyme activities and chyme metabolic profile in response to TBT supplementation. Our results indicated that TBT had no significant effects on the overall fish gut bacterial communities, digestive enzyme activities or metabolic profile when compared with non-supplemented controls. However, a more in-depth analysis into the most abundant taxa showed that diets at the highest TBT concentrations (0.2% and 0.4%) selectively inhibited members of the Enterobacterales order and reduced the relative abundance of a bacterial population related to Klebsiella pneumoniae, a potential fish pathogen. Furthermore, the predicted functional analysis of the bacterial communities indicated that increased levels of TBT were associated with depleted KEGG pathways related to pathogenesis. The specific effects of TBT on gut bacterial communities observed here are intriguing and encourage further studies to investigate the potential of this triglyceride to promote pathogen suppression in the fish gut environment, namely in the context of aquaculture.

Maternal dietary choline levels cause transcriptome shift due to genotype-by-diet interactions in rainbow trout (Oncorhynchus mykiss).

The objective of this study was to identify metabolic regulatory mechanisms affected by choline availability in rainbow trout (Oncorhynchus mykiss) broodstock diets associated with increased offspring growth performance. Three customized diets were formulated to have different levels of choline: (a) 0 % choline supplementation (Low Choline: 2065 ppm choline), (b) 0.6 % choline supplementation (Medium Choline: 5657 ppm choline), and (c) 1.2 % choline supplementation (High Choline: 9248 ppm choline). Six all-female rainbow trout families were fed experimental diets beginning 18 months post-hatch until spawning at 22 months post-hatch; their offspring were fed a commercial diet. Experimental broodstock diet did not affect overall choline, fatty acid, or amino acid content in the oocytes (p > 0.05), apart from tyrosine (p ≤ 0.05). Offspring body weights from the High and Low Choline diets did not differ from those in the Medium Choline diet (p > 0.05); however, family-by-diet and sire-by-diet interactions on offspring growth were detected (p ≤ 0.05). The High Choline diet did not improve growth performance in the six broodstock families at final harvest (520-days post-hatch, or dph). Numerous genes associated with muscle development and lipid metabolism were identified as affected by broodstock diet, including myosin, troponin C, and fatty acid binding proteins, which were associated with key signaling pathways of lipid metabolism, muscle cell development, muscle cell proliferation, and muscle cell differentiation. These findings indicate that supplementing broodstock diets with choline does regulate expression of genes related to growth and nutrient partitioning but does not lead to growth benefits in rainbow trout families selected for disease resistance.

Traditional Chinese medicine bufalin inhibits infectious hematopoietic necrosis virus infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis and severe economic losses to salmon and trout aquaculture worldwide. Currently, the only commercial vaccine against IHNV is a DNA vaccine with some biosafety concerns. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, 1,483 compounds were screened from a traditional Chinese medicine monomer library, and bufalin showed potential antiviral activity against IHNV. The 50% cytotoxic concentration of bufalin was >20 µM, and the 50% inhibitory concentration was 0.1223 µΜ against IHNV. Bufalin showed the inhibition of diverse IHNV strains in vitro, which confirmed that it had an inhibitory effect against all IHNV strains, rather than random activity against a single strain. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization. Bufalin also inhibited IHNV infection in vivo and significantly increased the survival of rainbow trout compared with the mock drug-treated group, and this was confirmed by in vivo viral load monitoring. Our data showed that the anti-IHNV activity of bufalin was proportional to extracellular Na+ concentration and inversely proportional to extracellular K+ concentration, and bufalin may inhibit IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout. IMPORTANCE: Infectious hematopoietic necrosis virus (IHNV) is the pathogen of infectious hematopoietic necrosis (IHN) which outbreak often causes huge economic losses and hampers the healthy development of salmon and trout farming. Currently, there is only one approved DNA vaccine for IHN worldwide, but it faces some biosafety problems. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, we report that bufalin, a traditional Chinese medicine, shows potential antiviral activity against IHNV both in vitro and in vivo. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization, and bufalin inhibited IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout.

Chronic cortisol stimulation enhances hypothalamus-specific enrichment of metabolites in the rainbow trout brain.

The hypothalamus is a key integrating center that is involved in the initiation of the corticosteroid stress response, and in regulating nutrient homeostasis. Although cortisol, the principal glucocorticoid in humans and teleosts, plays a central role in feeding regulation, the mechanisms are far from clear. We tested the hypothesis that the metabolic changes to cortisol exposure signal an energy excess in the hypothalamus, leading to feeding suppression during stress in fish. Rainbow trout (Oncorhynchus mykiss) were administered a slow-release cortisol implant for 3 days, and the metabolite profiles in the plasma, hypothalamus, and the rest of the brain were assessed. Also, U-13C-glucose was injected into the hypothalamus by intracerebroventricular (ICV) route, and the metabolic fate of this energy substrate was followed in the brain regions by metabolomics. Chronic cortisol treatment reduced feed intake, and this corresponded with a downregulation of the orexigenic gene agrp, and an upregulation of the anorexigenic gene cart in the hypothalamus. The U-13C-glucose-mediated metabolite profiling indicated an enhancement of glycolytic flux and tricarboxylic acid intermediates in the rest of the brain compared with the hypothalamus. There was no effect of cortisol treatment on the phosphorylation status of AMPK or mechanistic target of rapamycin in the brain, whereas several endogenous metabolites, including leucine, citrate, and lactate were enriched in the hypothalamus, suggesting a tissue-specific metabolic shift in response to cortisol stimulation. Altogether, our results suggest that the hypothalamus-specific enrichment of leucine and the metabolic fate of this amino acid, including the generation of lipid intermediates, contribute to cortisol-mediated feeding suppression in fish.NEW & NOTEWORTHY Elevated cortisol levels during stress suppress feed intake in animals. We tested whether the feed suppression is associated with cortisol-mediated alteration in hypothalamus metabolism. The brain metabolome revealed a hypothalamus-specific metabolite profile suggesting nutrient excess. Specifically, we noted the enrichment of leucine and citrate in the hypothalamus, and the upregulation of pathways involved in leucine metabolism and fatty acid synthesis. This cortisol-mediated energy substrate repartitioning may modulate the feeding/satiety centers leading to the feeding suppression.

Dietary sulfated polysaccharides extracted from Caulerpa sp. and Padina sp. modulated physiological performance, antibacterial activity and ammonia challenge test in juvenile rainbow trout (Oncorhynchus mykiss).

Nowadays, the use of seaweed derivatives in aquaculture has drawn attention for their potential as an immunostimulant and growth promotor. The sulfated polysaccharide extracted (SPE ) from green (Caulerpa sp.; SPC) and brown (Padina sp.; SPP) seaweeds with two concentrations (0.05% and 0.1%); nominated in four groups: SPC0.05 , SPC0.1 , SPP0.05 , SPP0.1 and control group (free of SPE ) were used for juvenile rainbow trout (Oncorhynchus mykiss) diet. Fish (N: 150; 8.5 ± 0.2 g) were selected aleatory distributed in 15 circular tanks (triplicate for the group) and fed test diets for 56 days. The outcomes revealed that the supplementation of SPE up to 1 g kg-1 failed to show significant differences in the organosomatic indices as compared to the control group. The most inferior protein value of dress-out fish composition was observed in the fish fed the control diet, which was statistically lower than the SCP0.1 group (p < 0.05), while no significant difference was observed in other macronutrient composition among the treatments. Total monounsaturated fatty acid (MUFA) had lower trend in the carcass of fish fed SPE supplemented diets, so that lowest MUFA were observed in SPC0.05 group (p < 0.05; 25.22 ± 4.29%). The lowest value of docosahexaenoic acid was observed in the control diet compared to the SPE -supplemented diets (p < 0.05). The serum alternative complement pathway levels in all treatments tend to promote compared to the control treatment. A similar trend was observed for lysozyme activity. According to the results, the superoxide dismutase (SOD) value were highest in SPC0.05 and SPC0.1 compared to the other treatments (p < 0.05), while a further elevation of the SPE Padina sp. extracted level (SPP0.1 ) leads to a decrease in SOD value. Thiobarbituric acid reactive substances of plasma was indicated not to influence by sulfated polysaccharide extracts in the refrigerated storage. The lowest serum stress indicators were observed in fish fed SPP0.05 group postchallenge test. Taken together, our outcomes revealed that SPE of two species of seaweeds bestows benefits in some of the immunity and antioxidant system. Also, notable elevations in HUFA were observed in juvenile rainbow trout fed supplemented with SPE .

Dietary chestnut bee pollen as an immunostimulant for rainbow trout (Oncorhynchus mykiss): Effects on the growth, haematological values, immune response, oxidant/antioxidant status, and survival against Aeromonas salmonicida subsp. achromogenes.

The present study was designed to assess the influence of dietary supplementation with chestnut bee pollen at various levels in rainbow trout, Oncorhynchus mykiss. For two weeks feeding period, a total of 300 fish were allocated into 12 fiberglass tanks and divided into four equal groups, three replicates each, with chestnut bee pollen (BP) dietary inclusion as follows; the fish group was given a basal diet (C); fish group fed a diet supplemented with BP 1% (BP-1); fish group fed a diet supplemented with BP 2% (BP-2); and fish group fed a diet supplemented with BP 4% (BP-3). At the end of the experiment, growth, haematological values, immune status, antioxidant status, and survival rate against Aeromonas salmonicida subsp. achromogenes were evaluated. Dietary supplementation with chestnut bee pollen significantly improves growth performance. Fish fed the diets containing chestnut bee pollen had higher the haematological values than those fed the control diet. The results showed that all the immunological parameters in the groups fed with chestnut bee pollen were significantly higher when compared to the control group. Moreover, dietary chestnut bee pollen increased disease resistance against Aeromonas salmonicida subsp. achromogenes compared to the control group. The tissue SOD, CAT and GSH-Px activities of groups fed with chestnut bee pollen significantly enhanced when compared with the control groups. In contrast, the tissue MDA levels in all groups fed with chestnut bee pollen were significantly decreased. The best values for the antioxidant parameters were determined in the groups fed with 2 and 4% of chestnut bee pollen. Overall, these findings suggest that dietary chestnut bee pollen enhances the growth, the haematological values, the immune and antioxidant response and increases disease resistance against rainbow trout.

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.

L-methionine supplementation modulates IgM+ B cell responses in rainbow trout.

The interest in dietary amino acids (AAs) as potential immunomodulators has been growing the recent years, since specific AAs are known to regulate key metabolic pathways of the immune response or increase the synthesis of some immune-related proteins. Methionine, tryptophan and lysine are among the ten essential AAs for fish, meaning that they cannot be produced endogenously and must be provided through the diet. To date, although dietary supplementation of fish with some of these AAs has been shown to have positive effects on some innate immune parameters and disease resistance, the effects that these AAs provoke on cells of the adaptive immune system remained unexplored. Hence, in the current study, we have investigated the effects of these three AAs on the functionality of rainbow trout (Oncorhynchus mykiss) IgM+ B cells. For this, splenic leukocytes were isolated from untreated adult rainbow trout and incubated in culture media additionally supplemented with different doses of methionine, tryptophan or lysine in the presence or absence of the model antigen TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The survival, IgM secreting capacity and proliferation of IgM+ B cells was then studied. In the case of methionine, the phagocytic capacity of IgM+ B cells was also determined. Our results demonstrate that methionine supplementation significantly increases the proliferative effects provoked by TNP-LPS and also up-regulates the number of cells secreting IgM, whereas tryptophan or lysine have either minor or even negative effects on rainbow trout IgM+ B cells. This increase in the number of IgM-secreting cells in response to methionine surplus was further verified in a feeding experiment, in which the beneficial effects of methionine on the specific response to anal immunization were also confirmed. The results presented demonstrate the beneficial effects of dietary supplementation with methionine on the adaptive immune responses of fish.

Effects of plant-derived protein and rapeseed oil on growth performance and gut microbiomes in rainbow trout.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is becoming popular with the increased demand for fish protein. However, the limited resources and expense of fish meal and oil have become restrictive factors for the development of the rainbow trout related industry. To solve this problem, plant-derived proteins and vegetable oils have been developed as alternative resources. The present study focuses on evaluating the effects of two experimental diets, FMR (fish meal replaced with plant-derived protein) and FOR (fish oil replaced with rapeseed oil), through the alteration of the gut microbiota in triploid rainbow trout. The commercial diet was used in the control group (FOM). RESULTS: Amplicon sequencing of the 16S and 18S rRNA genes was used to assess the changes in gut bacteria and fungi. Our analysis suggested that the α-diversity of both bacteria and fungi decreased significantly in the FMR and FOR groups, and ß-diversity was distinct between FOM/FMR and FOM/FOR based on principal coordinate analysis (PCoA). The abundance of the Planctomycetota phylum increased significantly in the FMR group, while that of Firmicutes and Bacteroidetes decreased. We also found that the fungal phylum Ascomycota was significantly increased in the FMR and FOR groups. At the genus level, we found that the abundance of Citrobacter was the lowest and that of pathogenic Schlesneria, Brevundimonas, and Mycoplasma was highest in the FMR and FOR groups. Meanwhile, the pathogenic fungal genera Verticillium and Aspergillus were highest in the FMR and FOR groups. Furthermore, canonical correspondence analysis (CCA) and network analysis suggested that the relatively low-abundance genera, including the beneficial bacteria Methylobacterium, Enterococcus, Clostridium, Exiguobacterium, Sphingomonas and Bacteroides and the fungi Papiliotrema, Preussia, and Stachybotrys, were positively correlated with plant protein or rapeseed oil. There were more modules that had the above beneficial genera as the hub nodes in the FMR and FOR groups. CONCLUSIONS: Our study suggested that the FMR and FOR diets could affect the gut microbiome in rainbow trout, which might offset the effects of the dominant and pathogenic microbial genera. This could be the underlying mechanism of explaining why no significant difference was observed in body weight between the different groups.

RNA-Seq analysis of the pyloric caecum, liver, and muscle reveals molecular mechanisms regulating fillet color in rainbow trout.

BACKGROUND: The characteristic pink-reddish color in the salmonids fillet is an important, appealing quality trait for consumers and producers. The color results from diet supplementation with carotenoids, which accounts for up to 20-30% of the feed cost. Pigment retention in the muscle is a highly variable phenotype. In this study, we aimed to understand the molecular basis for the variation in fillet color when rainbow trout (Oncorhynchus mykiss) fish families were fed an Astaxanthin-supplemented diet. We used RNA-Seq to study the transcriptome profile in the pyloric caecum, liver, and muscle from fish families with pink-reddish fillet coloration (red) versus those with lighter pale coloration (white). RESULTS: More DEGs were identified in the muscle (5,148) and liver (3,180) than in the pyloric caecum (272). Genes involved in lipid/carotenoid metabolism and transport, ribosomal activities, mitochondrial functions, and stress homeostasis were uniquely enriched in the muscle and liver. For instance, the two beta carotene genes (BCO1 and BCO2) were significantly under-represented in the muscle of the red fillet group favoring more carotenoid retention. Enriched genes in the pyloric caecum were involved in intestinal absorption and transport of carotenoids and lipids. In addition, the analysis revealed the modulation of several genes with immune functions in the pyloric caecum, liver, and muscle. CONCLUSION: The results from this study deepen our understanding of carotenoid dynamics in rainbow trout and can guide us on strategies to improve Astaxanthin retention in the rainbow trout fillet.

The effects of dietary microbial 6-phytase on growth parameters, intestinal morphometric properties and selected intestinal genes expression in rainbow trout (Oncorhynchus mykiss, Walbaum 1876).

This study investigated the effects of dietary 6-phytase, produced by a genetically modified Komagataella phaffii, on growth performance, feed utilisation, flesh quality, villus morphometric properties, and intestinal mRNA expression in rainbow trout. Six iso-nitrogenous, iso-lipidic, and iso-caloric diets were formulated and fed to triplicate groups of juvenile rainbow trout weighing 32.57 ± 0.36 g (mean ± SD) for 90 days. The dietary treatments included two positive controls (PC), one formulated with 400 g/kg of fish meal named T1, and the other formulated with 170 g/kg of fish meal plus 1% avP derived from monocalcium phosphate named T2. The remaining dietary treatments consisted of a negative control (NC) formulated with 170 g/kg of fish meal (T3), NC+ 750, NC+ 1500, and NC+ 3000 OTU/kg levels of phytase designated as T4, T5, and T6 diets respectively. Compared to T1, weight gain (WG) increased by 16.29, 13.71 and 11.66% in T4, T5 and T6, respectively (p < 0.05). Feed conversion ratio (FCR) was lowered by 3.2 and 0.8% in T4 and T5 compared to T1 (p < 0.05). WG, feed intake (FI), FCR, final body length, bone ash, bone ash P, and intestinal morphometry were negatively affected in T3 fed fish (p < 0.05). Whole-body fish nutrient, bone ash, bone ash phosphorus (P) compositions and mucosal villus morphometric properties improved in rainbow trout fed diets supplemented with phytase dose ranging from 750-3000 OTU. Bone ash increased by 6.12% in T5 compared to T1 (p < 0.05). Phytase inclusion enhanced the profitability of feeding juvenile rainbow trout such diets as it reduced the feed price and economic conversion rate. Dietary inclusion of phytase down-regulated mRNA expression of genes responsible for fatty acid synthesis and lipogenesis in juvenile rainbow trout. Dietary phytase up-regulated the mRNA expression of genes (SLC4A11 and ATP1A3A) responsible for nutrient uptake and down-regulated intestinal expression of MUCIN 5AC-like genes (mucus secreting genes) in juvenile rainbow trout. Along with improving performance parameters, the inclusion of phytase in rainbow trout diet containing plant-based protein sources, can preserve intestinal morphology by regulating the mRNA expression of genes responsible for fatty acid synthesis, lipogenesis and nutrient uptake and transport.

The synergistic effects of four medicinal plant seeds and chelated minerals on the growth, immunity, and antioxidant capacity of rainbow trout (Oncorhynchus mykiss).

Trace minerals are essential for the normal functioning of all living organisms. In addition, the positive effects of several medicinal plants have been demonstrated in aquaculture. In the present study, we aimed to investigate the effects of a mixture of medicinal plants and test the synergistic effects of medicinal plants and chelated minerals on fish growth and immunity. Thus, in the present experiment we evaluated the combined effects of a commercial chelated mineral source (BonzaFish®) and a mixture of 4 medicinal plants including caraway (Carum carvi), green cumin (Cuminum cyminum), dill (Anethum graveolens), and anise (Pimpinella anisum). Rainbow trout (Oncorhynchus mykiss) fingerlings (n = 225) were fed with five formulated diets, including a control diet (basal diet), Bonza (basal diet + 1 g/kg BonzaFish®), Z-5 (basal diet + 1 g/kg BonzaFish® + 5 g/kg mixture of plant seeds), Z-10 (basal diet + 1 g/kg BonzaFish®+10 g/kg mixture of plant seeds), Z-20 (basal diet+1 g/kg BonzaFish®+20 g/kg mixture of plant seeds) for 6 weeks. In diets including BonzaFish®, 50% of the inorganic mineral premix was replaced by BonzaFish®. Results revealed that fish receiving the Z-20 diet showed the best performance in terms of growth parameters, followed by the Bonza treatment (P < 0.05). The highest protease activity was found in Z-5 and Z-10. RBCs were highest in Z-5, while the highest WBCs and hemoglobin were found in the Bonza treatment followed by Z-20. Stress biomarkers were lowest in the Z-20 treatment. Results showed that Z-20 could elicit the most robust immunological responses of lysozyme activity, ACH50, total Ig, C3, and C4. In conclusion, chelated minerals could be successfully used to replace 50% of mineral premix with no negative impacts on fish growth and together with four medicinal plants, could enhance rainbow trout overall growth performance and immunity.

Expanding non-invasive approaches for fish-health monitoring: A survey of the epidermal mucous metabolomes of phylogenetically diverse freshwater fish species.

There is a pressing need for more-holistic approaches to fisheries assessments along with growing demand to reduce the health impacts of sample collections. Metabolomic tools enable the use of sample matrices that can be collected with minimal impact on the organism (e.g., blood, urine, and mucus) and provide high-throughput, untargeted biochemical information without the requirement of a sequenced genome. These qualities make metabolomics ideal for monitoring a wide range of fish species, particularly those under protected status. In the current study, we surveyed the relative abundances of 120 endogenous metabolites in epidermal mucus across eight freshwater fish species belonging to seven phylogenetic orders. Principal component analysis was used to provide an overview of the data set, revealing strong interspecies relationships in the epidermal mucous metabolome. Normalized relative abundances of individual endogenous metabolites were then used to identify commonalities across multiple species, as well as those metabolites that showed notable species specificity. For example, taurine was measured in high relative abundance in the epidermal mucus of common carp (Cyprinus carpio), northern pike (Esox lucius), golden shiner (Notemigonus crysoleucas), rainbow trout (Oncorhynchus mykiss), and rainbow smelt (Osmerus mordax), whereas γ-amino butyric acid (GABA) exhibited a uniquely high relative abundance in flathead catfish (Pylodictis olivaris). Finally, hierarchical cluster analysis was used to evaluate species relatedness as characterized by both the epidermal mucous metabolome (phenotype) and genetic phylogeny (genotype). This comparison revealed species for which relatedness in the epidermal mucous metabolome composition closely aligns with phylogenetic relatedness (e.g., N. crysoleucas and C. carpio), as well as species for which these two measures are not well aligned (e.g., P. olivaris and Polyodon spathula). These, and other findings reported here, highlight novel areas for future research with fish, including development of epidermal mucous-based markers for non-invasive health monitoring, sex determination, and hypoxia tolerance.

High immunogenicity of virus-like particles (VLPs) decorated with Aeromonas salmonicida VapA antigen in rainbow trout.

The Gram-negative bacterium A. salmonicida is the causal agent of furunculosis and used to be one of the most loss-causing bacterial infections in the salmonid aquaculture industry with a mortality rate of about 90% until the 1990s, when an inactivated vaccine with mineral oil as adjuvant was successfully implemented to control the disease. However, the use of this vaccine is associated with inflammatory side effects in the peritoneal cavity as well as autoimmune reactions in Atlantic salmon, and incomplete protection has been reported in rainbow trout. We here aimed at developing and testing a recombinant alternative vaccine based on virus-like particles (VLPs) decorated with VapA, the key structural surface protein in the outer A-layer of A. salmonicida. The VLP carrier was based on either the capsid protein of a fish nodavirus, namely red grouper nervous necrotic virus (RGNNV) or the capsid protein of Acinetobacter phage AP205. The VapA and capsid proteins were expressed individually in E. coli and VapA was fused to auto-assembled VLPs using the SpyTag/SpyCatcher technology. Rainbow trout were vaccinated/immunized with the VapA-VLP vaccines by intraperitoneal injection and were challenged with A. salmonicida 7 weeks later. The VLP vaccines provided protection comparable to that of a bacterin-based vaccine and antibody response analysis demonstrated that vaccinated fish mounted a strong VapA-specific antibody response. To our knowledge, this is the first demonstration of the potential use of antigen-decorated VLPs for vaccination against a bacterial disease in salmonids.

Genetic architecture of acute hyperthermia resistance in juvenile rainbow trout (Oncorhynchus mykiss) and genetic correlations with production traits.

BACKGROUND: Selective breeding is a promising solution to reduce the vulnerability of fish farms to heat waves, which are predicted to increase in intensity and frequency. However, limited information about the genetic architecture of acute hyperthermia resistance in fish is available. Two batches of sibs from a rainbow trout commercial line were produced: the first (N = 1382) was phenotyped for acute hyperthermia resistance at nine months of age and the second (N = 1506) was phenotyped for main production traits (growth, body length, muscle fat content and carcass yield) at 20 months of age. Fish were genotyped on a 57 K single nucleotide polymorphism (SNP) array and their genotypes were imputed to high-density based on the parent's genotypes from a 665 K SNP array. RESULTS: The heritability estimate of resistance to acute hyperthermia was 0.29 ± 0.05, confirming the potential of selective breeding for this trait. Since genetic correlations of acute hyperthermia resistance with the main production traits near harvest age were all close to zero, selecting for acute hyperthermia resistance should not impact the main production traits, and vice-versa. A genome-wide association study revealed that resistance to acute hyperthermia is a highly polygenic trait, with six quantitative trait loci (QTL) detected, but explaining less than 5% of the genetic variance. Two of these QTL, including the most significant one, may explain differences in acute hyperthermia resistance across INRAE isogenic lines of rainbow trout. Differences in mean acute hyperthermia resistance phenotypes between homozygotes at the most significant SNP was 69% of the phenotypic standard deviation, showing promising potential for marker-assisted selection. We identified 89 candidate genes within the QTL regions, among which the most convincing functional candidates are dnajc7, hsp70b, nkiras2, cdk12, phb, fkbp10, ddx5, cygb1, enpp7, pdhx and acly. CONCLUSIONS: This study provides valuable insight into the genetic architecture of acute hyperthermia resistance in juvenile rainbow trout. We show that the selection potential for this trait is substantial and selection for this trait should not be too detrimental to improvement of other traits of interest. Identified functional candidate genes provide new knowledge on the physiological mechanisms involved in acute hyperthermia resistance, such as protein chaperoning, oxidative stress response, homeostasis maintenance and cell survival.

Dietary flaxseed (Linum usitatissimum) oil supplementation affects growth, oxidative stress, immune response, and diseases resistance in rainbow trout (Oncorhynchusmykiss).

This paper describes the effects of flaxseed (Linum usitatissimum) oil (FSO) as a feed additive on growth performance, oxidative stress, immunity, and disease resistance in rainbow trout (Oncorhynchus mykiss). Eight-hundred-and-forty rainbow trout individuals (mean weight: 25.66 ± 1.33 g) were fed with different doses of FSO (0.5, 1, and 1.5%) ad libitum two times a day for 9 weeks. At the end of the feeding, growth performance was evaluated and the fish were challenged with two different bacteria (Yersinia ruckeri and Aeromonas hydrophila). At the end of the 3rd, 6th, and 9th weeks, blood and tissue samples were taken from 9 fish per treatment to evaluate innate immune response, cytokine gene expression levels, antioxidant enzyme activities and lipid peroxidation levels, and digestive enzyme activities. Determination of haematological parameters and histological examination was also carried out to evaluate the general health status of the fish. Results showed that the final weight and specific growth rate of FSO-supplemented fish increased significantly (p < 0.05). FSO-supplemented fish showed higher resistance to Y. ruckeri than the control group (p < 0.05). However, survival rates of all groups in A. hydrophila challenge test were similar (p > 0.05). Among the investigated innate immune response parameters, the potential killing activity of phagocytes, myeloperoxidase activity, and lysozyme activity increased in the FSO-supplemented groups (p < 0.05). Almost all cytokine gene expression levels in the experimental groups up-regulated especially after 9 weeks of feeding in the head kidney and intestine (p < 0.05). Similarly, superoxide dismutase and catalase activities were found to be significantly higher in the FSO group than in the control (p < 0.05) whereas, the lipid peroxidation levels drastically declined as a result of the FSO supplementation (p < 0.05). These results suggest that FSO can improve growth, enhance immune response, and lower oxidative damage in rainbow trout when supplemented at the rates of 0.5-1.5% for 9 weeks.

Dietary effect of grape (Vitis vinifera) seed extract mitigates hepatic disorders caused by oxidized fish oil in rainbow trout (Oncorhynchus mykiss).

The major goal of this study was to determine the effect of grape seed extract (GSE) on liver damage in rainbow trout (Oncorhynchus mykiss) that was caused by the consumption of dietary oxidized fish oil (OFO). Rainbow trout were fed six different experimental diets coded OX-GSE 0 (OFO diet), OX-GSE 1 (OFO and 0.1% GSE), OX-GSE 3 (OFO and 0.3% GSE), GSE 0 (fresh fish oil and 0.0% GSE), GSE 1 (fresh fish oil and 0.1% GSE), and GSE 3 (fresh fish oil and 0.3% GSE) for 30 days. The lowest % hepatosomatic index (HSI) result was calculated in fish fed with OX-GSE 0 and the highest HSI was determined in fish fed with GSE 1 diets (p < 0.05). Histopathologically, hydropic degeneration in hepatocytes significantly increased OX-GSE 0 and GSE 3 compared to GSE 1 diets (p < 0.05). Deposition of lipid droplets in hepatocytes was significantly increased in OX-GSE 0 and OX-GSE 3 groups than others (p < 0.05). Liver biochemistry parameters such as superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) were significantly affected by OX and GSE treatments (p < 0.05). There were significant differences in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) among the liver enzymes analyzed in serum in OX and GSE (p < 0.05), meanwhile no difference was observed in lactate dehydrogenase (LDH) values between groups (p > 0.05). In conclusion, liver biochemistry and histopathology of rainbow trout consuming diets containing oxidized fish oil were negatively affected. However, it was determined that the supplementation of 0.1% GSE to the diet had a significant ameliorative role in these adverse effects.

Oral administration of wild plant-derived minerals and red ginseng ameliorates insulin resistance in fish through different pathways.

Many kinds of fish are characterized by a limited efficiency to use carbohydrates. For this reason, raw fish and mixed feed containing a lot of fish meal have been used as feed for fish farming. However, continuing to use high-protein diets not only increases the cost of fish farming, but may also fuel animal protein shortages. Furthermore, carbohydrates are added to improve the texture of the feed and act as a binding agent and are usually contained at 20% in the feed. It makes sense, therefore, to find ways to make good use of carbohydrates rather than wasting them. The physiological mechanisms of glucose intolerance in fish are not yet well understood. Therefore, we investigated the glucose utilization of fish, omnivorous goldfish Carassius auratus and carnivorous rainbow trout Oncorhynchus mykiss. Furthermore, the effects of oral administration of wild plant-derived minerals and red ginseng on the glucose utilization in these fish muscle cells were investigated. As a result, we found the following. (1) An extremely high insulin resistance in fish muscle and the symptom was more pronounced in carnivorous rainbow trout. (2) Administration of wild plant-derived minerals promotes the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface of white muscle via activation of the PI3 kinase axis, whereas administration of red ginseng not only promotes GLUT4 transfer and translocation to the cell surface of white muscle via AMPK activation as well as promoting glucose uptake into muscle cells via a pathway separate from the insulin signaling system. (3) In fish, at least goldfish and rainbow trout, both PI3K/Akt and AMPK signaling cascades exist to promote glucose uptake into muscle cells, as in mammals.