In vitro fish mucosal surfaces producing mucin as a model for studying host-pathogen interactions.

Current prophylactic and disease control measures in aquaculture highlight the need of alternative strategies to prevent disease and reduce antibiotic use. Mucus covered mucosal surfaces are the first barriers pathogens encounter. Mucus, which is mainly composed of highly glycosylated mucins, has the potential to contribute to disease prevention if we can strengthen this barrier. Therefore, aim of this study was to develop and characterize fish in vitro mucosal surface models based on commercially available cell lines that are functionally relevant for studies on mucin regulation and host-pathogen interactions. The rainbow trout (Oncorhynchus mykiss) gill epithelial cell line RTgill-W1 and the embryonic cell line from Chinook salmon (Oncorhynchus tshawytscha) CHSE-214 were grown on polycarbonate membrane inserts and chemically treated to differentiate the cells into mucus producing cells. RTGill-W1 and CHSE-214 formed an adherent layer at two weeks post-confluence, which further responded to treatment with the γ-secretase inhibitor DAPT and prolonged culture by increasing the mucin production. Mucins were metabolically labelled with N-azidoacetylgalactosamine 6 h post addition to the in vitro membranes. The level of incorporated label was relatively similar between membranes based on RTgill-W1, while larger interindividual variation was observed among the CHSE in vitro membranes. Furthermore, O-glycomics of RTgill-W1 cell lysates identified three sialylated O-glycans, namely Galß1-3(NeuAcα2-6)GalNAcol, NeuAcα-Galß1-3GalNAcol and NeuAcα-Galß1-3(NeuAcα2-6)GalNAcol, resembling the glycosylation present in rainbow trout gill mucin. These glycans were also present in CHSE-214. Additionally, we demonstrated binding of the fish pathogen A. salmonicida to RTgill-W1 and CHSE-214 cell lysates. Thus, these models have similarities to in vivo mucosal surfaces and can be used to investigate the effect of pathogens and modulatory components on mucin production.

Characterization of the rainbow trout (Oncorhynchus mykiss) mucosal glycosphingolipid repertoire and Aeromonas salmonicida binding to neutral glycosphingolipids.

Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.

Pharmacokinetics, bioavailability and plasma protein binding of tolfenamic acid in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Although research on the mechanism and control of pain and inflammation in fish has increased in recent years, the use of analgesic drugs is limited due to the lack of pharmacological information about analgesic drugs. Tolfenamic acid is a non-steroidal anti-inflammatory drug and can be used in fish due to its low side effect profile and superior pharmacokinetic properties. OBJECTIVES: The pharmacokinetics, bioavailability and plasma protein binding of tolfenamic acid were investigated following single intravascular (IV), intramuscular (IM) and oral administration of 2 mg/kg in rainbow trout at 13 ± 0.5°C. METHODS: The experiment was carried out on a total of 234 rainbow trout (Oncorhynchus mykiss). Tolfenamic acid was administered to fish via IV, IM and oral route at a dose of 2 mg/kg. Blood samples were taken at 13 different sampling times until the 72 h after drug administration. The plasma concentrations of tolfenamic acid were quantified using high pressure liquid chromatography-ultraviolet (UV) and pharmacokinetic parameters were assessed using non-compartmental analysis. RESULTS: The elimination half-life (t1/2ʎz) of tolfenamic acid for IV, IM and oral routes was 3.47, 6.75 and 9.19 h, respectively. For the IV route, the volume of distribution at a steady state and total body clearance of tolfenamic acid were 0.09 L/kg and 0.03 L/h/kg, respectively. The peak plasma concentration and bioavailability for IM and oral administration were 8.82 and 1.24 µg/mL, and 78.45% and 21.48%, respectively. The mean plasma protein binding ratio of tolfenamic acid in rainbow trout was 99.48% and was not concentration dependent. CONCLUSIONS: While IM route, which exhibits both the high plasma concentration and bioavailability, can be used in rainbow trout, oral route is not recommended due to low plasma concentration and bioavailability. However, there is a need to demonstrate the pharmacodynamic activity of tolfenamic acid in rainbow trout.

Transcriptomic and Epigenomic Responses to Cortisol-Mediated Stress in Rainbow Trout (Oncorhynchus mykiss) Skeletal Muscle.

The production and release of cortisol during stress responses are key regulators of growth in teleosts. Understanding the molecular responses to cortisol is crucial for the sustainable farming of rainbow trout (Oncorhynchus mykiss) and other salmonid species. While several studies have explored the genomic and non-genomic impacts of cortisol on fish growth and skeletal muscle development, the long-term effects driven by epigenetic mechanisms, such as cortisol-induced DNA methylation, remain unexplored. In this study, we analyzed the transcriptome and genome-wide DNA methylation in the skeletal muscle of rainbow trout seven days after cortisol administration. We identified 550 differentially expressed genes (DEGs) by RNA-seq and 9059 differentially methylated genes (DMGs) via whole-genome bisulfite sequencing (WGBS) analysis. KEGG enrichment analysis showed that cortisol modulates the differential expression of genes associated with nucleotide metabolism, ECM-receptor interaction, and the regulation of actin cytoskeleton pathways. Similarly, cortisol induced the differential methylation of genes associated with focal adhesion, adrenergic signaling in cardiomyocytes, and Wnt signaling. Through integrative analyses, we determined that 126 genes showed a negative correlation between up-regulated expression and down-regulated methylation. KEGG enrichment analysis of these genes indicated participation in ECM-receptor interaction, regulation of actin cytoskeleton, and focal adhesion. Using RT-qPCR, we confirmed the differential expression of lamb3, itga6, limk2, itgb4, capn2, and thbs1. This study revealed for the first time the molecular responses of skeletal muscle to cortisol at the transcriptomic and whole-genome DNA methylation levels in rainbow trout.

Activin Signaling Pathway Specialization During Embryonic and Skeletal Muscle Development in Rainbow Trout (Oncorhynchus mykiss).

Activin signaling is essential for proper embryonic, skeletal muscle, and reproductive development. Duplication of the pathway in teleost fish has enabled diversification of gene function across the pathway but how gene duplication influences the function of activin signaling in non-mammalian species is poorly understood. Full characterization of activin receptor signaling pathway expression was performed across embryonic development and during early skeletal muscle growth in rainbow trout (RBT, Oncorhynchus mykiss). Rainbow trout are a model salmonid species that have undergone two additional rounds of whole genome duplication. A small number of genes were expressed early in development and most genes increased expression throughout development. There was limited expression of activin Ab in RBT embryos despite these genes exhibiting significantly elevated expression in post-hatch skeletal muscle. CRISPR editing of the activin Aa1 ohnolog and subsequent production of meiotic gynogenetic offspring revealed that biallelic disruption of activin Aa1 did not result in developmental defects, as occurs with knockout of activin A in mammals. The majority of gynogenetic offspring exhibited homozygous activin Aa1 genotypes (wild type, in-frame, or frameshift) derived from the mosaic founder female. The research identifies mechanisms of specialization among the duplicated activin ohnologs across embryonic development and during periods of high muscle growth in larval and juvenile fish. The knowledge gained provides insights into potential viable gene-targeting approaches for engineering the activin receptor signaling pathway and establishes the feasibility of employing meiotic gynogenesis as a tool for producing homozygous F1 genome-edited fish for species with long-generation times, such as salmonids.

Quality Assessment of Fish Oil Obtained after Enzymatic Hydrolysis of a Mixture of Rainbow Trout (Oncorhynchus mykiss) and Atlantic Salmon (Salmo salar) Rest Raw Material Pretreated by High Pressure.

Utilization of fish rest raw material for fish oil extraction has received interest with the increasing demand for sustainable food sources. Enzymatic hydrolysis is an efficient method for the extraction of value-added compounds, but its effectiveness may be enhanced by high-pressure processing (HPP). However, HPP can induce lipid oxidation, affecting the quality of the oil. This study aimed to evaluate the quality of fish oil obtained after enzymatic hydrolysis of a mixture of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) rest raw material pretreated by HPP. Six pretreatments were tested prior to enzymatic hydrolysis; 200 MPa × 4 min, 200 MPa × 8 min, 400 MPa × 4 min, 400 MPa × 8 min, 600 MPa × 4 min, and 600 MPa × 8 min. The oil samples were analyzed for lipid oxidation parameters, free fatty acid content, fatty acid composition, and color changes over 8 weeks. The results confirmed that HPP may induce lipid oxidation and revealed significant influence of HPP parameters on lipid oxidation, with higher pressures leading to increased oxidation. Fatty acid composition varied among samples, but it was not substantially affected by HPP.

Constitutive Neurogenesis and Neuronal Plasticity in the Adult Cerebellum and Brainstem of Rainbow Trout, Oncorhynchus mykiss.

The central nervous system of Pacific salmon retains signs of embryonic structure throughout life and a large number of neuroepithelial neural stem cells (NSCs) in the proliferative areas of the brain, in particular. However, the adult nervous system and neurogenesis studies on rainbow trout, Oncorhynchus mykiss, are limited. Here, we studied the localization of glutamine synthetase (GS), vimentin (Vim), and nestin (Nes), as well as the neurons formed in the postembryonic period, labeled with doublecortin (DC), under conditions of homeostatic growth in adult cerebellum and brainstem of Oncorhynchus mykiss using immunohistochemical methods and Western Immunoblotting. We observed that the distribution of vimentin (Vim), nestin (Nes), and glutamine synthetase (GS), which are found in the aNSPCs of both embryonic types (neuroepithelial cells) and in the adult type (radial glia) in the cerebellum and the brainstem of trout, has certain features. Populations of the adult neural stem/progenitor cells (aNSPCs) expressing GS, Vim, and Nes have different morphologies, localizations, and patterns of cluster formation in the trout cerebellum and brainstem, which indicates the morphological and, obviously, functional heterogeneity of these cells. Immunolabeling of PCNA revealed areas in the cerebellum and brainstem of rainbow trout containing proliferating cells which coincide with areas expressing Vim, Nes, and GS. Double immunolabeling revealed the PCNA/GS PCNA/Vim coexpression patterns in the neuroepithelial-type cells in the PVZ of the brainstem. PCNA/GS coexpression in the RG was detected in the submarginal zone of the brainstem. The results of immunohistochemical study of the DC distribution in the cerebellum and brainstem of trout have showed a high level of expression of this marker in various cell populations. This may indicate: (i) high production of the adult-born neurons in the cerebellum and brainstem of adult trout, (ii) high plasticity of neurons in the cerebellum and brainstem of trout. We assume that the source of new cells in the trout brain, along with PVZ and SMZ, containing proliferating cells, may be local neurogenic niches containing the PCNA-positive and silent (PCNA-negative), but expressing NSC markers, cells. The identification of cells expressing DC, Vim, and Nes in the IX-X cranial nerve nuclei of trout was carried out.

Generation of Porcine and Rainbow Trout 3D Intestinal Models and Their Use to Investigate Astaxanthin Effects In Vitro.

Astaxanthin (AST) is a natural compound derived from shellfish, microorganisms, and algae, with several healthy properties. For this reason, it is widely used in the diet of humans and animals, such as pigs, broilers, and fish, where its addition is related to its pigmenting properties. Moreover, AST's ability to reduce free radicals and protect cells from oxidative damage finds application during the weaning period, when piglets are exposed to several stressors. To better elucidate the mechanisms involved, here we generate ad hoc pig and rainbow trout in vitro platforms able to mimic the intestinal mucosa. The morphology is validated through histological and molecular analysis, while functional properties of the newly generated intestinal barriers, both in porcine and rainbow trout models, are demonstrated by measuring trans-epithelial electrical resistance and analyzing permeability with fluorescein isothiocyanate-dextran. Exposure to AST induced a significant upregulation of antioxidative stress markers and a reduction in the transcription of inflammation-related interleukins. Altogether, the present findings demonstrate AST's ability to interact with the molecular pathways controlling oxidative stress and inflammation both in the porcine and rainbow trout species and suggest AST's positive role in prevention and health.

Valine administration in the hypothalamus alters the brain and plasma metabolome in rainbow trout.

Central administration of valine has been shown to cause hyperphagia in fish. Although mechanistic target of rapamycin (mTOR) is involved in this response, the contributions to feed intake of central and peripheral metabolite changes due to excess valine are unknown. Here, we investigated whether intracerebroventricular injection of valine modulates central and peripheral metabolite profiles and may provide insights into feeding response in fish. Juvenile rainbow trout (Oncorhynchus mykiss) were administered an intracerebroventricular injection of valine (10 µg·µL-1 at 1 µL·100·g-1 body wt), and the metabolite profile in plasma, hypothalamus, and rest of the brain (composing of telencephalon, optic tectum, cerebellum, and medulla oblongata) was carried out by liquid chromatography-mass spectrometry (LC/MS)-based metabolomics. Valine administration led to a spatially distinct metabolite profile at 1 h postinjection in the brain: enrichment of amino acid metabolism and energy production pathways in the rest of the brain but not in hypothalamus. This suggests a role for extrahypothalamic input in the regulation of feed intake. Also, there was enrichment of several amino acids, including tyrosine, proline, valine, phenylalanine, and methionine, in plasma in response to valine. Changes in liver transcript abundance and protein expression reflect an increased metabolic capacity, including energy production from glucose and fatty acids, and a lower protein kinase B (Akt) phosphorylation in the valine group. Altogether, valine intracerebroventricular administration affects central and peripheral metabolism in rainbow trout, and we propose a role for the altered metabolite profile in modulating the feeding response to this branched-chain amino acid.NEW & NOTEWORTHY Valine causes hyperphagia in fish when it is centrally administered; however, the exact mechanisms are far from clear. We tested how intracerebroventricular injection of valine in rainbow trout affected the brain and plasma metabolome. The metabolite changes in response to valine were more evident in the rest of the brain compared with the hypothalamus. Furthermore, we demonstrated for the first time that central valine administration affects peripheral metabolism in rainbow trout.

Interactions of binary mixtures of metals on rainbow trout (Oncorhynchus mykiss) heart mitochondrial H2O2 homeodynamics.

For continuous pumping of blood, the heart needs a constant supply of energy (ATP) that is primarily met via oxidative phosphorylation in the mitochondria of cardiomyocytes. However, sustained high rates of electron transport for energy conversion redox reactions predisposes the heart to the production of reactive oxygen species (ROS) and oxidative stress. Mitochondrial ROS are fundamental drivers of responses to environmental stressors including metals but knowledge of how combinations of metals alter mitochondrial ROS homeodynamics remains sparse. We explored the effects and interactions of binary mixtures of copper (Cu), cadmium (Cd), and zinc (Zn), metals that are common contaminants of aquatic systems, on ROS (hydrogen peroxide, H2O2) homeodynamics in rainbow trout (Oncorhynchus mykiss) heart mitochondria. Isolated mitochondria were energized with glutamate-malate or succinate and exposed to a range of concentrations of the metals singly and in equimolar binary concentrations. Speciation analysis revealed that Cu was highly complexed by glutamate or Tris resulting in Cu2+ concentrations in the picomolar to nanomolar range. The concentration of Cd2+ was 7.2-7.5 % of the total while Zn2+ was 15 % and 21 % of the total during glutamate-malate and succinate oxidation, respectively. The concentration-effect relationships for Cu and Cd on mitochondrial H2O2 emission depended on the substrate while those for Zn were similar during glutamate-malate and succinate oxidation. Cu + Zn and Cu + Cd mixtures exhibited antagonistic interactions wherein Cu reduced the effects of both Cd and Zn, suggesting that Cu can mitigate oxidative distress caused by Cd or Zn. Binary combinations of the metals acted additively to reduce the rate constant and increase the half-life of H2O2 consumption while concomitantly suppressing thioredoxin reductase and stimulating glutathione peroxidase activities. Collectively, our study indicates that binary mixtures of Cu, Zn, and Cd act additively or antagonistically to modulate H2O2 homeodynamics in heart mitochondria.

Deep learning models with optimized fluorescence spectroscopy to advance freshness of rainbow trout predicting under nonisothermal storage conditions.

This study established long short-term memory (LSTM), convolution neural network long short-term memory (CNN_LSTM), and radial basis function neural network (RBFNN) based on optimized excitation-emission matrix (EEM) from fish eye fluid to predict freshness changes of rainbow trout under nonisothermal storage conditions. The method of residual analysis, core consistency diagnostics, and split-half analysis of parallel factor analysis was used to optimize EEM data, and two characteristic components were extracted. LSTM, CNN_LSTM, and RBFNN models based on characteristic components of EEM used to predict the freshness indices. The results demonstrated the relative errors of RBFNN models with an R2 above 0.96 and relative errors less than 10% for K-value, total viable counts, and volatile base nitrogen, which were better than those of LSTM and CNN_LSTM models. This study presents a novel approach for predicting the freshness of rainbow trout under nonisothermal storage conditions.

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.

Acute exposure and biomarkers assessment in rainbow trout exposed to selected pharmaceuticals.

Pharmaceuticals released from municipal effluents discharges pose a risk to aquatic organisms. The toxicity of 5 pharmaceuticals with distinct therapeutic actions were assessed in rainbow trout: olanzapine (antipsychotic), erythromycin (antibiotic), mycophenoate (immunosuppression), pinaverium (anti-inflammatory) and trazodone (sedative). Juveniles were exposed to these drugs for 96 h at concentrations between 64 µg/L up to 40 mg/L to reach lethality. Survival was determined and a suite of biomarkers was analyzed for drug biotransformation, oxidative stress/damage and metabolic activity at sublethal concentrations. The data revealed the following toxicity: olanzapine >trazodone>mycophenolate>pinaverium∼erythromycin based on mortality. The data also revealed that toxicity was associated to mass, pKa and hydrophobicity and the following sublethal effects: GST, LPO and DNA strand breaks. Pharmaceuticals with lower molecular weight, physiological pKa, moderate hydrophobicity, low biotransformation and DNA strand breaks were generally more toxic to fish. However, this should be considered as a general guide in identifying toxic pharmaceuticals in non-target organisms.

Dynamics of pax7 expression during development, muscle regeneration, and in vitro differentiation of satellite cells in rainbow trout (Oncorhynchus mykiss).

Essential for muscle fiber formation and hypertrophy, muscle stem cells, also called satellite cells, reside beneath the basal lamina of the muscle fiber. Satellite cells have been commonly identified by the expression of the Paired box 7 (Pax7) due to its specificity and the availability of antibodies in tetrapods. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. Based on the development of a highly sensitive in situ hybridization (RNAScope®) for pax7, we showed that pax7+ cells were detected in the undifferentiated myogenic epithelium corresponding to the dermomyotome at day 14 post-fertilization in rainbow trout. Then, from day 24, pax7+ cells gradually migrated into the deep myotome and were localized along the muscle fibers and reach their niche in satellite position of the fibres after hatching. Our results showed that 18 days after muscle injury, a large number of pax7+ cells accumulated at the wound site compared to the uninjured area. During the in vitro differentiation of satellite cells, the percentage of pax7+ cells decreased from 44% to 18% on day 7, and some differentiated cells still expressed pax7. Taken together, these results show the dynamic expression of pax7 genes and the follow-up of these muscle stem cells during the different situations of muscle fiber formation in trout.

Low Dietary Uptake Efficiencies and Biotransformation Prevent Biomagnification of Octamethylcyclotetrasiloxane (D4) and Decamethylcyclopentasiloxane (D5) in Rainbow Trout.

With octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) being considered for evaluation under the UN Stockholm Convention on Persistent Organic Pollutants, which specifically acknowledges risks of biomagnification of persistent organic pollutants in traditional foods, a study into the mechanism of the biomagnification process of D4 and D5 in Rainbow trout was conducted by combining the absorption-distribution-metabolism-excretion for bioaccumulation (ADME-B) approach to determine intestinal and somatic biotransformation rates and radiochemical analyses to identify metabolite formation. High rates of intestinal biotransformation of D4 and D5 (i.e., 2.1 (0.70 SE) and 0.88 (0.67 SE) day-1, respectively) and metabolite formation [i.e., 52.0 (17 SD)% of D4 and 56.5% (8.2 SD)% of D5 were metabolized] were observed that caused low dietary uptake efficiencies of D4 and D5 in fish of 15.5 (2.9 SE)% and 21.0 (6.5 SE)% and biomagnification factors of 0.44 (0.08 SE) for D4 and 0.78 (0.24 SE) kg-lipid·kg-lipid-1 for D5. Bioaccumulation profiles indicated little effect of growth dilution on the bioaccumulation of D4 and D5 in fish and were substantially different from those of PCB153. The study highlights the importance of intestinal biotransformation in negating biomagnification of substances in organisms and explains differences between laboratory tests and field observations of bioaccumulation of D4 and D5.

Effects of swimming activity and feed restriction on antioxidant and digestive enzymes in juvenile rainbow trout: Implications for nutritional and exercise strategies in aquaculture.

BACKGROUND: In this study, we investigated the effects of swimming activity and feed restriction on digestion and antioxidant enzyme activities in juvenile rainbow trout (average body weight of 26.54 ± 0.36 g). METHODS: The stomach, liver and kidney tissues were obtained from four distinct groups: the static water group (fish were kept in static water and fed to satiation), the feeding restricted group (fish were kept in static water with a 25% feed restriction), the swimming exercised group (fish were forced to swimming at a flow rate of 1 Body Length per second (BL/s)) and the swimming exercised-feed restricted group (subjected to swimming exercise at a 1 BL/s flow rate along with a 25% feed restriction). We determined the levels of glutathione, lipid peroxidation and the activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase, as well as the presence of reactive oxygen species in the tissues obtained from the fish. Additionally, the activities of pepsin, protease, lipase and arginase in these tissues were measured. RESULTS: Swimming activity and feed restriction showed different effects on the enzyme activities of the fish in the experimental groups. CONCLUSION: It can be concluded that proper nutrition and exercise positively influence the antioxidant system and enzyme activities in fish, reducing the formation of free radicals. This situation is likely to contribute to the fish's development.

Developing rainbow trout (Oncorhynchus mykiss) lose branchial plasma accessible carbonic anhydrase expression with hatch and the transition to pH-sensitive, adult hemoglobin polymorphs.

Salmonids possess a unique respiratory system comprised of three major components: highly pH-sensitive hemoglobins, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma accessible carbonic anhydrase (paCA), specifically with absence of paCA at the gills. These characteristics are thought to have evolved to enhance oxygen unloading to the tissues while protecting uptake at the gills. Our knowledge of this system is detailed in adults, but little is known about it through development. Developing rainbow trout (Oncorhynchus mykiss) express embryonic RBCs containing hemoglobins that are relatively insensitive to pH; however, availability of gill paCA and RBC pHi protection is unknown. We show that pre-hatch rainbow trout express gill paCA, which is lost in correlation with the emergence of highly pH-sensitive adult hemoglobins and RBC pHi protection. Rainbow trout therefore exhibit a switch in respiratory strategy with hatch. We conclude that gill paCA likely represents an embryonic trait in rainbow trout and is constrained in adults due to their highly pH-sensitive hemoglobins.

Dietary lipid sensing through fatty acid oxidation and chylomicron formation in the gastrointestinal tract of rainbow trout.

In mammals, physiological processes related to lipid metabolism, such as chylomicron synthesis or fatty acid oxidation (FAO), modulate eating, highlighting the importance of energostatic mechanisms in feeding control. This study, using rainbow trout (Oncorhynchus mykiss) as model, aimed to characterize the role of FAO and chylomicron formation as peripheral lipid sensors potentially able to modulate feeding in fish. Fish fed with either a normal- (24%) or high- (32%) fat diet were intraperitoneally injected with water alone or containing etomoxir (inhibitor of FAO rate-limiting enzyme carnitine palmitoyl-transferase 1). First, feed intake levels were recorded. We observed an etomoxir-derived decrease in feeding at short times, but a significant increase at 48 h after treatment in fish fed normal-fat diet. Then, we evaluated putative etomoxir effects on the mRNA abundance of genes related to lipid metabolism, chylomicron synthesis and appetite-regulating peptides. Etomoxir treatment upregulated mRNA levels of genes related to chylomicron assembly in proximal intestine, while opposite effects occurred in distal intestine, indicating a clear regionalization in response. Etomoxir also modulated gastrointestinal hormone mRNAs in proximal intestine, upregulating ghrl in fish fed normal-fat diet and pyy and gcg in fish fed high-fat diet. These results provide evidence for an energostatic control of feeding related to FAO and chylomicron formation at the peripheral level in fish.

Effect of three unsaturated fatty acids on the protein oxidation and structure of myofibrillar proteins from rainbow trout (Oncorhynchus mykiss).

In this study, the impact of three unsaturated fatty acids (Oleic acid: OA, Eicosapentaenoic acid: EPA, Docosahexaenoic acid: DHA) on the oxidation and structure of rainbow trout myofibrillar protein (MP) was explored. The findings revealed a notable increase in carbonyl content (P < 0.05) and a significant decrease in total sulfhydryl content (P < 0.05) of MP with the concentration increase of the three unsaturated fatty acids. Endogenous fluorescence spectroscopy and surface hydrophobicity analyses showed that unsaturated fatty acids can cause unfolding and exposure of hydrophobic groups in MP. In addition, SDS-PAGE showed that disulfide bonds were associated with MP cross-linking and aggregate size induced by unsaturated fatty acids. Overall, three unsaturated fatty acid treatments facilitated the oxidation of myofibrillar proteins, and the extent of protein oxidation was closely associated with the concentration of unsaturated fatty acids.

Growth performance, health status, gut microbiome, and expression of immune and growth-related genes of rainbow trout (Oncorhynchus mykiss) fed diets with pea protein replacement of fish meal.

The need for fish meal constrains fish farming and significantly impacts sustainability of the aquaculture industry. Hence, it is important to investigate the use of plant-based protein sources in fish diets. The present study was conducted to determine the effects of different levels of fish meal (FM) replacement by pea protein (PP) in a 60-day feeding experiment in rainbow trout, Oncorhynchus mykiss. Effects on growth performance, body composition, hematology, serum biochemistry and immunology, and immune (TNF-α, IL1-ß and Il-8) and growth-related (GH and IGFI) gene expression were investigated. Five experimental diets (45% protein and 20% lipid) differed in replacement level of FM by PP at rates of 0% (control (PP0)), 25% (PP25), 50%(PP50), 75%(PP75) and 100%(PP100). Fish were fed with experimental diets in triplicate twice daily. The best growth performance was obtained in PP0 and PP25 groups. While fat ratios of fish fillets significantly differed (p < 0.05), there was no significant effects on protein ratios (p < 0.05). There was no significant change in the hematological values of fish, except those fed the PP100 diets, which displayed a reduction in eyrthocyte counts, hemoglobin content and hematocrit. As PP supplementation increased fish showed elevated serum glucose, total protein, cholesterol and myeloperoxidase activity and decreased glutamic pyruvic transaminase and alkaline phosphatase activity. Fish fed diets with between 25 and 75% replacement showed a decline in lactic acid bacteria in the gut. Significant increases in expression were observed in the liver of the PP25 fish relative to the 0% control for all immune and growth-related genes except for IL1-ß. These data suggest that up to 25% of FM can be replaced by PP without any adverse effects on rainbow trout.