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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.

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 (t 1/2ʎz ) 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., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The aim of this study was to reveal the effects of foodborne fluoxetine on morphological and condition profile, hematological profile, biochemical and oxidative stress indices on juvenile rainbow trout. The study was performed according to OECD Guideline No. 215. Fluoxetine was incorporated into Biomar 921 pellets at a dose of 0.047 mg/kg (environmental concentration), 0.577 mg/kg and 6.7 mg/kg. There was statistically significant change in hematological profile, including an increasing trend in neutrophil/lymphocyte ratio and a decreasing trend in the number of lymphocytes. Measurements of oxidative stress indicated decreased activity of the detoxifying enzyme glutathione-S-transferase in the liver and kidney. However, the measurement of GR, GPx, CAT, SOD activity, and TBARS showed no changes. Histopathological examination revealed damage to the proximal tubules of caudal kidney in exposed groups. This study confirms that fluoxetine has a significant effect on immune response., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Chronic elevation of circulating cortisol is known to have deleterious effects on fish, but information about the consequences of prolonged cortisol elevation on the metabolism of fish is scarce. To test the effects of chronic cortisol elevation on the aerobic performance of rainbow trout, we examined how two severities of chronically elevated plasma cortisol levels affected the oxygen uptake during rest and after exhaustive exercise using a high (HC) and a medium cortisol (MC) treatment. High cortisol doses significantly affected standard (SMR) and maximum metabolic rates (MMR) compared to control fish. In comparison, the medium cortisol treatment elevated maximum metabolic rates (MMR) but did not significantly influence SMR compared to a sham group (S) and control group (C). The medium cortisol treatment resulted in a significantly increased metabolic scope due to an elevation of MMR, an effect that was abolished in the HC group due to co-occuring elevations in SMR. The elevated SMR of the HC-treated fish could be explained by increased in vitro oxygen uptake rates (MO 2 ) of specific tissues, indicating that the raised basal metabolism was caused, in part, by an increase in oxygen demand of specific tissues. Haematological results indicated an increased reliance on anaerobic metabolic pathways in cortisol-treated fish under resting conditions., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Postoperative antinociception control in fish is currently suboptimal, as commonly used antiinflammatory drugs last for only a few hours at tested temperatures. Therefore, long-acting anti-inflammatory drugs, such as robenacoxib, could improve the welfare of fish. The pharmacokinetics, duration of antinociceptive action, and potential adverse effects of robenacoxib were evaluated through two prospective randomized blinded trials in rainbow trout ( Oncorhynchus mykiss ). Six healthy rainbow trout received a single IM administration of robenacoxib (2 mg/kg), and two control fish received the same volume of saline IM. Blood samples were collected at predetermined time points for 5 d. Plasma robenacoxib concentrations were measured using high-performance liquid chromatography-high-resolution hybrid orbitrap mass spectrometry and noncompartmental pharmacokinetic analysis. Ten additional rainbow trout received an intralabial injection of 0.05 ml of 2% acetic acid following a previously validated nociceptive model. The treated group ( n = 6) received 2 mg/kg of robenacoxib IM and the control group ( n = 4) received an equivalent volume of saline IM. The behavior, appetite, and opercular rate of the fish were evaluated every hour for 5 h, then once daily for 3 d. All 12 treated trout and 6 controls underwent histopathologic evaluation. Average maximum plasma concentration ( C max ) was 329.9 ± 137.3 ng/ml observed at 2.1 ± 0.7 h ( T max ) and terminal half-life was 12.6 ± 2.27 h. Plasma concentrations described as antinociceptive in domestic carnivores were measured for 3-4 d. This dose was associated with a significant decrease in rocking behavior ( P = 0.017). No adverse effects were detected clinically nor on histopathology. Robenacoxib administered IM at 2 mg/kg appears to be safe and may provide an antinociceptive effect in rainbow trout. This study presents a new therapeutic option to provide long-lasting antinociception in rainbow trout.

In this study, changes in the blood tissue of rainbow trout ( Oncorhynchus mykiss, Walbaum, 1792 ) caused by Fipronil (FP) insecticide were investigated using different biomarkers (Hematology parameters, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), paraoxonase (PON), arylesterase (ARE), myeleperoxidase (MPO), micronucleus (MN), 8-hydroxy-2-deoxyguanosine (8-OHdG)) level and caspase-3 activity. Statistically significant alterations in hematology parameters occurred with FP effect. In blood tissue, dose-dependent inhibition was determined in SOD-CAT-GPX-PON and ARE enzyme activities, but MDA and MPO were induced statistically significant. The results of MN assay were compared with the control group and it was obtained that genotoxicity of different dose groups was similar. The level of 8-OHdG and the activity and caspase-3 examined in blood tissue was increased depending on the dose. It was determined with different biomarkers that this insecticide caused physiological stress changes in the tissues examined.

In the present study, the effect of polycyclic musk compound tonalide (AHTN) in two concentrations was studied in male rainbow trout (Oncorhynchus mykiss, Walbaum 1792). A feeding trial was conducted with AHTN incorporated into feed granules. One concentration was environmentally relevant (854 µg/kg); the second one was 10× higher (8699 µg/kg). The fish were fed twice a day with the amount of feed at 1 % of their body weight. After an acclimatization period, the experimental phase in duration of six weeks followed. At the end of the experiment, fish were sampled and the biometrical data were recorded. Subsequently, hematological and biochemical tests, histopathological examination, analysis of oxidative stress markers and evaluation of endocrine disruption using plasma vitellogenin were performed. In conclusion, an increase of hematocrit for both AHTN concentrations was found, but no significant changes were observed in biochemical profile. Moreover, AHTN caused lipid peroxidation in caudal kidney tissue, which was confirmed by histopathological images. The long-lasting AHTN exposure could thus be harmful for maintaining homeostasis in the rainbow trout organism. However, the vitellogenin concentration seemed not to be affected by AHTN.

Effluents from on-site wastewater treatment systems can influence surface water quality, particularly when infrastructure is aging, malfunctioning, and improperly installed. Municipal wastewater often contains chemical compounds that can lead to adverse biological effects, such as reproductive impairment, in organisms that are chronically exposed. A significant number of these compounds are endocrine-disrupting chemicals. Water quality influences of on-site systems are poorly studied in semi-arid regions where instream flows are seasonally dependent on snowmelt, and when instream dilution of wastewater effluents is minimal during other times of the year. Here we examined surface water estrogenicity in low order tributaries of two unique semi-arid streams with on-site wastewater treatment systems, for which seasonal instream flow fluctuations occur in Park City, UT, USA. Water samples were collected from a total of five locations along two lotic systems downstream from active on-site treatment systems. Samples were extracted for targeted chemical analyses and to perform in vivo and in vitro bioassays with juvenile rainbow trout. Estrogenic activity was measured by quantifying the concentration and expression of vitellogenin (VTG) in plasma and liver, respectively. Plasma VTG presented elevated levels in fish exposed to water samples collected at the two sites in close proximity to on-site systems and during seasons with low stream discharge, though the levels observed did not suggest severe endocrine disruption. However, long-term exposure to these surface water could compromise the fish populations. While the sensitivity of in vitro bioassays was low and targeted chemical analyses did not identify causative compounds, the use of complementary lines of evidence (e.g., in vivo biological models) was advantageous in identifying estrogenic activity in waters influenced by effluents from on-site wastewater systems.

The authors studied the effect of hot pepper ( capsicum sp.) oil on the growth performance and blood parameters in rainbow trout fed. Hot pepper oil was added to rainbow trout feeds at the rates of HPO 0‰ (0 mg/kg) (control), HPO 1‰ (1 mg/kg), HPO 2‰ (2 mg/kg), HPO 4‰ (4 mg/kg) and HPO 6‰ (6 mg/kg), and the fish were fed with experimental feeds for 60 days. The group fed with HPO 4‰ showed the highest percentage growth rate and the lowest feed conversion rate. Our results showed the significant differences serum biochemical parameters, a decrease of serum liver enzymes, glucose, cholesterol and triglyceride levels and an increase of total protein and albumin levels compared with the control. The use of HPO 1‰ in rainbow trout showed a positively affects the growth performance, haematological and serum biochemical parameters.[Formula: see text].

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout ( Oncorhynchus mykiss , Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments., (Copyright © 2020 Bakke, Monte, Stead, Causey, Douglas, Macqueen and Dooley.)

The GH/IGF-I axis influences many aspects of salmonid life history and is involved in a variety of physiological processes that are related to somatic growth (e.g., reproduction, smoltification, and the response to fasting and stress). As such, fisheries studies utilize GH/IGF-I axis components as indicators of growth and metabolic status. This study established time-resolved fluoroimmunoassays (TR-FIAs) for rainbow trout plasma GH and IGF-I using commercially available reagents. For the GH TR-FIA, the ED 80 and ED 20 were 0.6 and 28.1 ng/mL, the minimum detection limit was 0.2 ng/mL, and the intra- and inter-assay coefficients of variation (%CV) were 4.1% and 13.4%, respectively. Ethanol remaining from acid-ethanol cryoprecipitation (AEC) of plasma samples to remove IGF binding proteins reduced binding and increased variability in the IGF-I TR-FIA. Drying down and reconstituting extracted samples restored binding and reduced variability. The extraction efficiency of IGF-I standards through AEC, drying down, and reconstitution did not vary over the working range of the assay. For the IGF-I TR-FIA, the ED 80 and ED 20 were 0.2 and 6.5 ng/mL, the minimum detection limit was 0.03 ng/mL, and the intra- and inter-assay %CV were 3.0% and 6.5%, respectively. Biological validation was provided by GH injection and fasting studies in rainbow trout. Intraperitoneal injection with bovine GH increased plasma IGF-I levels. Four weeks of fasting decreased body weight, increased plasma GH levels, and decreased plasma IGF-I levels. The GH and IGF-I TR-FIAs established herein provide a cost-comparable, non-radioisotopic method for quantifying salmonid plasma GH and IGF-I using commercially available reagents., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Medicinal plants play an important role in aquaculture as feed additives. This study aimed to investigate effects of alcoholic extract of acorn on growth performance, body composition, digestive enzymes activity and blood biochemical parameters of rainbow trout ( O. mykiss ) as a commercially important fish. Five dietary treatments were supplemented: 100, 200, 400 and 600 mg.kg -1 of the extract. Fishes were fed twice per day for 8 weeks, and results showed that acorn extract positively affected all investigated parameters in rainbow trout fishes. Digestive enzymes activity and growth performance were increased, while activity of liver enzymes and cortisol were lowed in comparison to control individuals. Body composition of treated animals was also enhanced. Comparison between treated groups together with integrative biomarker response (IBR) values indicated greatest effects in animals fed with 400 and 600 mg.kg -1 of the extract. Positive effects of the acorn represent promising start point for further studies.

3-Trifluoromethyl-4-nitrophenol (TFM) has been used for more than 60 yr to control the invasive parasitic sea lamprey (Petromyzon marinus) in the Great Lakes Basin (USA/Canada). In the early 1990s, researchers reported that TFM induced vitellogenin in fish and that TFM was an agonist for the rainbow trout estrogen receptor. To support continued registration of TFM for sea lamprey control, regulatory agencies required further testing to evaluate potential endocrine disruption effects. Fathead minnow (Pimephales promelas) were exposed to TFM at measured concentrations of 0.0659, 0.181, 0.594, 1.79, and 5.11 mg active ingredient (a.i.)/L for 21 d. No-observable- and lowest-observable-effect concentrations (NOEC and LOEC, respectively) were determined to be 1.79 mg/L or greater for each endpoint. Male survival in the highest treatment group was reduced relative to the controls. Percentage of egg fertility was reduced in the highest treatment group, resulting in an estimated NOEC of 1.79 mg/L. Whereas no effect on the gonadosomatic index (GSI) was observed for males, female GSI was increased in the 5.11-mg/L treatment. Vitellogenin production was not altered relative to the controls for all TFM treatment groups. However, female testosterone was elevated in the 5.11-mg/L treatment. The results suggest that prolonged exposure to TFM at concentrations exceeding 1.79 mg/L has the potential to disrupt endocrine function. Biologically relevant effects were found at the highest exposure concentration following a 21-d exposure. However, the duration of exposure in our study is not consistent with typical treatment durations (12 h) for sea lamprey control. Environ Toxicol Chem 2020;39:1599-1607. © 2020 SETAC., (© 2020 SETAC.)

The aim of this study was to assess the effects of T-2 toxin-contaminated feed (at concentrations of 1.0 and 1.8 mg/kg) on the rainbow trout immune system by studying non-specific cellular and humoral immune responses and its effect on red and white blood cells. Consumption of T-2 toxin at both concentrations resulted in significantly increased erythrocyte counts and a decrease in mean corpuscular volume. While a significant decrease in mean corpuscular haemoglobin was observed at both experimental concentrations, the decrease in plasma haemoglobin was only significant at the higher T-2 toxin concentration. Higher T-2 toxin concentrations resulted in a significant increase in leukocyte and lymphocyte count, while absolute phagocyte count and counts of less mature neutrophil granulocyte forms remained unchanged at both concentrations. Non-specific humoral immunity (bactericidal activity measured as complement activation) decreased significantly in both experimental groups when compared with the control. The results of this study show that T-2 toxin in feed at a concentration range of 1.0-1.8 mg/kg influences the immunological defence mechanisms of rainbow trout.Trial registration number, MSMT-3876/2014-14; date of registration, 31/1/2014.

In this study, we examined changes occurred in blood parameters, immune responses, antioxidant enzyme activities, and growth performance of rainbow trout (Oncorhynchus mykiss) administered with ribwort plantain (RP) through feed. Fish (mean weight 36.56 ± 1.99 g) were fed a diet supplemented with an aqueous methanolic extract of RP at variable doses, 0 (control), 1 (RP1), 2 (RP2), and 3 g kg -1 (RP3) for 90 days. The final weight, weight gain, and specific growth rate were significantly increased in RP1, RP2, and RP3 treatment groups compared to that of the control. Among examined blood parameters, hemoglobin value in RP1 group (9.77 ± 0.10 g dl -1 ) only was significantly high on the 30th day of the study. When immune response parameters were evaluated, we observed that oxidative radical production and lysozyme activities were affected positively in experimental groups (P < 0.05). The highest oxidative radical production was determined in fish of RP3 group. Glutathione peroxidase and glucose 6 phosphate dehydrogenase were increased in RP3 group compared to control and other treatment groups. Based on these results, it is concluded that ribwort plantain promotes growth, enhances immune responses and antioxidant enzyme activities in rainbow trout, and therefore, may be used in aquaculture.

Grape seed, as a main source of polyphenols, has many nutritional and medicinal properties in humans. In the current study, the effects of dietary ethanolic grape seed extract (GSE) on the growth performance, antioxidant activity, and some biochemical parameters in rainbow trout were investigated. Ninety fish (initial weight 78.47 g) were randomly distributed among nine cement tanks (1.8 m × 0.22 m × 0.35 m) with 10 fish per tank. Three experimental diets containing either 0, 10, or 50 g kg -1 GSE were prepared and each diet was randomly assigned to three tanks of fish for 60 days. Results showed that feeding GSE enhanced some growth parameters including the specific growth rate and condition factor in comparison with the control group. Among different serum metabolites, the glucose levels in treatment groups significantly decreased compared to the control group. The total product of lipid peroxidation indicated as malondialdehyde significantly decreased in both the GSE-added treatment groups. The gene expression related to the antioxidant enzymes, catalase, glutathione peroxidase 1, and glutathione S-transferase A, were upregulated in the intestine of fish that received a low dose of GSE. The results of the current study suggest that GSE, especially at 10 g kg -1 , diet had the potential to improve (1) specific growth rate and condition factor, (2) biochemical parameters including glucose and lipid peroxidation product, and (3) upregulated the expression of antioxidant genes including catalase, glutathione peroxidase 1, and glutathione S-transferase A in rainbow trout.

The long-term effects of three dietary probiotics on rainbow trout during grow-out (mean body weight = 250 ± 50 g) were investigated by feeding for 130 days on eight diet treatments supplemented with Lactobacillus buchneri, L. fermentum and Saccharomyces cerevisiae at 10 7  CFU/g, singularly or in combination. Fish samples were taken for biochemical and immunological analysis in addition to growth performance indices at days 30 and 130 of the experiment. The expression levels of TNF-α and IL-1β genes were also measured at day 130. A positive effect on food conversion was observed in rainbow trout with dietary inclusion of S. cerevisiae (P < 0.05) over 130 days. Also, the total number of white blood cells and their differential count (blood neutrophils, lymphocytes and monocytes), as well as respiratory burst activity were all significantly affected by different treatments at 130 days (P < 0.05). Moreover, at 130 days there was a significant increase in the expression of TNF-α and IL-1β in yeast present treatment compared to the control group (P < 0.05), but no significant difference in the combined probiotic treatments from control group. Yeast and L. buchneri showed a contrary effect on the immune gene expression regulation. Serum cholesterol was significantly lower in all treatments receiving yeast as a dietary probiotic, either alone or in combination with other probiotics. However, none of the probiotic treatments had a significant effect on trout growth performance, or total protein, albumin, globulin, triglyceride and the red blood cell count after 30 or 130 days. Overall, the results suggest that inclusion of a single dietary probiotic, especially S. cerevisiae, in rainbow trout during grow-out has a greater positive effect than combinations of probiotics on the immune system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, growth and development. Arginine is a FAA with crucial roles in protein deposition and the immune response. In mammals, supplementation of arginine's precursor amino acid, citrulline, is known to increase circulating arginine to levels beyond direct arginine supplementation, however, citrulline supplementation is poorly studied in fish. To address this knowledge gap, we supplemented the diet of rainbow trout with arginine and its precursor amino acids, ornithine and citrulline, at 3 levels (0.5%, 1% and 2% of the total diet) during a 14-week experiment. We sampled fish at 3 h and 24 h post-feeding to investigate immediate and steady-state effects, respectively. There were no differences in fish growth for any of the diets across a range of indicators. In blood plasma, out of 26 amino acids detected, 11 and 6 displayed significant changes 24 h and 3 h post-prandial, respectively. Arginine, ornithine and citrulline levels were all significantly increased by the citrulline supplemented diets. In muscle, 8 amino acids were significantly altered by supplemented diets, while there were no significant changes in liver. Arginine was increased by 2% citrulline supplementation in muscle tissue. We also investigated the transcriptional responses of urea cycle, nitric oxide cycle and rate-limiting polyamine synthesis enzymes, related to arginine's metabolism, in liver. At both time points, only 2 enzymes were significantly altered by the supplemented diets, however several significant changes were observed comparing 3 h and 24 h post-prandial expression levels. Of these, the paralogous polyamine synthesis enzyme encoding genes ODC1 and ODC2 displayed the largest increases in 3 h post-prandial fish. These findings demonstrate that endogenous synthesis of arginine is possible from a citrulline supplemented diet and improve our understanding of arginine metabolism in fish., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cardiac stroke volume (S V ) is compromised in Atlantic cod and rainbow trout following acclimation to hypoxia (i.e., 40% air saturation; ~8 kPa O 2 ) at 10-12°C, and this is not due to changes in heart morphometrics or maximum achievable in vitro end-diastolic volume. To examine if this diminished S V may be related to compromised myocardial contractility, we used the work-loop method to measure work and power in spongy myocardial strips from normoxic- and hypoxic-acclimated steelhead trout when exposed to decreasing Po 2 levels (21 to 1.5 kPa) at several frequencies (30-90 contractions/min) at 14°C (their acclimation temperature). Work required to lengthen the muscle, as during filling of the heart, was strongly frequency dependent (i.e., increased with contraction rate) but was not affected by hypoxic acclimation or test Po 2 . In contrast, although shortening work was less frequency dependent, this parameter and network (and power) 1 ) were consistently lower (by ~30-50 and ~15%, respectively) in strips from hypoxic-acclimated fish and 2 ) fell by ~40-50% in both groups from 20 to 1.5 kPa Po 2 , despite the already-reduced myocardial performance in the hypoxic-acclimated group. In addition, strips from hypoxic-acclimated trout showed a poorer recovery of net power (by ~15%) when returned to normoxia. These results strongly suggest that hypoxic acclimation reduces myocardial contractility, and in turn, may limit S V (possibly by increasing end-systolic volume), but that this diminished performance does not improve the capacity to maintain myocardial performance under oxygen limiting conditions.

The present study was performed to investigate the effects of various levels of dietary ellagic acid (EA) on growth performance, haematological values, immune response, protection against Yersinia ruckeri infection, and oxidant/antioxidant status in rainbow trout, Oncorhynchus mykiss. Fish were fed with the control diet and three different experimental diets containing three graded levels of EA (50, 100 and 200 mg kg -1 diet) for 8 weeks. At the end of the experiment, the growth performance [weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR)], haematological values [the red blood cell (RBC) count, haemoglobin (Hb) concentration, haematocrit (Ht) level and erythrocyte indices: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)], immune response [white blood cell (WBC) count, oxidative radical production (nitroblue tetrazolium (NBT) assay), phagocytic activity (PA) and phagocytic index (PI), total protein (TP) and immunoglobulin M (IgM) levels, serum bactericidal activity (BA), lysozyme (LYZ) and myeloperoxidase (MPO) activities] and oxidant/antioxidant status [tissue malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities] were analysed. In addition, fish were challenged by Y. ruckeri and survival rate was recorded for 14 days. In the groups fed with EA the growth parameters such as WG, SGR, and FCR did not vary significantly. The RBC count, Hb concentration, and Ht level increased in the groups fed with EA when compared with the control group. However, there were no significant differences in the MCV, MCH and MCHC values among the groups. The results demonstrated enhancement in all the immunological parameters in the groups fed with EA compared to the control group. The results obtained from challenge with Y. ruckeri revealed reduction in the mortalities in the groups fed with EA. The dietary EA stimulated the SOD, CAT and GSH-Px activities in liver, head kidney and spleen as compared to the control group; however, a reverse trend was observed in the MDA levels of tissues. The present study suggest that EA can effectively enhance the haematological values, immune response, antioxidant capacity, and disease resistance in rainbow trout., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Long-term immunity is of great importance for protection against pathogens and has been extensively studied in mammals. Successive heterologous infections can affect the maintenance of immune memory, inducing attrition of T memory cells and diminishing B cell mediated protection. In fish, the basis of immune memory and the mechanisms of immunization to heterologous pathogens remain poorly understood. We sequentially immunized isogenic rainbow trout with two immunologically distinct viruses, VHSV and IPNV, either with one virus only or in combination, and analyzed the antibody responses and repertoires. Neutralizing antibodies and ELISPOT did not reveal an effect of heterologous immunization. Using a consensus read sequencing approach that incorporates unique barcodes to each cDNA molecule, we focused on the diversity expressed by selected responding VH/C combinations. We identified both public and private responses against VHSV and/or IPNV in all groups of fish. In fish immunized with two viruses, we registered no significant reduction in the persistence of the response toward the primary immunization. Similarly, the response to the second immunization was not affected by a prior vaccination to the other virus. Our data suggest that heterologous immunization does not enforce attrition of pre-existing antibody producing cells, which may impair the protection afforded by multiple successive vaccinations. These observations are potentially important to improve vaccination strategies practiced in aquaculture., (Copyright © 2019 Navelsaker, Magadan, Jouneau, Quillet, Olesen, Munang'andu, Boudinot and Evensen.)

Most pesticides used in agriculture end up in the aquatic environment through runoff and leaching of treated crops. One of the most commonly used herbicides is glyphosate. This compound or its metabolites are frequently detected in surface water in Europe. In the present study, in vivo and in vitro studies were carried out using the early life stages of rainbow trout (Oncorhynchus mykiss) and the cell line RTL-W1 (a liver cell line from rainbow trout) to characterize the toxic effects of glyphosate at environmentally-realistic concentrations. Both studies were performed using the commercial formulation Roundup® GT Max, and technical-grade glyphosate for the in vitro study. Eyed-stage embryos were exposed for 3 weeks to sub-lethal concentrations (0.1 and 1 mg/L) of glyphosate using Roundup. Numerous toxicity endpoints were recorded such as survival, hatching success, larval biometry, developmental abnormalities, swimming activity, genotoxicity (formamidopyrimidine DNA-glycosylase Fpg-modified comet assay), lipid peroxidation (TBARS), protein carbonyls and target gene transcription. Concentrations neither affected embryonic or larval survival nor increased developmental abnormalities. However, a significant decrease was observed in the head size of larvae exposed to 1 mg/L of glyphosate. In addition, a significant increase in mobility was observed for larvae exposed to glyphosate at 0.1 mg/L. TBARS levels were significantly decreased on larvae exposed to 1 mg/L (a.i.), and cat and cox1 genes were differently transcribed from controls. DNA damage was detected by the Fpg-modified comet assay in RTL-W1 cell line exposed to the technical-grade glyphosate and Roundup formulation. The results suggest that chronic exposure to glyphosate, at environmental concentrations, could represent a potential risk for early life stages of fish., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Recent studies have demonstrated that white sturgeon are more sensitive to acute exposure to Cu than rainbow trout (Oncorhynchus mykiss), especially during early life-stages. However, the physiological mechanisms underlying this difference in sensitivity to Cu is not known. In the present study, we first confirmed the higher sensitivity (lower 96 h LC 50 values) of white sturgeon to Cu at three different life stages (larva, swim-up, and juvenile) relative to their counterparts in rainbow trout. We also demonstrated that acute exposure to Cu (50 μg/L for 4.5 h) caused a significantly greater reduction in the rate of waterborne Na uptake in white sturgeon relative to that in rainbow trout across all three life-stages. In agreement with this observation, we also found that acute exposure to Cu (20 μg/L for 48 h) elicits a significantly greater decrease in whole body Na level in all life stages of white sturgeon compared to rainbow trout. In contrast, white sturgeon demonstrated a higher or similar level of Cu body burden relative to rainbow trout during acute Cu exposure (20 μg/L for 24 h), thereby indicating that Cu bioaccumulation is not a good indicator of its toxicity in these species. Overall, our study demonstrated that the differences in sensitivity to acute Cu exposure between white sturgeon and rainbow trout can be explained on the basis of differential effects of Cu on Na homeostasis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Steelhead Trout (Oncorhynchus mykiss) display a varied life-history, including precocious male maturation at age-1 or age-2. In wild fish, precocious male maturation represents an important component of a diverse life-history portfolio. In hatchery programs, however, it is undesirable if rearing practices increase rates of early male maturation and reduce numbers of anadromous male adults. Our study aimed to develop endocrine and molecular markers for identifying males at early stages of maturation in the spring (prior to smolt release) and evaluated the potential use of these markers for quantifying early male maturation rates at a hatchery scale. In a laboratory study, Skookumchuck winter-run Steelhead Trout were reared at a high growth rate in order to increase the occurrence of precocious male maturation. Fish were lethally sub-sampled in February, prior to the time of smolt release; in May, at the time of smolt release; and in September, when 1+ age maturing males that would spawn the following spring were clearly identifiable based solely on gonadosomatic index (GSI). In February and May samples, we measured GSI, plasma 11-ketotestosterone (11KT), mRNAs for pituitary follicle stimulating hormone (fshb) and luteinizing hormone (lhb) beta subunits, and analyzed stage of spermatogenesis by testis histology. Additionally, in May, we measured testis anti-Müllerian hormone (amh) and insulin-like growth factor 3 (igf3) mRNA. Our primary goal was to evaluate the aforementioned maturation indices for their efficacy in forecasting the proportion of fish initiating early male maturation in the spring (approximately 1 year prior to spermiation), compared to the proportion that actually matured. Combining measures of GSI, plasma 11KT, and pituitary fshb and lhb mRNA expression provided a useful, but conservative, estimate of the proportion of males initiating maturation in the spring (21%) compared to the proportion that were ultimately destined to mature (37%) the following spring. These results suggest that maturation may be less synchronous than previously appreciated and some males may have initiated maturation after our census in May., (Published by Elsevier Inc.)

The relationship between stress and immunosuppression was investigated in peripheral blood leucocytes (PBL) in rainbow trout, with reference to corticosteroid receptor (CR) expression and responses to cortisol- and/or lipopolysaccharide (LPS)-administration. Confinement stress in shallow water resulted in a sustained elevation of plasma cortisol, whereas lysozyme and immunoglobin levels were suppressed. Significant increases in mRNA levels of caspase-6 and insulin-like growth factor (IGF)-I were observed in PBL isolated from stressed fish. Confinement stress also suppressed proinflammatory cytokine, interleukin (IL)-1β, expression in PBL. There were decreasing tendencies for the mRNA levels of CRs in PBL of stressed fish. In-vitro treatment of cortisol and LPS on isolated PBL from unstressed trout increased both IL-1 β and CR mRNA expression. However, in PBL from stressed fish, cortisol and LPS treatment increased IL-1 β but not CR mRNA levels. Proliferative activities estimated as in-vitro incorporation of bromodeoxyuridine (BrdU) were decreased by cortisol in PBL from the unstressed and stressed fish groups; however, LPS-stimulated proliferation was observed only in the unstressed fish. Ratios of apoptotic PBL quantified as cell fragmentation using an automated cell counter were increased by cortisol in both groups; however, LPS-stimulated apoptosis was observed only in the stressed fish. Our study reveals cortisol has immune-suppressive effects in stressed fish, irrespective of CR down-regulation and desensitization. The complexity of immune-endocrine interaction is shown by the stress-induced attenuation of LPS effects., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Flowback and produced water (FPW) is a complex, often brackish, solution formed during the process of hydraulic fracturing. Despite recent findings on the short-term toxicity of FPW on aquatic biota, longer-term impacts of FPW on fish have not yet been investigated and the mechanisms of chronic effects remain unknown. The aim of the present study was to observe the effect of a diluted FPW on ionoregulatory endpoints in the rainbow trout Oncorhynchus mykiss, following a 28-d sub-chronic exposure. A salinity-matched control solution (SW), recreating the salt content of the FPW, was used to differentiate the specific effect of the salts from the effects of the other FPW components (i.e. organics and metals). Overall, fish ionoregulation was not impacted by the chronic exposure. An accumulation of strontium (Sr) and bromide (Br) occurred in the plasma of the FPW-exposed fish only, however no change of plasma ions (Na, K, Cl, Ca, Mg) was observed in SW- or FPW-exposed fish. Similarly, exposures did not alter branchial activity of the osmoregulatory enzymes sodium/potassium ATPase and proton ATPase. Finally, FPW exposure resulted in modifications of gill morphology over time, with fish exposed to the fluid displaying shorter lamellae and increased interlamellar-cell mass. However, these effects were not distinct from morphological changes that also occurred in the gills of control groups., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lactate ions are involved in several physiological processes, including a direct stimulation of the carotid body, causing increased ventilation in mammals. A similar mechanism eliciting ventilatory stimulation in other vertebrate classes has been demonstrated, but it remains to be thoroughly investigated. Here, we investigated the effects of lactate ions on the cardiorespiratory system in swimming rainbow trout by manipulating the blood lactate concentration. Lactate elicited a vigorous, dose-dependent elevation of ventilation and bradycardia at physiologically relevant concentrations at constant pH. After this initial confirmation, we examined the chiral specificity of the response and found that only l-lactate induced these effects. By removal of the afferent inputs from the first gill arch, the response was greatly attenuated, and a comparison of the responses to injections up- and downstream of the gills collectively demonstrated that the lactate response was initiated by branchial cells. Injection of specific receptor antagonists revealed that a blockade of serotonergic receptors, which are involved in the hypoxic ventilatory response, significantly reduced the lactate response. Finally, we identified two putative lactate receptors based on sequence homology and found that both were expressed at substantially higher levels in the gills. We propose that lactate ions modulate ventilation by stimulating branchial oxygen-sensing cells, thus eliciting a cardiorespiratory response through receptors likely to have originated early in vertebrate evolution.

In this study, 1% and 2% of macerated fenugreek oil was added to the feeds of rainbow trout with an average weight of 25.79 ± 1.5 g. At the end of the study, growth rate, blood parameters and NBT (Nitroblue Tetrazolium) level of rainbow trout were determined. The best feed ratio (FCR) was observed in the control group (0.77). Statistically significant differences were found only in MID values (P&lt;0.05), although there was a numerical increase in all blood parameters. There was no statistically significant difference between NBT levels (P&gt; 0.05). Although the best weight gain was in the control group as in the FCR values, the maximum elongation was measured at D1 and then at D2 (P &lt;0.05). The best survival rate was obtained with 96.66% in D1 while the worst was observed in D2 with 60% (p&lt;0,05).

This study evaluates changes in the pharmacokinetic behavior of a single oral dose of florfenicol in rainbow trouts experimentally infected with Lactococcus garvieae or Streptococcus iniae. One hundred and fifty fish were randomly divided into three equal groups: 1-healthy fish, 2-fish inoculated with S. iniae (2.87 × 10 7 CFU/ml, i.p.), and 3-fish inoculated with L. garvieae (6.8 × 10 5 CFU/ml, i.p.). Florfenicol was administered to all groups at 15 mg/kg by oral gavage. Blood sampling was performed at 0, 2, 3, 6, 8, 12, 24, 48, 72, and 120 hr after drug administration to each group, and plasma concentration of florfenicol was assayed by HPLC method. The MICs of florfenicol were 1.2 μg/ml and 5 μg/ml against L. garviae and S. iniae, respectively. Healthy fish showed higher values for most of the PK/PD parameters as compared to fish infected with L. garvieae which was reversed in fish infected with S. iniae. Fish infected with L. garvieae showed decreased relative bioavailability accompanied by increased volume of distribution at steady-state (Vd ss ) and total body clearance (Cl B ) . Infection with S. iniae increased the peak concentration of drug after administration (Cmax) and decreased elimination half-life (T 1/2 β ) , central compartment volume (V c ), and Vd ss . In conclusion, infection with these bacteria can affect the pharmacokinetic behavior of florfenicol in rainbow trouts as shown by decreased bioavailability and increased total body clearance and volume of distribution in L. garvieae infection and decreased volume of distribution accompanied by increased Cmax in S. iniae-infected fish., (© 2018 John Wiley & Sons Ltd.)

Because of their intrinsic biological activity and ubiquitous environmental occurrence, human pharmaceutical compounds have received increasing attention from health and environmental agencies. In the present study, all-female juvenile rainbow trout (Oncorhynchus mykiss) were exposed to environmentally-realistic concentrations of a mixture of nonsteroidal pharmaceuticals for 42 days, and the effects on plasma levels of sex-steroids and the expression of genes encoding key proteins involved in ovarian development were assessed. Paracetamol, carbamazepine, diclofenac, irbesartan and naproxen were selected, as these have been detected in the Meuse River in Belgium. Fish were exposed to three concentrations of the mixture including the environmental concentration, 10- and 100-times the environmental concentration. Plasma levels of sex-steroid hormones, particularly 11-ketotestosterone, increased in a concentration-dependent way in exposed females. In addition, some key genes involved in ovarian steroidogenesis were significantly overexpressed after 7 days of exposure, such as key genes involved in the maintenance of the ovary. The steady-state mRNA level of genes implicated in germ cell fate were especially affected, such as that of foxl3 which increased by 5 fold at the highest concentration of the mixture. In conclusion, this study highlights that combined occurrence of common pharmaceutical drugs at concentrations present in surface water environments may act as endocrine-disrupting compounds in rainbow trout., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Biotransformation may substantially impact the toxicity and accumulation of xenobiotic chemicals in fish. However, this activity can vary substantially within and among species. In this study, liver S9 fractions from rainbow trout (4-400 g) were used to evaluate relationships between fish body mass and the activities of phase I and phase II metabolic enzymes. An analysis of log-transformed data, expressed per gram of liver (g liver -1 ), showed that total cytochrome P450 (CYP) concentration, UDP-glucuronosyltransferase (UGT) activity, and glutathione S-transferase (GST) activity exhibited small but significant inverse relationships with fish body weight. In contrast, in vitro intrinsic clearance rates (CL IN VITRO,INT ) for three polycyclic aromatic hydrocarbons (PAHs) increased with increasing body weight. Weight normalized liver mass also decreased inversely with body weight, suggesting a need to express hepatic metabolism data per gram of body weight (g BW -1 ) in order to reflect the metabolic capabilities of the whole animal. When the data were recalculated in this manner, negative allometric relationships for CYP concentration, UGT activity, and GST activity became more pronounced, while CL IN VITRO,INT rates for the three PAHs showed no significant differences across fish sizes. Ethoxyresorufin O-deethylase (EROD) activity normalized to tissue weight (g liver -1 ) or body weight (g BW -1 ) exhibited a non-monotonic pattern with respect to body weight. The results of this study may have important implications for chemical modeling efforts with fish., (Published by Elsevier Inc.)

MicroRNAs (miRNAs) are a class of small non-coding RNAs which are known to posttranscriptionally regulate the expression of most genes in both animals and plants. Meanwhile, studies have shown that numbers of miRNAs are present in body fluids including the plasma. Despite the mode of action of these circulating miRNAs still remains unknown, they have been found to be promising biomarkers for disease diagnosis, prognosis and response to treatment. In order to evaluate the potential of miRNAs as non-invasive biomarkers in aquaculture, a time-course experiment was implemented to investigate the postprandial regulation of miRNAs levels in liver and plasma as well as the hepatic expression of genes involved in cholesterol metabolism. We showed that miR-1, miR-33a, miR-122, miR-128 and miR-223 were expressed in the liver of rainbow trout and present at detectable level in the plasma. We also demonstrated that hepatic expression of miR-1, miR-122 and miR-128 were regulated by feed intake and reached their highest levels 12 hours after the meal. Interestingly, we observed that circulating levels of miR-128 and miR-223 are subjected to postprandial regulations similar to that observed in their hepatic counterparts. Statistical correlations were observed between liver and plasma for miR-128 and miR-223 and between hepatic and circulating miR-122, miR-128 and miR-223 and expression of genes related to cholesterol synthesis and efflux or glucose phosphorylation. These results demonstrated that circulating miR-122, miR-128 and miR-223 are potential biomarkers of cholesterol metabolism in rainbow trout., Competing Interests: The authors have declared that no competing interests exist.

The aim of the present study was to investigate beneficial effects of dietary 1,8-cineole (cineole) supplementation on physiological, immunological and antioxidant responses of rainbow trout (Oncorhynchus mykiss) to crowding stress. The fish were fed for 50 days with diets containing 0 (control), 0.05, 0.1, 0.25, 0.5 and 1% cineole prior to exposure to a 14-day crowding stress. Serum stress markers (cortisol, glucose, lactate, T 4 and T 3 ), antioxidant responses [catalase (CAT) and superoxide dismutase (SOD) activities and total antioxidant capacity (TAC) and malondyaldehyde (MDA) levels] and immune responses [lysozyme and alternative complement (ACH50) activity, and total immunoglobulin (Ig) levels], and blood leukocyte (WBC) and differential counts were measured before and after crowding stress. Results showed that 1% cineole was capable to reduce the basal and stress-induced cortisol elevation and increase the serum T 3 levels after stress. Increase in dietary cineole levels significantly decreased serum cortisol, glucose and lactate levels. Increase in dietary cineole levels significantly increased serum CAT, SOD, lysozyme and ACH50 activities, and TAC and eosinophil levels, and decreased MDA and monocyte levels. After the stress, there was no significant difference in the control group CAT and SOD activities compared to the basal values; however, CAT activities decreased and SOD activities increased in the cineole-treated groups compared to the basal values. Nevertheless, the control group had significantly lower CAT and SOD activities compared to the fish treated with 0.1-1% cineole. Cineole significantly increased blood WBC and serum lysozyme, ACH50 and total Ig. Moreover, cineole administration significantly mitigated the stress-induced decrease in total Ig levels as well as increase in leukocyte count. The cineole-treated fish had higher survival and growth performance compared to the control group. Although all levels of cineole (0.05-1%) showed beneficial effects on different tested factors, 0.5 and 1% levels had beneficial effects on most of the tested factors; thus, are recommended for dietary inclusion to suppress adverse effects of stress in trout., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O 2 ) unloading for cardiovascular O 2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (β-NHE) create H + gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O 2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pH i ) must recover during venous transit (31-90 s) to enable O 2 loading at the gills. The halftimes ( t 1/2 ) and magnitudes of RBC β-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pH i , were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O 2 as a sensitive indicator for changes in RBC pH i . In addition, the recovery rate of RBC pH i was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t 1/2 of β-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t 1/2 of RBC pH i recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H + gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O 2 unloading from pH-sensitive Hb at the tissues, while protecting O 2 loading at the gills; the resulting increase in O 2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

Juvenile rainbow trout ( Oncorhynchus mykiss) confined in pairs form social hierarchies in which socially subordinate fish display characteristic traits, including reduced growth rates and altered glucose metabolism. These effects are, in part, mediated by chronically elevated cortisol levels and/or reduced feeding. To determine the effects of social status on lipid metabolism, trout were held in pairs for 4 days, following which organismal and liver-specific indexes of lipid metabolism were measured. At the organismal level, circulating triglycerides were elevated in dominant trout, whereas subordinate trout exhibited elevated concentrations of circulating free fatty acids (FFAs) and lowered plasma total cholesterol levels. At the molecular level, increased expression of lipogenic genes in dominant trout and cpt1a in subordinate trout was identified, suggesting a contribution of increased de novo lipogenesis to circulating triglycerides in dominant trout and reliance on circulating FFAs for β-oxidation in the liver of subordinates. Given the emerging importance of microRNAs (miRNA) in the regulation of hepatic lipid metabolism, candidate miRNAs were profiled, revealing increased expression of the lipogenic miRNA-33 in dominant fish. Because the Akt-TOR-S6-signaling pathway is an important upstream regulator of hepatic lipid metabolism, its signaling activity was quantified. However, the only difference detected among groups was a strong increase in S6 phosphorylation in subordinate trout. In general, the changes observed in lipid metabolism of subordinates were not mimicked by either cortisol treatment or fasting alone, indicating the existence of specific, emergent effects of subordinate social status itself on this fuel.

The present study investigated the effects of dietary trans-cinnamic acid (CA) on pre- and post-challenge haematological, serum biochemical, non-specific immune and head kidney gene expression responses of rainbow trout, Oncorhynchus mykiss juveniles. In this regard, fish with an average weight of 17.01 ± 0.05 g were divided into five groups, and fed daily with an additive free basal diet (control); 250, 500, 750 or 1500 mg kg -1  CA for a 60-day period. Fish were sampled every 20 days during the experiment. On days 20, 40 and 60 (the pre-challenge period), the dietary CA especially at 250 and/or 500 mg kg -1 significantly increased blood granulocyte percentage, and serum total protein, globulin, lysozyme and total immunoglobulin values. Furthermore, dietary CA increased activities of phagocytic activity, respiratory burst and potential killing, and increased the expression levels of immune related genes [serum amyloid A (SAA), interleukin 8 (IL-8), interleukin 1, beta (IL-1β), transforming growth factor beta (TGF-β), tumor necrosis factor (TNF-α), and immunoglobulin T (IgT)] in the head kidney of fish fed with 250 and/or 500 mg kg -1  CA. Following 60 days of feeding, fish were challenged with Yersinia ruckeri and mortality was recorded for 20 days. Highest percentage survival (%) rate was found in the 250 and/or 500 mg kg -1 CA-supplemented feeding groups. During the post-challenge period, red blood cell (RBC) count, hematocrit (%), respiratory burst activity, and total antiprotease activity increased in fish fed with feed containing 500 mg kg -1 content. Moreover, markedly up-regulated the expression of related genes (SAA, IL-8, IL-1β, TGF-β, TNF-α, IFN-γ and IgM) in fish fed 250, 500 and/or 750 mg kg -1  CA. Therefore, feeding O. mykiss for 60 days with dietary CA at 250-500 mg kg -1  CA incorporation levels can be suggested as optimal to enhance the immunity and disease resistance against Y. ruckeri., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study evaluates the effects of red beet (RB) and betaine on rainbow trout submitted to an acute stress challenge. A control diet was compared with four experimental diets in which red beet (14 and 28%) and betaine (0.9 and 1.63%) were incorporated in different concentrations according to a factorial design. Cortisol in plasma and fin, glucose and lactate plasma levels, and malondialdehide (MDA) in muscle were all measured before the stress challenge and 30 min and 6 and 12 h after the stress challenge as parameters to determine the diet effects. RB and betaine had no effect on cortisol, glucose, and MDA basal levels. However, lactate basal levels were significantly lower on fish fed with RB and betaine. Thirty minutes after the stress challenge, there was a significant increase in plasma and fin cortisol, glucose and lactate concentrations, although fish fed with diets containing RB and betaine showed significantly higher plasma cortisol values. MDA values of fish fed with 14% RB and 0.9% betaine were significantly higher than MDA values from fish fed with 28% RB and 1.63% betaine. After 6 and 12 h, plasma and fin cortisol and lactate levels recovered in a similar trend. Glucose plasma levels recovered in almost all groups 12 h after the stress. Also, MDA values recovered basal levels after 6 and 12 h. RB and betaine did not enhance the tolerance to the stress challenge compared to the control group, although the presence of these ingredients had no negative effect on any of the stress indicators.

Lean muscle mass plays an important role in overall health, as altered skeletal muscle metabolism can impact both the incidence and prevention of conditions related to metabolic health. Intriguingly, dietary methionine restriction (MR) has been shown to ameliorate this phenotype over time potentially through mechanisms related to changes in myogenic precursor cell (MPC) differentiation status. Recently the role of micro-RNAs (miRs) in regulating the expression of muscle specific transcription factors myoD and myogenin as well as signaling molecules involved in skeletal muscle differentiation has been reported in vitro. We performed an 8week feeding trial to determine if MR in vivo could alter miR abundance as well as change metabolic markers. Results show changes in muscle miR abundance for miR-133a at 4weeks with no significant difference seen in miR-210 or miR-206. After 8weeks of MR feeding fish demonstrated increased clearance of glucose, increased fat accumulation in the liver, and decreased fat accumulation in the muscle. These data demonstrate conservation of MR effects on fish metabolism, and suggest, for the first time, that miR-133a might play a role in tissue response to MR., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of the present study was to investigate anesthetic efficacy of myrcene in rainbow trout (Oncorhynchus mykiss) along with the fish biochemical response to anesthesia in comparison with eugenol. In the first experiment, 240 fish were stocked in 12 tanks and acclimatized to experimental conditions for 2 weeks. Then, the fish of each tank were subjected to one concentration of either eugenol (12, 20, 30, 50, 80, and 130 μL/L) or myrcene (100, 150, 200, 300, 400, and 500 μL/L) concentrations. Induction time of and recovery time from anesthesia were recorded for each fish separately. Using these results, desired concentrations to induce anesthesia within 60, 180, 300, and 600 s were determined, being 81, 30, 19, and 10 μL/L eugenol and 531, 251, 177, and 111 μL/L myrcene. In the second experiment, 96 fish were stocked in 8 tanks. Six fish were netted from each tank and exposed to the calculated eugenol or myrcene concentrations. Blood samples were taken after the fish reached anesthesia. The results showed that there was no significant difference in serum lactate, alanine transaminase, and alkaline phosphatase. Increase in the induction time of anesthesia resulted in increased serum glucose with no significant difference between the anesthetics. Increase in induction time of anesthesia led to increase in serum lactate dehydrogenase activity in the eugenol-anesthetized fish and aspartate transaminase activity in myrcene-anesthetized fish. In conclusion, myrcene is capable to anesthetize rainbow trout, but at higher concentrations compared to eugenol. In addition, biochemical analysis showed that increase in induction time of anesthesia leads to hyperglycemia and increase in AST and LDH activities depending on anesthetic type.

Silver nanoparticles (Ag NPs) are known for their antibacterial properties and are used in a growing number of nano-enabled products, with inevitable concerns for releases to the environment. Nanoparticles may also be antigenic and toxic to the haematopoietic system, but the immunotoxic effect of Ag NPs on non-target species such as fishes is poorly understood. This study aimed to assess the effect of Ag NP exposure via the water on the haematopoietic system of rainbow trout, Oncorhynchus mykiss, and to determine whether or not the hazard from Ag NPs was different from that of AgNO 3 . Fish were exposed for 7 days to a control (dechlorinated Plymouth freshwater), dispersant control, 1µgl -1 Ag as AgNO 3 or 100µgl -1 Ag NPs. Animals were sampled on days 0, 4 and 7 for haematology, tissue trace metal concentration, biochemistry for evidence of oxidative stress/inflammation in the spleen and histopathology of the blood cells and spleen. The Ag NP treatment significantly increased the haematocrit, but the haematological changes were within the normal physiological range of the animal. Thrombocytes in spleen prints at day 4, and melanomacrophage deposits at day 7 in the spleen, of Ag NP exposed-fish displayed significant increases compared to all the other treatments within the time point. A dialysis experiment confirmed that dissolution rates were very low and any pathology observed is likely from the NP form rather than dissolved metal released from it. Overall, the data showed subtle differences in the effects of Ag NPs compared to AgNO 3 on the haematopoietic system. The lack of pathology in the circulating blood cells and melanomacrophage deposits in the spleen suggests a compensatory physiological effort by the spleen to maintain normal circulating haematology during Ag NP exposure., (Copyright © 2018. Published by Elsevier Inc.)

To study the expression of four estrogen receptor genes (erα1, erα2, erβ1, erβ2) of female rainbow trout (Oncorhynchus mykiss) during first ovarian development, trouts were sampled from different ovarian stages. Serum E 2 (estradiol) was measured by ELISA and estrogen receptors mRNA expression were examined by qRT-PCR. Our results showed a close association between increased erα1 and vitellogenin mRNA expression during ovarian maturation and increased erα2 mRNA expression in mature ovarian stages. Correlation analysis revealed that a negative relationship between serum E 2 and ovarian erβ1 (or hepatic erβ2), but ovarian erβ2 mRNA expression was relatively unchanged during first ovarian development. Trout were also reared in different densities as stocking density 1, 2 and 3 (SD1, 4.6-31.1 kg/m 3 ; SD2, 6.6-40.6 kg/m 3 ; SD3, 8.6-49.3 kg/m 3 ) to elucidate effects of high density on estrogen receptor expression. Histology observation showed ovarian development of trout in higher densities were retard with a relatively early stage and fewer vitellogenin accumulation. Trout in high densities showed significantly decreased serum E 2 , erα mRNA expression and increasing trends of erβ mRNA expression. A noticeable increase of ovarian erβ2 mRNA expression was seen in trout when density is approaching to 50 kg/m 3 . In conclusion, we may hypothesize that increased erβ mRNA expression triggered by high density result in decreased erα mRNA expression and vitellogenesis. As a result, ovarian development in higher densities was retard., (Copyright © 2017. Published by Elsevier Inc.)

Genomic actions of estrogens in vertebrates are exerted via two intracellular estrogen receptor (ER) subtypes, ERα and ERβ, which show cell- and tissue-specific expression profiles. Mammalian immune cells express ERs and are responsive to estrogens. More recently, evidence became available that ERs are also present in the immune organs and cells of teleost fish, suggesting that the immunomodulatory function of estrogens has been conserved throughout vertebrate evolution. For a better understanding of the sensitivity and the responsiveness of the fish immune system to estrogens, more insight is needed on the abundance of ERs in the fish immune system, the cellular ratios of the ER subtypes, and their autoregulation by estrogens. Consequently, the aims of the present study were (i) to determine the absolute mRNA copy numbers of the four ER isoforms in the immune organs and cells of rainbow trout, Oncorhynchus mykiss , and to compare them to the hepatic ER numbers; (ii) to analyse the ER mRNA isoform ratios in the immune system; and, (iii) finally, to examine the alterations of immune ER mRNA expression levels in sexually immature trout exposed to 17β-estradiol (E2), as well as the alterations of immune ER mRNA expression levels in sexually mature trout during the reproductive cycle. All four ER isoforms were present in immune organs-head kidney, spleen-and immune cells from head kidney and blood of rainbow trout, but their mRNA levels were substantially lower than in the liver. The ER isoform ratios were tissue- and cell-specific, both within the immune system, but also between the immune system and the liver. Short-term administration of E2 to juvenile female trout altered the ER mRNA levels in the liver, but the ERs of the immune organs and cells were not responsive. Changes of ER gene transcript numbers in immune organs and cells occurred during the reproductive cycle of mature female trout, but the changes in the immune ER profiles differed from those in the liver and gonads. The correlation between ER gene transcript numbers and serum E2 concentrations was only moderate to low. In conclusion, the low mRNA numbers of nuclear ER in the trout immune system, together with their limited estrogen-responsiveness, suggest that the known estrogen actions on trout immunity may be not primarily mediated through genomic actions, but may involve other mechanisms, such as non-genomic pathways or indirect effects., Competing Interests: The authors declare no conflict of interest.

Red blood cells (RBCs) possess methemoglobin reductase activity that counters the ongoing oxidation of hemoglobin (Hb) to methemoglobin (metHb), which in circulating blood is caused by Hb autoxidation or reactions with nitrite. We describe an assay for determining metHb reductase activity in intact RBCs in physiological saline at normal Pco 2 and pH. After initial loading of oxygenated RBCs with nitrite (partly oxidizing Hb to metHb), the nitrite is removed by three washes of the RBCs in nitrite-free physiological saline to enable the detection of RBC metHb reductase activity in the absence of counteracting oxidation. This assay was used to compare metHb reduction in rainbow trout and carp RBCs under both oxygenated and deoxygenated conditions. Washing resulted in effective wash-out of nitrite to low and safe values (~2μM). The subsequent decline in [metHb] with time followed first-order kinetics, allowing characterization of metHb reductase activity through the first order rate constant k. In oxygenated RBCs at 25°C, the k values for rainbow trout and carp were slightly below or above 0.01min -1 , respectively; which is double the value reported for mammals at 37°C. We conclude the higher metHb reductase activity in fish offsets their higher Hb autoxidation and higher likelihood of encountering elevated nitrite. Deoxygenation significantly raised the rates of RBC metHb reduction, and more so in rainbow trout than in carp. The temperature sensitivity of metHb reduction in rainbow trout RBCs was high (Q 10 ~2.8) and instrumental in handling increased Hb autoxidation with temperature., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Studies were conducted to determine the distribution and elimination of imidacloprid (IMI) in rainbow trout. Animals were injected with a low (47.6 μg/kg), medium (117.5 μg/kg) or high (232.7 μg/kg) dose directly into the bloodstream and allowed to depurate. The fish were then sampled to characterize the loss of IMI from plasma and its appearance in expired water (all dose groups) and urine (medium dose only). In vitro biotransformation of IMI was evaluated using trout liver S9 fractions. Mean total clearance (CL T ) values determined by non-compartmental analysis of plasma time-course data were 21.8, 27.0 and 19.5 mL/h/kg for the low, medium and high dose groups, respectively. Estimated half-lives for the same groups were 67.0, 68.4 and 68.1 h, while fitted values for the steady-state volume of distribution (V SS ) were 1.72, 2.23 and 1.81 L/kg. Branchial elimination rates were much lower than expected, suggesting that IMI is highly bound in blood. Renal clearance rates were greater than measured rates of branchial clearance (60% of CL T in the medium dose group), possibly indicating a role for renal membrane transporters. There was no evidence for hepatic biotransformation of IMI. Collectively, these findings suggest that IMI would accumulate in trout in continuous waterborne exposures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Our goals were: (i) to determine whether sublethal concentrations of water-borne ammonia would prevent the formation of a dominance hierarchy, or alter its structure, in groups of 4 juvenile trout; (ii) to investigate the behavioral and physiological responses of individuals of different social rank exposed to a concentration of ammonia that still allowed hierarchy formation. Social hierarchies were created by using a technique in which a food delivery system that created competition also served to isolate individual fish for respirometry. Groups of 4 fish were exposed to elevated ammonia (NH 4 HCO 3 ) 12 h before first feeding; aggression was recorded by video camera during morning feedings. Experimental ammonia concentrations were 700, 1200 and 1500 μmol L -1 at pH 7.3, 12 °C (9.8, 16.8, and 21.0 mg L -1 as total ammonia-N, or 0.0515, 0.0884, and 0.1105 mg L -1 as NH 3 -N). Aggression was severely reduced by 1200 and abolished by 1500 μmol L -1 total ammonia, such that hierarchies did not form. However, groups exposed to 700 μmol L -1 total ammonia still formed stable hierarchies but displayed lower levels of aggression in comparison to control hierarchies. Exposure continued for 11 days. Physiological parameters were recorded on day 5 (end of period 1) and day 10 (end of period 2), while feeding and plasma cortisol were measured on day 11. In control hierarchies, dominant (rank 1) trout generally exhibited higher growth rates, greater increases in condition factor, higher food consumption, and lower cortisol levels than did fish of ranks 2, 3, and 4. In comparison to controls, the 700 μmol L -1 total ammonia hierarchies generally displayed lower growth, lower condition factor increases, lower O 2 consumption, lower cortisol levels, but similar feeding patterns, with smaller physiological differences amongst ranks during period 1. Effects attenuated during period 2, as aggression and physiological measures returned towards control levels, indicating both behavioral and physiological acclimation to ammonia. These disturbances in social behavior and associated physiology occurred at an ammonia concentration in the range of regulatory significance and relevance to aquaculture., (Copyright © 2017 Elsevier B.V. All rights reserved.)

As a part of their unique life cycle, most salmonids undergo a transition from fresh water to salt water requiring various adjustments in metabolism, osmoregulation and ion regulation. Exposure to pesticides may affect the acclimation of juvenile salmonids to salt water during downstream migration to estuaries. Using the Caspian Sea as a model waterbody, the present study aimed to determine how the toxicity of the organophosphate pesticide chlorpyrifos (CPF) impacts saline acclimation of rainbow trout (Oncorhynchus mykiss). We pre-exposed 4-month-old fish to nominal concentrations of 0, 20, 40, 80, 160 μg/L of CPF for seven days, and then gradually to salinity (12 ppt) for another seven days. Mortality, levels of cortisol, T3 and T4 in serum, and expression of genes involved in gill ion transport (Na + /K + ATPase α1a and α1b) and liver xenobiotic detoxification (Glutathione-S-Transferase pi, GST) were measured at day fourteen. Cortisol concentrations in serum were not changed by CPF exposure in freshwater, but serum T3 increased up to three fold relative to controls in freshwater. Following salinity acclimation, T3 and T4 concentrations in the serum were both increased up to 2.5 and 8.8 fold in animals treated with CPF followed by saltwater. Na+/K + ATPase α1a and α1b mRNA in gill were unchanged by CPF treatment in freshwater but trended higher in CPF-treated animals after salinity acclimation. Hepatic mRNA of GST was significantly increased following exposure to CPF but was unchanged after saltwater exposure. Although saltwater treatment reduced the acute lethality of CPF, changes in T3/T4 suggest sublethal impacts may occur in CPF-treated fish after they acclimate to Caspian seawater., (Copyright © 2017 Elsevier B.V. All rights reserved.)

When compared with fish meal and fish oil, plant ingredients differ not only in their protein content and amino acid and fatty acid profiles but are also devoid of cholesterol, the major component of cell membrane and precursor of several bioactive compounds. Based on these nutritional characteristics, plant-based diets can affect fish physiology and cholesterol metabolism. To investigate the mechanisms underlying cholesterol homeostasis, rainbow trout were fed from 1 g body wt for 6 mo with a totally plant-based diet (V), a marine diet (M), and a marine-restricted diet (MR), with feed intake adjusted to that of the V group. The expression of genes involved in cholesterol synthesis, esterification, excretion, bile acid synthesis, and cholesterol efflux was measured in liver. Results showed that genes involved in cholesterol synthesis were upregulated in trout fed the V diet, whereas expression of genes related to bile acid synthesis ( cyp7a1) and cholesterol elimination ( abcg8) were reduced. Feeding trout the V diet also enhanced the expression of srebp-2 while reducing that of lxrα and miR-223. Overall, these data suggested that rainbow trout coped with the altered nutritional characteristics and absence of dietary cholesterol supply by increasing cholesterol synthesis and limiting cholesterol efflux through molecular mechanisms involving at least srebp-2, lxrα, and miR-223. However, plasma and body cholesterol levels in trout fed the V diet were lower than in fish fed the M diet, raising the question of the role of cholesterol in the negative effect of plant-based diet on growth.

Although conventional wastewater treatment technologies are effective at removing many contaminants of emerging concern (CECs) from municipal wastewater, some contaminants are not removed efficiently. Ozonation may be a treatment option for reducing the concentrations of recalcitrant CECs in wastewater, but this process may generate toxic transformation products. In the present study, we conducted semibatch experiments to ozonate municipal wastewater effluent spiked with 5 commonly detected CECs. The purpose of the present study was to evaluate whether ozonation increased or decreased biological responses indicative of sublethal toxicity in juvenile rainbow trout (Oncorhynchus mykiss) injected intraperitoneally (i.p.) with extracts prepared from ozonated and nonozonated wastewater effluent. Blood, liver, and brain tissues were collected from the fish at 72 h post injection for analysis of a battery of biomarkers. In fish i.p. injected with the extracts from nonozonated wastewater effluent, significant induction of plasma vitellogenin (VTG) was observed, but ozonation of the municipal wastewater effluent spiked with CECs significantly reduced this estrogenic response. However, in fish injected with extracts from spiked municipal wastewater effluent after ozonation, the balance of hepatic glutathione in its oxidized (glutathione disulfide [GSSG]) form was altered, indicating oxidative stress. Levels of the neurotransmitter serotonin were significantly elevated in brain tissue from trout injected with the extracts from ozonated spiked municipal wastewater effluent, a biological response that has not been previously reported in fish. Other in vivo biomarkers showed no significant changes across treatments. These results indicate that ozonation reduces the estrogenicity of wastewater, but may increase other sublethal responses. The increase in biomarker responses after ozonation may be because of the formation of biologically active products of transformation of CECs, but further work is needed to confirm this conclusion. Environ Toxicol Chem 2018;37:274-284. © 2017 SETAC., (© 2017 SETAC.)

Cd is known for its carcinogenic effects, however its mechanism of toxicity and in particular its ability to promote oxidative stress is debated. In fact, although it is considered a redox-inactive metal, at high concentration Cd was shown to promote indirectly oxidative stress. In this study we investigated metal accumulation in ex vivo exposed trout (Oncorhynchus mykiss) erythrocytes and Cd dose-dependent effect in terms of RBC viability, cytosolic and mitochondrial ROS levels as well as its effects on mitochondrial membrane depolarization, hemoglobin stability and precipitation. In the concentration range used, Cd did not affect cell viability. However, metal accumulation was associated with an increase in all oxidative indexes evaluated, except mitochondrial superoxide anion production that, on the contrary, was significantly decreased, probably due to a lowered respiration rate associated with interference of Cd with complex I, II and III, as suggested by the observed Cd-dependent mitochondrial membrane depolarization. On the other hand, hemoglobin destabilisation seems to be the major trigger of oxidative stress in this cell type., (Copyright © 2017. Published by Elsevier Inc.)

The eradication of viral haemorrhagic septicaemia virus (VHSV Id) from Finnish brackish-water rainbow trout Oncorhynchus mykiss farms located in the restriction zone in the Province of Åland, Baltic Sea, failed several times in the 2000s. The official surveillance programme was often unable to find VHSV-positive populations, leading to the misbelief in the fish farming industry that virus eradication could be achieved. The ability of 3 other surveillance programmes to detect infected fish populations was compared with the official programme. One programme involved syndromic surveillance based on the observation of clinical disease signs by fish farmers, while 2 programmes comprised active surveillance similar to the official programme, but included increased sampling frequencies and 2 additional tests. The syndromic surveillance concentrated on sending in samples for analysis when any sign of a possible infectious disease at water temperatures below 15°C was noticed. This programme clearly outperformed active surveillance. A real-time reverse transcriptase-polymerase chain reaction method proved to be at least as sensitive as virus isolation in cell culture in detecting acute VHSV infections. An ELISA method was used to test fish serum for antibodies against VHSV. The ELISA method may be a useful tool in VHSV eradication for screening populations during the follow-up period, before declaring an area free of infection.