Search

The Root effect is a pH-dependent reduction in hemoglobin-O2 carrying capacity. Specific to ray-finned fishes, the Root effect has been ascribed specialized roles in retinal oxygenation and swimbladder inflation. We report that when rainbow trout are exposed to elevated water carbon dioxide (CO2), red muscle partial pressure of oxygen (PO2) increases by 65%--evidence that Root hemoglobins enhance general tissue O2 delivery during acidotic stress. Inhibiting carbonic anhydrase (CA) in the plasma abolished this effect. We argue that CA activity in muscle capillaries short-circuits red blood cell (RBC) pH regulation. This acidifies RBCs, unloads O2 from hemoglobin, and elevates tissue PO2, which could double O2 delivery with no change in perfusion. This previously undescribed mechanism to enhance O2 delivery during stress may represent the incipient function of Root hemoglobins in fishes.

Rainbow trout (Oncorhynchus mykiss) exposed to an acute heat shock (1 h at 25 °C after raising water temperature from 13 °C to 25 °C over 4 h) mount a significant catecholamine response. The present study investigated the proximate mechanisms underlying catecholamine mobilization. Trout exposed to heat shock in vivo exhibited a significant reduction in arterial O(2) tension, but arterial O(2) concentration was not affected by heat shock, nor was catecholamine release during heat shock prevented by prior and concomitant exposure to hyperoxia (to prevent the fall in arterial O(2) tension). Thus, catecholamine mobilization probably was not triggered by impaired blood O(2) transport. Heat-shocked trout also exhibited an elevation of arterial CO(2) tension coupled with a fall in arterial pH, but these factors are not expected to trigger catecholamine release. The changes in blood O(2) and CO(2) tension occurred despite a significant hyperventilatory response to heat shock. Future studies should investigate whether catecholamine mobilization during heat shock in rainbow trout is triggered by a specific effect of high temperature activating the sympathetic nervous system via a thermosensitive transient receptor potential channel., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In this study, the annual cycle of the gonadal steroids testosterone (T), 11-ketotestosterone (11-KT), 17β-estradiol (E2) and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was determined using radioimmunoassay and then compared for two populations of rainbow trout, XX diploid females (n = 40) and XXX triploid females (n = 15). In females, E2 and DHP levels were found to be significantly related to body weight (r = 0.22513; p < 0.0001 and r = 0.15831; p > 0.001, respectively). In this group, E2 concentrations peaked in November (25.05 ng/ml), while maximum DHP levels, only measurable from October to April, were attained in February (64.14 ng/ml). No significant differences in hormone ranges related to egg output ability were observed. Finally, sex steroid concentrations were low in the triploid female XXX fish compared to the female XX population. Nevertheless, maximum T (33.85 ng/ml) and 11-KT (32.35 ng/ml) levels were recorded in January, for XXX. The levels for these two hormones are relatively high and are also significantly associated (r = 0.8430; p < 0.0001). Diploid females showed significantly higher levels of E2 than triploids over the 12-month study period. The female triploid fish produced the lowest steroid hormone levels, such that these would be the most suitable for human consumption., (© 2012 Blackwell Verlag GmbH.)

There have been suggestions that analgesics be used by fish researchers. But in the absence of dose-response data for morphine, this suggestion seems imprudent. The purpose of the present study was to develop a dose-response relationship in fish using six doses of morphine. The response (movement of the fins or tail) to a noxious stimulus (electrical shock to the face region) was monitored before and after a dose of morphine intraperitoneally (i.p.). The i.p. dose of morphine ED(50) in rainbow trout was 6.7 ± 0.8 mg/kg (n = 12 at each dose). The plasma morphine concentration EC(50) was 4.1 ± 1.5 mg/L. In a second experiment, rainbow trout tested with equal amounts of morphine and naloxone (30 mg/kg) showed that the antinociceptive effect of morphine was blocked by naloxone. It has been suggested that stress-induced analgesia has been a confounding factor in some fish studies. However, plasma cortisol levels in our study indicated that stress was not a confounding factor in the present experiments. The ED(50) for morphine in fish was higher than that reported for humans or other mammals. Our observation showing a dose-response relation for morphine using a noxious stimulus supports arguments for its effectiveness as an antinociceptive drug in fish., (© 2012 Blackwell Publishing Ltd.)

Melamine and its triazine analogs, such as cyanuric acid, have been used to artificially inflate protein content both in animal feed ingredients, as well as in milk products produced for human consumption. We report here a LC-MS/MS method to quantify and confirm melamine and cyanuric acid in serum from channel catfish and rainbow trout with a limit of quantification of 0.8 μg/mL. The method was applied to serum samples from a residue depletion study in which fish were given a single oral dose of 20 mg/kg body weight melamine, cyanuric acid, or both compounds together. Samples were taken at 1, 3, 7, 14, and 28 days (an additional 42 day was added for trout). When given alone or in combination with cyanuric acid, melamine residues were highest on day 1 in both catfish and trout. Cyanuric acid was only quantifiable at day 1 in trout when given alone, and not at all in catfish. The serum half life of melamine in catfish was 1.50-1.62 days and 3.09-3.67 days in trout. This work highlights the differences of depletion kinetics in fish, which can be measured in days, as compared to the depletion in mammals, measured in hours., (Published by Elsevier Ltd.)

To assess whether the glucosensing capacity in peripheral (liver and Brockmann bodies) and central (hypothalamus and hindbrain) locations of rainbow trout displays day-night variations in its response to changes in circulating glucose levels, we evaluated the response of parameters related to glucosensing [glucose, glycogen and glucose 6-phosphate levels, activities of glucokinase (GK), glycogen synthetase (GSase) and pyruvate kinase (PK), and mRNA abundance of GK, glucose transporter 2 (GLUT2), and K(ATP) channel subunits Kir6.x-like and sulfonylurea receptor (SUR)-like] in fish subjected to hyperglycemic treatment under night or day conditions. No day-night significant variations were noticed in the glucosensing capacity of the hypothalamus, hindbrain and Brockmann bodies. In contrast, a clear differential response was noticed in the liver, where glucose levels, GK activity (and mRNA levels) and GSase activity displayed increased values during the day in hyperglycemic fish compared with controls, and lower (GK mRNA levels) or non-existent (glucose, GK and GSase activities, and Kir6.x-like mRNA levels) values during the night. A similar decrease in parameters related to glucosensing in the liver was observed when fish under day conditions were treated with melatonin, suggesting a modulatory role of melatonin in day-night changes of the glucosensing response in the same tissue.

Present study examined the effects of Ergosan on growth performance, digestive enzyme activities, hematological parameters and gastrointestinal structure of rainbow trout. Rainbow trout (mean weight 100-110 g) were fed basal diet (control) and diet treated with Aquavac Ergosan (5 g kg⁻¹ of diet) for 50 days. Results of this study showed that Ergosan supplementation significantly increased weight gain (94.27 g vs. 65.04 g), specific growth rate (4.09 vs. 3.10) and feed intake (136.85 g vs. 111.22 g) and decreased feed conversion ratio (1.43 vs. 2.03) compared to control (P<0.05). Lipase activity and leukocyte and erythrocyte count also increased in juvenile fish fed Ergosan-treated diet compared to control (P<0.05). Light microscopy demonstrated that both groups of fish displayed normal morphology of proximal intestine and pyloric caeca. In Ergosan-treated group, higher percentage of goblet cell was shown in proximal intestine and pyloric caeca. Present study suggests that Ergosan effectively promotes growth performance, lipase activity and gastrointestinal structure in rainbow trout.

Previous studies in two rainbow trout lines divergently selected for lean (L) or fat (F) muscle suggested that they differ in their ability to metabolise glucose. In this context, we investigated whether genetic selection for high muscle fat content led to a better capacity to metabolise dietary carbohydrates. Juvenile trout from the two lines were fed diets with or without gelatinised starch (17.1%) for 10 weeks, after which blood, liver, muscle and adipose tissues were sampled. Growth rate, feed efficiency and protein utilisation were lower in the F line than in the L line. In both lines, intake of carbohydrates was associated with a moderate post-prandial hyperglycaemia, a protein sparing effect, an enhancement of nutrient (TOR-S6) signalling cascade and a decrease of energy-sensing enzyme (AMPK). Gene expression of hepatic glycolytic enzymes was higher in the F line fed carbohydrates compared with the L line, but concurrently transcripts for the gluconeogenic enzymes was also higher in the F line, possibly impairing glucose homeostasis. However, the F line showed a higher gene expression of hepatic enzymes involved in lipogenesis and fatty acid bioconversion, in particular with an increased dietary carbohydrate intake. Enhanced lipogenic potential coupled with higher liver glycogen content in the F line suggests better glucose storage ability than the L line. Overall, the present study demonstrates the changes in hepatic intermediary metabolism resulting from genetic selection for high muscle fat content and dietary carbohydrate intake without, however, any interaction for an improved growth or glucose utilisation in the peripheral tissues.

This study was conducted to evaluate the effects of Echinacea purpurea (EP) on growth and some hematological and blood biochemical indices of rainbow trout fingerlings. A basal diet was supplemented with 0 (control), 0.25, 0.5, 1, and 2 g EP kg⁻¹ to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish with an initial average weight of approximately 8 g. After 8 weeks of feeding trial, fish fed diets with 0.25 and 0.5 g EP kg⁻¹ showed highest final weight and SGR and fish fed with the control diet indicated the lowest final weight and SGR. There was a significant (P < 0.05) decrease in FCR between the control group and the groups fed with diets of 0.25 and 0.5 g EP kg⁻¹. Biochemical parameters such as serum total protein content, albumin content, globulin content, and albumin/globulin ratio in the fish were evaluated. There were significant differences between hematological parameters including RBC, WBC, HB, lymphocyte, and neutrophil percentage in fish fed with dietary nucleotide compared with control treatment (P < 0.05). The results suggest that EP administration at 0.25 and 0.5 g EP kg⁻¹ exerted positive effects on growth and biochemical and hematological indices in rainbow trout.

Blood-O(2) affinity, Hill number and predicted arterial-O(2) saturation did not differ between diploid (2N) and triploid (3N) rainbow trout Oncorhynchus mykiss blood when compared under various temperature and CO(2) partial pressure combinations. These results suggest that reduced hypoxia and warm-water tolerance and aerobic capacity of 3N fishes are not due to altered blood-O(2) affinity. Further investigation into O(2) transport-diffusion capacity and intracellular pH within 3N fishes may further unravel the mechanisms behind reduced 3N tolerance of suboptimal environments., (© 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.)

The aim of this study was to assess the effect of the different aquatic conditions on anion transport of fish erythrocytes through the measurement of the sulphate uptake operating from band 3, the determination of reduced glutathione (GSH) and oxidized glutathione (GSSG). To this purpose, blood samples of 30 Liza aurata and 30 Salmo irideus fishes were collected via caudal vein and washed with physiological buffer. Successively, erythrocytes suspended at 3% haematocrit were used to measure the SO(4) (=) influx by atomic absorption spectrophotometry at 425 nm wavelength and the GSH concentration using an immunodiagnostic assay intended for the quantitative determination of glutathione in ethylenediaminetetraacetic acid in blood. All results were analyzed using unpaired Student's t-test and a P < 0.05 was considered statistically significant. The results of this study showed statistically significant differences about the sulphate uptake and the glutathione levels in S. irideus with respect to L. aurata (P < 0.0001). In conclusion, the different aquatic conditions play an important role on anion transport in fish erythrocytes, showing that environmental differences induce perturbations in erythrocyte membrane and should be evaluated to prevent physiological damages in fishes., (Copyright © 2012 John Wiley & Sons, Ltd.)

The aim of this study was to evaluate the influence of a synbiotic (Biomin IMBO) on serum parameters and feeding efficiency in rainbow trout (Oncorhynchus mykiss) fingerlings. The fish with initial average weight of 4.59 ± 0.2 g were randomly assigned to four dietary treatments for two months. The dietary treatment (0.5, 1 and 1.5 g/kg of diet) was supplemented with basal diet and non-supplemented basal diet was used as control. After two months, all treatments supplemented with synbiotic showed significant (p < 0.05) increase in final mean weight, weight gain percentage, specific growth rate, condition factor, food conversion efficiency and survival rate, compared to the control group. Among all supplemented treatments, the best result in terms of growth factors and survival was observed in the treatment supplemented with 1 g synbiotic per kilogram of diet. Furthermore, supplementation with symbiotic, specifically 1 and 1.5 g/kg, significantly (p < 0.05) increased the total serum protein, but there was no significant (p > 0.05) difference in globulin content, albumin/globulin ratio, and triglyceride content among experimental treatments. In terms of body composition, carcass protein content of fish fed with synbiotic significantly (p < 0.05) increased compared to the control. These results revealed that a feeding regime with synbiotic for two months led to a significant increase in growth performance, survival rate and feeding efficiency in rainbow trout fingerlings., (© 2011 Blackwell Verlag GmbH.)

Glucose and lipid metabolism in pancreatic islet organs is poorly characterized. In the present study, using as a model the carnivorous rainbow trout, a glucose-intolerant fish, we assessed mRNA expression levels of several genes involved in glucose and lipid metabolism (including ATP-citrate lyase; carnitine palmitoyltransferase-1 isoforms, CPT; the mitochondrial isoform of the phosphoenolpyrutave carboxykinase, mPEPCK and pyruvate kinase, PK) and glucosensing (glucose transporter type 2, Glut2; glucokinase, GK and the potassium channel, K(ATP)) in Brockmann bodies. We evaluated the response of these parameters to changes in feeding status (food deprived vs. fed fish) as well as to changes in the amount of carbohydrate (dextrin) in the diet. A general inhibition of the glycolytic (including the glucosensing marker GK) and β-oxidation pathways was found when comparing fed versus food-deprived fish. When comparing fish feeding on either low- or high-carbohydrate diets, we found that some genes related to lipid metabolism were more controlled by the feeding status than by the carbohydrate content (fatty acid synthase, CPTs). Findings are discussed in the context of pancreatic regulation of glucose and lipid metabolism in fish, and show that while trout pancreatic metabolism can partially adapt to a high-carbohydrate diet, some of the molecular actors studied seem to be poorly regulated (K(ATP)) and may contribute to the glucose intolerance observed in this species when fed high-carbohydrate diets.

The in vivo effects of the herbicide active substance (AS) pendimethalin (alone and with Prowl 400® adjuvant) were evaluated on sanitary status i.e. the health status with regard to chemical pollution and on the physiological state via the immune system in rainbow trout, Oncorhynchus mykiss. Four nominal exposure conditions were tested: i) control (C), ii) AS at 500 ng L(-1) (P500), iii) AS at 800 ng L(-1) (P800) and iv) Prowl 400® at 500 ng L(-1) (Pw). After a 28 day exposure period (D28), 10 fish were sampled for each condition and 10 other after a 15 day recovery period in clean fresh water (D43). Pendimethalin concentrations in the exposure water and muscles were followed. White blood cell counts, differential leucocyte counts, cell mortality and phagocytosis activity were measured. Haemolytic alternative complement activity, lysozyme concentration and stress parameters were analyzed. The resulting concentration of pendimethalin in the exposure water was lower than the expected concentration. At D28, the concentration quantified in the contaminated fish was negligible in comparison with the Reference Dose for Oral Exposure estimated by US-EPA's Integrated Risk Information System. Leucopenia was noted in all contaminated fish. A decrease in phagocytosis activity and ACH(50) was also observed in contaminated fish by P800 and Pw. Disturbed lysozyme activity was noted only in fish exposed to Pw. Furthermore, during exposure to a similar concentration of pendimethalin, the commercial product seemed to be more immunotoxic than the AS alone. Finally, at D43, the effects proved reversible for sanitary status while immunity was still disturbed in contaminated fish by P800 and Pw., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Boldness, a measure of an individual's propensity for taking risks, is an important determinant of fitness but is not necessarily a fixed trait. Dependent upon an individual's state, and given certain contexts or challenges, individuals may be able to alter their inclination to be bold or shy in response. Furthermore, the degree to which individuals can modulate their behaviour has been linked with physiological responses to stress. Here we attempted to determine whether bold and shy rainbow trout, Oncorhynchus mykiss, can exhibit behavioural plasticity in response to changes in state (nutritional availability) and context (predation threat). Individual trout were initially assessed for boldness using a standard novel object paradigm; subsequently, each day for one week fish experienced either predictable, unpredictable, or no simulated predator threat in combination with a high (2% body weight) or low (0.15%) food ration, before being reassessed for boldness. Bold trout were generally more plastic, altering levels of neophobia and activity relevant to the challenge, whereas shy trout were more fixed and remained shy. Increased predation risk generally resulted in an increase in the expression of three candidate genes linked to boldness, appetite regulation and physiological stress responses - ependymin, corticotrophin releasing factor and GABA(A) - but did not produce a significant increase in plasma cortisol. The results suggest a divergence in the ability of bold and shy trout to alter their behavioural profiles in response to internal and exogenous factors, and have important implications for our understanding of the maintenance of different behavioural phenotypes in natural populations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Effects of commercial source for astaxanthin (Haematococcus pluvialis) (H.p) on antioxidant power, specific marker enzymes, and some metabolites were examined in rainbow trout (Oncorhynchus mykiss). Fish were fed on diets containing 1, 3, and 10 g microalga kg(-1) feed for 30 days. Serum total antioxidant activity and lipid peroxidation product, indicated by malondialdehyde (MDA), significantly enhanced with different doses of administration, indicating the elevated antioxidant status in all treatment groups. In group fed with high dose of alga, significantly elevated aspartate aminotransferase activity (AST) was noted, indicating damage of normal liver function in this group. Alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were not affected in all groups. Although serum total protein remained unaffected, serum glucose level was decreased significantly in lower doses of administration. Furthermore, triglyceride and cholesterol levels showed significant decrease in 3 g kg(-1) microalga group by modulation of lipid metabolism in this group. On the other hand, in highest dose, significant increase in lipids was observed, indicating the slight dysfunction in lipid metabolism in this treatment group. The present study suggests that Haematococcus pluvialis especially in dose of 3 g kg(-1) feed administration may effectively enhance the antioxidant system and some biochemical parameters in rainbow trout.

Rainbow trout myoglobin (Mb) is characterized by an unusually low affinity for oxygen, having a P(50) of 4.92±0.29 mm Hg at 25 °C which is the highest ever reported for any vertebrate Mb at the same temperature (Helbo and Fago, (2011) Am. J. Physiol. Regul. Integr. Comp. Physiol. 300, R101-R108). In order to gain insight into the structural factors of the heme pocket that may be important determinants for this atypical oxygen affinity, we have carried out an electronic absorption and resonance Raman characterization of the ferric and ferrous protein with and without exogenous ligands (O(2), CO, F(-)) and compared the results with those of other Mbs. While the ν(Fe-His) stretch appears at a frequency similar to other vertebrate Mbs, the resonance Raman frequencies of the Fe-ligand stretching modes reveal significant variations in the interaction of iron-bound ligands with distal residues. In particular, the spectroscopic characterization highlights two exceptional properties of rainbow trout Mb, a significantly higher level of reversed heme and reduced hydrogen bonding interactions between ligands and the distal HisE7 residue compared with other Mbs. The weakening of the hydrogen bond interaction is proposed to be the primary cause of the significantly reduced oxygen affinity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Here we examined the use of stable isotopes, [¹³C]starch and [¹⁵N]protein, as dietary tracers to study carbohydrate assimilation and distribution and protein utilisation, respectively, by rainbow trout (Oncorhynchus mykiss). The capacity of glucose uptake and use by tissues was studied, first, by varying the digestibility of carbohydrate-rich diets (30 % carbohydrate), using raw starch and gelatinised starch (GS) and, second, by observing the effects of two regimens of activity (voluntary swimming, control; sustained swimming at 1·3 body lengths/s, exercise) on the GS diet. Isotopic ratio enrichment (¹³C and ¹⁵N) of the various tissue components (protein, lipid and glycogen) was measured in the liver, muscles, viscera and the rest of the fish at 11 and 24 h after a forced meal. A level of 30 % of digestible carbohydrates in the food exceeded the capacity of rainbow trout to use this nutrient, causing long-lasting hyperglycaemia that raises glucose uptake by tissues, and the synthesis of glycogen and lipid in liver. Total 13C recovered 24 h post-feeding in the GS group was lower than at 11 h, indicating a proportional increase in glucose oxidation, although the deposition of lipids in white muscle (WM) increased. Prolonged hyperglycaemia was prevented by exercise, since sustained swimming enhances the use of dietary carbohydrates, mainly through conversion to lipids in liver and oxidation in muscles, especially in red muscle (RM). Higher recoveries of total 15N for exercised fish at 24 h, mainly into the protein fraction of both RM and WM, provide evidence that sustained swimming improves protein deposition, resulting in an enhancement of the protein-sparing effect.

The aim of this study was to determine the effects of chronic waterborne copper (Cu) exposure on the acute stress-induced cortisol response and associated physiological consequences in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 30 μg Cu/L in moderately hard water (120 mg/L as CaCO(3)) for 40 days, following which time the acute cortisol response was examined with a series of stressors. At 40 days, a 65% increase in Cu was observed in the gill, but no accumulation was observed in the liver, brain or head kidney. Stressors such as air exposure or confinement did not elicit an increase in circulating cortisol levels for Cu-exposed fish, in contrast to controls. However, this inhibitory effect on the acute cortisol response appeared to have few implications on the ability of Cu-exposed fish to maintain ion and carbohydrate homeostasis. For example, plasma Na(+), Ca(2+) and glucose levels as well as hepatic glycogen levels were the same post-stress in control and Cu-exposed fish. Trout were also challenged with exposure to 50% seawater for 48 h, where Cu-exposed trout maintained plasma Na(+), glucose and hepatic glycogen levels. However, Cu-exposed fish experienced decreased plasma K(+) levels throughout the Cu exposure and stress tests. In conclusion, chronic Cu exposure resulted in the abolition of an acute cortisol response post-stress. There was no Cu accumulation in the hypothalamus-pituitary-interrenal axis (HPI axis) suggesting this was not a direct toxic effect of Cu on the cortisol regulatory pathway. However, the lack of an acute cortisol response in Cu-exposed fish did not impair the ability of the fish to maintain ion and carbohydrate homeostasis. This effect on cortisol may be a strategy to reduce costs during the chronic stress of Cu exposure, and not endocrine disruption as a result of toxic injury., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Acute temperature stress in animals results in increases in heat shock proteins (HSPs) and stress hormones. There is evidence that stress hormones influence the magnitude of the heat shock response; however, their role is equivocal. To determine whether and how stress hormones may affect the heat shock response, we capitalized on two lines of rainbow trout specifically bred for their high (HR) and low (LR) cortisol response to stress. We predicted that LR fish, with a low cortisol but high catecholamine response to stress, would induce higher levels of HSPs after acute heat stress than HR trout. We found that HR fish have significantly higher increases in both catecholamines and cortisol compared with LR fish, and LR fish had no appreciable stress hormone response to heat shock. This unexpected finding prevented further interpretation of the hormonal modulation of the heat shock response but provided insight into stress-coping styles and environmental stress. HR fish also had a significantly greater and faster heat shock response and less oxidative protein damage than LR fish. Despite these clear differences in the physiological and cellular responses to heat shock, there were no differences in the thermal tolerance of HR and LR fish. Our results support the hypothesis that responsiveness to environmental change underpins the physiological differences in stress-coping styles. Here, we demonstrate that the heat shock response is a distinguishing feature of the HR and LR lines and suggest that it may have been coselected with the hormonal responses to stress.

Zearalenone (ZEA) is a mycoestrogen frequently found in food and animal feed materials all over the world. Despite its hydrophobic character, ZEA is also found in surface and ground waters which suggests an environmental risk for aquatic animals. Knowledge concerning mycotoxin-related mechanisms of toxicity is still incomplete, e.g. little is known about the influence of ZEA exposure on fish. The aim of this study was to investigate the effect of ZEA on selected biochemical parameters in juvenile rainbow trout after 24, 72, and 168 h of intraperitoneal exposure (10 mg/kg of body weight). The analysis showed a slight tendency towards prolonged blood clotting time and significant iron deficiency in the liver and ovary of exposed animals. However, no differences in aminotransferase (AlaAT, AspAT) activity or glucose levels in fish plasma was observed. The results of this study suggest that although trout exposed to ZEA did not exhibit any distinct symptoms of liver damage, the mycotoxin tested was able interfere with blood coagulation and iron-storage processes.

Objectives: Effect of long-term oral administration of three different concentrations (0.5, 1.0, and 2.0%) of micronized β-1.3/1.6-D-glucan derived from oyster mushroom (Pleurotus ostreatus, Hiratake) on biometrical, haematological, biochemical, and immunological indices of half-year-old rainbow trout (Oncorhynchus mykiss) was assessed in the study., Design: Rainbow trout were feed commercial feed pellets containing β-1.3/1.6-D-glucan in the concentrations of 0.5, 1.0, and 2.0% for 85 days. Biometrical indices consisted in total and standard length, body and liver weight, from which derived somatic parameters such as Fulton´s condition factor and hepatosomatic index were calculated. Haematological parameters were evaluated according to unified methods for haematological examination in fish. Plasma biochemical profile was analysed using biochemical analyser Konelab 20i and Easy Lyte Analyzer. A phagocyte cells metabolic activity (induced chemiluminescence of phagocytes) was determined as an immunological parameter by a microplate luminometric method on Immunotech LM-01T., Results: No clinical signs of behavioral, respiratory, or neurologic distress were observed in rainbow trout. Fish showed normal feeding behavior. As for biometric parameters, no significant changes in total and standard length, body weight, liver weight, as well as in condition factor and hepatosomatic index of experimental and control fish were found. In the course of the study, weight gains in rainbow trout were similar and continuous. Shifts in PCV (p<0.05), haemoglobin (p<0.05), and MCHC (p<0.01) were found within haematological indices. Plasma concentration of glucose, lactate, total protein, cholesterol, calcium, natrium, potassium (all p<0.05), albumins and chlorides (both p<0.01), as well as catalytic activities of ALT and AST (both p<0.05) were changed in the course of the study. A phagocyte cells metabolic activity (luminol-induced chemiluminescence) in rainbow trout was not altered by oyster mushroom β-1.3/1.6-D-glucan administration., Conclusion: After long-term oral administration of three concentrations of micronized β-1.3/1.6-D-glucan derived from oyster mushroom (Pleurotus ostreatus, Hiratake) shifts in haematological and biochemical profiling were found in half-year-old rainbow trout (O. mykiss) in environmental conditions of a commercial rainbow trout fishery. Biometrical indices were not found significantly altered. No specific effect of β-glucan on immune system response of rainbow trout was found in the study. The use of β-glucan in prosperous, clinically healthy aquaculture is still an issue, nevertheless, its use in breedings endangered by stress stimuli, infectious diseases or adverse environmental factors is indisputable.

Silymarin, an extract from "milk thistle" (Silybum marianum) plant is traditionally used as herbal medicine. The present study was conducted to investigate the clinical effects and possible side effects of silymarin on biochemical blood parameters of rainbow trout (Oncorhynchus mykiss). Fishes were treated with 0 (control), 100, 400, and 800 mg of silymarin per kg of food during 4 weeks. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), creatine kinase (CK), glucose, total protein, creatinine, triglyceride, cholesterol, urea, uric acid and liver cellular total antioxidant, and protein content were measured after 7, 14, and 28 days of silymarin treatment. The results showed that oral administration of silymarin in fish significantly reduced plasma glucose and cholesterol levels and relatively increased plasma total protein and globulin concentrations (P < 0.05). Increasing plasma albumin levels indicate the important role of albumin in drug transportation in circulatory system of fish. Silymarin also stabilized cellular membrane structure and regulated the levels of AST, ALT, ALP, CK, and LDH activity. In conclusion, on the basis of these results, oral administration of silymarin up to 400 mg per 1 kg of food has no side effect on blood biochemical and clinical parameters of fishes. However, oral administration of 800 mg/kg- of silymarin caused cytotoxicity and modifications in blood biochemical parameters of fish.

This study was conducted to evaluate the effect of ethanolic extract of propolis (EEP) on growth performance and plasma biochemical parameters of rainbow trout (Oncorhynchus mykiss). Graded levels of EEP [0 (control), 1, 2, and 4 g kg(-1) diet] were fed to trout juveniles (mean weight 7.73 ± 0.17 g) for 10 weeks. Dietary EEP supplementation regardless of inclusion level significantly improved the specific growth rate of fish. Similarly, supplemental EEP generally improved the feed efficiency ratio and protein efficiency ratio, but no significant differences were observed between the 1 g kg(-1) EEP group and the control group. In addition, dietary EEP supplementation generally increased the plasma superoxide dismutase, lysozyme, total antioxidant capacity, glutathione peroxidase, and catalase activities, but decreased the plasma malondialdehyde level. The plasma triglycerides level was significantly lower in the 1 or 4 g kg(-1) EEP group as compared with the control group. Dietary EEP supplementation generally decreased the plasma aspartate aminotransferase and alanine aminotransferase activities, but increased the hepatic aspartate aminotransferase and alanine aminotransferase activities. These results indicate the potential to use the EEP as a growth promoter, hepatoprotective agent, and immunostimulant for rainbow trout.

The present study sought to determine whether environmentally realistic mixtures of agriculturally important pesticides are stressful to fish. Juvenile rainbow trout were exposed for 96 h to concentrations of a pesticide mixture found in a waterway that is the focus of salmon restoration efforts (Nicomekl River, BC, Canada). This mixture contained organochlorine, organophosphorus, phenylurea, and triazine classes of pesticides. Fish given a realistic mixture exposure (total concentration, 1.01 µg/L) had increased plasma cortisol concentration, packed red cell volume, hematocrit (Hct), as well as decreased white cell volume, leukocrit (Lct). Similar changes in Hct and Lct were apparent after exposure to a lower concentration (0.186 µg/L). Interestingly, no changes in plasma cortisol concentration, Hct, or Lct were noted after exposure to a higher concentration (13.9 µg/L). This suggests that the exposure likely impaired the mechanisms enabling the stress response. Across all exposures, plasma glucose concentration was related to plasma cortisol concentration, not to pesticide mixture concentration. This suggests that a secondary stress response may be more related to variability in individual primary stress response than to differences in pesticide exposure concentrations. In summary, the present study indicates that salmon living in agrichemical-contaminated waterways may be experiencing stress, and this may pose a threat to their survival., (Copyright © 2011 SETAC.)

Maternal size, age, and allostatic load influence offspring size, development, and survival. Some of these effects have been attributed to the release of glucocorticoids, and individual variation in these stress hormones is related to a number of traits. Correlated traits are often clustered and used to define the proactive and reactive stress coping styles. Although stress coping styles have been identified in a number of animal groups, little is known about the coupling between stress coping style and offspring characteristics. In the present study, plasma cortisol levels in ovulated mothers and cortisol levels in non-fertilized eggs from two rainbow trout (Oncorhynchus mykiss) strains selected for high (HR) and low (LR) post-stress plasma cortisol levels were compared. Offspring characteristics such as egg size, larval growth, and energy reserves also were compared between the two strains. Maternal plasma and egg cortisol levels were correlated, but no difference between the HR and LR strains was detected in either parameter. LR females produced larger eggs, and larvae with larger yolk sacs compared to HR females, however no differences in larval body size (excluding the yolk) was detected between strains. Considering that the HR and LR strains have a number of correlated behavioral and physiological traits that resemble the reactive and proactive stress coping styles, respectively, the results suggest that proactive mothers invest more energy into their offspring, producing larvae with larger energy reserves. It is possible that larger energy reserves in proactive larvae support the energy requirement for establishing and defending territory in salmonid fish. Furthermore, in the present study we found a positive relationship between mother plasma cortisol and egg cortisol; however neither mother plasma cortisol nor egg cortisol differed between strains. These results indicate that cortisol endowment from the mother to the offspring plays a minor role in the transfer of the behavioral and physiological traits which separates these strains., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Diclofenac is a nonsteroidal anti-inflammatory drug frequently found in the aquatic environment. Previous studies have reported histological changes in the liver, kidney, and gills of fish at concentrations similar to those measured in treated sewage effluents (approximately 1 µg/L). Analyses or predictions of blood plasma levels in fish allow a direct comparison with human therapeutic plasma levels and may therefore be used to indicate a risk for pharmacological effects in fish. To relate internal exposure to a pharmacological interaction, we investigated global hepatic gene expression together with bioconcentration in blood plasma and liver of rainbow trout (Oncorhynchus mykiss) exposed to waterborne diclofenac. At the highest exposure concentration (81.5 µg/L), the fish plasma concentration reached approximately 88% of the human therapeutic levels (C(max) ) after two weeks. Using an oligonucleotide microarray followed by quantitative PCR, we found extensive effects on hepatic gene expression at this concentration, and some genes were found to be regulated down to the lowest exposure concentration tested (1.6 µg/L), corresponding to a plasma concentration approximately 1.5% of the human C(max) . Thus, at concentrations detected in European surface waters, diclofenac can affect the expression of multiple genes in exposed fish. Functional analysis of differentially expressed genes revealed effects on biological processes such as inflammation and the immune response, in agreement with the mode of action of diclofenac in mammals. In contrast to some previously reported results, the bioconcentration factor was found to be stable (4.02 ± 0.75 for blood plasma and 2.54 ± 0.36 for liver) regardless of the water concentration., (Copyright © 2011 SETAC.)

Previous studies have demonstrated that chronic dietary salt loading causes hypertension and a decreased sensitivity of the systemic vasculature to α-adrenergic stimulation and other hypertensive stimuli (e.g. hypercapnia) in rainbow trout (Oncorhynchus mykiss). This reduced sensitivity to hypertensive stimuli is consistent with a possible blunting of homeostatic responses normally aimed at raising blood pressure. To test this idea, we examined the consequences of long-term salt feeding and the associated hypertension on the interactive capacities of the renin angiotensin system (RAS) and adrenergic systems to elevate blood pressure in trout. Secretion of catecholamines in response to a range of doses of homologous ANG II in vivo and in situ (using a perfused posterior cardinal vein preparation) was reduced in the salt-fed fish. The reduced sensitivity to ANG II could not be explained by alterations in stored catecholamine (adrenaline or noradrenaline) levels or the general responsiveness of the chromaffin cells to depolarizing stimuli (60 mmol/l KCl). Despite the decreased responsiveness of the chromaffin cells to ANG II, plasma catecholamines were increased to a greater extent in the salt-fed fish during acute hypoxia (a condition that activates the RAS). Interestingly, the pressor effects of ANG II in vivo were actually heightened in the salt-fed fish. The increased pressor response to exogenous ANG II was likely attributable to its direct interaction with vascular ANG II receptors because the effect persisted even after blockade of α-adrenergic receptors. Treating fish with the vascular smooth muscle relaxant papaverine caused similar reductions in blood pressure and increases in plasma ANG II levels regardless of diet. Similarly, inhibition of angiotensin converting enzyme with lisinopril reduced blood pressure equally in control and salt-fed fish. These results indicate that, while long-term dietary salt loading blunts the response of trout chromaffin cells to ANG II, the RAS itself appears to be unaffected. Indeed, the capacity of ANG II to elevate blood pressure is not compromised nor do fish exhibit a reduced capacity to mount an acute humoral adrenergic stress response during acute hypoxia.

Ammonia, the third respiratory gas in teleost fish, acts as an acute stimulant to ventilation in ammoniotelic rainbow trout. We investigated whether this sensitivity is maintained in trout chronically exposed (1+ months) to high environmental ammonia [HEA, 250 μmol l(-1) (NH(4))(2)SO(4)] in the water, and whether gill neuroepithelial cells (NECs) are involved in ammonia sensing. Hyperventilation was induced both by acute external (NH(4))(2)SO(4) exposure [250 or 500 μmol l(-1) (NH(4))(2)SO(4)] and by intra-arterial (NH(4))(2)SO(4) injection (580 μmol kg(-1) of ammonia) in control trout, but these responses were abolished in chronic HEA animals. Hyperventilation in response to acute ammonia exposure persisted after bilateral removal of each of the four gill arch pairs separately or after combined removal of arches III and IV, but was delayed by removal of gill arch I, and eliminated by combined removal of arches I and II. NECs, identified by immunolabeling against 5-HT, were mainly organized in two lines along the filament epithelium in all four gill arches. In control trout, NECs were slightly smaller but more abundant on arches I and II than on arches III and IV. Chronic HEA exposure reduced the density of the NECs on all four arches, and their size on arches I and II only. Fura-2 fluorescence imaging was used to measure intracellular free calcium ion concentration ([Ca(2+)](i)) responses in single NECs in short-term (24-48 h) culture in vitro. [Ca(2+)](i) was elevated to a comparable extent by perfusion of 30 mmol l(-1) KCl and 1 mmol l(-1) NH(4)Cl, and these [Ca(2+)](i) responses presented in two different forms, suggesting that ammonia may be sensed by multiple mechanisms. The [Ca(2+)](i) responses to high ammonia were attenuated in NECs isolated from trout chronically exposed to HEA, especially in ones from gill arch I, but responses to high K(+) were unchanged. We conclude that the hyperventilatory response to ammonia is lost after chronic waterborne HEA exposure, and that NECs, especially the ones located in gill arches I and II, are probably ammonia chemoreceptors that participate in ventilatory modulation in trout.

The intestine of marine teleosts produces carbonate precipitates from ingested calcium as part of their osmoregulatory strategy in seawater. The potential for estrogens to control the production of intestinal calcium carbonate and so influence osmoregulation was investigated in seawater-acclimated rainbow trout following intraperitoneal implantation of 17β-estradiol (E2) at two doses (0.1 and 10 μg E2 g(-1)). Levels of plasma vitellogenin provided an indicator of estrogenic effect, increasing significantly by three and four orders of magnitude at the low and high doses, respectively. Plasma osmolality and muscle water content were unaffected, whereas E2-treated fish maintained lower plasma [Na(+)] and [Cl(-)]. Plasma [Ca(2+)] and [Mg(2+)] and muscle [Ca(2+)] increased with vitellogenin induction, whereas the intestinal excretion of calcium carbonate was reduced. This suggests that elevated levels of circulating E2 may enhance Ca(2+) uptake via the gut and simultaneously reduce CaCO(3) formation, which normally limits intestinal availability of Ca(2+). Increasing E2 caused an elevation of [Na(+)] and [Cl(-)] and a reduction of [HCO(3(-))] in intestinal fluid. We speculate that E2 may influence a number of intestinal ion transport processes that ultimately may influence water absorption: (1) reduced NaCl cotransport, (2) reduced Cl(-) uptake via Cl(-)/HCO(3(-)) exchange and (3) reduced precipitation of Ca(2+) and Mg(2+) carbonates. Despite these effects on intestinal ion and water transport, overall osmoregulatory status was not compromised in E2-treated fish, suggesting the possibility of compensation by other organs.

During a generalized acidosis in rainbow trout, catecholamines are released into the blood, activating red blood cell (RBC) Na(+)/H(+) exchange (βNHE), thus protecting RBC intracellular pH (pH(i)) and subsequent O(2) binding at the gill. Because of the presence of a Root effect (a reduction in oxygen carrying capacity of the blood with a reduction in pH), the latter could otherwise be impaired. However, plasma-accessible carbonic anhydrase (CA) at the tissues (and absence at the gills) may result in selective short-circuiting of RBC βNHE pH regulation. This would acidify the RBCs and greatly enhance O(2) delivery by exploitation of the combined Bohr-Root effect, a mechanism not previously proposed. As proof-of-principle, an in vitro closed system was developed to continuously monitor extracellular pH (pH(e)) and O(2) tension (P(O(2))) of rainbow trout blood. In this closed system, adding CA to acidified, adrenergically stimulated RBCs short-circuited βNHE pH regulation, resulting in an increase in P(O(2)) by >30 mmHg, depending on the starting Hb-O(2) saturation and degree of initial acidification. Interestingly, in the absence of adrenergic stimulation, addition of CA still elevated P(O(2)), albeit to a lesser extent, a response that was absent during general NHE inhibition. If plasma-accessible CA-mediated short-circuiting is operational in vivo, the combined Bohr-Root effect system unique to teleost fishes could markedly enhance tissue O(2) delivery far in excess of that in vertebrates possessing a Bohr effect alone and may lead to insights about the early evolution of the Root effect.

Cortisol and glucocorticoid receptors (GRs) play an important role in fish osmoregulation, whereas the involvement of the mineralocorticoid receptor (MR) and its putative ligand 11-deoxycorticosterone (DOC) is poorly investigated. In this study, we assessed the implication of DOC and MR in rainbow trout (Oncorhynchus mykiss) osmoregulation during hypo- and hypersaline acclimation in parallel with the cortisol-GR system. A RIA for DOC was developed to measure plasma DOC levels, and a MR-specific antibody was developed to localize MR protein in the gill, intestine, and kidney. This is the first study to report DOC plasma levels during salinity change and MR localization in fish osmoregulatory tissue. Corticosteroid receptor mRNA abundance was investigated in osmoregulatory tissue during salinity acclimation, and the effect of cortisol and DOC on ionic transporters gene expression was assayed using an in vitro gill incubation method. Differential tissue-, salinity-, and time-dependent changes in MR mRNA levels during both hyper- and hyposaline acclimations and the ubiquitous localization of MR in osmoregulatory tissue suggest a role for the MR in osmoregulation. Presumably, DOC does not act as ligand for MR in osmoregulation because there were no changes in plasma DOC levels during either freshwater-seawater (FW-SW) or SW-FW acclimation or any effect of DOC on gill ionic transporter mRNA levels in the gill. Taken together, these results suggest a role for MR, but not for DOC, in osmoregulation and confirm the importance of cortisol as a major endocrine regulator of trout osmoregulation.

Blood is an indicator of physiological condition of an animal. Therefore, the chronic effects of propiconazole, a triazole fungicide present in aquatic environment, on hematology of rainbow trout were investigated in this study. Fish were exposed at various concentrations of PCZ (0.2, 50 and 500 μg L(-1)) for 7, 20 and 30 d. Multiple biomarkers were measured, including hematological indices (hemoglobin concentration, red blood cells count, hematocrit, leukocyte count, mean erythrocyte hemoglobin, mean erythrocyte volume and mean color concentration) and plasma biochemical parameters (ammonia, glucose, total proteins, creatine kinase, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase). Through principal component analysis and integrated biomarker response assessment, influence extent induced by PCZ-stress of each test group was distinguished. Additional, all parameters measured in this study displayed different dependent patterns to PCZ concentrations and exposure time by two-way ANOVA. The results of this study indicate that chronic exposure of PCZ has altered multiple physiological indices in fish hematology and CK activity may be an early biomarker of PCZ toxicity; however, before these parameters are used as special biomarkers for monitoring residual PCZ in aquatic environment, more detailed experiments in laboratory need to be performed in the future., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The effects of acute stressor exposure on proximal (growth hormone [GH]) and distal (insulin-like growth factor-I [IFG-I] and insulin-like growth factor-binding proteins [IFGBPs]) components of the somatotropic axis are poorly understood in finfish. Rainbow trout (Oncorhynchus mykiss) were exposed to a 5-min handling disturbance to mimic an acute stressor episode, and levels of plasma GH, IGF-I, and IGFBPs at 0, 1, 4, and 24 h post-stressor exposure were measured. An unstressed group was also sampled at the same clock times (09:00, 10:00, 13:00, and 08:00 [the following day]) as acute stress sampling to determine temporal changes in the above somatotropic axis components. The acute stressor transiently elevated plasma cortisol and glucose levels at 1 and 4 h post-stressor exposure, whereas no changes were seen in the unstressed group. Plasma GH levels were not affected by handling stress or sampling time in the unstressed animals. Plasma IGF-I levels were significantly depressed at 1 and 4 h post-stressor exposure, but no discernible temporal pattern was seen in the unstressed animals. Using a western ligand blotting technique, we detected plasma IGFBPs of 21, 32, 42, and 50 kDa in size. The plasma levels of the lower-molecular-weight IGFBPs (21 and 32 kDa) were unaffected by handling stressor, nor were there any discernible temporal patterns in the unstressed animals. By contrast, the higher-molecular-weight IGFBPs (42 and 50 kDa) were affected by stress or time of sampling. Levels of the 42-kDa IGFBP levels significantly decreased over the sampling period in unstressed control animals, but this temporal drop was eliminated in stressed animals. Levels of the 50-kDa IGFBPs also decreased significantly over the sampling time in unstressed trout, whereas handling disturbance transiently increased levels of this IGFBP at 1 h but not at 4 and 24 h post-stressor exposure compared with the control group. Overall, our results suggest that acute stress adaptation involves modulation of plasma IGF-1 and high-molecular-mass IGFBP levels (42 and 50 kDa) in rainbow trout., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Melatonin has been suggested to play a role in fish osmoregulation, and in salmonids has been related to the timing of adaptive mechanisms during smolting. It has been described that acclimation to different environmental salinities alters levels of circulating melatonin in a number of fish species, including rainbow trout. However, nothing is known regarding salinity effects on melatonin synthesis in the pineal organ, which is the main source of rhythmically produced and secreted melatonin in blood. In the present study we have evaluated, in rainbow trout, the effects of acclimation to different salinities on day and night plasma melatonin values and pineal organ melatonin synthesis. Groups of freshwater (FW)-adapted rainbow trout were placed in tanks with four different levels of water salinity (FW, 6, 12, 18 p.p.t.; parts per thousand) and maintained for 6 h or 5 days. Melatonin content in plasma and pineal organs, as well as the pineal content of serotonin (5-HT) and its main oxidative metabolite (5-hydroxyindole-3-acetic acid; 5-HIAA) were measured by high performance liquid chromatography. In addition, day-night changes in pineal organ arylalkylamine N-acetyltransferase (AANAT2) activity and aanat2 gene expression were studied. Plasma osmolalities were found to be higher in rainbow trout exposed to all salinity levels compared with the control FW groups. A salinity-dependent increase in melatonin content was found in both plasma and pineal organs. This effect was observed during the night, and was related to an increase in aanat2 mRNA abundance and AANAT2 enzyme activity, both of which also occurred during the day. Also, the levels of indoles (5-HT, 5-HIAA) in the pineal organ were negatively affected by increasing water salinity, which seems to be related to the higher recruitment of 5-HT as a substrate for the increased melatonin synthesis. A stimulatory effect of salinity on pineal aanat2 mRNA expression was also identified. These results indicate that increased external salinity promotes melatonin synthesis in the pineal organ of rainbow trout by enhancing synthesis of AANAT protein independently of its regulation by light. The possibility that pineal melatonin is a target for hormones involved in the response of fish to osmotic challenge is discussed, as well as the potential role of melatonin in the timing of osmoregulatory processes.

Corticotropin-releasing factor (CRF) is central in the stress response but also modulates several behaviors including anxiety-related behaviors and aggression. In this study, juvenile rainbow trout (Oncorhynchus mykiss) were tested for competitive ability, determined during dyadic fights for dominance, after intracerebroventricular (i.c.v.) administration of CRF, urotensin I (UI), the non-specific CRF antagonist α-helical RF(9-41) (ahCRF) or the CRF receptor subtype 1-specific antagonist antalarmin, when paired with a mass-matched con-specific injected with saline. In addition, isolated fish received the same substances. Plasma cortisol and brain monoamines were monitored in all fish. Most fish receiving CRF showed a conspicuous behavior consisting of flaring the opercula, opening the mouth and violent shaking of the head from side to side. When this occurred, the fish immediately forfeited the fight. Similar behavior was observed in most fish receiving UI but no effect on outcome of dyadic fights was noted. This behavior seems similar to non-ambulatory motor activity seen in rats and could be anxiety related. Furthermore, fish receiving CRF at a dose of 1000 ng became subordinate, whereas all other treatments had no effects on the outcome of dyadic fights. In addition, isolated fish receiving ahCRF had lower brain stem concentrations of 5-hydroxyindoleacetic acid, serotonin, 3,4-dihydroxyphenylacetic acid and dopamine. In conclusion, CRF seems to attenuate competitive ability, and both CRF and UI seem to induce anxiety-like behavior.

Awareness of residual pharmaceutically active compounds (PhACs) in the aquatic environment is growing as investigations into these pollutants are increasing and analytical detection techniques are improving. However, the toxicological effects of PhACs have not been adequately researched. In this study, the toxic effects of carbamazepine (CBZ), an anticonvulsant drug commonly present in surface and groundwater, was studied in juvenile rainbow trout, Oncorhynchus mykiss, by acute semi-static bioassay. Blood parameters, liver xenobiotic-metabolizing response and tissue antioxidant status were evaluated. Compared to the control group, fish exposed to CBZ (96 h LC50) showed significantly higher Er, Hb, MCHC, monocytes, neutrophil granulocytes and plasma enzymes activity, and significantly lower MCV and lymphocytes. CF and HSI were not significantly different among groups such as hepatic EROD. SOD, CAT, GPx and GR activity was significantly higher in liver of experimental groups, but decreased significantly in brain and gill. In general, antioxidant enzyme activity in intestine and muscle was less evident than in liver. Oxidative stress indices (levels of LPO and CP) were significantly higher in gill and brain, despite a trend to increased values were manifested in the remaining tissues. In short, CBZ-induced stress responses in different tissues were reflected in the oxidant stress indices and hematological parameters. However, before those parameters are used as special biomarkers for monitoring residual pharmaceuticals in aquatic environment, more detailed experiments in laboratory need to be performed in the future., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Long-term effects of propolis administration on serum biochemical parameters of rainbow trout (Oncorhynchus mykiss) were investigated. To determine the possible toxicity and side effects of propolis, fish were fed on diets containing 0, 0.5, 1.5, 4.5 and 9 g propolis/kg diet for 8 weeks. At the end of the experiment, various seric biochemical parameters were determined. Our results showed that all dosages induced no significant alterations in growth parameters and the seric levels of total protein, albumin, globulin, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides and activities of glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, alkaline phosphatase and lactate dehydrogenase, when compared to the control group. On the basis of our findings, propolis is a non-toxic substance for rainbow trout and its long-term administration might not have any side effects., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Treated sewage effluents contain complex mixtures of micropollutants, raising concerns about effects on aquatic organisms. The addition of advanced treatment steps has therefore been suggested. However, some of these could potentially produce effluents affecting exposed organisms by unknown modes of action. Here, (1)H NMR (proton nuclear magnetic resonance spectroscopy) metabolomics of fish blood plasma was used to explore potential responses not identified by more targeted (chemical or biological) assays. Rainbow trout was exposed in parallel to six differently treated effluents (e.g., conventional activated sludge, addition of sand filter, further addition of ozonation and/or a moving bed biofilm reactor or a separate membrane bioreactor line). Multivariate data analysis showed changes in the metabolome (HDL, LDL, VLDL and glycerol-containing lipids, cholesterol, glucose, phosphatidylcholine, glutamine, and alanine) between treatment groups. This formed the basis for postulating a hypothesis on how exposure to effluent treated by certain processes, including ozonation, would affect the metabolic profiles of exposed fish. The hypothesis withstood testing in an independent study the following year. To conclude, (1)H NMR metabolomics proved suitable for identifying physiological responses not identified by more targeted assays used in parallel studies. Whether these changes are linked to adverse effects remains to be tested.

In this study, the annual cycle of the gonadal steroids testosterone (T), 11-ketotestosterone (11-KT), 17β-oestradiol (E2) and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was determined using radioimmunoassay and then compared, for XY males (n=35) and sex-reversed XX males (n=27) rainbow trout, to establish possible endocrinology differences. Both in XY males and sex-reversed XX males, significant correlation was shown between body weight and T (r=0.5046 and 0.34078, respectively; p<0.0001) or KT (r=0.52494 and 0.43545, respectively; p<0.0001) concentrations. Plasma androgen levels in XY and sex-reversed XX males were similar and showed an intense seasonal variation. The highest levels for T and 11-KT were detected from December to April with a peak in January (51.67 ± 5.11 and 61.95 ± 4.25 ng/ml, for XY males and 57.1 ± 5.82 and 59.27 ± 4.84 ng/ml, respectively, for XX males). In addition, there was a positive correlation (p<0.0001) between T and 11-KT levels for XY males (r=0.7533) and sex-reversed XX males (r=0.6019). Concentrations of DHP in XY males also showed seasonal variation with a peak in February (25.18 ± 12.99 ng/ml). However, DHP levels in sex-reversed XX males were undetectable (<0.1 ng/ml) over the year. Levels of E2 were undetectable through the year in both groups of trout. In conclusion, the androgenic and oestrogenic profiles of sex-reversed XX males were similar to those observed in XY males. The only difference in the annual gonadal steroid cycle between XY and sex-reversed XX males was in the DHP profile., (© 2009 Blackwell Verlag GmbH.)

To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of (15)N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R(d)), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R(d) values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R(d) values ranged from 0.9 μmol 100 g(-1) h(-1) (lysine) to 22.1 μmol 100 g(-1) h(-1) (threonine) with most values falling between 2 and 6 μmol 100 g(-1) h(-1). There was a significant correlation between R(d) and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.

An in vitro gut-sac technique and (64)Cu as a radiotracer were used to characterize gastric copper (Cu) transport. Cu transport was stimulated by low luminal pH (4.0 vs. 7.4), to a greater extent than explained by the increased availability of the free Cu(2+) ion. At pH = 4.0, uptake kinetics were indicative of a low affinity (K (m) = 525 μmol L(-1)), saturable carrier-mediated component superimposed on a large linear (diffusive and/or convective) component, with about 50% occurring by each pathway at Cu = 50 μmol L(-1). Osmotic gradient experiments showed that solvent drag via fluid transport may play a role in Cu uptake via the stomach, in contrast to the intestine. Also unlike the intestine, neither the Na(+) gradient, high Ag, nor phenamil had any influence on gastric Cu transport, and a tenfold excess of Fe and Zn failed to inhibit Cu uptake. These findings indicate that neither Na(+)-dependent pathways nor DMT1 are likely candidates for carrier-mediated Cu transport in the stomach. We have cloned a partial cDNA sequence for the copper transporter Ctr1, and show its mRNA expression in all segments of the trout gastrointestinal tract, including the stomach. Based on the fact that this transporter is functional at low pH conventionally found in the stomach lumen, we suggest Ctr1 is a pathway for gastric Cu transport in trout. Extreme hypoxia inhibited Cu uptake. High P(CO₂) levels (7.5 torr) increased Cu uptake and acetazolamide (100 μmol L(-1)) significantly inhibited Cu uptake, indicating carbonic anhydrase activity was involved in gastric Cu transport. Transport of Cu was insensitive to bafilomycin (10 μmol L(-1)) suggesting a V-ATPase did not play a direct role in the process. Expression (mRNA) of H (+) , K (+)-ATPase, carbonic anhydrase 2, and the α-3 isoform of Na (+)-K (+)-ATPase were observed in the stomach. We suggest these enzymes facilitate Cu transport in the stomach indirectly as part of a physiological mechanism exporting H(+) to the cell exterior. However, pre-treatment with the H (+) , K (+)-ATPase proton pump blocker omeprazole did not affect gastric Cu transport, suggesting that other mechanisms must also be involved.

A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells. Fish are the most primitive gnathostomes (jawed vertebrates) possessing an adaptive immune system. Vaccination of rainbow trout against enteric redmouth disease (ERM) by immersion in Yersinia ruckeri bacterin confers a high degree of protection to the fish. The immune mechanisms responsible for protection may comprise both cellular and humoral elements but the role of specific immunoglobulins in this system has been questioned and not previously described. The present study demonstrates significant increase in plasma antibody titers following immersion vaccination and significantly reduced mortality during Y. ruckeri challenge.Rainbow trout were immersion-vaccinated, using either a commercial ERM vaccine (AquaVac™ ERM vet) or an experimental Y. ruckeri bacterin. Half of the trout vaccinated with AquaVac™ ERM vet received an oral booster (AquaVac™ ERM Oral vet). Sub-groups of the fish from each group were subsequently exposed to 1 x 10⁹ CFU Y. ruckeri/ml either eight or twenty-six weeks post vaccination (wpv). All vaccinated groups showed 0% mortality when challenged, which was highly significant compared to the non-vaccinated controls (40 and 28% mortality eight and twenty-six weeks post vaccination (wpv), respectively) (P<0.0001). Plasma samples from all groups of vaccinated fish were taken 0, 4, 8, 12, 16 and 26 wpv. and Y. ruckeri specific IgM antibody levels were measured with ELISA. A significant increase in titers was recorded in vaccinated fish, which also showed a reduced bacteremia during challenge. In vitro plasma studies showed a significantly increased bactericidal effect of fresh plasma from vaccinated fish indicating that plasma proteins may play a role in protection of vaccinated rainbow trout.

Two studies to examine the effect of waterborne clofibric acid (CA) on growth-rate and condition of rainbow trout were conducted using accepted regulatory tests (Organisation for Economic Co-operation and Development [OECD] 215). The first study (in 2005) showed significant reductions after 21 d of exposure (21-d growth lowest-observed-effect concentration [LOEC] = 0.1 µg/L, 21-d condition LOEC = 0.1 µg/L) that continued to 28 d. Growth rate was reduced by approximately 50% (from 5.27 to 2.67% per day), while the condition of the fish reduced in a concentration-dependant manner. Additionally, in a concentration-dependent manner, significant changes in relative liver size were observed, such that increasing concentrations of CA resulted in smaller livers after 28-d exposure. A no-observed-effect concentration (NOEC) was not achieved in the 2005 study. An expanded second study (in 2006) that included a robust bridge to the 2005 study, with four replicate tanks of eight individual fish per concentration, did not repeat the 2005 findings. In the 2006 study, no significant effect on growth rate, condition, or liver biometry was observed after 21 or 28 d (28-d growth NOEC = 10 µg/L, 28-d condition NOEC = 10 µg/L), contrary to the 2005 findings. We do not dismiss either of these findings and suggest both are relevant and stand for comparison. However, the larger 2006 study carries more statistical power and multiple-tank replication, so probably produced the more robust findings. Despite sufficient statistical power in each study, interpretation of these and similar studies should be conducted with caution, because much significance is placed on the role of limited numbers of individual and tank replicates and the influence of control animals., (Copyright © 2010 SETAC.)

This paper reports the effect of feeding probiotic diets on blood profiles in rainbow trout. Two experiments were performed: in the first, fish of average weight 75 g were offered either a commercial feed or the same incorporated with 10(9) CFU g(-1) of lactic acid bacteria Lactobacillus rhamnosus for 30 days; in the second study performed for a similar duration, fish of average weight 126 g were offered formulated diets that either contained the same bacteria in heat-killed or freeze-dried form (nearly 10(11) CFU g(-1)), or the basal diet without the bacteria. Blood samples were collected at different times after commencement of probiotic feeding to determine the total cholesterol, triglyceride contents, the plasma alkaline phosphatase activity, plasma protein and hematocrit value. The plasma cholesterol significantly increased upon probiotic feeding in the first experiment. A significant elevation (P<0.05) of plasma cholesterol and triglyceride and alkaline phosphatase activity level was found in the freeze-dried probiotic fed groups at 20 and 30 days postfeeding. This was concomitant with the increased plasma protein and hematocrit values in FD group at 20 and 30 days. Likewise, the heat-killed probiotic fed group registered significantly high values of triglycerides, alkaline phosphatase activity, and plasma protein compared to the control diet fed groups after 20 days of feeding. Thus, alterations in the blood profiles could serve as supplementary information when examining the benefits of probiotics for fish.

To assess mechanisms relating to food intake and glucosensing in fish, and their interaction with stress, we evaluated changes in the expression of orexigenic (NPY) and anorexigenic (POMC, CART and CRF) peptides in central glucosensing areas (hypothalamus and hindbrain) of rainbow trout subjected to normoglycaemic (control), hypoglycaemic (4 mg insulin kg(-1)) or hyperglycaemic (500 mg glucose kg(-1)) conditions for 6 h under normal stocking density (NSD; 10 kg fish mass m(-3)) or under stress conditions induced by high stocking density (HSD; 70 kg fish mass m(-3)). Hyperglycaemic NSD conditions resulted in decreased mRNA levels of NPY and increased levels of CART and POMC in the hypothalamus as well as increased mRNA levels of CART and CRF in the hindbrain compared with hypo- and normoglycaemic conditions. HSD conditions in normoglycaemic fish induced marked changes in the expression of all peptides assessed: mRNA levels of NPY and CRF increased and mRNA levels of POMC and CART decreased in the hypothalamus, whereas the expression of all four peptides (NPY, POMC, CART and CRF) decreased in the hindbrain. Furthermore, HSD conditions altered the response to changes in glycaemia of NPY and POMC expression in the hypothalamus and CART expression in the hypothalamus and the hindbrain. The results are discussed in the context of food intake regulation by glucosensor systems and their interaction with stress in fish.

Current understanding of lactate metabolism in fish is based almost entirely on the interpretation of concentration measurements that cannot be used to infer changes in flux. The goals of this investigation were: (1) to quantify baseline lactate fluxes in rainbow trout (Oncorhynchus mykiss) under normoxic conditions; (2) to establish how changes in rates of lactate appearance (R(a)) and disposal (R(d)) account for the increase in blood lactate elicited by hypoxia; and (3) to identify the tissues responsible for lactate production. R(a) and R(d) lactate of rainbow trout were measured in vivo by continuous infusion of [U-(14)C]lactate in trout exposed to 25% O(2) saturation or maintained in normoxia for 90 min. In normoxic fish, R(a) lactate decreased from 18.2 to 13.1 μmol kg(-1) min(-1) and R(d) lactate from 19.0 to 12.8. R(a) and R(d) were always matched, thereby maintaining a steady baseline blood lactate concentration of ∼0.8 mmol l(-1). By contrast, the hypoxic fish increased blood lactate to 8.9 mmol l(-1) and R(a) lactate from 18.4 to 36.5 μmol kg(-1) min(-1). This stimulation of anaerobic glycolysis was unexpectedly accompanied by a 52% increase in R(d) lactate from 19.9 to 30.3 μmol kg(-1) min(-1). White muscle was the main producer of lactate, which accumulated to 19.2 μmol g(-1) in this tissue. This first study of non-steady-state lactate kinetics in fish shows that the increase in lactate disposal elicited by hypoxia plays a strategic role in reducing the lactate load on the circulation. Without this crucial response, blood lactate accumulation would double.