Dietary Na does not reduce dietary Cu uptake by juvenile rainbow trout

Rainbow trout Oncorhynchus mykiss fry in moderately hard water were exposed to control or high levels of dietary Cu (c. 6 and 580 ug Cu g food^?1) at one of three levels of Na (1·5, 3·0 or 4·5%) in the diet, i.e. six experimental groups. Fish were fed a 4% body mass ration daily for 28 days and 10 individuals from each group were sampled every 7 days. Concentrations of Cu and Na were measured in the gills, liver, gut and remaining carcass of sampled fish. Growth was not affected and no consistent differences were found in mass, total lengths (L_T) or indices of body condition among any of the groups on any sampling day. Copper concentration was significantly higher in tissues of Cu‐exposed groups, although within treatment types (control Cu v. high Cu diet), it did not differ consistently among groups that received different levels of dietary Na. Tissue Na concentration did not differ among any of the groups and did not show any marked changes over time. In Cu‐exposed groups, the proportion of total body Cu burden contained in the liver approximately doubled over time, from c. 30% on day 7 to c. 60% on day 28. In unexposed fish, the liver maintained c. 25% of the total Cu burden throughout the experiment. In contrast, the proportion of the total body Cu burden contained in the gut decreased somewhat over time in Cu‐exposed fish, from c. 40% on day 7 to c. 30% on day 28, and remained fairly stable at c. 25–30% in control groups, i.e. approximately equal to liver values. In all groups, the carcass contained by far the largest portion of the total Na content (>80%). Measurements made 36 h post‐feeding indicated that all six groups had much higher Na efflux relative to influx, suggesting that the fish were eliminating excess Na taken up from the diet, and differences in Na influx rates were small. Na efflux rate was significantly higher in the high Cu and high Na group than in the high Cu and low Na group. The results indicate that at the concentrations used in this experiment, dietary Na has little effect on dietary Cu uptake by juvenile rainbow trout, and dietary Cu has little effect on Na homeostasis.     

Uptake and accumulation of zinc in juvenile rainbow trout,Salmo gairdneri

Synopsis The toxicity of zinc to rainbow trout was determined and the 72 h median lethal concentration was found to be 2.00 mg l^–1 in freshwater, hardness 7.50 mg l^–1 as calcium. An insignificant increase in zinc concentration of internal tissues occurred in fish exposed to 1.52 mg l^–1 in freshwater for 72 h. However, there was a significant uptake of zinc by gills and the body surface. Fish exposed to 10 mg l^–1 zinc for 72 h in two-thirds sea water showed significant zinc uptake by liver, rectum and muscle, when compared to control fish. Drinking rate decreased from 1.43 to 0.26 ml kg^–1 h^–1 when zinc sulphate was added to freshwater. Trout adapted to two-thirds sea water showed no decrease in drinking, about 7 ml kg^–1 h^–1 when zinc was added to the water.     

Exhaustive exercise does not affect the preferred temperature for recovery in juvenile rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that juvenile rainbow trout (Oncorhynchus mykiss) would select a temperature colder than their acclimation temperature (16 deg +/-1 deg C) to minimize postexhaustive exercise metabolic demands and enhance oxygen availability. After an initial 3-h exploratory period in a thermal gradient (6 degrees -25 degrees C), fish selected a temperature of approximately 14 degrees C and had a baseline exploratory swimming activity of approximately 60 cm min(-1). Subsequently, experimental (chased) fish were individually removed, exhaustively exercised for 1.5 min, and replaced. Both control (unchased) and experimental fish were allowed to explore the thermal gradient for another 2 h. Immediately after being chased, trout had a metabolic profile that was consistent with being exhausted; levels of plasma and muscle lactate were 4.38+/-0.25 mmol L(-1) and 28.0+/-2.0 mmol kg(-1), respectively, and levels of muscle glycogen, adenosine triphosphate, and phosphocreatine were 3.89+/-0.95, 4.23+/-0.62, and 3.07+/-0.73 mmol kg(-1), respectively. Although exploratory swimming activity of the chased fish was significantly lower (by 81%) as compared with control fish during the first 5 min postchase, differences in the mean, median, and mode values for selected temperatures during the next 2 h were neither large (<1 degrees C) nor significant (P>0.05). Contrary to our initial hypothesis, these findings suggest that juvenile rainbow trout do not select a colder temperature to decrease metabolic rate following exhaustive exercise. Instead, rainbow trout selected a temperature marginally cooler than their acclimation temperature (16 degrees C) regardless of whether they had been previously exhausted.     

ACTH does not mediate divergent stress responsiveness in rainbow trout.

Two lines of rainbow trout selected for high (HR) and low (LR) responsiveness to a standardised confinement stressor displayed a sustained divergence in plasma cortisol levels during a 3-h period of confinement (max.: HR: 167+/-13 ng ml(-1); LR: 103+/-8 ng ml(-1); P<0.001). However, no significant difference in plasma ACTH levels was evident (max: HR: 153+/-9 pg ml(-1); LR: 142+/-7 pg ml(-1)). Dexamethasone (DEX) was administered to HR and LR fish to block endogenous adrenocorticotropin (ACTH) release. Administration of a weight-adjusted dose of ACTH to the DEX-blocked fish elevated plasma cortisol levels to a significantly greater extent in HR (233+/-24 ng ml(-1)) than LR (122+/-14 ng ml(-1)) fish (P<0.001). Plasma cortisol levels in DEX-blocked HR and LR fish after sham injection were low but also significantly different (HR: 6.7+/-1 ng ml(-1); LR: 2.2+/-0.2 ng ml(-1); P<0.001). These results indicate that modulation of cortisol responsiveness to stressors in HR and LR fish resides, at least in part, downstream of the hypothalamic-pituitary axis.     

Cryopreservation‐induced alterations in protein composition of rainbow trout semen

The aim of this study was to detect cryopreservation‐induced alterations in the protein composition of rainbow trout semen using two independent methods 1DE SDS‐PAGE prefractionation combined with LC‐MS/MS and 2D difference gel electrophoresis followed by MALDI‐TOF/TOF identification. Here, we show the first comprehensive dataset of changes in rainbow trout semen proteome after cryopreservation, with a total of 73 identified proteins released from sperm to extracellular fluid, including mitochondrial, cytoskeletal, nuclear, and cytosolic proteins. Our study provides new information about proteins released from sperm, their relation to sperm structure and function, and changes of metabolism of sperm cells as a result of cryopreservation. The identified proteins represent potential markers of cryoinjures of sperm structures and markers of the disturbances of particular sperm metabolic pathways. Further studies will allow to decipher the precise function of the proteins altered during rainbow trout cryopreservation and are useful for the development of extensive diagnostic tests of sperm cryoinjures and for the successful improvement of sperm cryopreservation of this economically important species.     

Dietary fructose does not specifically induce hepatic glucokinase expression in rainbow trout

Glucokinase (GK) catalyses the first step of hepatic glycolysis to store the excess dietary glucose. After rainbow trout Oncorhynchus mykiss were fed a single meal with dietary glucose and fructose, there was induction of GK expression, although no specific effect of dietary fructose was observed. This suggested that dietary fructose is not essential to improve dietary glucose Utilization.     

Intersexual diVerences in aggression in juvenile rainbow trout

Agonistic interactions were observed in dyadic contests between size-matched rainbow trout. The results suggest that male juveniles are more aggressive than females.     

Characterization of p53 expression in rainbow trout

The tumour suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. Given the high incidence of p53 mutations in human cancers, it has been extensively studied, though only a small fraction of these investigations have been in non-mammalian systems. For the present study, an anti-rainbow trout p53 polyclonal antibody was generated. A variety of rainbow trout (Oncorhynchus mykiss) tissues and cell lines were examined through western blot analysis of cellular protein extracts, which revealed relatively high p53 levels in brain and gills. To evaluate the checkpoint response of rainbow trout p53, RTbrain-W1 and RTgill-W1 cell lines were exposed to varying concentrations of the DNA damaging agent bleomycin and ribonucleotide reductase inhibitor hydroxyurea. In contrast to mammals, these checkpoint-inducing agents provoked no apparent increase in rainbow trout p53 levels. These results infer the presence of alternate DNA damage checkpoint mechanisms in rainbow trout cells.     

Chromosome studies of progenies of tetraploid female rainbow trout

Summary Nine induced tetraploid females were artificially inseminated by UV-irradiated sperm collected from diploid males, in order to induce the gynogenetic development of their ova. Most of the resulting embryos were diploid (or minor aneuploids). Several gynogenetic tetraploids, likely to issue from unreduced ova, were also detected in these progenies. The same females fertilized by normal sperm of diploid males gave a majority of triploids and several pentaploids, while the fertilization by normal sperm of tetraploid males gave rise to a majority of tetraploids and one hexaploid. The same crosses, after the eggs had been heat-shocked to double the maternal genetic contribution, yielded about three-quarters pentaploids and one quarter haploids (normal sperm of diploids), or three-quarters hexaploids and one quarter diploids (normal sperm of tetraploids). These haploids and diploids are likely to result from androgenesis.     

Sublethal ammonia and urea concentrations inhibit rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase

In vitro and in vivo effects of sublethal ammonia and urea concentrations were assayed on glucose-6-phosphate dehydrogenase (G6PD) of rainbow trout (Oncorhynchus mykiss) erythrocyte. G6PD was purified from erythrocytes with a specific activity of 16.7 EU (mmol NADP+/min)/mg protein and approximately 1600-fold in a yield of approximately 60% by ammonium sulphate precipitation and 2',5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was confirmed using SDS polyacrylamide gel electrophoresis. Experiments with ammonia (2.2-5.5 microM) and urea (20-50 microM) showed the inhibitory effects on the enzyme, in vitro. Inhibition effects were determined in vitro by Lineweaver-Burk and regression graphs. The dissociation constant of the enzyme inhibitor complex (Ki) and 50% inhibitory values were 2.26+/-1.21 and 2.86+/-3.51 microM for ammonia and 18.69+/-6.75 and 23.77+/-4.58 microM for urea, respectively. In vivo studies in rainbow trout erythrocytes showed significant (p < 0.01) inhibition of G6PD by ammonia and urea. However, ammonia inhibited more than urea since there were significant differences between the final values of erythrocyte G6PD activities.     

Creatine supplementation affects sprint endurance in juvenile rainbow trout

Fingerling rainbow trout were supplemented with equal amounts of creatine (Cr) by two routes: dietary (12.5 mg Cr per g food); or intraperitoneal injection (0.5 mg Cr per g fish). Endurance in a fixed velocity sprint test (at a speed of 7 BL s(-1)), and resting levels of white muscle metabolites (total creatine [a measure of free creatine plus phosphocreatine (PCr), ATP, lactate and glycogen] were assessed following 7 days of supplementation and compared to controls. None of the treatments had a significant effect on growth, muscle total creatine, percent phosphorylation of creatine, ATP or lactate. However, resting muscle glycogen was elevated in creatine-supplemented fish. Higher muscle glycogen corresponded to significantly greater endurance in creatine-supplemented fish. Although fish do not actively transport additional creatine into the muscle, a mechanism whereby circulating creatine acts to enhance muscle glycogen is present. These results suggest that the improved endurance may be due to an insulin-dependent mechanism (similar to that elucidated in mammalian studies) that allows fish to supercompensate muscle glycogen stores, thus extending endurance through enhanced glycolytic flux.     

Effect of induced triploidy on fin regeneration of juvenile rainbow trout, Oncorhynchus mykiss

Triploidy was induced in the rainbow trout in order to evaluate whether the altered numbers and sizes of triploid cells could modify fin regeneration. Amputation of one lobe of the tail fin of diploid and triploid juveniles resulted in regeneration for experimentals and controls. Nevertheless, both rate and frequency of regeneration in triploids were significantly increased as compared with diploids, a fact that can be attributed to the increased nuclear and cellular volume in a wide range of tissues, whereas the cell numbers were reduced. These data suggest that a great deal of interesting and important research could be done using triploid animals as experimental models for studying the regeneration of appendages.     

Observations of poor swimming performance among hatchery-reared rainbow trout,Salmo gairdneri

Synopsis The swimming performance, as judged by maximum sustained swimming speed, of rainbow trout from a fish farm in S.W. England, is low when compared to previously published values for this species. This may be a localised peculiarity resulting from hatchery selection and rearing procedures.     

Relating intramuscular fuel use to endurance in juvenile rainbow trout

This study examined fuel depletion in white muscle of juvenile rainbow trout sprinted to fatigue to determine whether the onset of fatigue is associated with a measurable metabolic change within the muscle and whether muscle glycogen levels influence endurance. In this study, "fuels" refer to any energy-supplying compounds and include glycogen, phosphocreatine (PCr), and ATP. Fuel depletion in white muscle was estimated by the calculation of the anaerobic energy expenditure (AEE; in micromol ATP equivalents g(-1)) from the reduction of PCr and ATP and the accumulation of lactate. Progression of fuel use during sprinting was examined by sampling fish before they showed signs of fatigue and following fatigue. Most of the AEE before fatigue was due to PCr depletion. However, at the first signs of fatigue, there was a 32% drop in ATP. Similarly, when fish were slowly accelerated to a fatiguing velocity, the only significant change at fatigue was a 30% drop in ATP levels. Muscle glycogen levels were manipulated by altering ration (1% vs. 4% body weight ration per day) combined with either daily or no exercise. Higher ration alone led to significantly greater muscle glycogen but had no effect on sprint performance, whereas sprint training led to higher glycogen and an average threefold improvement in sprint performance. In contrast, periodic chasing produced a similar increase in glycogen but had no effect on sprint performance. Taken together, these observations suggest that (i) a reduction in ATP in white muscle could act as a proximate signal for fatigue during prolonged exercise in fish and (ii) availability of muscle glycogen does not limit endurance.     

Subclinical Listonella anguillarum infection does not impair recovery of swimming performance in rainbow trout Oncorhynchus mykiss

This study examines whether injections of the commonly used bacterial-challenge pathogen Listonella anguillarum (formerly Vibrio anguillarum) negatively impact the ability of rainbow trout Oncorhynchus mykiss Walbaum to perform repeat swimming trials. Fish were given intraperitoneal injections of either a sub-lethal (10(5) colony forming units; CFUs) or a lethal (10(7) CFUs) dose of L. anguillarum, held for 48 h, and then given 2 successive ramp critical swimming speed (Ucrit) tests separated by 45 min. Compared with saline-injected control fish, the low-dose injection did not significantly impair swimming performance and recovery. Similarly, Ucrit and re-performance for fish surviving the high-dose injection were comparable to control (2 of 6 fish died after injection and before testing). In contrast, a positive control test of seawater challenge did impair recovery of swimming performance. In view of these results and common use of L. anguillarum as a challenge pathogen for toxicological studies, it seems unlikely that the consequences of pathogenesis impact the important cardiorespiratory changes associated with exercise.     

The dynamics of oocyte growth during vitellogenesis in the rainbow trout (Oncorhynchus mykiss)

This report describes the dynamics of oocyte growth during vitellogenesis in a population of virgin female rainbow trout. Indices of ovarian development increased dramatically during the period of study: the gonadosomatic index (GSI) increased over 50-fold, reaching a peak of 20 just before ovulation; the mean oocyte diameter increased from less than 1 mm to 5.4 mm; and plasma levels of vitellogenin increased from less than 1.5 mg/ml to 25 mg/ml. There were no changes in the numbers of developing oocytes (measuring 0.5 mm or greater in diameter) from the time when the majority of oocytes undergoing secondary development had entered vitellogenesis in August to ovulation in February (averaging 4000 oocytes per fish). The increase in ovary weight during vitellogenesis was, therefore, due to an increase in the size of oocytes rather than to recruitment of more maturing oocytes. The numbers of vitellogenic oocytes in the ovary during the entire study also suggested that atresia of vitellogenic oocytes does not play a prominent role in determining fecundity. During early vitellogenesis, the volume of maturing oocytes within an ovary varied by as much as 250-fold. From September onwards, when all oocytes to be ovulated that season had entered vitellogenesis, a gradual uniformity in size began to develop, such that at ovulation, in February, all the eggs were very similar in size (there was less than a 2-fold variation in volume). The pattern of growth of oocytes in an ovary during vitellogenesis suggests that growth between oocytes is closely coordinated.     

Multiple biomarker response in rainbow trout during exposure to hexavalent chromium

This research investigated hexavalent chromium toxicity in rainbow trout using a panel of biomarkers at different levels of biological organization. A time-course experiment in which rainbow trout were exposed in hard water (63.5 mg/L CaCO3) to a sublethal concentration of hexavalent chromium (10 mg/L) for a period of 28 days was conducted. The responses of multiple biomarkers were measured in gill and liver tissues at varying time points. Significant differences in metallothionein induction, superoxide dismutase activity, lipid peroxidation, cellular morphology, and growth were observed. Results indicated that gill tissues were more sensitive than hepatic tissues to chromium toxicity, yet hepatic tissues appeared to play a larger role in the organism's adaptive response to chromium compared to gill tissues. This study highlights the importance of using a set of integrated biomarkers to assess contaminant exposure and effects.     

Stress responsiveness affects dominant-subordinate relationships in rainbow trout.

The magnitude by which plasma cortisol levels increase following exposure to a stressor is a heritable trait in rainbow trout. The relative growth in coculture of F1 lines selected for high responsiveness (HR) and low responsiveness (LR) to a confinement stressor suggested that behavioral characteristics related to food acquisition, aggression, or competitive ability might differ between the two lines. This hypothesis was tested using the F2 generation of the selected lines. The F2 lines clearly exhibited the characteristics of the F1 parents, displaying significantly divergent plasma cortisol responses to a 1-h confinement stressor and a high heritability for the trait. Behavioral differences between the lines were assessed by observing the outcome of staged fights for dominance in size-matched pairs of HR and LR fish. The identification of dominant and subordinate fish within each pair on the basis of their behavior was supported by the levels of blood cortisol in the fish attributed to each group (dominant < subordinate). Fish from the LR line were identified as dominant in significantly more trials than were HR individuals. The results suggest that behavioral attributes that affect the outcome of rank-order fights are closely linked to the magnitude of the plasma cortisol response to stress in rainbow trout. Whether the link is causal or circumstantial is not yet evident.     

A single major chromosomal region controls natural killer cell-like activity in rainbow trout

We report the identification of a single major chromosomal region controlling natural killer (NK) cell-like activity in rainbow trout (Oncorhynchus mykiss). A genetic map based on 484 AFLP and 39 microsatellite genotypes from 106 doubled haploid fish was constructed. These fish were produced by androgenesis from a hybrid of two clonal lines divergent in NK-like activity. NK-like activities for 75 of the doubled haploids were quantified by an in vitro chromium release assay utilizing ⁵¹Cr-labeled YAC-1 target cells. Composite interval mapping revealed a single major quantitative trait locus (QTL) associated with NK-like activity in this rainbow trout model. Genetic mapping revealed this QTL to also be unlinked to: fragmented MHC class I and MHC class II regions, the leukocyte receptor cluster, the natural killer cell enhancement factor (NKEF) gene, the RAG-1 gene, and two QTL associated with resistance to infectious pancreatic necrosis virus in rainbow trout. Collectively, these results extend the utility of rainbow trout as an immunological model and are consistent with the idea that a single chromosomal region homologous to the natural killer cell complex (NKC) located on syntenic portions of mouse chromosome (Chr) 6, human Chr 12, and rat Chr 4 may exist in a lower vertebrate model.     

Ritonavir does not inhibit calpain in vitro

Ritonavir, an inhibitor of HIV-1 protease, has been reported to also inhibit the Ca2+-dependent cysteine protease, calpain. We have investigated these claims with an in vitro study of the effect of ritonavir on the m-calpain and mu-calpain isoforms. Ritonavir failed to block either autolytic or hydrolytic calpain activity, but remained fully capable of inhibiting the HIV-1 protease. Any calpain-related effects of ritonavir in cells must, therefore, arise by a mechanism other than direct inhibition of calpains.