Dietary carotenoid pigment supplementation influences hepatic lipid and mucopolysaccharide levels in rainbow trout (Oncorhynchus mykiss)

We assessed the effects of dietary carotenoid pigment supplementation on liver histochemistry in the rainbow trout. One hundred and eight rainbow trout (mean mass 266 ± 10 g) were assigned to each of three replicate tanks for each of three dietary treatments; astaxanthin, canthaxanthin, or control at a target dietary inclusion of 100 mg/kg, by top-coating a pigment-free commercially extruded basal diet (Trouw Aquaculture, U.K.). Fish were fed for 3 weeks at a ration of 1.2% body mass/day, in a recirculating freshwater system maintained at 16 °C. Frozen liver sections were stained for total lipids, unsaturated lipids, glycogen, mucopolysaccharides, glycogen phosphorylase and aspartate aminotransferase. Relative amounts were measured quantitatively by image analysis. Carotenoid treatment significantly (P < 0.05) altered the total lipid profile and hepatic mucopolysaccharide contents of livers of rainbow trout. Results are discussed in relation to the catabolic potential of the liver in carotenoid pigment metabolism.     

Tissue astaxanthin and canthaxanthin distribution in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

A comparative investigation of tissue carotenoid distribution between rainbow trout, Oncorhynchus mykiss, and Atlantic salmon, Salmo salar, was undertaken to identify the relative efficiency of utilization of astaxanthin and canthaxanthin. Higher apparent digestibility coefficients (ADCs) (96% in trout vs. 28-31% in salmon; P<0.05), and pigment retention efficiencies (11.5-12.5% in trout vs. 5.5% in salmon; P<0.05), for both astaxanthin and canthaxanthin, were observed for rainbow trout. Astaxanthin deposition was higher than canthaxanthin in rainbow trout, while the reverse was true for Atlantic salmon, suggesting species-specificity in carotenoid utilization. The white muscle (95% in trout vs. 93% in salmon) and kidneys (0.5% in trout vs. 0.2% in salmon) represented higher proportions of the total body carotenoid pool in rainbow trout than in Atlantic salmon (P<0.05), whereas the liver was a more important storage organ in Atlantic salmon (2-6% in salmon vs. 0.2% in trout; P<0.05). The liver and kidney appeared to be important sites of carotenoid catabolism based on the relative proportion of the peak chromatogram of the fed carotenoid in both species, with the pyloric caecae and hind gut being more important in Atlantic salmon than in the rainbow trout. Liver catabolism is suspected to be a critical determinant in carotenoid clearance, with higher catabolism expected in Atlantic salmon than in rainbow trout.     

Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products

The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial. Fish were fed four diets containing either beta-carotene or astaxanthin at 100 and 200 mg kg-1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids. Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation. Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control. On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments. As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control. These findings demonstrate that dietary carotenoids from both D. salina and P. rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.     

Response of serum carotenoid levels to dietary astaxanthin and canthaxanthin in immature rainbow trout Oncorhynchus mykiss

Rainbow trout were fed a diet supplemented with astaxanthin (89 mg/kg) or canthaxanthin (116 mg/kg) in two different experiments: experiment 1 was designed to measure the kinetics of the appearance and disappearance of carotenoids in the serum; experiment 2 was undertaken to establish the serum dose-response to synthetic astaxanthin and canthaxanthin for immature rainbow trout. The serum carotenoid concentrations of immature rainbow trout increased when fish were fed carotenoid supplemented feed and then reached a plateau after 1 day of intake for astaxanthin and after 2 days for canthaxanthin. Circulating astaxanthin represented a value 2.3 times that of canthaxanthin. After dietary supplementation was discontinued, the serum carotenoid concentrations decreased within 3 days for both carotenoids. The average decreasing slopes for the two carotenoid pigments were parallel, indicating a similarity in the rate of which astaxanthin and canthaxanthin are utilized by rainbow trout. The serum dose-response of trout that received dietary keto-carotenoids increased with increasing pigment levels. The hypothesis that absorption of dietary carotenoids in 12.5–200 mg/kg range of concentration across the gut wall may be by passive diffusion is proposed.     

Determination of carotenoid and vitamin A concentrations in everted salmonid intestine following exposure to solutions of carotenoid in vitro

Carotenoid (astaxanthin and canthaxanthin) concentrations in everted intestine from rainbow trout (Oncorhynchus mykiss, Walbaum) and Atlantic salmon (Salmo salar, L.) exposed to micelle solubilised carotenoid, have been determined. Following exposure (1 h) to astaxanthin solution (5 mg l(-1)), trout pyloric caeca and mid intestine had higher (P<0.05) mean tissue astaxanthin concentrations (0.50+/-0.08 microg g(-1) and 0.54+/-0.09 microg g(-1), respectively) compared to hind intestine (0.04+/-0.01 microg g(-1); n=11+/-S.E.). Furthermore, the astaxanthin concentration in pyloric caeca (0.50+/-0.08 microg g(-1)) was greater (P<0.05) than that of canthaxanthin (0.11+/-0.01 microg g(-1); n=11, +/-S.E.) when exposed to solutions of similar carotenoid concentration (5.11+/-0.16 mg l(-1) and 5.35+/-0.16 mg l(-1), respectively; n=3+/-S.E.). However, no differences (P>0.05) were recorded between trout and salmon intestinal tissue in terms of astaxanthin concentration following exposure. Trout caeca exposed to astaxanthin solution had significantly (P<0.05) more vitamin A (514.1+/-36.4 microg g(-1)) compared to control tissues (316.5+/-61.7 microg g(-1); n=8+/-S.E.). Vitamin A(1) concentrations in caeca (287.7+/-11.0 microg g(-1)) exposed to astaxanthin solution were significantly higher (P<0.05) compared to controls (174.9+/-26.9 microg g(-1)). However, vitamin A(2) concentrations were not significantly (P>0.05) different (226.3+/-28.2 microg g(-1) and 141.6+/-35.2 microg g(-1), respectively).     

Effect of partial substitution of dietary protein by a single gluconeogenic dispensable amino acid on hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss)

Our objective was to understand the influence of dietary gluconeogenic amino acids on hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss). We analyzed the effects of partial substitution of dietary protein by a single gluconeogenic dispensable amino acid (DAA: alanine, aspartic acid or glutamic acid), on the regulation of hepatic glycolytic and gluconeogenic enzymes. We fed juvenile rainbow trout with isonitrogenous and isoenergetic diets in which part of nitrogen from fishmeal was replaced by nitrogen from one of the three DAA. Fish were fed over 9 weeks and samples withdrawn 6 h after feeding or 5 days after food deprivation. Our data did not show a clear effect of an excess of DAA on activities of glycolytic enzymes (glucokinase and pyruvate kinase) compared to the control diet. In contrast, feeding caused a significant repression of gluconeogenic enzyme activities (glucose-6-phosphatase, fructose-1,6-bisphosphatase and mitochondrial phosphoenolpyruvate carboxykinase) only in fish fed the three DAA substituted diets. However, these differences were insufficient to affect postprandial glycemia significantly. In conclusion, an excess of dietary DAA tested does not seem to modify glycemia or to have a negative impact on dietary carbohydrate utilization in rainbow trout.     

Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.     

Effect of cellulase,phytase and pectinase supplementation on growth performance and nutrient digestibility of rainbow trout (Oncorhynchus mykiss,Walbaum 1792) fry fed diets containing canola meal

This experiment was conducted to evaluate the effects of supplementing exogenous enzymes on growth, feed conversion ratio (FCR) and apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fry diets containing 32% canola meal. Five experimental diets (including a control diet containing no enzymes) were prepared as isonitrogenous (44% crude protein) and isocaloric (4000 kcal DE kg¹). The four other diets contained either cellulase, phytase, pectinase or an enzyme mix (a mixture of cellulase, phytase and pectinase in the same ratio). The feeding trial was conducted in triplicate for 12 weeks in 15 tanks (100‐L). At the beginning of the experiment 20 rainbow trout fry (initial weight 1.23 g) were stocked into each tank. Mean water temperature in the rearing tanks was 11°C and water flow in each tank was 6 L min^?1. At the end of the experiment the growth parameters and FCR displayed no significant differences in enzyme supplementation (P > 0.05). In addition, no differences were observed in dry matter, protein, or lipid digestibility with enzyme supplementation (P > 0.05). The results of this study showed that the addition of pectinase, phytase, cellulase or an enzyme mix to a diet containing 32% canola meal had no effect on growth, feed efficiency or dry matter, protein, or lipid digestibility in rainbow trout fry.     

Constraints on energy intake in fish: the link between diet composition, energy metabolism, and energy intake in rainbow trout

The hypothesis was tested that fish fed to satiation with iso-energetic diets differing in macronutrient composition will have different digestible energy intakes (DEI) but similar total heat production. Four iso-energetic diets (2 × 2 factorial design) were formulated having a contrast in i) the ratio of protein to energy (P/E): high (H(P/E)) vs. low (L(P/E)) and ii) the type of non-protein energy (NPE) source: fat vs. carbohydrate which were iso-energetically exchanged. Triplicate groups (35 fish/tank) of rainbow trout were hand-fed each diet twice daily to satiation for 6 weeks under non-limiting water oxygen conditions. Feed intake (FI), DEI (kJ kg(-0.8) d(-1)) and growth (g kg(-0.8) d(-1)) of trout were affected by the interaction between P/E ratio and NPE source of the diet (P<0.05). Regardless of dietary P/E ratio, the inclusion of carbohydrate compared to fat as main NPE source reduced DEI and growth of trout by ~20%. The diet-induced differences in FI and DEI show that trout did not compensate for the dietary differences in digestible energy or digestible protein contents. Further, changes in body fat store and plasma glucose did not seem to exert a homeostatic feedback control on DEI. Independent of the diet composition, heat production of trout did not differ (P>0.05). Our data suggest that the control of DEI in trout might be a function of heat production, which in turn might reflect a physiological limit related with oxidative metabolism.     

Effects of dietary Ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss)

The effects of dietary Ergosan on the growth performance and mucosal immunity in rainbow trout skin were investigated. 60 rainbow trout (100-110 g) were randomly assigned to 2 groups in triplicates and fed one of the experimental diet formulated with 5 g kg⁻¹ Ergosan or control diet for 50 days. Results showed that on the 45th day of feeding trial, Ergosan supplementation significantly enhanced the growth performance compared to control group. Various enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase in treatment group were also enhanced on the 45th and 50th day. Skin mucus in Ergosan-fed fish showed the agglutination of erythrocytes while in control group, no visible agglutination was shown. In addition, skin mucus in treatment group showed strong antibacterial activity against Yersinia ruckeri. In conclusion, the major immune components of rainbow trout mucus that are involved in the non-specific immunity were enhanced by administration of Ergosan in 5 g kg⁻¹.     

Hilyses®, fermented Saccharomyces cerevisiae, enhances the growth performance and skin non-specific immune parameters in rainbow trout (Oncorhynchus mykiss)

Effects of Hilyses^®, fermented Saccharomyces cerevisiae (S. cerevisiae), on growth, body composition and skin mucus immune components in rainbow trout were quantified. Ninety rainbow trout (105 ± 5 g) were randomly assigned to 2 groups in triplicates and fed dietary Hilyses^® (5 g kg^?1) or control diet without Hilyses^® for 50 days. Results of this study demonstrated that growth performance increased significantly by the dietary yeast supplement; however body composition was not affected in treatment group. At the 45th and 50th day of feeding trial, results of mucus samples demonstrated that yeast supplementation in treatment group significantly promoted enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase compared to control group. Significant increases were also observed in hemagglutination and antibacterial activity against Yersinia ruckeri in fish fed treatment diet. The present study suggests that fermented S. cerevisiae may effectively promote the growth performance and skin non-specific immune parameters in rainbow trout.     

Urea production and transport in teleost fishes

Teleosts appear to have retained the genes for the urea cycle enzymes. A few species express the full complement of enzymes and are ureotelic (e.g., Lake Magadi tilapia) or ammoniotelic (e.g., largemouth bass), whereas most species have low or non-detectable enzyme activities in liver tissue and excrete little urea (e.g., adult rainbow trout). It was surprising, therefore, to find the expression of four urea cycle enzymes during early life stages of rainbow trout. The urea cycle may play a role in ammonia detoxification during a critical time of development. Exposure to alkaline water (pH 9.0-9.5) or NH4Cl (0.2 mmol/l) increased urea excretion by several-fold in trout embryos, free embryos and alevin. Urea transport is either by passive simple diffusion or via carried-mediated transport proteins. Molecular studies have revealed that a specialised urea transport protein is present in kidney tissue of elasmobranchs, similar to the facilitated urea transporter found in the mammalian inner medulla of the kidney.     

Induction of metallothionein gene expression by cadmium and the retention of the toxic metal in the tissues of rainbow trout (Salmo gairdneri)

1. When rainbow trout were exposed to cadmium by intraperitoneal injection, there was a rapid (within 3hr) and significant (approx. 63%) loss of the metal from the whole bodies of the fish.2. Of the metal retained in the bodies of the fish (approx. 37% of the injected dose), more than 98% was accounted for collectively among the liver, kidney and gills.3. Subsequent maintenance of the rainbow trout in fresh water for up to 98 days post-metal administration, indicated that there was no further loss of the cadmium accumulated in the organs studied and that the distribution of the metal among the liver, kidney and gills remained unchanged over that period.4. During this 98-day period of maintenance of the fish, tissue concentrations of metallothionein-specific mRNA and metallothionein protein were quantified using riboprobe and ELISA systems respectively. Metallothionein-specific mRNA concentrations increased rapidly (within 24 hr) before falling back to levels similar to, or slightly greater than, those found in control animals. The concentration of metallothionein protein also increased significantly (within 3 days) then remained elevated thereafter.5. Throughout the experimental period, the concentrations of zinc and copper were also monitored in the liver, kidney and gills of the rainbow trout. The concentrations of each ion differed between each of the organs but did not change during the experiment.6. The induction of metallothionein gene expression by cadmium in the liver, kidney and gill of rainbow trout and the subsequent sequestration of the toxic metal is discussed with regard to the relative levels of these other essential metal ions.     

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.     

Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp

Glucokinase (GK) plays a central role in glucose homeostasis in mammals. The absence of an inducible GK has been suggested to explain the poor utilization of dietary carbohydrates in rainbow trout. In this context, we analyzed GK expression in three fish species (rainbow trout, gilthead seabream, and common carp) known to differ in regard to their dietary carbohydrate tolerance. Fish were fed for 10 wk with either a diet containing a high level of digestible starch (>20%) or a diet totally deprived of starch. Our data demonstrate an induction of GK gene expression and GK activity by dietary carbohydrates in all three species. These studies strongly suggest that low dietary carbohydrate utilization in rainbow trout is not due to the absence of inducible hepatic GK as previously suggested. Interestingly, we also observed a significantly lower GK expression in common carp (a glucose-tolerant fish) than in rainbow trout and gilthead seabream, which are generally considered as glucose intolerant. These data suggest that other biochemical mechanisms are implicated in the inability of rainbow trout and gilthead seabream to control blood glucose closely.     

Effects of temperature, food deprivation and salinity on growth, RNA/DNA ratio and certain enzyme activities in rainbow trout (Salmo gairdneri Richardson)

1. The connection between feeding regime (food deprivation and restricted diet) and thermal acclimation (1-2, 6, 11 and 16 degrees C) was studied in rainbow trout held in diluted seawater (20% S). 2. At 1 degree C, food deprivation effects on all parameters are slight, and on RNA and certain enzymes they are masked by thermal acclimation effects. 3. At a salinity of 20% rainbow trout on a restricted diet and held at 11 degrees C have the highest growth rate. 4. Owing to increasing RNA levels, the RNA/DNA quotient is significantly higher than normal in rainbow trout held at 1 degree C although the fishes do not grow at this temperature. 5. Temperature and feeding both affect the enzymes we studied (liver: G1DH, AspT, arginase, G6PDH, and 6PGDH; kidney: G1DH, AspT, arginase, and Na/K-ATPase; white muscle: AspT and A1T; gill: Na/K-ATPase) differently. Interactions between these two factors also occur in some cases.     

Comparative studies of catalase and superoxide dismutase activity within salmon fish erythrocytes

1. Superoxide dismutase isolated from erythrocytes of several species of salmon and the rainbow trout exhibited single electrophoretic bands of activity which migrated anodally similar to the human erythrocyte enzyme; two discrete bands were observed for the coho salmon. 2. No polymorphism was observed for 30 samples from sockeye salmon and six samples from king salmon. Only one sample of rainbow trout (one of 12) exhibited an electrophoretic mobility difference. 3. Catalase migration on starch-gel resembled the human enzyme's electrophoretic mobility for all salmon species and rainbow trout. Catalase activity of the sockeye salmon (2929 +/- 895 mumol min-1 gHb-1) was determined to be lower than human catalase activity. 4. All samples differed from the human enzymes in that they required the presence of a detergent, Triton X-100, for solubilization.     

Effects of Haematococcus pluvialis in maternal diet on reproductive performance and egg quality in rainbow trout (Oncorhynchus mykiss)

The aim of this study was to clarify the effects of dietary Haematococcus pluvialis (H.p) on reproductive performance in female rainbow trout and egg quality in terms of antioxidant system and biochemical parameters. 60 rainbow trout (2475.5 ± 64.4 g) were randomly assigned to 2 groups in triplicates and fed diet containing 3 g H.p kg(-1) feed equivalent to 30 mg astaxanthin kg(-1) die or control diet for 30 days. On days 20 and 30 during feeding trial, mature fish were weighed and sampled for stripping. Results indicated that supplementation of H.p did not improve total egg weight, egg number per gram and fecundity. There were few changes in triglyceride and total protein content in fish eggs. Level of glucose decreased markedly on day 30 while on day 20 of feeding trial, a non-significant decrease was shown in treatment group. On day 20, the level of malondialdehyde (MDA) indicating lipid peroxidation product significantly decreased in eggs of the treatment group. The activities of enzymes of the antioxidant system did not change during this study, even though slight increase in glutathione peroxidase in treatment group was revealed during this study. In conclusion, this study showed that female rainbow trout appear to benefit from inclusion of H.p in diet during their reproductive stages in terms of improved egg quality.     

Glucokinase is highly induced and glucose-6-phosphatase poorly repressed in liver of rainbow trout (Oncorhynchus mykiss) by a single meal with glucose

The low dietary starch utilisation by rainbow trout (Oncorhynchus mykiss) may be attributed to a dysfunction of the nutritional regulation of the hepatic glucose/glucose-6-phosphate cycle. The present study was initiated to analyse the regulation of activity and gene expression of hepatic glucokinase (GK) and glucose-6-phosphatase (G6Pase) by dietary carbohydrates in this species. We found that even a single meal containing 24% of glucose is sufficient to induce the GK expression (mRNA and activity) as in mammals. In contrast, although the inhibitory effect of dietary glucose on G6Pase expression is observed at the molecular level, the G6Pase activity is not significantly inhibited by dietary glucose. Thus, in contrast to the gluconeogenic G6Pase enzyme, a rapid adaptation of the hepatic glycolytic GK enzyme to dietary glucose seems effective in rainbow trout. These results suggest that in carnivorous rainbow trout, the liver is capable to strongly regulate the utilisation of glucose but not the synthesis of glucose.