Cytochrome P-450-dependent metabolism in isolated liver cells from fed and starved rainbow trout,Salmo gairdneri

The cytochrome P-450 content in liver cells from rainbow trout was not affected by starvation for 12 weeks whereas the rate of cytochrome P-450-dependent deethylation of 7-ethoxycoumarin in liver cells from 6 or 12 weeks starved fish was 60% of the rate in fed fish. Treatment of fish with -naphthoflavone increased the 7-ethoxycoumarin metabolism several-fold in both starved and fed fish.Optimal cytochrome P-450 monooxygenation in liver cells from fed or starved fish was not affected by addition of glucose or 2-bromooctanoate, an inhibitor of fatty acid -oxidation which is the main source of metabolic fuel in trout liver. The cellular content of NADPH, an obligatory cofactor for cytochrome P-450 monooxygenation, was not affected by addition of substrate to cytochrome P-450, inhibition of fatty acid -oxidation or inhibition of the oxidative phosphorylation. This indicates a great capacity of rainbow trout liver cells to retain high NADPH/NADP⁺ ratios. These results suggest that the cytochrome P-450 mediated metabolism of xenobiotics in liver cells from fed or starved trout is not limited by the availability of reducing equivalents.     

Effects of starvation and feeding on canthaxanthin depletion in the muscle of rainbow trout (Salmo gairdneri Rich.)

An experiment was conducted to examine carotenoid depletion in the muscle of rainbow trout. After 4 weeks of ingestion of canthaxanthin, fish were divided into two groups: the first was fed the same diet but without the canthaxanthin, the second was starved. Lipid and canthaxanthin levels in epaxial muscle were followed for 38 days. The lipid content increased (× 1.84) in fed fish and remained stable in those which were starved. Fed fish lost more canthaxanthin than starved fish. The ratio of the relative weight gain of canthaxanthin to that of the lipids was higher in starved than in fed fish (0.91 and 0.69 respectively). It is concluded that an internal redistribution of canthaxanthin takes place in the absence of an intake of the pigment. These results also show that the muscle of rainbow trout has a high storage capacity.     

Toxicity and immunogenicity of Aeromonas salmonicida extracellular products to salmonids

Abstract. Aeromonas salmonicida extracellular products (ECP) were fractionated by gel filtration chromatography. Three protein fractions were found. The first and second fractions showed low and high haemolytic activity, respectively, whereas the third fraction showed protcolytic activity. By intramuscular injection into juvenile rainbow trout, the median lethal doses of the first, second and third fractions were calculated at >669, 152 and <1514μg/g body weight, respectively. Cytopathic effects of the fractionated ECP to coho salmon lymphocytes were observed in vitro. The first and second fractions caused cell deformation, nuclear granulation and cytoplasmic streaming after 3h. No cytologic effects with the third fraction were observed. A mixture of the first and third fractions, a mixture of the second and third fractions, and non-fractionated ECP caused nuclear granulation and cytoplasmic streaming after 3Qmin. Using immunodiffusion analysis, the first and second fractions formed a single precipitating line each against white-spotted char anti-ECP sera, but the third fraction did not formed a precipitating line against the antisera.     

Distribution and induction of cytochrome P450 1A1 in the rainbow trout brain

Cytochrome P450 (CYP) 1A1 participates in the activation as well as detoxification of environmental pollutants such as aromatic hydrocarbons. This CYP form is also efficiently induced by aromatic hydrocarbons. The presence of CYP 1A1 in the brain might thus be of physiological and toxicological importance. In the present investigation on rainbow trout, the distribution of 7-ethoxyresorufin-O-deethylase (EROD) activity, a cytochrome CYP 1A1 catalyzed reaction, was measured in whole tissue homogenates from brain parts. In control fish, a relatively high activity was found in the rainbow trout olfactory bulb compared to the other brain parts. Although an EROD induction (3 to 7-fold) by β-naphthoflavone (BNF) was recorded in all brain parts from the rainbow trout, the highest induced activity was measured in the olfactory bulbs. To ascertain the distribution of EROD activity in cells, whole brain tissue was subfractionated by differential centrifugation. The fractionation scheme separated mitochondria (P2 fraction) and microsomes (P3 fraction) as determined by marker enzymes and electron microscopy. In control rainbow trout, a low EROD activity could be measured in the P2 fraction. BNF induced the EROD activity in both P2 and P3 fractions. Western blotting showed the induction by BNF of a protein band in the P2 and P3 fractions with a molecular mass around 58,000 when highly specific anti-cod CYP 1A1 antibodies were used. ELISA measurements confirmed the induction of CYP 1A1 protein in the rainbow trout brain subcellular fractions.     

The effect of short-term starvation on ovarian development and egg viability in rainbow trout (Salmo gairdneri)

Adult female rainbow trout (Salmo gairdneri) were fed a commercial trout production diet until approximately 40 days prior to spawning at which time the fish were divided in two groups. One group continued to be fed while the other group was starved until spawning. Egg size, egg number, ovary weight, and egg proximate composition were examined periodically starting 5 months before spawning. Muscle tissues of the fish were sampled at spawning to measure somatic depletion of proximate consituents in the two groups. Egg hatchability was determined by incubating test lots from each spawning female. No significant differences (P > 0.05) were found in egg hatchability between the fed and starved groups. Changes in proximate composition were observed in the eggs during the months prior to spawning, but no differences were detected between the eggs of the starved and the fed groups. Short-term starvation did not significantly alter the proximate composition of muscle tissue of the spawned female trout.     

Effects of dietary zinc intake upon copper metabolism in rainbow trout (Salmo gairdneri)

Purified diets with copper levels of 2.5 and 500 mg/kg and dietary zinc levels ranging from 34 to 1000 mg/kg were fed for 20 weeks to rainbow trout of mean initial weight 8 g. Growth and feed conversion were similar in all groups of fish and no gross pathologies were observed. Liver copper levels were greatest in trout given the high copper diets; increasing dietary zinc intake reduced these hepatic copper concentrations. Subcellular fractionation of the livers showed that in all groups of trout 70–80% of the hepatic copper was present in the nuclear and mitochondrial fractions. In contrast, the cytosol and microsomal fractions contained, on average, 73% of the liver zinc. Total liver superoxide dismutase activity was constant in all groups of fish. However, in trout fed the high copper diets the relative proportions of the copper-zinc and the manganese superoxide dismutase were correlated with the dietary zinc intake. Increasing dietary zinc reduced the activity of the manganese enzyme and increased that of the copper-zinc metalloenzyme.     

Oxidative stress parameters during starvation and refeeding periods in Adriatic sturgeon (Acipenser naccarii) and rainbow trout (Oncorhynchus mykiss)

This work analyses the changes in the redox balance in two fish species: Adriatic sturgeon ( Acipenser naccarii ) and rainbow trout ( Oncorhynchus mykiss ) during starvation and refeeding period. The starvation period raised the lipid peroxidation (thiobarbituric-acid-reacting substances) levels in liver and blood, while a decline occurred in the antioxidant enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) in both fish species. In liver, after the refeeding period, SOD activity recovered in both species, whereas CAT activity recovered only in trout. Furthermore, in both tissues of the two species, the lipid peroxidation levels remained high after 2 months of refeeding. In white muscle and heart, the lipid peroxidation levels indicate that these tissues did not undergo oxidative stress during the 72-day period. During starvation, in the muscle of both fish the fall in the lipid peroxidation level coincided with a rise in CAT, GPX and GR. The refeeding period in this tissue raised the lipid peroxidation level, and the enzymatic activities reached the values of the first point of starvation. In heart, no oxidative damage was detected during starvation in either species. The CAT and SOD activities increased during the starvation period only in trout.     

Development of a protein binding assay for teleost insulin-like growth factor (IGF)-like: relationships between growth hormone (GH) and IGF-like in the blood of rainbow trout (Oncorhynchus mykiss)

Using rainbow trout plasma protein (IGF-BP) which specifically binds human insulin-like growth factor (IGF) (Niu and Le Bail 1993), we have developed an assay to measure plasma IGF-like levels in different teleost species. Before the assay and to prevent interference by IGF-BP, IGF-like was extracted from all samples, using SP Sephadex C-25 in acidic conditions. After this treatment, contamination of the IGF fraction by IGF-BP which was estimated by binding assay, was approximately 5%, and was not detectable by western ligand blot. Human IGF-I was used as standard and labelled hormone. Sensitivity of the assay was 0.15–0.40 ng/ml (ED90) and ED50 was 1–3 ng/ml. hIGF-II crossreaction was partial and no significant displacement was observed with Insulin from different species or with other hormones. Inhibition curves were obtained with plasma IGF fractions (but not with tissue extracts) from teleost and mammals and are parallel to the standard curve. These results suggest that the protein binding assay can quantify an IGF-like factor in the plasma of teleost and that the binding sites of IGF are well conserved during vertebrate evolution. Using this IGF binding assay, IGF-like was measured in parallel with growth hormone (GH) in plasma from young rainbow trout killed every 1.5h throughout one day. The daily profiles for both hormones, which appear pulsatile, are similar. A significant correlation was observed between GH levels and IGF-like levels with a 1.5h delay. Analogous observations were obtained in individual catheterized adult rainbow trout. Although plasma GH levels differ greatly between fish, less variability is found with IGF-like. In a third experiment, rainbow trout were starved or submitted to bovine GH treatment for four weeks. Starved fish, in which plasma GH levels increased, had plasma IGF-like level significantly lower than in fed fish. In bGH injected fish, plasma IGF-like level was significantly higher than in non-injected fish. These results suggest that, as in mammals, IGF-like secretion depends on plasma GH level and could be modulated by the nutritional status of fish.     

Indirect estimation of stomach volume of rainbow trout Oncorhynchus mykiss (Walbaum)

The main hypothesis of this study was that if stomach volume is correlated with food intake it can be estimated without laborious and destructive direct measurement. Rainbow trout, Oncorhynchus mykiss (Walbaum), ca. 500–1300 g, were starved for 1, 4, 8 or 16 days at 15°C after which they were fed in excess with dry pellets containing known amounts of X‐ray‐dense markers. Immediately after feeding the fish were killed, X rayed and weighed. Then the stomach was dissected, its contents removed and weighed, and stomach volume was measured. X‐ray plates were developed and feed intake was estimated based on the amount of marker. All measured variables correlated positively with stomach volume. The best fit for linear regression models was obtained for fish starved for 4 days, where stomach content (dry mass) explained 94%, food intake (based on X‐ray measurement) 77% and fish mass 62% of the variation in stomach volume. However, as stomach content measurement can be a lethal, or at least very stressful, event for the fish, the accuracy of food intake measurement (X‐ray) could be increased using multiple regression. In multiple linear regressions, R²‐values varied between 0.79 (16‐day starvation) and 0.91 (1‐day starvation) with food intake and fish mass as explanatory variables for stomach volume. These results indicate that the stomach volume in rainbow trout can be estimated satisfactorily using indirect methods, which are not detrimental to the fish, although feeding history may affect the accuracy of the estimates.     

Effect of increased liver glycogen and liver weight on liver function in rainbow trout, Salmo gairdneri Richardson: recovery from anaesthesia and plasma 35S-sulphobromophthalein clearance

Abstract. Juvenile rainbow trout (initial mean body weight 17 g) reared on practical trout diets containing different levels of available carbohydrate for 8–24 weeks were assayed for liver function based on the time required to recover from the anaesthetics tricaine methanesulphonate (MS222), 2-phenoxyethanol (2PE) and tertiary-amyl alcohol (TAA), as well as plasma clearance of ³⁵S-sulphobromo-phthalein (BSP), Liver/body weight ratios (LBW) and percentage liver glycogen content (LG) increased in a linear manner with increased available carbohydrate. The mean recovery time (MRT) of trout anaesthetized with 2PE and TAA increased linearly with increased LBW and LG. No difference was observed in the MRT of trout anaesthetized with MS222 which is not metabolized in the trout liver. There was a significant linear regression of the biological half-life of plasma BSP on LBW (MR²= 95.7) and LG (MR²= 99.6). The results indicate that increased liver glycogen and liver weight due to higher levels of available carbohydrate affect the liver function of rainbow trout. These liver function tests may be applicable in determining acceptable levels of available carbohydrate in practical trout diets.     

Liver damage in brown trout, Salmo trutta L., and rainbow trout, Oncorhynchus mykiss (Walbaum), following administration of the cyanobacterial hepatotoxin microcystin-LR via the dorsal aorta

Purified microcystin-LR (MC-LR) was administered via the dorsal aorta to brown trout, Salmo trutta L., or rainbow trout, Oncorhynchus mykiss (Walbaum), and, within 24 h, a dose of 300 μ g MC-LR kg^–1 caused increased activities in the blood by enzymes originating mainly from the liver, i.e. lactate dehydrogenase (LDH) and alanine transaminase (ALT). A dose of 75 μ g MC-LR kg^–1 significantly increased liver enzyme activities in the blood of brown trout at 24 h, but was without effect on rainbow trout, whereas 25 μ g MC-LR kg^–1 had no effect on blood LDH or ALT activities in either species. However, histopathological analysis of liver from both species following administration of the lowest toxin dose showed hepatocyte swelling and necrosis. Liver damage was more severe in brown trout compared to rainbow trout following administration of 300 μ g MC-LR kg^–1, showing disruption of the parenchymal architecture. After 48 h, there was a dose-dependent increase in the hepatosomatic index in both species. It is concluded that brown trout are less tolerant to MC-LR than rainbow trout.     

Metabolic adjustments of Dentex dentex to prolonged starvation and refeeding

The particular metabolic strategies of the common dentex (Dentex dentex) to face a period of prolonged starvation and subsequent refeeding were assessed. Plasma metabolites, endogenous reserves, and the activity of key enzymes of intermediary metabolism in liver, white muscle, and heart were evaluated. Plasma glucose, total lipid, triglycerides, total-, HDL- and LDL-cholesterol, and protein levels, liver, and white muscle glycogen, and perivisceral, and muscle fat were significantly reduced by starvation, whereas liver lipid content was surprisingly increased. Those enzymes involved in phosphorylation and oxidation of glucose and lipid synthesis, as well as alanine aminotransferase activity, were significantly depressed in liver of starved fish. The increase in β-hydroxyacyl-CoA dehydrogenase (HOAD) indicated an enhanced fatty acid oxidation during starvation. Part of the acetyl-CoA generated by β-oxidation was oxidized in the hepatic Krebs cycle, as reflected the increased citrate synthase (CS) activity. The oxaloacetate required for the reaction catalized by CS activity would be supplied by aspartate aminotransferase (ASAT) activity whose activity was also enhanced. Glutamate dehydrogenase also increased to deaminate the glutamate produced by transaminases, especially by the increased ASAT activity. Liver gluconeogenesis of starved fish was maintained at the same rate that in controls, with glycerol playing an important role as glucogenic substrate. The increased hepatic β-hydroxybutyrate dehydrogenase (β-OHBDH) activity indicates that part of the acetyl-CoA arriving from β-oxidation was being diverted for ketone bodies production with dentex liver playing an important role in providing ketone bodies as fuels for other tissues under such circumstances. Most enzyme activities in white muscle of starved dentex were significantly depressed. In heart, starvation induced an important inhibition of those enzymes involved in glucose and protein metabolism, whereas CS, HOAD, and β-OHBDH activities were maintained at control levels. Although several biomarkers assayed returned to control values after refeeding, many others did not, which indicate that after 3 weeks of refeeding, pre-starved dentex is still experiencing a transient period of metabolic adjustments directed toward the restoration of body mass.     

Tissue-specific responses of oxidative stress biomarkers and antioxidant defenses in rainbow trout Oncorhynchus mykiss during a vaccination against furunculosis

The present study was conducted to evaluate the effects of vaccination against furunculosis on responses of oxidative stress and antioxidant defenses in rainbow trout Oncorhynchus mykiss muscle, gills, liver, and brain tissues. The oxidative stress markers (malondialdehyde and carbonyl derivatives of protein oxidative destruction levels), antioxidant defenses (superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase), and total antioxidant capacity in different tissues of rainbow trout were measured. Our data showed that exposure of trout to vaccine against furunculosis produced changes (either increase or decrease) in oxidative stress and antioxidant enzymes responses, and these responses showed marked organ differences, associated with tissue patterns. Our study demonstrated that vaccinated trout showed alteration in antioxidant defenses and oxidative stress responses, with higher severity in the liver, compared with other tissues. Our data also suggest that vaccination against furunculosis induced lipid peroxidation in gill and liver tissues. However, muscle and brain tissue are capable of restoring its pro- and antioxidant balance after vaccination.     

Alterations in iron, zinc and major plasma proteins of rainbow trout, Oncorhynchus mykiss (Walbaum), and brook trout, Salvelinus fontinalis (Mitchill), with sterile peritonitis induced by oil-adjuvanted multivalent bacterin vaccination

The early humoral responses of rainbow trout, Oncorhynchus mykiss (Walbaum), and brook trout, Salvelinus fontinalis (Mitchill), with sterile inflammation induced by intraperitoneal Lipogen Triple vaccination were compared to determine if genetic differences in susceptibility to furunculosis in salmonids correlated with different acute phase responses to vaccination. Similar severe acute sterile peritonitis occurred in response to Lipogen Triple in both species. Both species also had a rapid transient reduction in plasma iron concentration at 3 days. Moderate hypoferraemia persisted to day 14 in brook trout, but returned to normal by day 7 in rainbow trout. Plasma zinc decreased sharply 3 days after vaccination in rainbow trout and returned nearly to control levels by day 10; however, plasma zinc did not change in brook trout. Two-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis of plasma proteins revealed that increased amounts of a 48-kDa protein group coincided with the hypoferraemic response in rainbow trout. In addition, a modest elevation in a 16-kDa protein group also occurred in rainbow trout. These studies demonstrated the rapid changes in plasma iron in both species and mild elevation of two putative acute phase plasma proteins associated with vaccine-induced inflammation in rainbow trout.     

Effect of varying dietary protein level in different families of rainbow trout

Groups of rainbow trout (Salmo gairdneri) fingerlings from five different families and five inbred groups were fed for 153 days on each of four diets similar in energy content, but differing in their percentage of protein. The protein contents were 24, 33, 42 and 51%, respectively. Significant differences between fish families were found for growth, condition factor, chemical composition of the carcass, and digestibility of protein. At the end of the experiment, interaction between diet fed and family was found close to significance for weight and relative growth rate, and significant for length and condition factor. This interaction indicates that prospects for selectively breeding strains of rainbow trout specifically better able to utilize protein are promising.Fish growth rate increased with increasing dietary protein content. The corresponding condition factor, content of dry matter and energy in the carcass decreased. The high protein diets gave more protein in the carcass and a more discoloured liver than the low protein diets. Apparent protein digestibility increased with increasing protein level.     

Experimental validation of geosmin uptake in rainbow trout,Oncorhynchus mykiss (Waldbaum) suggests biotransformation

The bioconcentration of waterborne geosmin in rainbow trout, Oncorhynchus mykiss (Waldbaum) was assessed. Fifty rainbow trout with a mean (SD) weight of 226.6 (29.0) g and lipid content of 6.2 (0.6) % (w/w) were exposed to geosmin in static water for 0, 2, 4, 6, 8, 12, 24, 36, 48 and 120 hr, with one tank containing five fish for each exposure period. Geosmin concentrations were measured in fish tissue and water samples collected over time. With time the geosmin concentration in the fish increased and decreased in the water. However, the total absolute amount of geosmin in the system declined over time which could be explained by induction of biotransformation. This is in accordance with the decreasing lipid normalized geosmin levels in the liver compared with the liver‐free carcass. Geosmin distribution within rainbow trout clearly is not exclusively governed by the lipid content of tissues. In vivo geosmin bioconcentration in rainbow trout is slower and the body burden reached is lower than the generally accepted theoretical model predicts.     

The optimum dietary essential amino acid pattern for rainbow trout (Oncorhynchus mykiss), to maximize nitrogen retention and minimize nitrogen excretion

The optimum dietary essential amino acid (EAA) pattern for a given animal species is considered to be that EAA pattern which results in maximum nitrogen (N) retention for protein growth and minimum N excretion. In a series of two 8-week experiments, we investigated the optimum dietary EAA pattern for rainbow trout (Oncorhynchus mykiss). Experimental diets were fed to quadruplicate tanks of fish, using the equalized satiation feeding method. In the first experiment, we used the amino acid deletion method to arrive at an estimate of optimum dietary EAA pattern for rainbow trout. There were 11 dietary treatments: 1 diet with a control EAA pattern, and 10 other diets with 40% deletions of a single EAA from the control pattern. Based on N utilization data, an estimate of optimum dietary EAA pattern for rainbow trout was made. In the second experiment, we compared this dietary EAA pattern with three other estimates of optimum dietary EAA pattern for rainbow trout, based on 1) amino acid composition of rainbow trout whole-body protein, 2) EAA requirements for rainbow trout published by the National Research Council, and 3) EAA requirements for rainbow trout based on nonlinear regression analysis. Response variables included growth rate, feed efficiency ratio and N retention and excretion. The EAA pattern associated with EAA requirements as published by the National Research Council was found to result in the highest mean N retention and lowest mean N excretion, and so was considered the best estimate of optimum EAA pattern of those compared.