Effect of selenium deficiency on hydroperoxide-stimulated release of glutathione from isolated perfused liver of rainbow trout (Salmo gairdneri).

1. Duplicate groups of rainbow trout (Salmo gairdneri) were each given partially purified diets which were either adequate or depleted in selenium for 40 weeks. 2. Although there was no significant difference in weight gain, liver Se concentration was significantly lower in fish given the deficient diet. 3. Glutathione (GSH) peroxidase (EC 1.11.1.9) activity was significantly reduced in liver of Se-deficient fish but a differential assay did not indicate the presence of a non-Se-dependent GSH peroxidase activity, although liver GSH S-transferase (EC 2.5.1.18) was significantly increased. 4. Perfusion of livers from trout given Se-adequate diets with t-butyl hydroperoxide (BuOOH) or hydrogen peroxide caused an increase in the rate of release of glutathione disulphide (GSSG) into the perfusate. 5. Perfusion of livers from Se-deficient trout with BuOOH or H2O2 did not result in any change in rate of release of GSSG into the perfusate. 6. These findings confirm the absence of any compensatory non-Se-dependent peroxidase activity in Se-depleted trout.

Some effects of selenium deficiency on glutathione peroxidase (EC 1.11.1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri).

1. Two duplicate groups of rainbow trout (Salmo gairdneri; mean weight 27 g) were given diets of differing selenium content (deficient 0.025 mg Se/kg; supplemented 1.022 mg Se/kg) for 30 weeks. 2. There were no significant differences between treatments in weight gain but packed cell volume, liver vitamin E and liver and plasma Se concentrations were all significantly lower in the Se-deficient trout. 3. Ataxia occurred in about 10% of the Se-deficient trout and histopathologies were evident in nerve cord (damage to axon sheath) and liver (loss of integrity in endoplasmic reticulum and mitochondria with appearance of increased vesiculation). 4. Glutathione peroxidase (EC 1.11.1.9) activity was significantly reduced in liver and plasma of Se-deficient fish but there was no indication, from differential assay, of any non-Se-dependent glutathione peroxidase activity. Glutathione transferase (EC 2.5.1.18) activity was significantly increased in Se-deficient trout.

Dietary requirements of rainbow trout for tryptophan, lysine and arginine determined by growth and biochemical measurements.

Three separate studies were performed to determine the dietary requirements of rainbow troutSalmo gairdneri for tryptophan (Trp), lysine (Lys) and arginine (Arg) from both growth and biochemical data. The growth studies were carried out over a 12 week period. From graphical plots of % mean weight gain against % amino acid in diet the following requirement values were obtained, Trp 0.25% diet (0.4% dietary crude protein); Lys 1.9% diet (4.3% dietary protein); and Arg 1.6-1.8% diet (3.6-4% dietary protein). Plasma and liver amino acid concentrations measured 20h after feeding did not prove useful for determination of requirement values. Hepatic activities of Trp pyrrolase (TP), Lys α ketoglutarate reductase (LKGR) and arginase were not significantly affected by varying levels of Trp, Lys and Arg respectively in the diet. TP has a cytosolic location and a Km of 0.2 mM for Trp; LKGR is mitochondrial and the Km for Lys is 7.3 mM; arginase is also mitochondrial and has a Km of 4.9 mM for arginine. Measurements of expired(14)CO2, after injection of a tracer dose of(14)C amino acid, did allow estimates of requirement levels to be made. The values obtained from the oxidation studies reinforced the values obtained from the growth data but were not precise enough to justify using this method on its own.

Some effects of vitamin E and selenium deprivation on tissue enzyme levels and indices of tissue peroxidation in rainbow trout (Salmo gairdneri).

Duplicate groups of rainbow trout (Salmo gairdneri) (mean weight 11 g) were given for 40 weeks one of four partially purified diets that were either adequate or low in selenium or vitamin E or both. Weight gains of trout given the dually deficient diet were significantly lower than those of trout given a complete diet or a diet deficient in Se. No mortalities occurred and the only pathology seen was exudative diathesis in the dually deficient trout. There was significant interaction between the two nutrients both with respect to packed cell volume and to malondialdehyde formation in the in vitro NADPH-dependent microsomal lipid peroxidation system. Tissue levels of vitamin E and Se decreased to very low levels in trout given diets lacking these nutrients. For plasma there was a significant effect of dietary vitamin E on Se concentration. Glutathione (GSH) peroxidase (EC 1.11.1.9) activity in liver and plasma was significantly lower in trout receiving low dietary Se but was independent of vitamin E intake. The ratios of hepatic GSH peroxidase activity measured with cumene hydroperoxide and hydrogen peroxide were the same for all treatments. This confirms the absence of a Se-independent GSH peroxidase activity in trout liver. Se deficiency did not lead to any compensatory increase in hepatic GSH transferase (EC 2.5.1.18) activity; values were essentially the same in all treatments. Plasma pyruvate kinase (EC 2.7.1.40) activity increased significantly in the trout deficient in both nutrients. This was thought to be due to leakage of the enzyme from the muscle and may be indicative of incipient (subclinical) muscle damage.

The effect of dietary lysine levels on growth and metabolism of rainbow trout (Salmo gairdneri).

Groups of rainbow trout (Salmo gairdneri; mean weight 5 g) were given diets containing 10, 12, 14, 17, 21, 24 and 26 g lysine/kg diet for 12 weeks. By analysis of the growth values the dietary requirement of lysine in this experiment was found to be 19 g/kg diet. A similar requirement value was obtained from a dose-response curve of expired 14CO2 (following an intraperitoneal injection of L-[U-14C]lysine) v. dietary lysine concentration. Liver concentrations of total lipid and carnitine and activities of lysine-alpha-ketoglutarate reductase (saccharopine dehydrogenase (NADP+, lysine-forming), EC 1.5.1.8) in the liver were not significantly different in fish from the different dietary treatments. Hepatosomatic index, however, was higher in those fish given low levels of dietary lysine.

The effects of dietary tryptophan levels on growth and metabolism of rainbow trout (Salmo gairdneri).

Groups of rainbow trout (Salmo gairdneri) (mean weight 14 g) were given diets containing 0.8, 1.3, 2, 3, 4 or 6 g tryptophan/kg diet for 12 weeks. By analysis of the growth results, the dietary requirement of tryptophan was found to be 2.5 g/kg diet (equivalent to 50 mg/kg biomass per d). Carbon dioxide expired by trout following intraperitoneal injection of [14COOH]tryptophan contained little radioactivity when dietary tryptophan level was low but, above 2.0 g/kg diet, it increased rapidly with increasing dietary tryptophan level. The break point in the dose-response curve did not, however, coincide with that from the growth results. Changes in concentrations of free tryptophan in blood and liver and activity of hepatic tryptophan pyrrolase (EC 1. 13. 11. 11) in response to changes in dietary tryptophan concentration did not provide reliable indicators for quantifying dietary requirement. Unlike the situation in mammals, blood tryptophan was not protein-bound to any appreciable extent. Tryptophan pyrrolase of trout has properties which suggest it has no apoenzyme form. In fish given adequate levels of tryptophan injected intraperitoneally with a tracer dose of [14COOH]tryptophan, 60% of the dose was incorporated into body protein within 1 d. The turnover of the label in this protein is very slow. Those trout given diets deficient in tryptophan suffered from severe scoliosis and lordosis as well as having increased liver and kidney levels of calcium, magnesium, sodium and potassium.

Methionine metabolism in rainbow trout fed diets of differing methionine and cystine content.

The metabolism of methionine was studied in rainbow trout fed diets containing different levels of methionine and cystine. Growth data indicated that methionine requirement was between 0.5 and 1% dry diet in the absence of dietary cystine, but 0.5% was adequate when dietary cystine was 2%. In fish fed diets deficient in sulfur-containing amino acids, elevated hepatic activities of glutathione reductase were found, whereas glutathione peroxidase and glutathione levels were unaffected. Plasma and liver concentrations (18 hours after feeding) of methionine and cystine were affected by dietary methionine, but dietary cystine had little effect. Cystine appeared to be converted to taurine in the liver. In fish injected intraperitoneally with [14COOH]- and [14CH3]methionine, over 24 hours the carboxyl group was oxidized more than the methyl group and more was incorporated into protein. However, much more of the methyl group was incorporated into the lipid fraction. The results suggest the operation of the transsulfuration pathway of methionine catabolism. Oxidation of methionine was related to its concentration in the tissues, and little affected by dietary cystine. A 28-day experiment on the metabolism of injected [14COOH]methionine showed that its turnover was slow, and much of the radioactivity was associated with protein.

The effect of low dietary manganese intake on rainbow trout (Salmo gairdneri).

1. Rainbow trout (Salmo gairdneri) of mean initial weight 15 g were given either a low-manganese or control diet containing 1.3 and 33 mg Mn/kg dry diet respectively. 2. Weight gains over a 24-week feeding period were the same for both groups of trout. 3. Hepatosomatic index, blood packed cell volume and haemoglobin concentration, plasma protein and the activities of aspartic aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) were unaffected by dietary Mn intake. 4. Plasma potassium and iron levels were increased in the trout given the low-Mn diet. 5. The hepatic levels of magnesium, sodium, K, zinc, copper, Mn and phosphorus were significantly reduced in the fish given the low-Mn diet. 6. In those trout given the low-Mn diet the levels of Mn and calcium in the vertebral ash were significantly reduced. 7. The hepatic activity of Cu-Zu superoxide dismutase (EC 1.15.1.1; Cu-ZnSOD) and of Mn superoxide dismutase (EC1.15.1.1; MnSOD) in cardiac muscle and liver was reduced in the group of trout given the low-Mn diet. The fall in Cu-ZnSOD and MnSOD activities coincided with reduced tissue levels of their respective metal components.

Tissue distribution, uptake, and requirement for alpha-tocopherol of rainbow trout (Salmo gairdneri) fed diets with a minimal content of unsaturated fatty acids.

The metabolism of and requirements for alpha-tocopherol in rainbow trout fed diets containing 1% linolenic acid as sole source of unsaturated fat and graded levels of tocopherol (0.06-10 mg/100 g) were examined. Fish grew 5-fold over a 16-week period. In liver, tocopherol was concentrated in mitochondria with little in cytosol. Orally administered [3H]-tocopherol was rapidly taken up by plasma and liver but uptake into erythrocytes and white muscle was much slower; in most tissues radioactivity reached a plateau after about 3 days but in red muscle radioactivity increased over a 10-day period. Activities of enzymes that prevent free radical initiated tissue damage did not change in tocopherol deficiency. Tocopherol-deficient trout had no gross or subcellular pathologies even though liver and muscle were severely depleted of the vitamin. Ascorbic acid-stimulated lipid peroxidation in liver organelles indicated a tocopherol requirement of 2-3 mg/100 g diet; the molar ratios of polyunsaturated fatty acids to tocopherol in livers of trout fed diets lacking or supplemented with tocopherol (100 mg/100 g) were 980 and 170, respectively.

Effects of quantity and quality of dietary protein on certain enzyme activities in rainbow trout.

The effects of variation in quality and quantity of dietary protein on certain tissue enzymes in rainbow trout (Salmo gairdneri) were examined. Trout were given for 9 weeks diets containing proteins of different quality (fish-meal, casein and corn gluten) and with protein energy levels ranging from 26 to 74% of total metabolizable energy. In the first experiment, activities of a number of enzymes were monitored by only hepatic serine pyruvate transaminase (SPT) activity changed in response to the dietary treatments--increasing as protein energy level was raised. In the second experiment, opposing glycolytic an gluconeogenic enzyme activities [pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK); phosphofructokinase (PFK) and fructose diphosphatase (FDP)] were measured. Gluconeogenic enzyme activities correlated positively and significantly with dietary protein energy level; glycolytic enzymes correlated negatively and significantly with this parameter for all three proteins. There was no consistent relationship between presumed equilibrium point of opposing enzyme activities and maximum weight gain for the three proteins. It is suggested that hepatic activities of SPT, PFK, PK, FDP and PEPCK will provide useful indices of protein status in trout.

Studies on the nutrition of salmonid fish. The magnesium requirement of rainbow trout (Salmo gairdneri).

1. Rainbow trout (Salmo gairdneri) of mean initial weight 35 g were given one of five experimental diets for 20 weeks. The diets contained (g/kg dry diet) 15 calcium, 10 phosphorus and graded levels of magnesium from 0.04 (diet no. 1) to 1.0 (diet no. 5). In a second experiment rainbow trout of mean initial weight 16 g were given one of six experimental diets for 20 weeks. The diets contained (g/kg dry diet): Ca (40), P (30) and levels of Mg from 0.06 (diet no. 6) to 2.0 (diet no. 11). 2. In both experiments weight gains were lowest in those trout given diets containing the basal levels of Mg (diet no. 1 and diet no. 6) but increased with increasing dietary Mg concentration. In neither experiment was there any further increase in weight gain once the Mg concentration reached 0.25-0.5 g/kg dry diet; weight gain reached a plateau at this dietary MG level. 3. The following trends occurred in serum electrolyte concentrations as dietary Mg increased. Mg increased in both experiments, in Expt 2 it reached a maximum of 1 mmol/l when the diet contained 0.5 g Mg/kg and did not increase further; sodium was positively correlated in both experiments; potassium decreased and in Expt 2 reached a plateau minimum of 1.7 mmol/l at a dietary Mg concentration of 0.5 g/kg; Ca and P altered little in either experiment. 4. In both experiments renal Ca concentrations were greatly increased in trout given diets lacking supplementary Mg; they fell to low levels (3-5 mmol/kg) when diets contained 0.15 g Mg/kg or more. Renal K and P concentrations were negatively correlated with dietary Mg in Expt 2; other electrolytes measured were not altered in concentration by the treatments used. 5. Extracellular fluid volume (ECFV) of muscle was negatively correlated with dietary Mg. In Expt 2 it reached a minimal or normal value at 0.5 g Mg/kg diet and did not decrease further. Muscle Mg concentration increased with diet Mg in both experiments and muscle K concentration was also correlated with diet Mg in Expt 2. These changes were related to the shift in muscle water. In Expt 1, P concentration was decreased with increasing diet Mg but in Expt 2 its concentration increased, these changes may have been connected with the three-fold difference in dietary P in the two experiments. 6. By contrast with skeletal muscle, Mg levels in cardiac muscle increased at low dietary Mg intakes. 7. Concentrations of electrolytes in liver did not alter with the dietary treatments used. 8. The results show that Mg requirement of rainbow trout is met by a diet containing 0.5 gMg/kg diet.

Studies on the nutrition of marine flatfish. The pyridoxine requirement of turbot (Scophthalmus maximus).

1. Diets containing graded levels of pyridoxine hydrochloride (to supply 0.26--30 mg pyridoxine/kg) were given to seven duplicate groups of turbot (Scophthalmus maximus) for 12 weeks and their growth rate was measured during this period. 2. Good growth was obtained on all treatments except those groups given less than 1.0 mg pyridoxine/kg diet. These fish grew normally until weeks 8--10 but thereafter their weight gain was significantly less than that for other treatments. 3. Measurements of aspartate aminotransferase (EC 2.6.1.1) in muscle and liver and of alanine amino-transferase (EC 2.6.1.2) in liver of the turbot showed that the activities of these enzymes increased with increasing dietary pyridoxine intake up to a level of 2.5 mg pyridoxine/kg. The activities of these enzymes were not further enhanced by additional dietary pyridoxine. 4. Percentage stimulation of these enzymes by pre-incubation of extracts with pyridoxal phosphate was minimal with those groups of turbot given 2.5 mg pyridoxine/kg diet or more. 5. It is concluded that the dietary requirement of turbot for vitamin B6 can be safely met with a diet containing between 1.0 and 2.5 mg pyridoxine/kg. 6. An eighth group of turbot given the pyridoxine antagonist 4-deoxypyridoxine hydrochloride (20 mg/kg) showed retarded growth after 2 weeks, together with a high mortality rate.

Studies on the nutrition of marine flatfish. The effect of different dietary fatty acids on the growth and fatty acid composition of turbot (Scophthalmus maximus).

1. Five groups of juvenile turbot (Scophthalmus maximus) which had been given a diet free of fat for 12 weeks were given diets in which the lipid component (g/kg) was: oleic acid alone 50, oleic acid 40+linoleic acid 10, oleic acid 40+linolenic acid 10, oleic acid40+arachidonic acid 10 or oleic acid 40+cod-liver oil 10. These five experimental diets were given for 16 weeks. 2. Weight gains were highest in the group given the diet containing cod-liver oil and lowest in the groups given diets containing oleic acid alone or oleic acid+linoleic acid. Weight gains in the groups given oleic acid+arachidonic acid or linolenic acid were markedly inferior to those of the group given oleic acid+cod-liver oil. It is concluded that arachidonic acid is inferior to polyunsaturated fatty acids of the omega3 series in maintaining growth rate in turbot. 3. Fatty acid analyses of neutral lipids and phospholipids of liver and extrahepatic tissues did not suggest any evidence of desaturation of dietary oleic acid, linoleic acid or linolenic acid by the turbot. These experiments confirm previous isotopic evidence that turbot lack the necessary microsomal desaturases to perform this metabolic transformation.

Studies on the nutrition of marine flatfish. The thiamin requirement of turbot (Scophthalmus maximus).

1. Seven groups of young turbot (Scophthalmus maximus) were given diets containing graded levels of thiamin (0-19--50 mg/kg) for 16 weeks and their growth rate was measured during this period. 2. Good growth was obtained on all these treatments except in the group given the lowest dietary thiamin level (0-19 mg/kg). These fish grew normally until the 12th week but thereafter their weight did not increase. 3. Measurements of erythrocyte transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase; EC 2.2.1.1) activity at the end of the experiment and of percentage stimulation of erythrocyte transketolase by thiamin pyrophosphate indicated that the apoenzyme was saturated with coenzyme at a dietary thiamin level of 2-6 mg/kg, but not at 1-1 mg/kg. 4. An 8th group of turbot given the thiamin antagonist pyrithiamin (40 mg/kg diet) grew normally for 6 weeks. Thereafter mortalities began to occur and all fish died by the 10th week. No clear-cut signs of thiamin deficiency were observed. 5. The dietary thiamin requirement of turbot is much lower than published requirements of freshwater fish other than carp (Cyprinus carpio) and appears to be between 0-6 and 2-6 mg/kg diet.

Elongation and desaturation of dietary fatty acids in turbot Scophtalmus maximus L., and rainbow trout, Salmo gairdnerii rich.

Turbot and rainbow trout, which had previously recieved diets free of fat, were fed [1-14C] fatty acids. The distribution of radioactivity in the tissue fatty acids was examined 6 days later. In rainbow trout fed [1-14C] 18:3omega3, 70% of the radioactivity was present in 22:6omega3 fatty acid. In contrast, turbot fed [1-14C] 18:1omega9, 18:2omega6, or 18:3omega3 converted only small amounts of labeled fatty acids (3-15%) into fatty acids of longer chain length. The major product of the limited modification found in turbot was the dietary acid elongated by 2 carbon atoms.