Intraperitoneal and dietary administration of astaxanthin in rainbow trout (Oncorhynchus mykiss)--plasma uptake and tissue distribution of geometrical E/Z isomers

Astaxanthin enters circulation in salmonid fishes upon intraperitoneal injection (IP) of small doses. Blood uptake and tissue distribution of geometrical E/Z astaxanthin isomers were determined in tissues and plasma of duplicated groups of rainbow trout (Oncorhynchus mykiss, initial weight 550 g) some of which were administered high doses of astaxanthin by IP in a trial lasting for 8 weeks. Doses of 10 (IP10), 50 (IP50) or 100 mg (IP100) astaxanthin (Lucantin Pink, BASF, Germany), respectively, dispersed in phosphate buffered saline were tested in comparison with diets containing 10 (Control) or 60 (Fed 60) mg astaxanthin kg(-1). Astaxanthin concentrations in all examined tissues and plasma were significantly higher in IP50 and IP100 than in controls and Fed 60 (p<0.05). In IP50, 11 mg astaxanthin kg(-1) muscle was detected after 4 weeks, compared to 4 mg kg(-1) in rainbow trout fed 60 mg kg(-1). Concentrations up to 80 and 100 mg astaxanthin kg(-1) were detected in liver and kidney after IP, respectively, whereas fish only fed astaxanthin contained about 2 mg astaxanthin kg(-1). No increase in muscle astaxanthin concentration was found between 4 and 8 weeks in fish given IP, and the muscle astaxanthin concentration in IP50 and IP100 were similar. Muscle concentration and injected dose were curvilinearly correlated and the proportion of ingested dose retained by the muscle was negatively correlated with the amount of injected astaxanthin. Plasma and muscle concentrations of astaxanthin were highly correlated (p<0.0001). Astaxanthin Z-isomers accumulated selectively in the various tissues after IP, whereas all-E-astaxanthin was preferably absorbed into plasma when administered via the diet. There was a selective uptake of all-E-astaxanthin in the muscle of all fish. Mortality was not affected by treatment, but a dose-dependent reduction in SGR was evident after IP. In conclusion, a more rapid and higher uptake of astaxanthin in plasma, muscle, kidney and liver of rainbow trout takes place after IP compared to when astaxanthin is fed via the diet.     

Maslinic acid as a feed additive to stimulate growth and hepatic protein-turnover rates in rainbow trout (Onchorhynchus mykiss)

Maslinic acid is a triterpene present in a considerable proportion in solid residues from olive-oil production. In the present work the effects of maslinic acid on growth, protein-turnover rates and nucleic-acid concentration on liver were investigated in the rainbow trout. Five groups of 120 fish of a mean body mass of 20 g were fed for 225 days with diets containing 0, 1, 5, 25 and 250 mg of maslinic acid per kg diet. At the end of the experiment, whole-body and liver weight and growth rate of trout fed with maslinic acid were higher than controls. The highest weight increase was registered for the group fed 250 mg kg(-1), representing a 29% increase over controls. The total hepatic DNA or liver cell hyperplasia levels in trout fed with 25 and 250 mg of maslinic acid kg(-1) were 37% and 68% higher than controls. Also in these same groups of trout, fractional and absolute hepatic protein-synthesis rates were significantly higher than in control, and significant increments in hepatic protein-synthesis efficiency and protein-synthesis capacity were reported. In close agreement with these results, microscopy studies showed that trout fed on 25 and 250 mg kg(-1) hepatocytes appeared to be more compact, with a larger rough-endoplasmic reticulum and larger glycogen stores than controls. These results suggest that maslinic acid can act as a growth factor when added to trout diet.     

Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout,Oncorhynchus mykiss

The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (¹²⁵iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (K _d=8.2·10^-9 mol·1^-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin.Abbreviations B _max maximum number of binding sites available - BSA bovine serum albumin - bt-VTG brown trout vitellogenin - c-VTG earp vitellogenin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - K _d dissociatian constant - NCM nitrocellulose membranes - PMSF phenylmethylsulphonylfluoride - rt-VTG rainbow trout vitellogenin - VTG vitellogenin     

The preparation and characterization of vitellogenin messenger RNA from rainbow trout (Salmo gairdneri).

Agarose-gel electrophoresis of polyadenylated RNA from livers of oestrogen-treated male rainbow trout revealed a major high-Mr species (7200 nucleotides), which is absent from the polyadenylated RNA isolated from hormonally unstimulated male trout liver. Translation in vitro of the RNA from oestrogen-treated males in a mRNA-dependent rabbit reticulocyte lysate produced a protein (Mr 200 000) that could be immunoprecipitated with antibodies against trout serum vitellogenin, but no immunoprecipitable protein was synthesized with RNA from control animals. DNA complementary to the RNA from oestrogen-stimulated and control male trout liver was synthesized and back-hybridized, with R0t1/2 of 3.8 X 10(-2) and 1 X 10(-1) mol X litre-1 X s for RNA from hormone-treated and control animals respectively. The 9% increase in the abundant mRNA after oestrogen stimulation is due to the induction of vitellogenin mRNA.     

Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta)

Vitellogenin synthesis is induced in juvenile rainbow trout (Salmo gairdneri) and juvenile sea trout (Salmo trutta) by estradiol-17 beta. A purification procedure for vitellogenin from trout plasma by precipitation with MgCl2-EDTA and subsequent anion exchange chromatography on DEAE-Sephacel is described. The total lipid contents of purified rainbow trout and sea trout vitellogenins are 18 and 19%, respectively. Approximately 2/3 of the lipids are phospholipids, while the remainder consists of triglycerides and cholesterol. Phosphorus determinations on delipidated vitellogenin yield a phosphorus content of 0.63% in rainbow trout and 0.58% in sea trout vitellogenin. Native (dimeric) vitellogenins from rainbow trout and sea trout both have an apparent molecular weight of 440,000, when estimated by gel filtration on Sepharose 6B.     

Uptake and distribution of radioiodine, and the effect of ambient nitrate, in some fish species

125I uptake by the thyroid was most pronounced in the smelt (Osmerus eperlanus), perch (Perca fluviatilis), rainbow trout (Salmo gairdneri), roach (Rutilus rutilus) and bleak (Alburnus alburnus). Unlike other tissues, only the muscle in the species studied no relative accumulation of 125I as compared to the ambient water. The Crusian carp (Cyprinus carassius) indicated the lowest levels of accumulation of 125I among the species tested. Thyroid radioiodine concentration was independent of the length of the fish in the rainbow trout and perch. In the rainbow trout, however, the liver and muscle radioiodine concentrations decreased significantly with increasing length of the fish. Exposure of rainbow trout to water containing supranormal concentration of nitrate (1500 micrograms/l) resulted in lowered 125I uptake. The same phenomenon was observed for the perch and Crusian carp, with low nitrate concentrations, while with higher nitrate concentrations, the uptake levels were again increased. Thyroid function, as judged from the conversion ratio and T/S ratio, was not affected by nitrate.     

Serine dehydratase and tyrosine aminotransferase activities increased by long-term starvation and recovery by refeeding in rainbow trout (Oncorhynchus mykiss)

Here, we study a cycle of long-term starvation followed by refeeding in relation to the kinetics of serine dehydratase (SerDH) and tyrosine aminotransferase (TyrAT) in rainbow trout (Oncorhynchus mykiss). We determine SerDH- and TyrAT- specific activity at different substrate concentrations in liver and white muscle of juvenile trout starved for 70 days and then refed for 6 hr, 32 hr, 4 days, and 9 days. SerDH showed a hyperbolic kinetic with a K(m) for L-serine of 77.07+/-8.78 mM in the liver of control trout. After 70 days of starvation, the SerDH activity at saturate substrate concentration rose 100% over control. No significant changes were found in the K(m) values of the enzyme. After refeeding, the SerDH activity declined to control values. TyrAT also showed a hyperbolic kinetic with a K(m) for L-tyrosine of 1.86+/-0.12 and 2.55+/-0.57 mM in liver and white muscle, respectively. In starved trout, TyrAT activity in liver and white muscle was about 64 and 267%, respectively, higher than control. After 9 days of refeeding, the control values recovered, although, at 6 hr of refeeding, hepatic TyrAT activity was higher than that for starvation. This work shows that SerDH and TyrAT are present in rainbow trout and that the two enzymes have regulatory functions in the catabolism of their respective amino acids in this species.     

Influence of salinity and ratio of lipid to protein in diets on certain enzyme activities in rainbow trout (Salmo gairdneri Richardson)

The connection between metabolic and sea water adaptation of the rainbow trout was investigated. The rainbow trout were kept in fresh water and diluted sea water of 8 and 20 0/00 S at 16 degrees C and fed on three different diets for 51 days. Hyperosmotic salinity (20 0/00) tends to inhibit growth in rainbow trout by reducing the food conversion efficiency. A higher protein concentration in the diet can partly compensate for this effect. The liver IDH, G6PDH and 6PGDH activities of the rainbow trout are influenced only by food quality, whereas the liver G1DH, AspT and A1T activities, like the muscle A1T, are also affected by salinity. The salinity had no significant effect on the activities of the kidney enzymes we investigated (Na/K-ATPase, G1DH, A1T, AspT) or of the muscle AspT in these experiments.     

Rainbow trout, Oncorhynchus mykiss, as a model for aromatase inhibition.

The feasibility of utilizing rainbow trout, Oncorhynchus mykiss, as an alternative model for studying the inhibition of aromatase (CYP 19) was investigated. The suppression of estrogen-dependent tumors by aromatase inhibitors has been important in the treatment of breast cancer. Estrogens, estrogen precursors and xenoestrogens have been found to promote liver cancer in the trout model. A steroid, 4-hydroxy-4-androstene-3,17-dione (4-OHA), and non-steroids, aminoglutethimide (AG) and Letrozole (CGS 20267), all of which are known aromatase inhibitors in rats and humans, were examined in vitro for activity in trout ovarian microsomes. Aromatase activity was quantified as the release of 3H2O from the conversion of [3H]-4-androstene-3,17-dione to 17beta-estradiol and estrone. Trout ovarian microsomes exhibited activity between 39-60 fmol mg(-1) min(-1) with a calculated Vmax of 71.1 fmol mg(-1) min(-1) when incubated at 25 degrees C with 200 nM 4-androstene-3,17-dione (K(M) = 435 nM). Significant inhibition by 4-OHA up to 80% was seen at 1.5 microM. At 2000 microM, AG decreased aromatase activity by up to 82%. Letrozole reduced aromatase activity a maximum of 90% in a dose-dependent manner, but the Ki (2.3 microM) was 1000-fold higher than reported in human trials. Indole-3-carbinol and some of its derivatives, two DDE isomers and four flavones (except alpha-naphthoflavone) at 1000 microM did not significantly inhibit aromatase in vitro. Letrozole and clotrimazole, fed to juvenile rainbow trout at doses up to 1000 ppm for 2 weeks, were not effective in suppressing dehydroepiandrosterone (DHEA) induced increases in vitellogenin and 17beta-estradiol levels. These results document that trout aromatase is sensitive to inhibition in vitro by known inhibitors of the mammalian enzyme. The mechanism(s) for lack of inhibition in vivo is currently unknown and must be further investigated in order to develop a trout model for studying the role of aromatase in carcinogenesis.     

Occurrence and synthesis of a non-thionein, zinc-binding protein in the rainbow trout (Salmo gairdneri)

A non-thionein, Zn-binding protein (ZBP) was induced in Donaldson strain rainbow trout (Salmo gairdneri) by 7 mg/kg, i.p. injections of divalent Zn ion. The Sephacryl S-200 used for supernatant fractionation had to be saturated with Zn to recover quantitatively the Zn-ZBP complex. The ZBP was present in liver and kidney, but was absent from gill and spleen. The apparent molecular weights of the liver and kidney ZBP as estimated by gel filtration were 17,300 +/- 1300 (SD; N = 11) and 18,100 (N = 1), respectively. Starvation induced hepatic ZBP synthesis whereas cycloheximide inhibited hepatic ZBP synthesis. The quantity of hepatic ZBP synthesized varied with the temperature of the water in which the trout resided. The maximum quantity of ZBP in the liver following a single 7 mg/kg Zn injection (17 micrograms Zn/g liver wet weight) occurred at 24 hr.     

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.     

Identification of four ovarian receptor proteins that bind vitellogenin but not other homologous plasma lipoproteins in the rainbow trout,Oncorhynchus mykiss

Membrane proteins from ovarian follicles, testis and somatic tissues of rainbow trout, Oncorhynchus mykiss, were extracted by ultracentrifugation, separated on sodium dodecyl sulphate gels and isolated on polyvinyl difluoride membranes. Vitellogenin receptor proteins were visualised using protein staining and hybridisation with ¹²⁵I-vitellogenin Four follicle-membrane proteins, with molecular masses of 220, 210, 110 and 100 kDa, showed a strong affinity for vitellogenin and were specific to the ovary. Other homologous lipoproteins (very low density lipoprotein, low density lipoprotein and high density lipoprotein) had a very limited ability to displace ¹²⁵I-vitellogenin from its receptor, indicating that the ovarian receptor proteins were fairly specific for vitellogenin. Proteins with an affinity for very low density lipoprotein and low density lipoprotein were visualised in liver, spleen and muscle, eluting on sodium dodecyl sulphate gels with molecular masses of about 150 kDa. Peptides generated from trypsin digests of the receptor proteins with a high affinity for vitellogenin showed sequence homology with receptors in the lipoprotein family, including a sequence that is believed to act as the internalisation signal [Phe-Asp-Asn-Phe-Tyr-] and, a sequence identity with the recently characterised chicken vitellogenin/very low density lipoprotein receptor [Ser-Glu-Leu-Tyr-Glu-Pro-Ala-]. Together, the ligand blotting and peptide sequence data support the contention that the four ovarian membrane proteins isolated are receptor proteins specific for vitellogenin and they do not bind other plasma lipoproteins to any significant degree.Abbreviations BSA bovine serum albumin - HDL high density lipoprotein - LDL low-density lipoprotein - HPLC high performance liquid chromatograph - PVDF polyvinylidene difluoride - RIA radioimmunoassay - rt-VTG rainbow trout vitellogenin - SDS sodium dodecyl sulphate - VLDL very low density lipoprotein - VTG vitellogenin - VRP-1,-2,-3 or -4 vitellogenin receptor proteins     

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.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.     

Vitellogenin levels in male and female rainbow trout (Salmo gairdneri Richardson) at various stages of the reproductive cycle

The immunoassayable vitellogenin (VTG) in plasma from male rainbow trout had the same molecular weight as authentic VTG from female fish. The VTG level in male trout was low (usually nanograms, occasionally up to a few micrograms, per ml) and did not correlate with the stage of sexual maturity. The plasma VTG level of female trout that were two years from first spawning was 200-fold higher than males of the same strain and age. The plasma VTG level of female rainbow trout rose approximately a million-fold during the two or three years required to attain sexual maturity.     

Seasonal variations of lipid fatty acids of boreal freshwater fish species

1. The fatty acid levels of muscle and liver lipids of perch, vendace and rainbow trout (Salmo gairdneri) cultivated in the same area (for comparison) were monitored. 2. The total lipid content in the muscle of perch (Perca fluviatilis) and vendace (Coregonus albula) was less than 50% of that in rainbow trout and a seasonal variation was clear only in vendace. 3. The relative amounts of polyunsaturated fatty acids as well as omega-3 acids were higher in vendace and perch than in cultivated rainbow trouts. Arachidonic acid content was much higher in vendace and perch than in rainbow trout. The content of monoenes was considerably higher in rainbow trout than in free freshwater fish. 4. The seasonal variations in the degree of unsaturation were small in fish muscle. 5. In the muscle of rainbow trout the relative amount of polyunsaturated fatty acids diminished with the increase of total lipids.     

Maslinic acid added to the diet increases growth and protein-turnover rates in the white muscle of rainbow trout (Oncorhynchus mykiss)

Maslinic acid (2-alpha, 3-beta-dihydroxiolean-12-en-28-oic acid) is a triterpenoid compound present in fruit and leaves of Olea europaea that can be used as an additive in the diet of trout. The present work investigates the effects of maslinic acid on growth, protein-turnover rates and nucleic acid concentration in trout white muscle. Five groups of 180 trout of a mean body mass of 20 g were fed for 225 days with diets containing 0, 1, 5, 25 and 250 mg of maslinic acid per kg of diet. At the end of the experiment, white-muscle weight and protein-accumulation rate of trout fed with maslinic acid were higher than in control. The total content of DNA, RNA, and protein in trout fed with 25 and 250 mg of maslinic acid kg(-1) were significantly higher than in control. The protein:DNA ratio was also slightly higher than control. In the same groups of trout, fractional (K(S)) and absolute (A(S)) protein-synthesis rates increased to more than 80% over the control values while no differences were found in the fractional protein-degradation rate (K(D)). These results, similar to previous findings in liver, show that maslinic acid can act as a growth factor when added to a standard trout diet.     

Astaxanthin and canthaxanthin do not induce liver or kidney xenobiotic-metabolizing enzymes in rainbow trout (Oncorhynchus mykiss Walbaum)

This study was designed to assess the effects of dietary carotenoid supplementation on liver and kidney xenobiotic-metabolizing enzymes in the rainbow trout. Twelve rainbow trout (mean weight 266+/-10 g) were assigned to each of three replicate tanks for each of four dietary treatments; astaxanthin, canthaxanthin, negative control and positive control using beta-naphthoflavone, at a target dietary inclusion of 100 mg kg(-1) for each additive. Fish were fed for 3 weeks at a level of 1.2% body wt. day(-1). Serum carotenoid levels were used as indicators of exposure and were not significantly different (P>0.05) between carotenoid-fed trout. Livers and kidney were frozen separately in liquid N(2) by immersion and microsomal fractions from pooled samples (n=3) assayed for xenobiotic-metabolizing enzyme (cytochrome P450 monoxygenase) activities including ethoxyresorufin O-deethylase; methoxyresorufin O-demethylase; pentoxyresorufin O-dealkylase; benzoxyresorufin O-dearylase; and the conjugating enzymes glucuronosyl transferase; and glutathione-s-transferase. Results revealed that carotenoid treatment did not significantly (P>0.05) induce any enzyme system examined. Results are discussed in the context of metabolism of absorbed carotenoids.