Bile Of Rainbow Trout Research Articles

Bile components affect the functions and transcriptome of the rainbow trout intestinal epithelial cell line RTgutGC

The concomitant increase in cultivation of fish and decrease in supply of marine ingredients, have greatly increased the demand for new nutrient sources. This also regards so-called functional ingredients which may benefit health and welfare of the fish. In vitro cell line-based intestinal epithelial barrier models may serve as tools for narrowing down the broad range of ingredient options, to identify the most promising candidates before in vivo feeding trials are run. In vivo, differentiation of the various epithelial cells in the fish intestine, from the multipotent stem cells, takes place in the presence of a variety of substances from dietary and endogenous origin. Among these, bile salts have recently received attention as regulators of epithelial function in health and disease but have not, until now, been included in the medium when culturing fish gut epithelial cells in vitro. As bile salts are present at high levels in the chyme of the fish intestine, in particular in salmon and rainbow trout, mostly as taurocholate (>90%), their role for effects of diet ingredients on the in vitro gut cell model should be understood.With this study, we wanted to investigate whether inclusion of bile from rainbow trout or pure taurocholate in the culture media would modulate functions of the RTgutGC epithelial cells. Here, we demonstrated that the rainbow trout intestinal epithelial cell line RTgutGC responded significantly to the presence of bile components. Treatment with rainbow trout bile taken from the gall bladder (RTbile) or pure taurocholate (TC) at taurocholate concentrations of ≤0.5 mg/mL retained normal cell morphology, cell viability as in cell oxidation-reduction metabolic activity and membrane integrity, and barrier features, while high concentrations of bile salts (≥1 mg/mL) were cytotoxic to the cells. After long-term (4 days) bile treatment, transcriptome responses showed how bile salts play important roles in intestinal epithelial cell metabolism. qPCR data demonstrated that barrier function genes, brush border enzyme genes and immune genes were significantly affected. Although similar trends were seen, treatment with bile salt as a component of rainbow trout bile or pure taurocholate, induced somewhat different effects. In conclusion, this study clearly indicates that bile salts should be included in the cell medium when running in vitro studies of gut cell functions, not at least immune functions, preferably at the level of ∼0.5 mg/mL supplemented as pure taurocholate to ensure reproducibility.

In vitro assessment of some probiotic properties and inhibitory activity against Yersinia ruckeri of bacteria isolated from rainbow trout Oncorhynchus mykiss (Walbaum)

In the present study, we isolated bacteria from intestines of healthy rainbow trout, screened for their probiotic properties, and inhibitory activity against Y. ruckeri. A total of 21 out of 541 isolates from the intestine of adult rainbow trout (∼ 200–350 g), showed inhibition zones around at least one Y. ruckeri strains tested. Six of them were selected due to their ability to inhibit all the pathogenic strains on solid medium. They were identified as B. amyloliquefaciens 131and Paenibacillus spp (code 134, 1cc, 1d, 1k, and 2cc). Excluding the isolate 1k, selected strains were able to inhibit Y. ruckeri strains at 22 and 15 ± 0.5 °C when they were simultaneously cocultured, however, they did not show inhibition at 12 ± 0.5 °C. A pre incubation process of the selected strains for 24 h prior to co-culture with strains of Y. ruckeri enabled them to inhibit target pathogens at the lowest temperature tested. Furthermore, probiotic candidates survived to high concentrations of rainbow trout bile salts and acidic conditions. Selected bacteria were also able to produce amylolitic, proteolytic and lipolytic enzymes and showed variable hydrophobic values. Biosafety assays, performed by oral and intraperitoneal infection using B. amyloliquefaciens 131 and Paenibacillus spp (code 134, 1cc, 1d, 1k, and 2cc), showed no pathogenic effect in rainbow trout during 20-day experiment. Therefore, selected strains showed potential characteristics that are required to be evaluated in vivo for their possible subsequent use as probiotics in rainbow trout aquaculture.

Nisin Z Production by Lactococcus lactis subsp. cremoris WA2-67 of Aquatic Origin as a Defense Mechanism to Protect Rainbow Trout (Oncorhynchus mykiss, Walbaum) Against Lactococcus garvieae.

Probiotics represent an alternative to chemotherapy and vaccination to control fish diseases, including lactococcosis caused by Lactococcus garvieae. The aims of this study were (i) to determine the in vitro probiotic properties of three bacteriocinogenic Lactococcus lactis subsp. cremoris of aquatic origin, (ii) to evaluate in vivo the ability of L. cremoris WA2-67 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against infection by L. garvieae, and (iii) to demonstrate the role of nisin Z (NisZ) production as an anti-infective mechanism. The three L. cremoris strains survived in freshwater at 18 °C for 7 days, withstood exposure to pH 3.0 and 10 % (v/v) rainbow trout bile, and showed different cell surface hydrophobicity (37.93-58.52 %). The wild-type NisZ-producer L. cremoris WA2-67 and its non-bacteriocinogenic mutant L. cremoris WA2-67 ∆nisZ were administered orally (10(6) CFU/g) to rainbow trout for 21 days and, subsequently, fish were challenged with L. garvieae CLG4 by the cohabitation method. The fish fed with the bacteriocinogenic strain L. cremoris WA2-67 reduced significantly (p < 0.01) the mortality (20 %) compared to the fish treated with its non-bacteriocinogenic knockout isogenic mutant (50 %) and the control (72.5 %). We demonstrated the effectiveness of L. cremoris WA2-67 to protect rainbow trout against infection with the invasive pathogen L. garvieae and the relevance of NisZ production as an anti-infective mechanism. This is the first report demonstrating the effective in vivo role of LAB bacteriocin (NisZ) production as a mechanism to protect fish against bacterial infection. Our results suggest that the wild-type NisZ-producer strain L. cremoris WA2-67 could be used in fish farming to prevent lactococcosis in rainbow trout.

Marine Lactobacillus pentosus H16 protects Artemia franciscana from Vibrio alginolyticus pathogenic effects.

Vibrio alginolyticus is an opportunistic pathogen which may affect different aquatic organisms. The aim of this study was to assess the probiotic properties and the protective mode of action of Lactobacillus pentosus H16 against V. alginolyticus 03/8525, through in vitro and in vivo studies using Artemia franciscana (hereafter Artemia). This strain showed antimicrobial activity against V. alginolyticus 03/8525 and Aeromonas salmonicida subsp. salmonicida ATCC33658 possibly related to lactobacilli organic acid production. It was able to survive at high rainbow trout bile concentrations and showed high selective adhesion to rainbow trout mucus (1.2×10(5)±8.0×10(3) cells cm(-2)). H16 outcompeted V. alginolyticus 03/8525 and A. salmonicida subsp. salmonicida ATCC33658, greatly reducing their adherence to rainbow trout mucus (64.8 and 74.1%, respectively). Moreover, H16 produced a cell-bound biosurfactant which caused an important decrease in the surface tension. H16 also protected Artemia nauplii against mortality when it was administered previous to V. alginolyticus 03/8525 inoculation. Furthermore, H16 bioencapsulated in Artemia, suggesting that it is possible to use live carriers in its administration. We conclude that the ability of L. pentosus H16 to selectively adhere to mucosal surfaces and produce cell-bound biosurfactants, displacing pathogenic strains, in addition to its antimicrobial activity, confer H16 competitive advantages against pathogens as demonstrated in in vivo challenge experiments. Thus, L. pentosus H16, a marine bacterium from the intestinal tract of hake, is an interesting probiotic for Artemia culture and also has the potential to prevent vibriosis in other aquaculture activities such as larvae culture and fish farming.

Isolation of bacterial probiotic candidates from the gastrointestinal tract of rainbow trout, Oncorhynchus mykiss (Walbaum), and screening for inhibitory activity against Flavobacterium psychrophilum

In this study, 318 bacterial strains were isolated from the gastrointestinal (GI) tracts of 29 rainbow trout, Oncorhynchus mykiss (Walbaum). These bacteria were screened in vitro for their ability to inhibit growth of Flavobacterium psychrophilum, the causative agent of coldwater disease. Bacteria observed to inhibit F.psychrophilum growth were further screened against rainbow trout bile, as an indicator of their ability to survive in the GI tract. This screening resulted in narrowing the pool to 24 bacterial isolates. Those 24 isolates were then tested for pathogenicity in rainbow trout by intraperitoneal injection. Following a 28-day challenge, eight isolates were shown to cause direct mortality and were eliminated from further study. As a result, 16 bacterial isolates were identified as probiotic candidates with the potential to control or reduce disease caused by F.psychrophilum.

Characterization of probiotic properties of lactic acid bacteria isolated from an estuarine environment for application in rainbow trout (Oncorhynchus mykiss, Walbaum) farming

Bacterial attachment to fish surfaces and the capacity to compete with pathogens for adhesion sites are essential characteristics in order to select a candidate probiotic for aquaculture. Twelve lactic acid bacteria (LAB) isolated from fish and sediments from Bahía Blanca Estuary, Argentina, were examined for in vitro adhesion to rainbow trout mucus, cell surface properties and competitive exclusion against two salmonid pathogens, Yersinia ruckeri and Aeromonas salmonicida. In order to assess their survival through the digestive tract, pH and rainbow trout bile tolerance were evaluated. All LAB strains survived for 1.5h incubation in 10% rainbow trout bile. Most of the strains survived 1.5h at pH 3.0 and three of them showed a reduction of viable counts lower than 2 logarithms, with respect to control (pH 6.5). Only a few strains showed tolerate pH 2.0. All the strains were able to attach to rainbow trout skin mucus (10(4)-10(6)cells/cm(2)), to glass (10(4)-10(5)cells/cm(2)) and to stainless steel (10(3)-10(4)cells/cm(2)). Sixty percent of LAB strains were capable of competing with and successfully excluding Y. ruckeri and all strains were able to displace it. Against A. salmonicida, 75% of LAB strains competed successfully, 50% were capable of displacing and 60% excluded this pathogen. Our data suggest the potential of these strains as anti-infective agents for use in rainbow trout culture. This study is the first report on the probiotic potential of LAB strains isolated from an estuarine environment from Argentina.

Metabolites of the Aquatic Pollutant Diclofenac in Fish Bile

The uptake and metabolism of anti-inflammatory drug diclofenac (DCF) was studied by exposing rainbow trout (Oncorhynchus mykiss) to DCF intraperitoneally, and via water at concentration of 1.7 μg L(-1). The bile was collected and the formed metabolites were identified. The identification was based on the exact mass determinations by a time-of-flight mass analyzer and on the studies of fragments and fragmentation patterns of precursor ions by an ion trap mass analyzer. The main metabolites found were acyl glucuronides of hydroxylated DCFs. In addition, one ether glucuronide of hydroxylated DCF was found. Also, unmetabolized DCF was detected in the bile. The total bioconcentration factors (BCF(total-bile) for DCF and its metabolites) in rainbow trout bile, varied between individuals and was roughly estimated to range from 320 to 950. These findings suggest that fish living downstream the wastewater treatment plants (WWTPs) and which are chronically exposed to the drug may accumulate the drug and its metabolites in the bile.

KP3 Characterization of compounds from the bile of rainbow trout which inhibit infectious hematopoietic necrosis virus

KP3 Characterization of compounds from the bile of rainbow trout which inhibit infectious hematopoietic necrosis virus

Accumulation, depuration and hepatic mixed-function oxidase enzyme induction in juvenile rainbow trout and lake whitefish exposed to dietary 2,3,7,8-tetrachlorodibenzo -p-dioxin

Juvenile rainbow trout ( Oncorhynchus mykiss) and lake whitefish ( Coregonus clupeaformis) were exposed to three concentrations (40, 190, 400 pg g −1) of dietary 2,3,7,8-[ 3 H] tetrachlorodibenzo-p-dioxin (TCDD) to compare bioaccumulation and hepatic monooxygenase enzyme (MO) induction. Fish were exposed for 30 days followed by a 180 day depuration phase. Differences in the accumulation and depuration of TCDD were found between rainbow trout and lake whitefish, despite similar body size and lipid content. Assimilation efficiencies of TCDD were greater in lake whitefish (66–76%) than rainbow trout (43–58%), but TCDD half lives were shorter in lake whitefish (32–39 days) than in rainbow trout (73–83 days). Biomagnification factors (BMF) ranged from 1.6 to 1.8 in rainbow trout and from 0.8 to 0.9 in lake whitefish, confirming the known potential for biomagnification of TCDD in aquatic food webs. Reverse phase HPLC showed that a majority of the radioactivity in the rainbow trout bile was TCDD, with minor amounts present as a hydroxylated TCDD and as a glucuronide conjugate. MO enzyme induction, measured by ethoxyresorufin- O-deethylase (EROD), was observed in the rainbow trout after 10 days of exposure to 400 pg g −1 TCDD, and in the lake whitefish after 5 days of exposure to 380 pg g −1 TCDD. The whole fish threshold concentration for EROD induction by TCDD ranged between 15 and 45 pg g −1 (wet weight) for both species. EROD activity returned to control levels 120 and 80 days after the cessation of the treatments in the rainbow trout and lake whitefish, respectively. Growth rates were significantly reduced in trout and whitefish at whole fish concentrations (wet weight) of 150 ± 4.6 and 85 ± 8.3 pg g −1, respectively. Histological effects of the TCDD were found in the spleen and liver of the rainbow trout which had whole fish concentrations (wet weight) of 150 ± 4.6 pg mg −1and 72 ± 8.0 pg mg −1 TCDD, respectively.

Identification of Thyroid Hormone Conjugates Produced by Isolated Hepatocytes and Excreted in Bile of Rainbow Trout,Oncorhynchus mykiss

We studied thyroid hormone (TH) conjugation in fasted trout by incubating isolated hepatocytes with either [125I]T4or [125I]T3, and by analyzing bile from trout injected with either [125I]T4or [125I]T3. Glucuronide conjugates were identified by hydrolysis with β-glucuronidase and sulfate conjugates by acid solvolysis with ethyl acetate/trifluoroacetic acid (1%). We used Sephadex LH-20 chromatography to concentrate the conjugate fractions from hepatocyte incubates prior to HPLC analysis. Glucuronide conjugates of T4and T3were producedin vitroand glucuronides of T4, T3, and 3,3′-T2were foundin vivo.Sulfation of T4occurredin vitroandin vivo.T3sulfation was not establishedin vitro,but sulfate conjugates of T3and T2were found in bile. Significant proportions of unconjugated T4and T3also occurred in the bile. We conclude that (i) as in other vertebrates, iodothyronines undergo hepatic glucuronidation and are excreted as such in the bile, (ii) T4and T3undergo sulfation, and in contrast to mammals, are excreted in significant amounts in the bile, (iii) 3,3′-T2, a prominent deiodination product of T3, is excreted as both glucuronide and sulfate conjugates, and (iv) the isolated hepatocyte system is appropriate for studying aspects of TH metabolism in trout.

Assessing impacts on Baltic coastal ecosystems with mesocosm and fish biomarker tests: a comparison of new and old wood pulp bleaching technologies

This study has taken an advanced ecotoxicological approach to assess the effects on the aquatic environment of pulp bleaching effluents (representing different types of bleaching technology) using land-based model ecosystems which mimic the sublittoral zone of the Baltic Sea. Exposure was carried out at realistic, low concentrations over a time period of 4.5 months. The effluent from the conventional or oldest bleaching technology was most toxic to invertebrates, causing secondary effects on algal production. However, when assessing the overall effects on algae, invertebrates, fish growth and fish physiology caused by the effluents from newer bleaching technologies, no correlation was observed between concentration of adsorbable organic halogen (AOX) and level of effect. Nor was there any correlation between the levels of conjugated chlorophenolics or resin acids in the bile of rainbow trout and physiological effects in the fish. This lack of relationship between concentration of chlorinated organic substances and biological responses makes it necessary to search for alternative bioactive compounds. Possible candidates might be plant sterols originating from the pulped wood.

The biliary accumulation of corticosteroids in rainbow trout,Oncorhynchus mykiss, during acute and chronic stress

The accumulation of immunoreactive corticosteroids in the bile of rainbow trout during stress was monitored by radioimmunoassay and GUMS. Although plasma cortisol levels were elevated by confinement for 1 hour, biliary levels of free and conjugated steroids in the bile were unaffected. However, after 24 hours confinement, in addition to elevated plasma cortisol levels, free and conjugated steroids in the bile were also significantly higher than in control, unstressed fish. The time-course of change in plasma and biliary corticosteroid levels was determined in rainbow trout subject to 96 hours confinement stress. Free steroid levels in the bile of stressed fish were elevated within 2 hours of the onset of stress, while levels of conjugated steroids were significantly elevated within 4 hours of the onset of confinement. Analysis of bile from stressed fish, by GC/MS, established the major conjugated steroids present to be tetrahydrocortisone (230 μg ml(-1) bile), tetrahydrocortisol (75 μg ml(-1)), cortisone (33.5 μg ml(-1)), cortisol (25 μg ml(-1)) and β-cortolone (5 μg ml(-1)). The data are discussed with reference to the role of cortisone and conjugating enzymes in the clearance of cortisol, and further data are presented to suggest that the analysis of biliary steroid content may provide a suitable means of identifying stressed fish under conditions in which an additional sampling stress is unavoidable.

Simultaneous determination of sulfamonomethoxine, sulfadimethoxine and their N4-acetylated forms in fish tissues by high performance liquid chromatography.

A high performance liquid chromatographic (HPLC) method for simultaneous determination of sulfamonomethoxine (SMMX), sulfadimethoxine (SDMX), and their N4-acetylated forms in fish tissues was developed. The sulfa drugs and their N4-acetylated forms extracted from samples were adsorbed on Sep-Pak Alumina B cartridge and eluted with 20ml of 70% acetonitrile. The eluate was evaporated to dryness. The residue was dissolved in 30% acetonitrile. The solution obtained was analyzed by HPLC. The chromatographic conditions were as follows: analytical cloumn, YMC-Pack C18; mobile phase, 0.05M phosphate buffer, pH 2.5-acetonitrile (65:35); wavelength, 265nm. The average recoveries of SMMX, SDMX, and their N4-acetylated forms from serum, muscle, liver, kidney, and bile of rainbow trout were more than 80%. Their coefficients of variation were 1.1-6.0%. The detection limits for serum, muscle, liver, and kidney were 0.05ppm, and that for bile. was 0.1ppm.

Absorption, Distribution, and Excretion of Butylated Hydroxytoluene in Rainbow Trout and Common Carp.

The absorption, distribution, and excretion of 14C-BHT in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio were examined. The radioactivity was high, as measured by the method of whole-body autoradiography, in the gall bladder and adipose tissue of the two fish species injected intravenously. The radioactivity of the bile was high, as measured by the method of scintillation counting, only in the common carp administered orally. In the excretion profile of 14C-BHT in the bile of rainbow trout, the radioactivity was increasing with time, reached a maximum level at 4 days or one week and then decreased gradually. In the excretion profile of 14C-BHT in common carp, the radioactivity was quickly distributed in tissues at the beginning. After 4 days, the radioactivity was shown to decrease in bile, hepatopancreas, and kidney.

Metabolites of three structural isomers of butylbenzene in the bile of rainbow trout

In an ongoing study of the fate of petroleum hydrocarbons in fish, the authors have examined the petroleum derived metabolites which concentrate in the gall bladder bile of cunners, and rainbow trout. The metabolites obtained from the bile of rainbow trout exposed to a petroleum oil have been analyzed by GLC-MS, and several two- and three-ring aromatic alcohols were identified after hydrolysis with {beta}-glucuronidase. Another approach toward the elucidation of the structure of metabolites derived from No. 2 fuel oil relied on chemical synthesis. In another series of experiments, they investigated the type of metabolites obtained in the bile of trout exposed to three n-alkylbenzenes. Exposure of fish to a mixture containing equal amounts of these three n-alkylbenzenes showed their presence in the bile, in a ratio of 1:3:4, for n-butylbenzene, n-hexylbenzene and n-octylbenzene metabolites, respectively. The present exposure was undertaken to determine if, given aromatic hydrocarbons of same molecular weight and ring structure, a relationship could be found between the type of alkyl side chain and metabolites formed; and the influence of the amount of hydrocarbon administered during an exposure on the type of glucuronides formed. Three structural isomers of butylbenzene were chosen for the present experiment: n-butylbenzene, sec-butylbenzenemore » and tert-butylbenzene.« less

Bile pigments in the bile of freshwater fish, rainbow trout, tilapia, and pejerrey.

Bile pigments in the bile of the rainbow trout, Salmo gairdneri, tilapia, Tilapia nilotica, and pejerrey, Odonthetes bonariensis, were analyzed by HPLC and TLC.Major bile pigments of rainbow trout and pejerrey were bilirubin glucuronides and ditauro-bilirubin, respectively. Ditaurobilirubin was not detected in rainbow trout and bilirubin glucuronides were scarcely found in pejerrey. Biliverdin seemed to be the sole bile pigment in tilapia, but it was a minor component in the other fish. These results are in accord with the previous reports in which the diversity of bile pigment composition was demonstrated in some fish.

Metabolism of n-butyl, n-hexyl, and n-octyl benzene in the bile of rainbow trout.

Exposure of fish to petroleum can occur directly in the aquatic environment, via the water, or indirectly through the diet. In previous investigations of petroleum metabolites extracted from the bile of fish, the analysis indicated the presence of ..beta..-glucuronides enriched with unsaturated vs saturated carbons, when comparing the metabolites mixture to No. 2 fuel oil. The naphthenic carbons represented a significant fraction of the total carbons and were observed at different chemical shifts than in the petroleum oil, strongly indicating that metabolic oxidations took place on the aliphatic carbons. Mass spectra analysis of the hydrolyzed metabolites isolated from the bile of trout exposed to No. 2 fuel oil, using ..beta..-glucuronidase, enabled the identification of at least eight metabolites. These two- and three-ring aromatic alcohols had side chains of two to six carbons. In order to answer some of the questions raised from the above observations, the metabolism of n-butyl benzene, n-hexyl benzene and n-octyl benzene was considered. The present study was undertaken in order to answer three questions: (1) For a given ring system, does the number of carbons in the alkyl side chain influence the rate of hydrocarbon uptake. (2) For n-alkyl aromatic molecules, are primary, secondary or aromaticmore » alcohols formed preferentially. (3) Are long alkyl side chains attached to aromatic rings degraded to a relatively shorter number of carbon atoms prior to excretion. To the authors knowledge, these questions had not been previously considered in the study of fish metabolites.« less

Free and conjugated resin acids in the bile of rainbow trout, Salmo gairdneri.

Free and conjugated resin acids in the bile of rainbow trout, Salmo gairdneri.

Glucuronides in bile of rainbow trout ( Salmo gairdneri) injected with [ 3H]aflatoxin B 1 and the effects of dietary β-naphthoflavone

1. Rainbow trout Salmo gairdneri were fed a diet containing the mixed-function oxidase system inducer, β-naphthoflavone or were fed a control diet. For the two respective diets, as much as 50 and 12% of an i.p.-injected dose of [ 3H]aflatoxin B 1 was recovered in the bile. 2. The major product in the bile of β-naphthoflavone-fed trout was an aflatoxicol-M 1 glucuronide, whereas the major product in the control bile was an aflatoxicol glucuronide.

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.