The dynamics of in vitro 17 β-estradiol secretion by isolated ovarian follicles of the rainbow trout (Oncorhynchus mykiss)

This study investigated the effects of human chorionic gonadotropin (hCG), 17-hydroxyprogesterone (17P) and testosterone (T) on the in vitro production of 17-estradiol (E₂) by isolated ovarian follicles of the rainbow trout (Oncorhynchus mykiss). 17P at 100 ng ml^-1, and hCG at 100 IU ml^-1 stimulated E₂ production relative to controls, whereas lower doses were ineffective. T was the most effective in stimulating E₂ production, followed by 17P and hCG respectively. The timecourse of E₂ production was investigated for both static culture, and incubations with media replacement, with follicles being exposed to hormone treatment for 30 min, 1 or 3 h, or constantly. E₂ production was observed after 30 min, 3 and 3-6 h in response to T, 17P and hCG respectively. Under static culture, E₂ levels reached maximal levels in 6 h. Longer incubations resulted in further metabolism of E₂ to E₂-glucuronide, which resulted in the blurring of treatment effects after 18 h. Incubations with media replacement resulted in higher E₂ production than in static culture. The results indicate that a 6 h incubation period is sufficient to produce significant increases in E₂ production in response to hCG, 17P and T, and that incubations longer than 12 h result in losses E₂ from the incubation media. These findings have implications for the validity of using static cultures to examine the effects of hormone treatment on the activity of steroid converting enzymes in vitro.     

The role of cAMP in regulating the β-adrenergic response of rainbow trout (Oncorhynchus mykiss) red blood cells

The -adrenergic response of teleost red blood cells (RBCs) enables the fish to maintain or even enhance the oxygen affinity of haemoglobin during various stress situations. The role of CAMP in the pronounced -adrenergic response of hypoxic rainbow trout RBCs was studied. Rainbow trout RBCs were incubated with three different -agonists (noradrenaline, adrenaline and isoproterenol, 10^–9 - 10^–4 M) at two oxygen tensions (P_{O 2}, 155 and 8 mmHg), and thereafter cAMP accumulation and cellular water content were measured.The cAMP concentration of non-stimulated trout RBCs was ca. 1200 nmol/kg dw. Of the three -agonists used, isoproterenol was the most effective in formation of cAMP, followed by noradrenaline and adrenaline. Oxygen tension affected the accumulation of cAMP in two ways. At physiological catecholamine levels (1–100 nM) there was either no difference between normoxic and hypoxic cells or a slight increase in the normoxic ones. At high catecholamine concentrations the accumulation of cAMP was greater in the hypoxic than in the normoxic cells. Oxygen tension also affected the magnitude of cell swelling but had no effect on the catecholamine concentrations causing half-maximal swelling (EC₅₀-values). The results indicate that, at physiological catecholamine levels, the -adrenergic response of rainbow trout RBCs is mainly regulated on the level of the Na⁺/H⁺ exchange.     

In vitro hepatic monodeiodination of L-thyroxine and the temporal effect of 17β-estradiol on deiodination in rainbow trout (Salmo gairdneri Richardson)

Thein vitro hepatic monodeiodination of L-thyroxine (T4) to triiodo-L-thyronine (T3) in rainbow trout (Salmo gairdneri) was found to be pH- and temperature-dependent, and was related to the amount of homogenate in the reaction vessel, suggestive of an enzyme-regulated event. Dithiothreitol (DTT) introduced into the reaction medium stimulated T3 production in a dose-related manner, whilst 6n-propyl-2-thiouracil (PTU) inhibited T3 production, also in a dose-related manner. The conversion was stimulated in the presence of light and depressed at buffer concentrations of less than 0.1 M.Prior treatment of fish with an intraperitoneal slow-release implant containing 17-estradiol (E2), at doses which are known to induce chronic mild elevations in plasma E2 levels, elicited a biphasic response to E2 as regards hepatic T3 production from T4 with a depression of T4 to T3 conversion evident within 1–2 days after implantation, and a subsequent stimulation of T3 production evident 56 days after, implantation. This increased hepatic deiodinase activity after chronic exposure to E2 at physiological doses was accompanied by a 3.5 fold increase in Vmax without a significant change in Km, suggesting the presence of an increased amount of the enzyme.     

The effects of hypoxia,in vivo,on red blood cell β-adrenoceptors in the rainbow trout,Oncorhynchus mykiss

Rainbow trout, Oncorhynchus mykiss, were exposed to 48h of environmental hypoxia (water partial pressure of oxygen = 8.0 kPa) at either 5 or 15°C. Blood was sampled during hypoxia via a dorsal aorta cannula to measure arterial blood partial pressure of oxygen and plasma catecholamine concentrations. After 48h, the number (B_max) and dissociation constant (K_d) of red blood cell surface -adrenoceptors were determined using a radioligand-displacement binding assay. At 5°C, plasma catecholamine levels were elevated at 24h whereas at 15°C, levels were elevated at 48h. At either temperature, following 48h of hypoxia, there was no change in B_max or K_d of red blood cell surface -adrenoceptors, compared to normoxic control fish. This study demonstrates that chronic exposure to moderate hypoxia does not affect the number or affinity of cell surface -adrenoceptors on trout red blood cells.     

Comparison of 3,5,3′-triiodo-L-thyronine and L-thyroxine absorption from the intestinal lumen of the fasted rainbow trout,Oncorhynchus mykiss

The absorptions of 3,5,3-triiodo-L-thyronine (T₃) and L-thyroxine (T₄) from the intestinal lumen of the rainbow trout were compared in vivo. Tracer doses of [¹²⁵I]T₄ (⁺T₄) or [¹²⁵I]T₃ (^*T₃) were injected through an anal cannula into the duodenum of trout fasted for 3 days at 12°C, and radioactivity was measured in blood and tissues at 4–48 h. ^*T₃ was removed more extensively than ^*T₄ from the intestinal lumen and more radioactivity was absorbed into the blood and tissues of u+T₃-injected trout than ^*T₄-injected trout. HPLC analysis showed that a high proportion of the radioactivity in the plasma, liver, kidney and intestinal lumen of ^*T₃-injected trout remained as the parent ^*T₃. However, in ^*T₄-injected trout most plasma radioactivity was in the form of ¹²⁵I^–, and by 24 h a high proportion of luminal radioactivity was ¹²⁵I^–. By 48 h, over 4% of the injected ^*T₃ and 1% of the injected ^*T₄ dose resided in the gall bladder, primarily as derivatives of ^*T₃ or ^*T₄. We conclude that T₃ is absorbed more effectively than T₄ from the intestinal lumen of fasted trout, indicating the potential for an enterohepatic T₃ cycle.     

Characterization of hepatic and extrahepatic glutathione S-transferases in rainbow trout (Oncorhynchus mykiss) and their induction by 3,3′,4,4′-tetrachlorobiphenyl

Liver, kidney, gill and olfactory epithelium cytosolic fractions of rainbow trout (Oncorhynchus mykiss) were examined for glutathione S-transferase (GST) contents. Proteins retained on a glutathione (GSH)-affinity matrix were separated as monomers by reversed-phase HPLC and characterized by immunoblotting, mass spectrometry and partial amino acid sequence. For each organ concerned, a specific pattern of these proteins was determined and appeared similar for liver and kidney on one hand, and for gill and olfactory epithelium on the other hand. It was confirmed that the prominent hepatic GST is a class enzyme, also constitutively expressed as a major isoform in the four organs studied. Moreover, a class variant and two new class GST subunits were characterized in minor fractions. An unknown protein, which was found major in gills and olfactory epithelium, exhibited some characteristics of class GSTs. Occurrence of possible GSH-adduct formation observed on two distinct monomers in specific experimental conditions is discussed. These results and methods were used to investigate the effect of 3,3,4,4-tetrachlorobiphenyl (TCB), a polychlorinated biphenyl (PCB), on GST expression in trout liver. From HPLC-profiling, significant co-induction of the major class and the two minor class GST subunits was observed in trout after waterborne exposure to TCB which was followed by a slight increase in 1-chloro-2,4-dinitrobenzene (CDNB) activity. The present work allows qualitative evaluation of the specific detoxification potential of rainbow trout. The use of HPLC-profiling of GSTs as a possible tool for the biomonitoring of polluted aquatic environment is suggested.     

FTA® card tool for sampling and rapid diagnosis of bacterial diseases from rainbow trout (Oncorhynchus mykiss) tissue

The collection of farmed fish samples and subsequent diagnosis of microbial pathogens can be difficult and time-consuming procedure under field conditions. Correct diagnosis and identification of bacterial diseases agents (Yersinia ruckeri, Flavobacterium psychrophilum, Lactococcus garvieae, and Vagococcus salmoninarum) are crucial for the effective treatment. In this study, the feasibility of using FTA® card (Flinders Technology Associates filter papers) for sampling infected rainbow trout (Oncorhynchus mykiss) tissues and significantly accelerated the molecular diagnosis of enteric redmouth disease, rainbow trout fry syndrome, bacterial coldwater disease, lactococcosis associated with fatal hemorrhagic septicemia, and vagococcosis were evaluated. We also examined and compared a practical and reliable extraction obtained from immobilized DNA on FTA® card with one commonly used commercial kit to diagnose bacterial pathogens in fish tissues. Results proved that the active working time of extracting DNA from FTA® card is requiring only 30 min and the cost is less than USA $ 0.5 for each eluted sample. A total of hundred extracted DNA from tissues on FTA® card were tested and showed successful PCR amplification of the aroA, murG, pLG, and pSa1 genes. In conclusion, our finding indicates FTA® card as an appropriate tool for the simple collection, easy transfer, and stored at room temperature without contamination and retrieval of high-quality DNA for rapid molecular diagnosis of bacterial fish pathogens without isolation and culture.

     

Cortisol does not affect hepatic α- and β-adrenoceptor properties in rainbow trout (Oncorhynchus mykiss)

Distribution and function of hepatic - and -adrenoceptors were examined in rainbow trout (Oncorhynchus mykiss) injected with slow release hydrogenated coconut oil implants alone (sham) or containing cortisol. - and -Adrenoceptors were assayed on purified hepatic membranes 10–14 days post-implantation using ³H-prazosin () and ³H-CGP (). At 10–14 days, plasma cortisol values were significantly elevated to approximately 220 compared with 35.0 ng ml^-1 in cortisol implanted vs. sham trout. No significant differences were found between any of the experimental groups for either the affinity (K_d) or maximal number of binding sites (B_max) for either receptor type. Epinephrine significantly stimulated glucose release from hepatocytes isolated from sham injected trout, but not from cortisol-treated fish. Epinephrine-induced glucose release was blocked by both - and -antagonists. These studies do not support the hypothesis that rainbow trout exposed to chronic cortisol alter properties of hepatic adrenoceptors.     

Catalytic activity and immunochemical quantification of hepatic cytochrome P-450 in β-naphthoflavone and isosafrol treated rainbow trout (Oncorhynchus mykiss)

In addition to catalytical assays, immunochemical techniques have recently been employed to measure induction of the cytochrome P-450 (P450) monooxygenase system in fish with polyaromatic hydrocarbons (PAH). In the present study, polyclonal antibodies were raised against rainbow trout P450IA1. Levels of rainbow trout P450IA1 determined using protein blotting- and ELISA procedures were compared with levels of 7-ethoxyresorufin-O-deethylase (7-EROD) activity in liver microsomes from rainbow trout. These comparisons showed that values of P450A1 were positively correlated (r=0.99 and r=0.97) with 7-EROD activities. In addition, the effects of isosafrol (ISF) or -naphthoflavone (NF) treatments on P450 levels in rainbow trout liver were investigated using immunochemical and catalytical methods. ISF treatment induced 7-EROD activity as well as 7-methoxycoumarin-O-demethylase-, 7-ethoxycoumarin-O-deethylase-, 7-propoxy-coumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities, although to a lesser extent, compared with the NF treatment. In contrast, immunochemical quantification of rainbow trout P450IA1 protein revealed a slightly different pattern. ISF appeared to be a weak inducer of P450IA1 in rainbow trout compared with NF. In addition, the degree of inhibition of 7-alkoxycoumarin-O-dealkylase activities in ISF microsomes differed from that measured in control- and NF microsomes. The discrepancies between catalytic and immunochemical estimates of rainbow trout P450IA1 in ISF treated fish in addition to differencs between specific inhibitory pattern by specific polyclonal antibodies raised against rainbow trout P450IA1, indicate that important differences exists between the responses induced by NF- and ISF treatments in the rainbow trout liver.Part of this work was presented at the 6th International Conference on Biochemistry and Biophysics of Cytochrome P-450, Vienna, Austria, July 3–8, 1988.     

Can heat‐killed Gordonia bronchialis enhance growth and immunity in rainbow trout (Oncorhynchus mykiss)?

In this study, the efficacy of heat‐killed Gordonia bronchialis on growth performance, immune system and gastrointestinal structure in rainbow trout (Oncorhynchus mykiss) was evaluated. Fish (mean weight 30 g) were fed basal diet (control), or treatment diets containing 2.48 × 10⁸ (low dose) or 1.24 × 10⁹ (high dose) cells kg^?1 feed of heat‐killed G. bronchialis on a pulse basis (5 days on treatment diet; 10 days on basal diet) for 95 days. On days 95 and 105, some of the fish were sampled for analysis of mentioned parameters. On days 110 and 120, the remaining fish were injected intraperitoneally with a 20 mL L^?1 suspension of chicken red blood cells. Results showed that growth performance was significantly enhanced in both treatment groups compared with the control group. Serum complement and lysozyme activities and hemagglutination antibody titre were higher in both treatment groups compared with the control group. The length of the intestinal and pyloric caeca folds was increased in the high‐dose group. Meanwhile, the number of goblet cells was increased in both treatment groups. This study suggests that heat‐killed G. bronchialis has the potential to enhance growth, immunological parameters and the gastrointestinal structure in rainbow trout.     

Are dietary genistein and equol potent enhancers of eicosapentaenoic acid and docosahexaenoic acid levels in rainbow trout (Oncorhynchus mykiss)?

Phytoestrogens are putatively able to enhance the biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but have also been shown to affect fish growth dose dependently. The aim of the present study was to identify a concentration for the phytoestrogen genistein and the phytoestrogen metabolite equol that further increases the endogenous biosynthesis of EPA and DHA without impairing fish growth. Juvenile rainbow trout (87.2 ± 0.3 g) were fed seven diets on a fixed ratio for 8 weeks. A vegetable oil‐based diet served as a control diet and was supplemented with equol (EQ) and genistein (G), respectively, at 0.1%, 0.2% and 0.3% of feed dry matter (1, 2 and 3). Growth and nutrient composition of whole body homogenates were not affected by dietary treatments. EPA and DHA levels in liver, fillet and whole body samples were not significantly increased by EQ and G diets. Fish fed EQ diets showed dose dependently increased liver weights and C18:0 liver levels, indicating estrogen‐like effects at increased dietary dosages. In conclusion, the utilization of equol and genistein in plant oil‐based diets in order to enhance the biosynthesis of EPA and DHA seems not reasonable in rainbow trout.     

Effects of chronic exposure to γ-HCH (Lindane) on brain serotonergic and gabaergic systems,and serum cortisol and thyroxine levels of rainbow trout,Oncorhynchus mykiss

Rainbow trout (Oncorhynchus mykiss) were implanted intraperitoneally with 0.5 ml.100 g^–1 body weight of coconut oil alone (controls) or coconut oil contaning 0.05 mg of -HCH (Lindane). After 18 days, changes in brain serotonin and GABA metabolism, as well as in serum cortisol and thyroxine levels, were measured. A lower final body weight was observed in -HCH treated fish when compared with control fish. No significant differences were found for serum thyroxine levels between control and treated fish, but a significantly higher cortisol level was found in the -HCH-implanted trout. Although GABA levels did not differ significantly in any brain region in the two treatment groups, the activity of the serotonergic system was significantly altered by the pesticide in both the hypothalamus and the telencephalon.     

Tartrate resistant acid phosphatase as a marker for scale resorption in rainbow trout,Oncorhynchus mykiss: effects of estradiol-17β treatment and refeeding

In teleosts, a considerable part of the body calcium is found in the scales. Associated with the scales are osteoblasts and osteoclasts, and during periods of high calcium demand such as during sexual maturation or starvation, the scales can be resorbed and thereby act as an internal calcium reservoir. In mammalian bone tissue, the activity of an acid phosphatase (ACP) isoenzyme, tartrate resistant acid phosphatase (TRACP), can be used as a marker for osteoclastic activity. In the present study, an evaluation of TRACP as a marker for osteoclastic activity in teleost scales has been performed. ACP and TRACP was histologically localized at resorption sites around the edge of the scales as well as at resorption holes in the scales. The optimal conditions for biochemical measurements of ACP and TRACP activity were found to be pH 5.3, 10 mM paranitrophenylphosphate, incubated for 30 min at room temperature, and 10 mM tartrate added when required. Using TRACP as a marker, estradiol-17 (E₂) was found to increase the proportion of scales being resorbed, as well as the number and size of resorption sites per scale. Also, the scales of E₂-treated fish showed weaker staining for calcium. Together, the obtained data indicate that estradiol-17 induces osteoclastic activity in teleost scales, resulting in increased resorption of the scales. A period of refeeding following a period of starvation did not have detectable effects on the scale osteoclastic activity and scale resorption.     

Video surveillance methods to evaluate individual feeding response in rainbow trout (Oncorhynchus mykiss,Walbaum)—implications for feeding regime optimisation

Aquaculture International - Precisely analysing and optimising feeding regimes is central to salmonid growth performance and delivery of special diets. The current study developed novel video...     

Synthesis and regulation of 17α-hydroxy-20β-dihydroprogesterone in immature males of Oncorhynchus mykiss

Three experimental approaches were chosen to study the question if the progestin 17-hydroxy-20-dihydroprogesterone (1720OHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20-hydroxysteroid dehydrogenase activity (20HSD), testes homogenates were incubated with ³H-labeled 17OHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 1720OHP-³H, indicating that already immature testes contain 20HSD activity and are able to produce 20-reduced steroids. Second, 1720OHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 1720OHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20HSD is present in testicular cells other than spermatozoa. Furthermore, 1720OHP is indeed secreted at a very early stage of testicular development; 1720OHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.     

Are brown trout Salmo trutta fario and rainbow trout Oncorhynchus mykiss two of a kind? A comparative study of salmonids to temperature‐influenced Tetracapsuloides bryosalmonae infection

Proliferative kidney disease (PKD) of salmonids caused by Tetracapsuloides bryosalmonae causes high mortalities of wild brown trout (Salmo trutta fario) and farmed rainbow trout (Oncorhynchus mykiss) at elevated water temperatures. Here the aim was to compare the temperature‐dependent modulation of T. bryosalmonae in the two salmonid host species, which display different temperature optima. We used a novel experimental set‐up in which we exposed brown trout and rainbow trout to an identical quantified low concentration of T. bryosalmonae for a short time period (1 hr). We followed the development of the parasite in the fish hosts for 70 days. PKD prevalence and parasite kinetics were assessed using qPCR. Exposures were performed at temperatures (12°C and 15°C) that reflect an environmental scenario that may occur in the natural habitat of salmonids. T. bryosalmonae infection was confirmed earliest in brown trout kept at 15°C (day 7 post‐exposure) while, in all other groups, T. bryosalmonae was not confirmed until day 15 post‐exposure. Moreover, significantly greater infection prevalence and a faster increase of parasite intensity were observed in brown trout kept at 15°C than in all other groups. These results indicate that PKD is differentially modulated by water temperature in related host species.     

The Effects of Sodium Alginate on Quality of Rainbow Trout (Oncorhynchus mykiss) Fillets Stored at 4 ± 2°C

Edible coating is a novel food packaging technology for controlling the growth of spoilage bacteria and chemical changes in ready-to-eat food products. Fresh fish are highly perishable foodstuffs and are very susceptible to spoilage. A sodium alginate coating was used to maintain the quality of rainbow trout fillets in chilled storage over a period of 20 days. Fillet samples were coated with an aqueous solution of 3% sodium alginate and then stored at chilled temperature (4 ± 2°C). The control and coated samples were analyzed periodically (every 5 days) for microbial (aerobic plate count and psychrotrophic count), chemical (total volatile basic nitrogen, thiobarbituric acid, peroxide value), and sensory characteristics. The results indicated that the sodium alginate coating helped protect the quality characteristics of treated fresh fish fillets and prolonged the shelf life during chilled storage of rainbow trout fillets.     

Sulfation and uptake of the maturation-inducing steroid, 17α,20β-dihydroxy-4-pregnen-3-one by rainbow trout ovarian follicles

Rainbow trout ovarian follicles were incubated in vitro with tritiated 17,20-dihydroxy-4-pregnen-3-one (17,20-P; maturation-inducing steroid). Within 18–24 h, 56–66% had been converted to tritiated 17,20-dihydroxy-4-pregnen-3-one 20-sulfate (identification confirmed by HPLC) and 27% had been taken up (absorbed) by the follicles. Addition of 125 ng of cold (non-tritiated) 17,20-P to the incubations caused a decrease in the percentage of [³H]-17,20-P which was sulfated (56% 10%) and an increase in the percentage that was taken up (27% 57%). Seven steroids were tested for their effectiveness in decreasing the sulfation and increasing the uptake of tritiated [³H]-17,20-P. The order of effectiveness was in both cases the same: 17,20-P > cortisol > 11-deoxycortisol > 17,20,21-trihydroxy-4-pregnen-3-one > 17-hydroxy-4-pregnene-3,20-dione > 17-estradiol > testosterone. This indicated that the processes of sulfation and uptake of [³H]-17,20-P were related to each other and led to the hypothesis that, when cold 17,20-P is added to the medium, it reduces the proportion of [³H]-17,20-P which is sulfated and thus allows more free [³H]-17,20-P to enter the ovarian follicles. This hypothesis was supported by the finding that each ovarian follicle had the capacity in vitro to sulfate only ca. 2 ng of [³H]-17,20-P per 18h but a capacity to take up > 500 ng per 18h.Gonadotropin I, Gonadotropin II, forskolin and phorbol-12-myristate-13-acetate (which all have an affect on steroid biosynthesis) did not affect the amount of 17,20-P which was sulfated. Sulfating activity was localized in the thecal cell layer of the follicle. The yolk fraction was shown to be responsible for absorbing the [³H]-17,20-P.     

Effects of 17β-estradiol injection on the reproductive efficiency of freshwater crayfish,Astacus leptodactylus (Eschscholtz, 1823)

The effects of 17β-estradiol injection (10^?7mol/crayfish) in adult female Astacus leptodactylus on the reproductive efficiency (i.e., pleopodal egg and stage 1 juvenile numbers) were investigated. In addition, hepatosomatic index, gonodosomatic index, and 17β-estradiol (E2) levels of crayfish ovary, hepatopancreas, and heamolymph before and after injections were also determined. Results showed that E2 injection to females enhances the reproductive efficiency of A.leptodctylus. E2 injection significantly increased pleopodal egg and stage 1 juvenile numbers, GSI, HSI, and concentrations of E2 in heamolymph, ovary, and hepatopancreas. However, E2 injection did not accelerate time of mating and proportion of pleopodal egg-berried females at the first week of reproduction season in this species. This study highlights that to improve the reproductive efficiency of A.leptodctylus by using hormones to regulate the ovarian cycle, E2 should be used at least 1.5 months before the commencement of the breeding season.