Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss)

Abstract

Sublethal concentrations of copper in water cause the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Receptor cell loss has been correlated to the loss of olfaction in fish and may cause difficulties in olfactory mediated behaviors such as migration. This study investigated the effects of three levels of copper (100, 75 and 50 mg L^?1) on the olfactory epithelium of rainbow trout. Twenty fish randomly allocated between three exposure groups and one control were exposed for 24 hours under static renewal conditions. Light and scanning electron microscopic observations of olfactory tissue were taken to determine the extent of degeneration of receptors. In addition, levels of copper and zinc in the brain tissues were analyzed to determine if the olfactory route was a significant route of copper exposure and transfer to fish brain tissue. Results indicate that degeneration of receptors is related to the concentration of copper. Levels of copper in brain were found to be below detection of the instrument. Levels of zinc were extremely variable ranging from 52 to 132 ng zinc g^?1 brain tissue.     

Patterns of transgene inheritance in rainbow trout (Oncorhynchus mykiss).

There have been very few studies of the inheritance of introduced genes (transgenes) in fish. We have followed the inheritance of the mammalian fusion gene MTrGH from founder generation transgenics (originating from eggs microinjected with the MTrGH DNA) to offspring in crosses with control fish. Initial screening of the founder generation transgenics was by analysing DNA from blood samples. Only three out of six fish which carried the novel gene in blood DNA transmitted it to their offspring, despite the presence of the gene in DNA extracted from the sperm of all four male fish in this group. The frequency of transgenics in the progeny groups from the three fish which transmitted the gene varied widely: in one of these groups more than one type of MTrGH restriction pattern was found. These results suggest widespread mosaicism in founder generation transgenics.     

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.     

Haematological disturbances and osmotic shifts in rainbow trout,Oncorhynchus mykiss (Walbaum) under acid and aluminium exposure

Summary Elevated values of haematocrit were observed in rainbow trout (Oncorhynchus mykiss) during combined acid and aluminium exposure. Possible causes of this, i.e., decreased plasma volume, swelling of erythrocytes, and/or mobilization of erythrocytes into the blood were investigated. Slight haematocrit increases (10–20%) during mild acid stress (pH 5.0, 25 mol Ca²⁺·1^-1) were mostly caused by osmotic shifts. Both swelling of erythrocytes rocytes and a significant decrease of the plasma volume were demonstrated in fish at pH 5.0. These osmotic disturbined were greater during acid exposure (pH 5.0) combined with Al (60 g·1^-1; 200 g·1^-1). In addition, numbers of erythrocytes increased by 40% compared to acid exposure, which contributed to the severe haematocrit rise (35%) during Al exposure. A contraction of the spleen releasing erythrocytes into the blood is suggested to occur as an adrenergic response to hypoxia, which is observed in fish acutely exposed to Al.Abbreviations BV blood volume - ECFV extracellular fluid volume - ICFV intracellular fluid volume - ISFV interstitial fluid volume - MCHC mean cell haemoglobin concentration - MCV mean cell volume - PV plasma volume     

Intra-strain dioxin sensitivity and morphometric effects in swim-up rainbow trout (Oncorhynchus mykiss)

Inter and intra-specific differences in sensitivity of early life stage salmonids to 2,3,7,8-TCDD exposure have been reported, but intra-strain differences have not been found in the literature. Our results indicate that intra-strain variability in terms of embryo mortality (LD50) is small in Eagle Lake strain of rainbow trout, LD50 values ranging from 285 to 457 pg TCDD egg g(-1). These results confirm Eagle Lake as a less sensitive strain within rainbow trout, and do not indicate overlap with reported LD50 values for brook or lake trout. Our results also demonstrate that although generalized edema in regions including the yolk-sac are frequently associated with mortality following dioxin exposure, not all edematous fish die. We detected dose-dependent decreases in cranial length, eye diameter, mass, and total length (P < 0.05) in viable swim-up rainbow trout. These effects are presumed to indicate more subtle dose-dependent disruptions of the viteline vein vasculature and, therefore, in access to energy sources. A tendency for dose-dependent decrease in liver glycogen reserves concurred with previous results on salmonids and with the well described TCDD-induced alterations in intermediate metabolism of rats and chicken embryos (wasting syndrome). This syndrome could be contributing to the reduced growth that we observed.     

Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro

The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum     

Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum)

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n = 42) obtained with the application of 11β-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX < 25%; II XX 25-50% and III XX > 50%. The average percentage of sperm motility was 18 ± 7% n = 12 (group I XX); 42 ± 6% n = 15 (group II XX) and 65 ± 12% n = 15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3 × 10⁶ spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.     

The functional characterisation of CK-1, a putative CC chemokine from rainbow trout (Oncorhynchus mykiss)

Recently a number of cytokine homologs have been cloned in teleost fish, including several that resemble chemokines, but to date few have been confirmed using functional assays. Chemokines are a family of cytokines that are able to induce chemotaxis in leucocytes. In this study CK-1, a rainbow trout chemokine, was functionally characterised. Recombinant CK-1 is able to attract rainbow trout peripheral blood leucocytes (PBL) in a micro-chemotaxis chamber. A greater number of PBLs migrated in response to CK-1 than to negative controls, either media alone or equivalent concentrations of beta2M, while comparable numbers migrated to the positive control, recombinant human C5a. The tissue distribution of CK-1 mRNA was also assessed by Northern blotting of RT-PCR and showed that expression is constitutive in the liver and gut, and is inducible by intraperitoneal injection of phytohemagglutinin in PBL and the head-kidney. Continuous cell lines generated from the gut and pituitary gland of the rainbow trout also express CK-1 message, whilst Southern analysis shows that CK-1 is a single copy gene. Finally, CK-1 shows the greatest amino acid similarity CCL20/LARC/Mip-3alpha as well as similar gene structure and expression pattern.     

Polarized-light sensitivity in rainbow trout (Oncorhynchus mykiss): characterization from multi-unit responses in the optic nerve

Integrated spike activity of axons from the optic nerve was measured in an investigation of the e-vector sensitive mechanism underlying the ability of rainbow trout (Oncorhynchus mykiss) for orientation in downwelling, linearly-polarized light. In anaesthetized, immobilized fish, one eye was exposed to incremental light flashes which were superimposed over closely controlled background conditions. Under scotopic and various photopic conditions, intensity/response curves were generated from the on-response of the optic nerve. Relative sensitivity curves were then obtained as a function of e-vector direction for the 5 kinds of receptor cells in this trout's retina: rods, ultraviolet cones (UV), short wavelength cones (S), medium wavelength cones (M), and long wavelength cones (L).Under scotopic conditions, no sensitivity to e-vector was apparent: thus, rods do not mediate polarization sensitivity. Under photopic conditions, parr weighing 8–10 g exhibited e-vector sensitivity in two orthogonal channels. A UV stimulus (380 nm) on a white background evoked a three-peaked response (0°, 90°, and 180°) to the e-vector orientations presented in 30° increments between 0° and 180°. In contrast, when the background was illuminated with appropriate short and long wavelength cut-off filters, M-and L-cones showed maximum responses only to the horizontal (90°) plane whether they were stimulated at their -absorption band or their -absorption band in the near UV. Isolated UV-cones gave maximum responses to the vertical (0° and 180°) e-vector, thus corresponding to a second channel. The blue sensitive, S-cones, did not show evidence of polarization sensitivity. As well, no evidence of the polarization sensitivity was observed under UV isolating background conditions in larger individuals, 50–78 g smolts, although the other cone mechanisms responded as in smaller individuals.     

Effects of dietary Ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss)

The effects of dietary Ergosan on the growth performance and mucosal immunity in rainbow trout skin were investigated. 60 rainbow trout (100-110 g) were randomly assigned to 2 groups in triplicates and fed one of the experimental diet formulated with 5 g kg⁻¹ Ergosan or control diet for 50 days. Results showed that on the 45th day of feeding trial, Ergosan supplementation significantly enhanced the growth performance compared to control group. Various enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase in treatment group were also enhanced on the 45th and 50th day. Skin mucus in Ergosan-fed fish showed the agglutination of erythrocytes while in control group, no visible agglutination was shown. In addition, skin mucus in treatment group showed strong antibacterial activity against Yersinia ruckeri. In conclusion, the major immune components of rainbow trout mucus that are involved in the non-specific immunity were enhanced by administration of Ergosan in 5 g kg⁻¹.     

Salmon growth hormone is transported into the circulation of rainbow trout,Oncorhynchus mykiss,after intestinal administration

Summary Transport of recombinant salmon growth hormone (rsGH) into the circulation of rainbow trout following intragastric or rectal administration was investigated. Changes in plasma GH levels were analyzed by radioimmunoassay specific to chum salmon GH. Intragastric administration of rsGH by oral intubation resulted in elevation of GH immunoreactivity in plasma after 11h. The plasma GH increased maximally after 15h, and then declined rapidly to reach a normal level after 19h. On the other hand, oral intubation of rsGH to carp, which has no stomach, caused elevation of plasma GH levels after 1 h which lasted for 21 h. In sharp contrast, rectal administration of rsGH to rainbow trout significantly elevated plasma GH levels after 15 min, which reached a maximum after 30 min. The rsGH was labeled with fluorescein isothiocyanate (FITC) to distinguish exogenous from endogenous GH, and administered into rainbow trout rectally. Subsequent analysis of plasma samples on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two fluorescent bands at the same molecular weights as those of the monomeric and dimeric rsGHs. Intragastric and rectal administration of rsGH into juvenile trout resulted in significant increases in length and body weight compared to the control fish. This study strongly suggests that the rsGH is transported into the circulation of salmonids via the lower part of the intestine and takes part in growth stimulation.Abbreviations DCA deoxycholic acid - FITC fluorescein isothiocyanate - GH growth hormone - HPLC high-performance liquid chromatography - HRP horseradish peroxidase - RIA radioimmunoassay - rsGH recombinant salmon growth hormone - sGH chum salmon growth hormone - SEM standard error of mean - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TFA trifluoroacetic acid     

Isolation, characterization, and distribution of two cDNAs encoding for growth hormone receptor in rainbow trout (Oncorhynchus mykiss)

Growth hormone (GH) plays important roles in a vast array of physiological processes, including growth, metabolism, and reproduction. In this study, cDNAs for two unique growth hormone receptor variants were cloned and sequenced from rainbow trout. The two cDNAs, one consisting of 2920 bp and the other of 2820 bp, share 87.2% identity in nucleotide sequence and 85.5% identity in deduced amino acid sequence and presumably arose through gene duplication. The cDNAs encode for putative 593- and 594-amino acid growth hormone receptors (designated GHR1 and GHR2, respectively), each containing a single transmembrane domain and other motifs characteristic of the receptor family. Both GHR1 and GHR2 mRNAs were present in all tissues examined. Trout GHR mRNAs are differentially expressed, both in terms of abundance among tissues and in terms of abundance within selected tissues. GHR1 was more abundant than GHR2 in the brain, whereas GHR2 was more abundant than GHR1 in pancreas and spleen. These findings expand our understanding of the evolution of the GH receptor family and suggest that independent mechanisms serve to regulate the tissue-specific expression of GHR mRNAs.     

Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm

Cryopreservation causes several types of damage to spermatozoa, such as loss of plasma membrane integrity and functionality, loss of motility, and ATP content, resulting in decrease of fertility rates. This spermatozoal damage has been widely investigated for several marine and freshwater fish species. However, not much attention has been paid to the nuclear DNA. The objective of this study was to determine the degree to which cryopreservation induces spermatozoal DNA damage in two commercially cultured species, rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), both of which could benefit from the development of cryopreservation strategies on a large scale. We have used the single-cell gel electrophoresis, commonly known as Comet assay to detect strand breaks in DNA. This technique was performed on fresh and cryopreserved sperm from both species. In rainbow trout there was a significant increase in the averages of fragmented DNA and Olive tail moment after cryopreservation (11.19-30.29% tail DNA and 13.4-53.48% Olive tail moment in fresh and cryopreserved sperm, respectively), as well as in the proportion of cells with a high percentage of DNA fragmentation. For gilthead sea bream there were no significant differences in the percentage of tail DNA between the control samples and sperm diluted 1:6 and cryopreserved (28.23 and 31.3% DNA(t), respectively). However, an increase in the sperm dilution rate produced an increase in the percentage of DNA fragmentation (41.4%). Our study demonstrates that cryopreservation can induce DNA damage in these species, and that this fact should be taken into account in the evaluation of freezing/thawing protocols, especially when sperm cryopreservation will be used for gene bank purposes.     

Hepatic CYP1A induction in rainbow trout (Oncorhynchus mykiss) after exposure to benzo[a]pyrene in water

Juvenile rainbow trout were exposed to unlabelled benzo[a]pyrene BaP and ³H benzo a pyrene (³H BaP), in a static exposure system for 2 days. The initial concentration was 30 μg l^-1 and 0.625 μCi l^-1, corresponding to 6 mg kg^-1 body weight and 125 μCi kg^-1 body weight. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity was measured during the exposure and depuration periods, elucidating the time course pattern of CYP1A induction. Maximum induction (11-fold) of EROD activity was observed on day 2 after addition of BaP to the water. Tissue distribution of ³H-BaP was studied by liquid scintillation counting and whole body autoradiography. The concentration of ³H-BaP-derived radioactivity was highest in the bile at all sampling times. High levels of radiolabelled compound were also present in the gills, liver and the olfactory organ. There was an overall decrease in all tissues during the depuration period. The elimination of ³H-BaP-derived radioactivity from the gills, however, was slow compared with liver and blood (6.2 days vs 2.7 and 2.9 days, respectively).     

Hepatic CYP1A induction in rainbow trout (Oncorhynchus mykiss) after exposure to benzo[a]pyrene in water

Juvenile rainbow trout were exposed to unlabelled benzo[a]pyrene BaP and ³H benzo a pyrene (³H BaP), in a static exposure system for 2 days. The initial concentration was 30 μg l^-1 and 0.625 μCi l^-1, corresponding to 6 mg kg^-1 body weight and 125 μCi kg^-1 body weight. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity was measured during the exposure and depuration periods, elucidating the time course pattern of CYP1A induction. Maximum induction (11-fold) of EROD activity was observed on day 2 after addition of BaP to the water. Tissue distribution of ³H-BaP was studied by liquid scintillation counting and whole body autoradiography. The concentration of ³H-BaP-derived radioactivity was highest in the bile at all sampling times. High levels of radiolabelled compound were also present in the gills, liver and the olfactory organ. There was an overall decrease in all tissues during the depuration period. The elimination of ³H-BaP-derived radioactivity from the gills, however, was slow compared with liver and blood (6.2 days vs 2.7 and 2.9 days, respectively).     

Regulation of lipoprotein lipase gene expression by insulin and troglitazone in rainbow trout (Oncorhynchus mykiss) adipocyte cells in culture

Adipose tissue plays a central role regulating the balance between deposition and mobilization of lipid reserves. Lipoprotein lipase (LPL) is a key enzyme controlling lipid accumulation in mammals and fish. In the present study, we have examined the expression of LPL in rainbow trout cultured adipocytes and we have investigated the effect of troglitazone, a member of thiazolidinediones (TZDs), and insulin on its expression. LPL gene expression increased from day 1 until day 12 of culture, and the level was maintained up to day 21. The addition of insulin at 10 nM and 1.7 μM increased significantly LPL gene expression in undifferentiated cells (days 7 to 12 maintained in growth medium). Nevertheless, treatment of day 7 cells incubated in growth medium with troglitazone (5 μM) or troglitazone plus insulin (1 μM each), tended to enhance LPL expression. In addition, LPL mRNA levels increased significantly in the presence of 1 μM and 5 μM of troglitazone (days 7 to 12) when the cells were induced to differentiate by addition of differentiation medium. Although troglitazone alone (1 μM) did not stimulate lipid accumulation in the cells neither in growth nor in differentiation medium, the simultaneous presence of troglitazone (1 μM) and insulin (1 μM) increased significantly the content of triglycerides in adipocyte cells maintained in growth medium (days 7 to 12). These results indicate that insulin and troglitazone regulate LPL gene expression during adipocyte differentiation and suggest that both factors may have combined effects in the modulation of adipogenesis.