Assessment of oxidative DNA damage, oxidative stress responses and histopathological alterations in gill and liver tissues of Oncorhynchus mykiss treated with linuron.

We investigated changes in 8-hydroxy-2-deoxyguanosine (8-OHdG) activity which is a product of oxidative DNA damage, histopathological changes and antioxidant responses in liver and gill tissues of rainbow trout, following a 21-day exposure to three different concentrations of linuron (30 µg/L, 120 µg/L and 240 µg/L). Our results indicated that linuron concentrations caused an increase in LPO levels of liver and gill tissues (p < 0.05). While linuron induced both increases and decreases in GSH levels and SOD activity, CAT activity was decreased by all concentrations of linuron (p < 0.05). The immunopositivity of 8-OHdG was detected in the hepatocytes of liver and in the epithelial and chloride cells of the secondary lamellae of the gill tissues. Our results suggested that linuron could cause oxidative DNA damage by causing an increase in 8-OHdG activity in tissues, and it induces histopathological damage and alterations in the antioxidant parameters of the tissues.

A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300-2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.

Anatomical, histological and immunohistochemical study of the tongue in the rainbow trout (Oncorhynchus mykiss).

The rainbow trout (Oncorhynchus mykiss Walbaum, 1792) is a fish commercially farmed all over the world. These fish are usually fed, in aquaculture, with pellets rich in proteins and fat. It is well known that there are close relationships among the adaptation of vertebrates to their environment, the capacity and the modality of feeding and the oral cavity morphology, especially the tongue one. No data are so far available about the morphology of the rainbow trout tongue, and therefore, the aim of this study was to investigate by light, scanning electron and confocal laser microscopy, the morphological characteristics of the tongue. An apex, a body and a root can be distinguished in the tongue, and the presence of teeth, taste buds and fungiform-like papillae was demonstrated. Light microscopy shows the presence of an adipose tissue pad in the deeper layer of the apex and in the most superficial layer of the root. In the deeper layer of the body, a triangular-shaped pad consisting of fusiform cells immersed in abundant extracellular matrix of the mesenchymal tissue was observed. The confocal laser microscopy shows the presence of cells with a fibroblast-like morphology positive for vimentin. In the deepest layer of the tongue root, a large area of osteo-cartilaginous tissue was observed. The results, besides the description of the morphological characteristics of the tongue, related to studies regarding the feeding, could be considered for the eventual applications of the use of mesenchymal cells, observed in adult fish, in cell therapies in different pathologies.

Manual fish length measurement accuracy for adult river fish using an acoustic camera (DIDSON).

Total lengths (LT ) of 50 free-swimming fish in a tank, silver carp Hypophthalmichthys molitrix and rainbow trout Oncorhynchus mykiss, were measured using a DIDSON (Dual-frequency IDentification SONar) camera. Using Sound Metrics software, multiple measurements of each fish (LT , side aspect angle and distance from the camera) at different times were analysed by two experienced operators while a subset of data was analysed by two inexperienced operators. The main result showed high variability in intra-fish LT measurements. The number of measurements required to minimise errors and to obtain robust fish measurements (true LT ± 3 cm) was estimated by a bootstrap method. Three to five measurements per fish were recommended for fish surveys in rivers. In this experimental study, aiming to reproduce river conditions, no evidence of fish position (side aspect angle and distance from the camera) effect was detected, but an operator effect (partially explained by training) was observed. General linear mixed models also showed that lengths of the smallest fish (LT < 57 cm) were overestimated and lengths of the largest fish (LT > 57 cm) were underestimated in comparison with their true lengths. In conclusion, we highlight that this technology, like any monitoring methods, returns imperfect observations. We advise DIDSON users to ensure that measurements are carried out correctly in order to draw accurate conclusion from this new technology.

Evaluation of endocrine and transcriptomic markers of male maturation in winter-run Steelhead Trout (Oncorhynchus mykiss).

Steelhead Trout (Oncorhynchus mykiss) display a varied life-history, including precocious male maturation at age-1 or age-2. In wild fish, precocious male maturation represents an important component of a diverse life-history portfolio. In hatchery programs, however, it is undesirable if rearing practices increase rates of early male maturation and reduce numbers of anadromous male adults. Our study aimed to develop endocrine and molecular markers for identifying males at early stages of maturation in the spring (prior to smolt release) and evaluated the potential use of these markers for quantifying early male maturation rates at a hatchery scale. In a laboratory study, Skookumchuck winter-run Steelhead Trout were reared at a high growth rate in order to increase the occurrence of precocious male maturation. Fish were lethally sub-sampled in February, prior to the time of smolt release; in May, at the time of smolt release; and in September, when 1+ age maturing males that would spawn the following spring were clearly identifiable based solely on gonadosomatic index (GSI). In February and May samples, we measured GSI, plasma 11-ketotestosterone (11KT), mRNAs for pituitary follicle stimulating hormone (fshb) and luteinizing hormone (lhb) beta subunits, and analyzed stage of spermatogenesis by testis histology. Additionally, in May, we measured testis anti-Müllerian hormone (amh) and insulin-like growth factor 3 (igf3) mRNA. Our primary goal was to evaluate the aforementioned maturation indices for their efficacy in forecasting the proportion of fish initiating early male maturation in the spring (approximately 1 year prior to spermiation), compared to the proportion that actually matured. Combining measures of GSI, plasma 11KT, and pituitary fshb and lhb mRNA expression provided a useful, but conservative, estimate of the proportion of males initiating maturation in the spring (21%) compared to the proportion that were ultimately destined to mature (37%) the following spring. These results suggest that maturation may be less synchronous than previously appreciated and some males may have initiated maturation after our census in May.

Influence of macerated fenugreek (Trigonella foenum graecum) oil added to trout feed at the different rates on the Feed Conversion Rate (FCR), body length, blood parameters and Nitroblue Tetrazolium (NBT) values ​​of rainbow trout (Oncorhynchus mykiss Wal.

In this study, 1% and 2% of macerated fenugreek oil was added to the feeds of rainbow trout with an average weight of 25.79 ± 1.5 g. At the end of the study, growth rate, blood parameters and NBT (Nitroblue Tetrazolium) level of rainbow trout were determined. The best feed ratio (FCR) was observed in the control group (0.77). Statistically significant differences were found only in MID values (P<0.05), although there was a numerical increase in all blood parameters. There was no statistically significant difference between NBT levels (P> 0.05). Although the best weight gain was in the control group as in the FCR values, the maximum elongation was measured at D1 and then at D2 (P <0.05). The best survival rate was obtained with 96.66% in D1 while the worst was observed in D2 with 60% (p<0,05).

Formation of intramuscular connective tissue network in fish: first insight from the rainbow trout (Oncorhynchus mykiss).

The formation of the intramuscular connective tissue was investigated in rainbow trout Oncorhynchus mykiss by combining histological and in situ gene-expression analysis. Laminin, a primary component of basement membranes, surrounded superficial slow and deep fast muscle fibres in O. mykiss as soon as the hatching stage (c. 30 days post fertilization (dpf)). In contrast, type I collagen, the primary fibrillar collagen in muscle of vertebrates, appeared at the surface of individual slow and fast muscle fibres only at c. 90 and 110 dpf, respectively. The deposition of type I collagen in laminin-rich endomysium ensheathing individual muscle fibres correlated with the late appearance of collagen type 1 α 1 chain (col1α1) expressing fibroblasts inside slow and then fast-muscle masses. Double in situ hybridization indicated that coll1α1 expressing muscle resident fibroblasts also expressed collagen type 5 α 2 chain (col5α2) transcripts, showing that these cells are a major cellular source of fibrillar collagens within O. mykiss muscle. At c. 140 dpf, the formation of perimysium-like structure was manifested by the increase of type I collagen deposition around bundles of myofibres concomitantly with the alignment and elongation of some collagen-expressing fibroblasts. Overall, this study shows that the formation of O. mykiss intramuscular connective tissue network is completed only in aged fry when fibroblast-like cells expressing type I and V collagens arise inside of the growing myotome.

Characterization of the histologic appearance of normal gill tissue using special staining techniques.

Anatomic pathologists are familiar with stains used in light microscopy to identify cells, storage products, tissue deposits, and pathogens. Assessment of the surrounding tissue with special stains may reveal aspects of interest for the tissue or the species. We illustrate the expected staining characteristics of normal rainbow trout gill tissue with routine hematoxylin and eosin and 18 other histochemical stains.

Behavioral and physiological adaptations to high-flow velocities in chubs (Gila spp.) native to Southwestern USA.

Morphological streamlining is often associated with physiological advantages for steady swimming in fishes. Though most commonly studied in pelagic fishes, streamlining also occurs in fishes that occupy high-flow environments. Before the installation of dams and water diversions, bonytail (Cyprinidae, Gila elegans), a fish endemic to the Colorado River (USA), regularly experienced massive, seasonal flooding events. Individuals of G. elegans display morphological characteristics that may facilitate swimming in high-flow conditions, including a narrow caudal peduncle and a high aspect ratio caudal fin. We tested the hypothesis that these features improve sustained swimming performance in bonytail by comparing locomotor performance in G. elegans with that of the closely related roundtail chub (Gila robusta) and two non-native species, rainbow trout (Oncorhynchus mykiss) and smallmouth bass (Micropterus dolomieu), using a Brett-style respirometer and locomotor step-tests. Gila elegans had the lowest estimated drag coefficient and the highest sustained swimming speeds relative to the other three species. There were no detectible differences in locomotor energetics during steady swimming among the four species. When challenged by high-velocity water flows, the second native species examined in this study, G. robusta, exploited the boundary effects in the flow tank by pitching forward and bracing the pelvic and pectoral fins against the acrylic tank bottom to 'hold station'. Because G. robusta can station hold to prevent being swept downstream during high flows and G. elegans can maintain swimming speeds greater than those of smallmouth bass and rainbow trout with comparable metabolic costs, we suggest that management agencies could use artificial flooding events to wash non-native competitors downstream and out of the Colorado River habitat.

Cardiometabolic response of juvenile rainbow trout exposed to dietary selenomethionine.

Selenium (Se) is considered an essential trace element, involved in important physiological and metabolic functions for all vertebrate species. Fish require dietary concentrations of 0.1-0.5 µg Se/g dry mass (d.m.) to maintain normal physiological and selenoprotein function, however concentrations exceeding 3 µg/g d.m. have been shown to cause toxicity. As Se is reported to have a narrow margin between essentiality and toxicity, there is growing concern surrounding the adverse effects of elevated Se exposure caused by anthropogenic activities. Previous studies have reported that elevated dietary exposure of fish to selenomethionine (Se-Met) can cause significant cardiotoxicity and alter aerobic metabolic capacity, energy homeostasis and swimming performance. The goal of this study aims to further investigate mechanisms of sublethal Se-Met toxicity, particularly potential underlying cardiovascular and metabolic implications of chronic exposure to environmentally relevant concentrations of dietary Se-Met in juvenile rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were fed either control food (1.3 µg Se/g d.m.) or Se-Met spiked food (6.4, 15.8 or 47.8 µg Se/g d.m.) for 60 d at 3% body weight per day. Following exposure, ultrahigh resolution B-mode and Doppler ultrasound was used to characterize cardiac function in vivo. Chronic dietary exposure to Se-Met significantly increased stroke volume, cardiac output, and ejection fraction. Fish fed with Se-Met spiked food had elevated liver glycogen and triglyceride stores, suggesting impaired energy homeostasis. Exposure to Se-Met significantly decreased mRNA abundance of citrate synthase (CS) in liver and serpin peptidase inhibitor, clad H1 (SERPINH) in heart, and increased mRNA abundance of sarcoplasmic reticulum calcium ATPase (SERCA) and key cardiac remodelling enzyme matrix metalloproteinase 9 (MMP9) in heart. Taken together, these responses are consistent with a compensatory cardiac response to increased susceptibility to oxidative stress, namely a decrease in ventricular stiffness and improved cardiac function. These cardiac alterations in trout hearts were linked to metabolic disruption in other major metabolic tissues (liver and skeletal muscle), impaired glucose tolerance with increased levels of the toxic glucose metabolite, methylglyoxal, increased lipid peroxidation in skeletal muscle, development of cataracts and prolonged feeding behaviour, indicative of visual impairment. Therefore, although juvenile rainbow trout hearts were apparently able to functionally compensate for adverse metabolic and anti-oxidant changes after chronic dietary exposure Se-Met, complications associated with hyperglycemia in mammalian species were evident and would threaten survival of juvenile and adult fish.

The osmorespiratory compromise in rainbow trout (Oncorhynchus mykiss): The effects of fish size, hypoxia, temperature and strenuous exercise on gill diffusive water fluxes and sodium net loss rates.

In the context of the osmorespiratory compromise, hypoxia and temperature have been little studied relative to exercise, and diffusive water flux rates (as assessed by 3H2O efflux) have received almost no attention. We investigated the effects of fish size, hypoxia, exercise and acute temperature increase on diffusive water flux rates and net sodium loss rates in juvenile rainbow trout. Trout weighing 13-50 g were used to determine the effects of fish size under normoxia. Thereafter 25-50 g trout were selected to assess the effects of different hypoxia levels (3.15, 5.25 and 8.40 KPa), time course of hypoxia (1 h 8.40 KPa, 3 h 8.40 KPa, 1 h 8.40 KPa +1 h normoxic recovery, and 1 h 8.40 KPa + 3 h normoxic recovery), strenuous exercise (5 min) and acute temperature challenge (transfer from 8 °C to 13 °C or to 18 °C). Small fish (13 g) had higher diffusive water flux rates than larger fish, turning over >100% of their fractional body water pool per hour against 34% per hour for 50 g fish. Hypoxic exposure exerted a biphasic effect, increasing the diffusive water flux rate at 8.40 KPa and 5.25 KPa, while returning it to control levels at 3.15 KPa. All the levels of hypoxia increased net Na+ loss. One hour hypoxia (8.40 KPa) increased diffusive water flux rate while prolonged 3 h hypoxia (8.40 KPa), and short or prolonged normoxic recovery returned diffusive water flux rates to control levels. All the treatments over the time course of hypoxia and normoxic recovery increased net Na+ loss rates. Strenuous exercise increased both the diffusive water flux and net Na+ loss rates. Acute temperature rise increased diffusive water flux rates, with Q10 values of 4.03 for 8 to 13 °C and 2.16 for 8 to 18 °C, but the net Na+ loss rate did not change. There was no significant correlation between diffusive water flux rate and net Na+ loss rates at different hypoxia levels, over the course of hypoxia and normoxic recovery, or during acute temperature stress. In contrast, we observed a significant correlation between diffusive water flux and net Na+ loss rates following exercise. Overall, diffusive water flux and sodium loss were regulated differently during acute temperature challenge and hypoxia, while following exercise the two parameters were regulated in a similar fashion.

17ß-Estradiol Increases Non-CpG Methylation in Exon 1 of the Rainbow Trout (Oncorhynchus mykiss) MyoD Gene.

MyoD is an important myogenic transcription factor necessary for the differentiation of myogenic precursor cells (MPC) to form mature myotubes, a process essential for muscle growth. Epigenetic markers such as CpH methylation are known gene regulators that are important for the differentiation process. In this study, we investigated whether DNA methylation is a potential mechanism associated with the ability of 17ß-estradiol (E2) to reduce MyoD gene expression and muscle growth in rainbow trout. Rainbow trout received a single intraperitoneal injection of E2 or the injection vehicle (control). Skeletal muscle was collected 24 h post injection and analyzed for DNA methylation within the MyoD gene and the expression of DNA methyltransferases. CpG islands of the MyoD gene were predicted using MethPrimer software, and these regions were PCR amplified and analyzed using bisulfite sequencing. The percent methylation of the targeted CpG did not differ between control and E2-treated fish. However, percent CpH methylation in the MyoD exon 1 region was elevated with E2 treatment. Two of the methylated CpH sites were located in conserved transcription factor binding motifs, estrogen response element (ERE), and Myc binding site. Quantitative real-time PCR analysis revealed a significant increase in expression of DNA methyltransferases, Dnmt3a and Dnmt3b, in E2-treated muscle, suggesting an increased genome methylation. Differential CpH methylation in MyoD gene of control and E2-treated fish suggests an epigenetic mechanism through which E2 decreases MyoD gene expression and contributes to reduced muscle growth.

The effect of hydraulic flowback and produced water on gill morphology, oxidative stress and antioxidant response in rainbow trout (Oncorhynchus mykiss).

Hydraulic fracturing fluid are complex mixtures containing high concentrations of salts (up to 330,000 ppm), organic, and metal contaminants. However, little data exist on the potential mechanisms of toxicity of these flowback and produced wastewaters (FPW) on aquatic biota. Juvenile rainbow trout were exposed to either control, FPW (2.5 or 7.5%), FPW that had been treated with activated charcoal (AC), or a custom salt-matched control (SW; replicating only the salt content of FPW) for 48 hours. Gill histology revealed decreases in interlamellar cell mass (ILCM) and mean lamellar length in all treatments (FPW, AC and SW) compared to control, indicative of hyperosmotic stress. Liver CYP1A1 activity was significantly elevated by 7.5-fold in the FPW 7.5% treatment only, indicative of Phase I metabolism. Superoxide dismutase activity significantly decreased in the gills to all treatments with the lowest activity occurring in the 7.5% FPW group. Catalase activity increased in liver with the highest values noted in fish exposed to 7.5% FPW. No changes were observed with respect to glutathione-S-transferase, while increased lipid peroxidation was only observed in both FPW treatments (2.5, 7.5%). These data suggest a characteristic signature of FPW impact which may help in risk assessment and biomonitoring of FPW spills.

Hypoxia-reoxygenation differentially alters the thermal sensitivity of complex I basal and maximal mitochondrial oxidative capacity.

Hypoxia-reoxygenation (H-R) transitions and temperature fluctuations occur frequently in biological systems and likely interact to alter cell function. To test how H-R modulates mitochondrial function at different temperatures we measured the effects of H-R on isolated fish liver mitochondrial oxidation rates over a wide temperature range (5-25°C). Subsequently, the mechanisms underlying H-R induced mitochondrial responses were examined. H-R inhibited the complex I (CI) maximal (state 3) and stimulated the basal (state 4) mitochondrial oxidation rates with temperature enhancing both effects. As a result, the thermal sensitivity (Q10) for CI maximal respiration was reduced while that for basal respiration was increased by H-R. H-R reduced both the coupling and phosphorylation efficiencies more profoundly at high temperature suggesting that mitochondria were more resistant to H-R at low temperature. The H-R induced mitochondrial impairments were associated with increased reactive oxygen species (ROS) production and proton leak, dissipation of membrane potential, and loss of structural integrity of the organelles. Overall, our study provides insight into the mechanisms of H-R induced mitochondrial morphofunctional disruption and shows that the moderation of effects of H-R on oxidative phosphorylation by temperature depends on the functional state.

Growth of juvenile steelhead Oncorhynchus mykiss under size-selective pressure limited by seasonal bioenergetic and environmental constraints.

Increased freshwater growth of juvenile steelhead Oncorhynchus mykiss improved survival to smolt and adult stages, thus prompting an examination of factors affecting growth during critical periods that influenced survival through subsequent life stages. For three tributaries with contrasting thermal regimes, a bioenergetics model was used to evaluate how feeding rate and energy density of prey influenced seasonal growth and stage-specific survival of juvenile O. mykiss. Sensitivity analysis examined target levels for feeding rate and energy density of prey during the growing season that improved survival to the smolt and adult stages in each tributary. Simulated daily growth was greatest during warmer months (1 July to 30 September), whereas substantial body mass was lost during cooler months (1 December to 31 March). Incremental increases in annual feeding rate or energy density of prey during summer broadened the temperature range at which faster growth occurred and increased the growth of the average juvenile to match those that survived to smolt and adult stages. Survival to later life stages could be improved by increasing feeding rate or energy density of the diet during summer months, when warmer water temperatures accommodated increased growth potential. Higher growth during the summer period in each tributary could improve resiliency during subsequent colder periods that lead to metabolic stress and weight loss. As growth and corresponding survival rates in fresh water are altered by shifting abiotic regimes, it will be increasingly important for fisheries managers to better understand the mechanisms affecting growth limitations in rearing habitats and what measures might maintain or improve growth conditions and survival.

Sub-lethal effects and bioconcentration of the human pharmaceutical clotrimazole in rainbow trout (Oncorhynchus mykiss).

The aim of this study was to characterize biomarker responses, haematological profiles, structural changes and uptake in juvenile rainbow trout exposed to clotrimazole (CLO) at three concentrations (0.01 - [lowest environmentally relevant concentration], 1.0 [highest environmentally relevant concentration] and 10 µg L(-1)) in a semi-static system over a period of 42 days. Antioxidant defence enzymes, which responded to CLO exposure, changed the oxidative stress status of cells, but no differences were observed in lipid peroxidation. Clotrimazole triggered a biphasic response of CYP3A-like activity in liver microsomes, which may indicate a detoxification process in the liver. Histopathological alterations were most pronounced in kidneys and testes in the group exposed to 10 µg L(-1). Structural changes in the kidney included tubulonephrosis and hyaline droplet degeneration in the tubular epithelial cells. The relative proportions of germ cells in testes were changed: The number of spermatozoa was reduced, and the spermatogonia and spermatocytes were increased. The highest CLO concentration was detected in fish liver (3710 ng per gram wet tissue) and kidney (4280 ng per gram wet tissue). Depuration half-life was estimated to be 72, 159, and 682 h in liver, muscle, and kidney, respectively. Taken together, these results provide valuable toxicological data on the effects of CLO on aquatic non-target organisms, which could be useful for further understanding of the potential risks in the real aquatic environment.

Morphological arrangement of the coronary vasculature in a shark (Squalus sucklei) and a teleost (Oncorhynchus mykiss).

The coronary circulation is of great importance in maintaining cardiovascular function and consequently it has been extensively studied in many mammalian species. However, much less attention has been paid to the coronary circulation in other vertebrates. For example, while elasmobranch fishes are of special interest as they are the most ancient lineage of vertebrates to possess a coronary circulation, only qualitative studies exist on their coronary circulation and most concern the architecture of the large arteries. Our study tested the prediction that the coronary circulation of sharks is better developed than previously thought. However, to test this idea, a methodology was needed to quantify vascularity, vessel morphology and oxygen diffusion distances in a heart with predominantly spongy myocardium. Here, we describe this methodology using dogfish and rainbow trout and suggest that the dogfish spongy myocardium appears to rely predominantly on the coronary circulation for its oxygen supply, an arrangement that contrasts with the spongy myocardial tissue of rainbow trout. In support of this suggestion, the density of the microvasculature of the spongy myocardial tissue of dogfish exceeded that of their compact tissue. Although vascularity in the compact myocardium of dogfish was significantly lower than trout, intervascular distances were similar on account of a significantly larger vessel diameter in dogfish, which corresponds to a larger red blood cell size of the dogfish when compared to trout. J. Morphol. 277:896-905, 2016. © 2016 Wiley Periodicals, Inc.

Procedures for the reconstruction, primary culture and experimental use of rainbow trout gill epithelia.

This protocol describes how to reconstruct and culture the freshwater rainbow trout gill epithelium on flat permeable membrane supports within cell culture inserts. The protocol describes gill cell isolation, cultured gill epithelium formation, maintenance, monitoring and preparation for use in experimental procedures. To produce a heterogeneous gill epithelium, as seen in vivo, seeding of isolated gill cells twice over a 2-d period is required. As a consequence, this is termed the double-seeded insert technique. Approximately 5-12 d after cell isolation and seeding, preparations develop electrically tight gill epithelia that can withstand freshwater on the apical cell surface. The system can be used to study freshwater gill physiology, and it is a humane alternative for toxicity testing, bioaccumulation studies and environmental water quality monitoring.

Growth, immune responses and intestinal morphology of rainbow trout (Oncorhynchus mykiss) supplemented with commercial probiotics.

The influence of two commercial probiotics on the growth, innate immune parameters and intestinal morphology of rainbow trout (Oncorhynchus mykiss) juveniles (initial weight: 16.4 ± 0.4 g) was evaluated. Two probiotic types: A, multi-species (Bacillus sp., Pedicoccus sp., Enterococcus sp., Lactobacillus sp.) and B, mono-species (Pediococcus acidilactici) were tested at two levels each (A1: 1.5 g kg(-1), 8.6 × 10(5) CFU g(-1); A2: 3 g kg(-1), 1.6 × 10(6) CFU g(-1); B1: 0.1 g kg(-1), 2.6 × 10(4) CFU g(-1); B2: 0.2 g kg(-1), 7.2 × 10(4) CFU g(-1)) versus an unsupplemented diet (C). Diets were distributed to sextuplicate tanks, three times a day to visual satiation for 8 weeks. Growth performance and immune responses (plasma lysozyme, ACH50, peroxidase and head kidney respiratory burst) were determined at 4 and 8 weeks of feeding. Body composition and intestine morphology were determined at the end of the feeding trial. At 8 weeks, the lower dose of multi-species probiotic (A1) improved growth rate, while both probiotic types improved feed conversion rate compared to the control animals, at the lower dose of multi-species (A1) and at the higher dose of mono-species (B2) probiotics. Body composition did not vary between treatments. At 4 weeks, ACH50 activity was significantly higher in fish fed higher dose of B probiotic (B2, 123.7 ± 50.6 vs 44.1 ± 7.7 U.ml(-1) in control). At 8 weeks, lysozyme activity was higher in fish fed A1 (13.1 ± 5.2 µg ml(-1)) diet compared to fish fed control diet (7.8 ± 1 µg ml(-1)). Plasma peroxidase and head-kidney respiratory burst did not differ among the dietary treatments. Villi length and integrity and goblet cell counting of a cross section of the anterior intestine were not significantly different between groups. Results suggest benefits in zootechnical performance and immune humoral responses using both probiotic types, in a dose dependent manner, without apparent alterations in intestinal morphology.