Histopathology changes in the rainbow trout (Onchorhyncus mykiss) consuming boric acid supplemented fish fodder

ObjectiveEven though boron (B), as a trace micronutrient, occurs in natural waters and organisms, its high concentration could cause harmful and even toxic for organisms. The aim of present study was to investigate the effects of boric acid (BA) added to feed (0.01%, 0.05%, 0.10%, and 0.20% of B in feed) on Rainbow Trout (Oncorhynchus mykiss) by histopathological methods and compared to the control feed as without B.MethodologyAt the end of the feeding, after weighing the live weight of 9 rainbow trout from each group, tissue fragments were taken from the liver, gill, kidney, skeletal muscle, spleen and brains of fish, which systemic necropsies was done, and were fixed in 10 % buffered formalin solution.ResultsHistopathological examinations revealed degenerative and necrotic changes in the liver, gill, kidney, skeletal muscle, spleen and brain. Hydropic and vacuolar degenerations in liver parenchyma, lamellar edema in the gills, hyaline accumulation in the kidneys, degenerations in the muscles, necrosis in the spleen and hyperemia in the brain were observed in all groups except control group. It was observed that the destruction of boric acid on fish increased depending on the amount of boron supplemented to the feed, and the most effect was in the group fed with 0.20 % boron supplemented feed.ConclusionsThe findings aside from causing pathological changes in all organs in terms of histopathological findings of Boric Acid (BA), the most severe lesions were observed in the liver.     

Subcellular fractionation of rainbow trout gonads with emphasis on microsomal enzymes involved in steroid metabolism

Summary Rainbow trout gonads were subfractionated by differential centrifugation with emphasis on obtaining preparations suitable for the study of steroid-metabolizing enzymes. A fractionation scheme was evaluated for the mature testis and for 3 ovarian developmental stages. The distribution of cell organelles among the fractions was determined using enzyme-markers and electron microscopy. The fractionation scheme was found to be suitable for separating mitochondria and microsomes which were recovered at similar yields to those that had been reported for other extraheptic fish tissues. Fractionation of the mature ovary was fraught with problems probably because a large yolk protein cytosole fraction interfered with the recovery of microsomes. However, no difference in the specific activity of microsomal NADPH-cytochrome c-reductase between the various organ preparations was evident. The testis microsomes contained detectable amounts of cytochrome P450, whereas its content in the various ovary microsomes was too low to be detected. Progesterone 17-hydroxylase was detected in microsomes from testes and early developing ovaries, and microsomal aromatase activity was present in microsomes from early developing, mature and postovulatory ovaries. Furthermore, the testis microsomes contained a highly active UDP glucuronosyltransferase with testosterone used as a substrate.     

Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver

In fish, as in other aerobic organisms, glutathione and glutathione-related enzymes are important components in the defences against oxidative stress. To study if hepatic glutathione levels and/or activities of glutathione-related enzymes can act as indicators of oxidative stress in fish, we injected rainbow trout (Oncorhynchus mykiss) intraperitoneally with paraquat (PQ), menadione (MD), naphthazarin (DHNQ), or beta-naphthoflavone (beta-NF), all known to cause a rise in reactive oxygen species (ROS). After 2 and 5 days of exposure, we measured the activities of hepatic glutathione peroxidase (GPox), glutathione S-transferase (GST), gamma-glutamylcysteine synthetase (GCS), and glutathione reductase (GR). We also measured total glutathione (tGSH) and oxidised glutathione (GSSG) in the liver of fish treated with PQ and MD. All chemicals caused an increase in GR activity after 5 days, which ranged from 160% in fish treated with beta-NF to 1,500% in fish treated with PQ. All chemicals except beta-NF caused moderate elevation in GST activity; GPox activity was lower in fish treated with DHNQ and MD, while GCS activity increased twofold in the fish treated with DHNQ, without being affected by beta-NF, PQ or MD. After 5 days of treatment with PQ or MD, tGSH content was elevated. Our findings demonstrated that of the parameters included in the study, GR activity was the most responsive to treatment with redox cycling compounds, indicating that GR activity is a promising biomarker of such compounds and possibly indicating oxidative stress in rainbow trout.     

Serotonin, but not melatonin, plays a role in shaping dominant-subordinate relationships and aggression in rainbow trout

The aim of this study was to clarify to what extent the effects of elevated dietary L-tryptophan (Trp) on aggressive behavior and stress responsiveness in rainbow trout are mediated by circulating melatonin and central serotonin (5-HT), respectively. Isolated rainbow trout were paired for 1h a day for 7 days in order to create fish with experience of being dominant and subordinate. Following this week, the fish were tested for aggressive behavior using a resident-intruder test after which they were subjected to one of four treatments: (1) tryptophan, (2) the selective serotonin reuptake inhibitor (SSRI) citalopram, (3) melatonin, and (4) no treatment (controls). After 7 days of treatment, the fish were subjected to a second resident-intruder test. Trp-supplemented feed resulted in a suppression of aggressive behavior in fish with experience of being dominant. Moreover, fish fed Trp-supplemented feed, regardless of social experience, also displayed lower plasma cortisol levels than controls. These effects of elevated dietary Trp were closely mimicked by citalopram treatment, whereas exogenous melatonin had no effect on either aggressive behavior or plasma cortisol. Thus, the effect of elevated dietary Trp on aggressive behavior and stress responses does not appear to be mediated by melatonin even though elevated dietary intake of Trp resulted in an increase in plasma melatonin concentrations.     

The effects of diet composition and ration on biotransformation enzymes and stress parameters in rainbow trout, Oncorhynchus mykiss

Juvenile rainbow trout (Oncorhynchus mykiss) were fed one of three isoenergetic diets varying in protein (35-55%) and lipid content (8-18%), at full satiation levels or half rations for 6 weeks in order to investigate the effects of diet on baseline stress parameters and biotransformation enzyme activity. Growth was greatest in fish fed to satiation on a low protein and high lipid diet. Stress parameters, including plasma lactate, glucose and cortisol concentrations were not significantly affected by dietary treatment or ration. Basal biotransformation enzymes, including glutathione S-transferase (GST) and ethoxyresorufin O-deethylase (EROD) activity, were also unaffected by dietary treatment. Fish exposed to the biotransformation enzyme inducer beta-naphthoflavone did not exhibit an alteration in stress indicators or GST activity; however, EROD activity was increased (87- to 210-fold) in fish receiving all diets and rations. The results of the present study indicate that, unlike mammals, fish may be more recalcitrant to different levels of ingestion of isoenergetic diets varying in protein and lipid concentration with respect to stress responses and the maintenance of basal titers of biotransformation enzymes and their induction.     

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.     

Effect of damming on distribution of rainbow trout in Hokkaido,Japan

Rainbow trout introduced into Hokkaido in 1920 have become widely distributed due to extensive release into many reservoirs and lakes for sport-fishing; their presence often results in reductions of native fish populations. We analyzed and predicted the relationship between the probability of occurrence of rainbow trout and the proximity of dams (or attributed reservoirs), using a database of the presence or absence of rainbow trout collected during 1960–2004 in Hokkaido to clarify the spread patterns of exotic species (e.g., rainbow trout) due to large-scale damming over a long period. Rainbow trout were abundant in streams within approximately 10 km of dams in recent years, regardless of whether the stream was up- or down-stream from the dam and after accounting for the effects of other environmental variables (e.g. elevation, population density, and survey year). A delayed increase in trout occurrence below dams as compared with above dams suggests that the occurrence below dams may be largely due to escapement of stocked populations and a continuously increasing abundance since 1970. The management of dams and reservoirs is necessary to prevent further spread of rainbow trout because they can threaten habitats of native Japanese salmonids through various mechanisms.

The brain-pituitary-gonadal axis in the rainbow trout,Salmo gairdneri: Gonadal hormones and the maturation of gonadotropic cells

Summary Intact and castrated juvenile male rainbow trout (Salmo gairdneri) were treated with testosterone and gonadotropic hormone (GTH) to determine the maturational effects of these hormones on the GTH-cells. Electron-microscopic studies of the GTH-cells revealed that GTH and testosterone in intact animals, and testosterone in castrated fish, caused GTH-cell maturation: These cells now displayed the same appearance as GTH-cells in adult trout, including the presence of globules, a well-developed Golgi apparatus and rough endoplasmic reticulum, all of which were absent in GTH-cells of control animals. Animals with stimulated GTH-cells also had an increased GTH content of the pituitary; release of GTH could not be demonstrated. Animals treated with GTH exhibited an accelerated development of the testes, resulting in complete gametogenesis and elevated plasma testosterone levels. These results indicate that exogenous steroids as well as endogenous gonadal steroids can stimulate the full development of GTH-cells and accelerate GTH synthesis. The significance of this stimulating effect of the gonadal hormones with respect to the development of the brain-pituitary-gonadal axis and the onset of puberty is discussed.The results were presented as a poster at the 11th conference of the European Society of Comparative Endocrinology, Jerusalem, August 10–14, 1981     

The effects of chronic immune stimulation on muscle growth in rainbow trout

Successful production of aquaculture species depends on efficient growth with low susceptibility to disease. Therefore, selection programs have focused on rapid growth combined with disease resistance. However, chronic immune stimulation diminishes muscle growth (a syndrome referred to as cachexia), and decreases growth efficiency in production animals, including rainbow trout. In mammals, recent results show that increased levels of pro-inflammatory cytokines, such as those seen during an immune assault, specifically target myosin and MyoD and inhibit muscle growth. This suggests that increased disease resistance in fish, a desired trait for production, may actually decrease the growth of muscle, the main aquacultural commodity. To test this possibility, a rainbow trout model of cachexia was developed and characterized. A six-week study was conducted in which rainbow trout were chronically immune stimulated by repeated injections of LPS. Growth indices were monitored, and whole body and muscle proximate analyses, real-time PCR, and Western blotting were conducted to examine the resulting cachectic phenotype. Muscle ratio was decreased in fish chronically immunostimulated, however expression levels of MyoD2 and myosin were not decreased compared to fish that were not immunostimulated, indicating that while muscle accretion was altered, the mechanism by which it occurred was somewhat different than that characterized in mammals. Microarray analysis was used to compare gene expression in fish that had been chronically immunostimulated versus those that had not to identify possible alternative mechanisms of cachexia in fish.     

Membrane order conservation in raft and non-raft regions of hepatocyte plasma membranes from thermally acclimated rainbow trout

Homeoviscous adaptation (HVA), the thermal conservation of membrane fluidity/order at different body temperatures, has been observed to varying degrees in different membranes. However, HVA has not been studied in raft and non-raft regions of the plasma membrane (PM) separately. Rafts are ordered PM microdomains implicated in signal transduction, membrane traffic and cholesterol homeostasis. Using infrared spectroscopy, we measured order in raft-enriched PM (raft) and raft-depleted PM (RDPM) isolated from hepatocytes of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20 °C. We found approximately 130% and 90% order compensation in raft and RDPM, respectively, suggesting their independent regulation. Raft was more ordered than RDPM in the warm-acclimated trout, a difference fully explained by a 58% enrichment of cholesterol, compared to RPDM. Unexpectedly, raft and RDPM from cold-acclimated trout did not differ in cholesterol content or order. Freezing the membrane samples during preparation had no effect on order. Treatment with cyclodextrin depleted cholesterol by 36%, 56%, and 55%, producing significant decreases in order in raft and RDPM from warm-acclimated trout and RDPM from cold-acclimated trout, respectively. However, a 69% depletion of cholesterol from raft from cold-acclimated trout had no significant effect on order. This result, and the lack of a difference in order between raft and RDPM, suggests that raft and non-raft PM in cold-acclimated trout are not spatially segregated by phase separation due to cholesterol.     

Cellular and functional adaptation to thermal stress in ovarian granulosa cells in mammals

Climate change represents a significant environmental challenge to human welfare. One of many negative impacts may be on animal reproduction. Elevated ambient temperature unfavourably influences reproductive processes in mammals. High temperature can affect reproductive processes such as follicle development and may alter follicular fluid concentrations of amino acids, fatty acids, minerals, enzymes, antioxidants defence and growth factors. These impacts may lead to inferior oocyte competence and abnormal granulosa cell (GCs) function. Mammalian oocytes are enclosed by GCs that secret hormones and signalling molecules to promote oocyte competence. GCs are essential for proper follicular development, oocyte maturation, ovulation, and luteinization. Many environmental stressors, including thermal stress, affect GC function and alter oocyte development and growth. Several studies documented a link between elevated ambient temperature and increased generation of cellular reactive oxygen species (ROS). ROS can damage DNA, reduce cell proliferation, and induce apoptosis in GCs, thus altering oocyte development. Additionally, thermal stress induces upregulation of thermal shock proteins, such as HSP70 and HSP90. This review provides an update on the influence of thermal stress on GCs of mammals. Discussions include impacts to steroidogenesis (estradiol and progesterone), proliferation and cell cycle transition, apoptosis, oxidative stress (ROS), antioxidants related genes, heat shock proteins (HSPs) and endoplasmic reticulum responses.     

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility,velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), l-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), l-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, l-methionine, SOD, l-carnitine, α-tocopherol and l-reduced glutathione (p < 0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, l-methionine, SOD, α-tocopherol and l-reduced glutathione (p < 0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

The effect of stress and exercise on post-mortem biochemistry of Atlantic salmon and rainbow trout

Freshwater Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss responded similarly to increase in water flow (exercise), reduction in holding tank water level (stress), or 30 min chasing with water level reduction (stress and exercise). Stress generally resulted in elevated plasma cortisol, above the control. Fish responded to stress and exercise combined, with elevated lactate and [H⁺] which was sometimes associated with elevated plasma cortisol. These changes were combined with a depletion of the muscle adenylate pool. Post-mortem, this resulted in an increase in the rate of onset of rigor, and a higher and sometimes sustained muscle proton load. Both species produced predominantly inosine as opposed to hypoxanthine, for up to 72 h of ice storage. This study shows that the physiological disruption in Atlantic salmon and rainbow trout caused by simulated harvest conditions of stress and exercise, results in mostly transient changes in post-mortem muscle biochemistry. These changes lead to an earlier onset and resolution of rigor, and lower post-mortem muscle pH in comparison to the control.     

Relationship between stress, feeding and plasma ghrelin levels in rainbow trout, Oncorhynchus mykiss

Sexually immature rainbow trout were acclimated to small-volume (1 m³) holding tanks and then exposed to short-term stress to examine the relationship between feeding, stress, plasma ghrelin levels and other plasma stress parameters. Plasma ghrelin levels showed an increase 24 h after a single feed, plasma lactate and glucose levels decreased over the same period and plasma cortisol levels were low and constant. One hour of confinement stress resulted in elevations of plasma cortisol, glucose and lactate and depression of plasma ghrelin levels. In a separate experiment, 2 h of confinement stress also depressed feeding immediately after stress, concomitant with increases in plasma cortisol, lactate and glucose; however, in this case there was no change in plasma ghrelin concentrations. A repeat of the 2-h confinement experiment using fish that had not been acclimated to small-volume holding tanks produced a more marked elevation in plasma cortisol and a stronger suppression of feeding post-stress but in this case also, there was no change in plasma ghrelin levels. The results of this study confirm that feeding in rainbow trout is suppressed by confinement stress although the effect is transitory in this domesticated stock. Similar to that in other fishes, plasma ghrelin levels appear to be modulated by feeding status and may be influenced by stress, suggesting an orexigenic role for ghrelin in rainbow trout.     

Pleiotropic mutants hypersensitive to heavy metals and to oxidative stress in Chlamydomonas reinhardtii

Insertional mutagenesis was used in Chlamydomonas reinhardtii to isolate original mutants hypersensitive to multiple drugs and physical agents. Out of 5200 transformants analyzed, 13 mutants belonging to seven phenotypic classes were isolated. Five were exclusively sensitive to cadmium and represented two loci. The other mutants were pleiotropic and presented a cross sensitivity to several (2--6) of the following agents: cadmium, copper, lead, paraquat, hydrogen peroxide, UVC and light. In all mutants analyzed, the hypersensitive phenotype was most probably due to a single mutational event. The sensitivity of several pleiotropic mutants to a broad range of physical and chemical agents suggests that the disrupted genes are involved in multiple stress responses.