Production of short-chain fatty acids and gas from various oligosaccharides by gut microbes of carp (Cyprinus carpio L.) in micro-scale batch culture

We studied the metabolism of various oligosaccharides by carp (Cyprinus carpio) hindgut microbes by measuring gas productivity and organic acid production in gut contents using a 50-microl-scale batch culture system. Carp hindgut contents were incubated with 500 microg each of raffinose, lactosucrose, kestose, lactulose, gentiobiose, 4'-galactosyllactose and 6'-galactosyllactose and soybean-, xylo-, and isomalto-oligosaccharides or none (blank culture) at 25 degrees C for 6 h. The time-course of gas release from the culture (Y microl/culture) was expressed as an exponential function of incubation time (t) [Y=A+Bx(1-e(-kt))]; A, B and k are constants). Potential production of gas (A+B) from soybean-oligosaccharide and raffinose was larger than for the other saccharides except for kestose, and blank culture. The rate constant of gas (k) for lactosucrose was larger than that for isomalto- and xylo-oligosaccharide, lactulose, kestose or blank culture. Net production of total SCFA (sum of acetic, propionic and n-butyric acid weights) from cultures with soybean- and isomalto-oligosaccharides, raffinose, gentiobiose and lactosucrose was greater than that from blank culture. These results suggested that soybean-oligosaccharide and raffinose were potentially highly fermentable oligosaccharides for carp hindgut microbes. Chemical structures of oligosaccharides seem to play an important role in the fermentability. It is also likely that oligosaccharide utilization differs between mammals and teleosts.     

Variations in plasma melatonin levels of the rainbow trout (Oncorhynchus mykiss) under various light and temperature conditions

Daily variations in plasma melatonin levels in the rainbow trout Oncorhynchus mykiss were studied under various light and temperature conditions. Plasma melatonin levels were higher at mid-dark than those at mid-light under light-dark (LD) cycles. An acute exposure to darkness (2 hr) during the light phase significantly elevated the plasma melatonin to the level that is comparable with those at mid-dark, while an acute exposure to a light pulse (2 hr) during the dark phase significantly suppressed melatonin to the level that is comparable with those at mid-light. Plasma melatonin kept constantly high and low levels under constant darkness and constant light, respectively. No circadian rhythm was seen under both conditions. When the fish were subjected to simulative seasonal conditions (simulative (S)-spring: under LD 13.1:10.9 at 13 degrees C; S-summer: under LD 14.3:9.7 at 16.5 degrees C; S-autumn: under LD 11.3:12.7 at 13 degrees C; S-winter: under LD 10.1:13.9 at 9 degrees C), melatonin levels during the dark phase were significantly higher than those during the light phase irrespective of simulative seasons. The peak melatonin level in each simulative season significantly correlated with temperature but not with the length of the dark phase employed. In addition, the peak melatonin level in S-autumn was significantly higher than those in S-spring although water temperature was the same under these conditions. These results indicate that the melatonin rhythm in the trout plasma is not regulated by an endogenous circadian clock but by combination of photoperiod and water temperature.     

Primary culture and histological characterization of phagocytic cells from rainbow trout Oncorhynchus mykiss

In this study the pronephros of rainbow trout Oncorhynchus mykiss was explanted and cultured. The morphology of cultured cells suggested they were leukocyte derived. To confirm this, cells were incubated with non-opsonized fluorescent beads and human apoptotic polymorphonuclear leukocytes (PMN) to establish that they were capable of phagocytosis and that they became activated following exposure to apoptotic bodies. The cells were studied using light microscopy, transmission electron microscopy and a live cell observer system. Cells in culture were also stained with phalloidin to identify actin reorganization following activation. Cultured cells ingested the inert beads and apoptotic PMNs and c . 80% of cells became activated following exposure to the apoptotic PMN, evidenced by enhanced filopodial extensions. This methodology may play a role in future studies, in particular, the competence of macrophages following exposure to radiation can now be tested.     

Protein kinase C and CYPlAl induction in rainbow trout (Oncorhynchus mykiss) hepatocyte culture

1. The role of protein kinase C (PKC) in B-naphthoflavone (BNF) induction of CYP1A1 in rainbow trout hepatocytes was investigated.2. Primary cultures of rainbow trout hepatocytes treated with BNF for 24 hr showed an increase in microsomal 7-ethyoxyresorufm-O-deethylase (EROD) activity compared to cells treated with vehicle (DMSO) only.3. Increases in EROD activities were proportional to increased concentrations of BNF from 1 to 10 nM reaching a plateau at higher concentrations (20–100 nM) of BNF.4. Western blot analysis using specific antibody (LM4b) against CYP1A1 showed that changes in microsomal CYP1A1 protein paralleled that of EROD activity.5. The induction of EROD activity by BNF required both protein and RNA synthesis since the process was blocked by both cycloheximide and actinomycin D.6. Pretreatment of hepatocytes with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to a dose dependent suppression of BNF-induced EROD activity and CYP1A1 content. TPA alone had no effect on hepatic EROD activity and CYP1A1 protein level.7. Pretreatment with sn-1,2 didecanoylglycerol, a PKC activator, had no effect on BNF-induced EROD activity in these cells.8. Pretreatment of cells with staurosporine, a PKC inhibitor, effectively blocked BNF-induced EROD activity.9. PKC may play a role in the induction of CYP1A1 gene expression in fish liver by BNF.     

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss)

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.     

Individual condition and stream temperature influence early maturation of rainbow and steelhead trout, Oncorhynchus mykiss

Alternative male phenotypes in salmonine fishes arise from individuals that mature as larger and older anadromous marine-migrants or as smaller and younger freshwater residents. To better understand the processes influencing the expression of these phenotypes we examined the influences of growth in length (fork length) and whole body lipid content in rainbow trout (Oncorhynchus mykiss). Fish were sampled from the John Day River basin in northeast Oregon where both anadromous (“steelhead”) and freshwater resident rainbow trout coexist. Larger males with higher lipid levels had a greater probability of maturing as a resident at age-1+. Among males, 38% were maturing overall, and the odds ratios of the logistic model indicated that the probability of a male maturing early as a resident at age-1+ increased 49% (95% confidence interval (CI) = 23–81%) for every 5 mm increase in length and 33% (95% CI = 10–61%) for every 0.5% increase in whole body lipid content. There was an inverse association between individual condition and water temperature as growth was greater in warmer streams while whole body lipid content was higher in cooler streams. Our results support predictions from life history theory and further suggest that relationships between individual condition, maturation, and environmental variables (e.g., water temperature) are shaped by complex developmental and evolutionary influences.     

Tissue-specific distribution patterns of retinoids and didehydroretinoids in rainbow trout Oncorhynchus mykiss

Retinoids (vitamin A) are known to be involved in many key biological functions in mammals, such as embryonic development, reproduction or vision. Besides standard vitamin A forms, freshwater fish tissues contain high levels of didehydroretinoids or vitamin A₂ forms. However, the tissue distribution, metabolism and function of both standard and particularly the didehydroretinoids are still poorly known in fish. In this study, we have quantified the levels of retinoids, including retinol, retinaldehyde, retinyl palmitate and their corresponding didehydro forms, as well as the levels of the active polar retinoids all-trans-, 9-cis- and 13-cis-retinoic acid in distinct tissues of juvenile rainbow trout. Our results indicate that the liver is clearly the main retinoid storage tissue in juvenile rainbow trout. Didehydroretinoids were dominant over retinoids in all analyzed tissues with the exception of plasma. Additionally, significant differences among tissues were observed between retinoids and didehydroretinoids, such as differences in the ester profiles and the proportions between free and esterified forms, suggesting that mechanisms that favor the utilization or storage of one of the other groups of compounds might exist in fish. Our data also show the presence of polar retinoids in different tissues of fish at the fmol/g scale. Overall, this study clearly demonstrates the presence of tissue-specific patterns of accumulation of both polar and nonpolar retinoids in fish tissues. The biological relevance of these findings should be the focus of future studies.     

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout (Oncorhynchus mykiss) leukocytes

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout (Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19°C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5°C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A₂ is involved in the heat shock response.     

Quantitative evaluation of macrophage aggregates in brook trout Salvelinus fontinalis and rainbow trout Oncorhynchus mykiss

Macrophage aggregates (MAs) occur in various organs of fishes, especially the kidney, liver and spleen, and contain melanin, ceroid/lipofuscin and hemosiderin pigments. They have been used as indicators of a number of natural and anthropogenic stressors. Macrophage aggregates occur in salmonids but are poorly organized, irregularly shaped, and are generally smaller than those in derived teleosts. These features complicate quantification, and thus these fishes have seldom been used in studies correlating MAs with environmental stressors. To alleviate these complications, we developed color filtering algorithms for use with the software package ImagePro Plus (Media Cybernetics) that select and quantify pigmented area (i.e. colors ranging from gold to brown to black) in tissue sections. Image analysis results compared well with subjective scoring when tested on brook trout Salvelinus fontinalis and rainbow trout Oncorhynchus mykiss captured from high-elevation lakes or hatcheries. Macrophage aggregate pigments correlated positively with age and negatively with condition factor. Within individual fish, pigmentation correlated positively among organs, suggesting that the kidney, liver or spleen are suitable indicator organs. In age-matched fishes, MA pigments were not different between hatcheries and lakes in the organs examined. Between lakes, differences in pigments were observed in the kidney and spleen, but were not explained by age, condition factor, sex or maturation state. Our results indicate that quantification of the area occupied by MA pigments is an efficient and accurate means of evaluating MAs in salmonid organs and that organ pigmentation correlates with age and condition factor, as seen in studies with more derived fishes.     

Effects of hypoxia on the venous circulation in rainbow trout (Oncorhynchus mykiss)

Hypoxia in fish is generally associated with bradycardia while cardiac output (Q) remains unaltered or slightly increased due to a compensatory increase in stroke volume (SV). Rainbow trout (Oncorhynchus mykiss) were subjected to severe (P(W)O2=7.3+/-0.2 kPa) or mild (P(W)O2=11.5+/-0.2 kPa) hypoxia. Central venous pressure (P(ven)), dorsal aortic pressure (P(da)), heart rate (f(H)) and Q, were recorded in vivo. Both levels of hypoxia triggered a significant increase in P(ven). Severe hypoxia was associated with bradycardia and unaltered Q, whereas mild hypoxia was associated with a small but significant increase in Q and no bradycardia. These findings indicate that an increase in P(ven) promotes an increase in SV during hypoxia. Since mild hypoxia increased P(ven), Q and SV without bradycardia or reduced systemic resistance (R(sys)), we hypothesize that an active increase in venous tone serving to mobilize blood to the central venous compartment in order to increase cardiac preload and consequently SV, is an important cardiovascular trait associated with hypoxia. Pharmacological pre-treatment with prazosin (1 mg kg(-1)) did not conclusively reveal the underlying mechanisms to the observed changes in P(ven). This study discusses the influence of venous pooling, reduced R(sys) and altered venous tone on changes in P(ven) observed during hypoxia.     

Studies on the infectivity of Diplostomum spathaceum in rainbow trout (Oncorhynchus mykiss)

The infectivity of Diplostomum spathaceum (Digenea: Trematoda) cercariae to rainbow trout and the efficacy of the diplostomule migration to the lens following different routes of administration was examined. The optimum age of infectivity for cercariae was between 0-5 h after liberation from the snail and for intraperitoneally injected diplostomules, 5 h post-transformation in vitro through fish skin. After exposure of the entire fish body or head to cercariae, metacercariae first appeared in the lens at 5 h and their numbers gradually increased until 22 h. Following exposure of the tail region of rainbow trout to cercariae, metacercariae first appeared in the lens at 14 h. Significantly more metacercariae established in the lens of fish following exposure of the fish head compared with the tail region; 40% of penetrating cercariae reached the lens of fish following exposure of the head or entire body, 20% of cercariae or diplostomules injected either intraperitoneally, intramuscularly or intracardially reached the lens while only 5% of cercariae established as metacercariae following exposure of the tail region.     

Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalisation of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In nonimmunised fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and nonimmunised trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.     

The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation

The influence of permeating and of non-permeating cryoprotectants on motility, eosin permeability (sperm cells viability test) and leaking lactate dehydrogenase activity of spermatozoa of the rainbow trout ( Oncorhynchus mykiss ) was investigated. Correlations between sperm motility rate, percentage of eosinimpermeable (viable) cells and LDH activities established the three parameters as indicators for damages to rainbow trout spermatozoa. High sperm motility rates, velocities, and numbers of linear swimming sperm cells, high numbers of eosinimpermeable spermatozoa and a low extracellular LDH activity were evaluated as positive quality characteristics of semen. With a buffered physiological saline solution as basic extender we found a mixture of 0.67 mol L (5%) dimethylsulfoxide (DMSO) and 0.13 mol/L (1%) glycerol to be the most effective cryoprotectant, followed by DMSO (0.67 mol/L [5%]-1.85 mol/L [15%]) and glycerol (0.65 mol/L [5%]-1.78 mol/L [15%]) and propanediol (1.24mol L [10%]-1.78 mol/L [15%]). Addition of hen egg yolk (7%, 20%) and of 15 mmol/L (0.5%) sucrose significantly increased the semen quality in comparison to the same extender without these additives. Bovine serum albumin (1.5%, 3%) had no influence on the investigated semen parameters.     

Effect of turpentine oil on C-reactive protein (CRP) production in rainbow trout (Oncorhynchus mykiss)

The effect of turpentine oil on C-reactive protein (CRP) production was studied in rainbow trout (Oncorhynchus mykiss). Serum CRP concentration was estimated by sandwich enzyme-linked immunosorbent assay using anti-rainbow trout CRP monoclonal antibody (mAb) AC4 and polyclonal antibody. Intracellular CRP was demonstrated by flow cytometry using anti-trout CRP mAb. Hepatocytes, head kidney macrophages, spleen lymphocytes and peripheral blood lymphocytes showed reaction against AC4, but RTG-2 fibroblastic line cells, derived from rainbow trout gonad did not. This is the first report on the detection of intracellular CRP in fish. CRP levels decreased significantly 1 day after intramuscular injection of turpentine oil and remained low for 14 days. Significant decreases in the expression of CRP in hepatocytes, head kidney macrophages and spleen lymphocytes after injection of turpentine oil were found. The reduction of serum CRP concentration after turpentine oil injection may be attributed to decreases in intracellular CRP synthesis.     

An in vitro system for the long-term tissue culture of juvenile rainbow trout (Oncorhynchus mykiss) testis

In vitro cultivation of fish gonad fragments continues to be an important experimental approach to answer both fundamental and applied scientific questions. The aim of the present investigation was to test whether juvenile rainbow trout (Oncorhynchus mykiss) testes cultured for a week or more were physiologically competent. Trout testis fragments (approximately 1 mm2) were placed on pieces of flat, culture plate insert filter, in a drop of liquid medium (modified Leibovitz's L-15), and floated on the medium surface in a multi-well culture plate. Culture plates were covered and incubated in air at 15 degrees C. Three different endpoints were used to test whether cultured testis fragments remained healthy and functional. First, a comparison of the histological appearance of testis fragments cultured in vitro for different periods up to 8 days with intact testes indicated that no differences were evident. Secondly, testis fragments incubated in medium supplemented with bovine calf serum (BCS) at concentrations of 2.5% and 25% BCS had significantly greater proliferation of interstitial and spermatocyst cells as measured by nuclear 5-bromo-2'-deoxyuridine incorporation. Finally, testis fragments cultured for 5 days and transplanted back into the donor fish underwent precocious sexual maturity in response to a regime of salmon pituitary extract injections and produced fertile sperm as determined by their ability to successfully fertilize eggs. These experiments demonstrate the utility of this method for in vitro culture of juvenile rainbow trout testis fragments.