Development of a rainbow trout intestinal epithelial cell line and its response to lipopolysaccharide

A cell line, RTgutGC, was developed from the intestine of Oncorhynchus mykiss. RTgutGC has an epithelial‐like shape, been passaged over 100 times, and cryopreserved successfully. A rainbow trout origin was confirmed by sequencing a 652 bp region of the mitochondrial cytochrome c oxidase I gene. RTgutGC is grown routinely in Leibovitz’s L15 without glutamine supplemented with 10% fetal bovine serum (FBS). Cell viability was evaluated with Alamar blue (AB) for metabolic activity and carboxyfluorescein diacetate acetoxymethyl ester (CFDA AM) for membrane integrity. Viability was unchanged by lipopolysaccharide (LPS) for cultures in FBS. For cultures at low cell densities in L15 without FBS or glutamine, cell viability declined in a LPS dose‐dependent manner, allowing calculation of the concentration causing a 50% decline in viability (EC₅₀). When glutamine was present, the EC₅₀ was increased for both AB and CFDA AM. As the cell density increased, LPS became much less cytotoxic and no EC₅₀ could be calculated for very confluent cultures. Only high‐density cultures had alkaline phosphatase (AP) activity. Thus, glutamine and possibly AP protect against LPS cytotoxicity. RTgutGC should be a useful in vitro tool for studying problems of nutrition and gastrointestinal health in fish.     

Responses of rainbow trout intestinal epithelial cells to different kinds of nutritional deprivation

In order to develop an in vitro system to study the cell biology of starvation in the fish intestine, rainbow trout intestinal epithelial cells were subjected to three kinds of nutrient deprivation and evaluated for 7 days. The RTgutGC cell line was grown into monolayers in Leibovitz’s basal medium supplemented with fetal bovine serum (L15/FBS) and then subjected to deprivation of serum (L15); of serum, amino acids, and vitamin (L15/ex); and of all nutrients (L15/salts). After 7 days of nutrient deprivation, the cells remained attached to the plastic surface as monolayers but changes were seen in shape, with the cells becoming more polygonal, actin and α-tubulin cytoskeleton organization, and in tight junction protein-1 (ZO-1) localization. Two barrier functions, transepithelial electrical resistance (TEER) and Lucifer Yellow (LY) retention, were impaired by nutrient deprivation. In L15/FBS, cells rapidly healed a gap or wound in the monolayer. In L15 and L15/ex, some cells moved into the gap, but after 7 days, the wound remained unhealed, whereas in L15/salts, cells did not even migrate into the gap. Upon nutrient replenishment (L15/FBS) after 7 days in L15, L15/ex, or L15/salts, cells proliferated again and healed a wound. After 7 days of nutrient deprivation, monolayers were successfully passaged with trypsin and cells in L15/FBS grew to again form monolayers. Therefore, rainbow trout intestinal epithelial cells survived starvation, but barrier and wound healing functions were impaired.     

Cryopreservation diluents for spermatozoa of Sakhalin taimen <Emphasis Type="Italic">Hucho perryi</Emphasis>

To develop a suitable cryopreservation diluent for spermatozoa of the endangered Sakhalin taimen Hucho perryi, all possible combinations of cryoprotectants (glycerol, dimethyl sulfoxide [DMSO], methanol) and extenders (fetal bovine serum [FBS], 300 mM glucose solution [GS], artificial seminal plasma for masu salmon) were examined by observing sperm motility 10 s after thawing. Spermatozoa cryopreserved with diluents such as mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS showed the highest motility. The maximal post-thaw motility was observed at 10% among all concentrations (0, 5, 10, 15 and 20%) of these three cryoprotectants. No significant difference among three diluents was observed in motility at 10 s. Mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS are suitable cryopreservation diluents for Sakhalin taimen spermatozoa.     

Changes of Non-Specific Immune Parameters in Rainbow Trout,Oncorhynchus mykiss,After Exposure to Antimicrobial Agents Used in Aquaculture

Changes of plasma lysozyme and serum bactericidal activity, ceruloplasmin, total plasma protein, serum glucose, hematocrit and leucocrit levels in rainbow trout (Oncorhynchus mykiss) were studied after exposure to formalin and chloramine-T, which are used in aquaculture as anti-ectoparasitic and antibacterial agents. Fish were exposed to formalin at a concentration of 250 ppm for 60 min and exposed to chloramine-T for 5 ppm for 3 h. The parameters were analyzed for rainbow trout exposed to formalin and chloramine-T soon after treatments and rainbow trout exposed to freshwater after 24 h (return) following the treatments. Plasma lysozyme activity decreased after exposure to formalin; however, it was not affected by chloramine-T exposure. Serum bactericidal activity reduced after both formalin and chloramine-T exposure and did not change after returning to freshwater. Ceruloplasmin and total plasma protein were not affected by both treatments. Serum glucose levels of exposed fish elevated and remained at higher levels during recovery period when compared to control values. Hematocrit values of fish exposed to formalin increased; however, no significant change was observed in fish exposed to chloramine-T. Leucocrit levels of fish treated with both formalin and chloramine-T were higher than that of controls.     

Severity of summer drought as predictor for smolt recruitment in migratory brown trout (Salmo trutta)

The recruitment of anadromous brown trout smolt from a small Baltic stream, frequently exposed to summer drought, was quantified for 9 years, and the effect from drought on smolt number and age composition analysed. Drought was quantified by, (a) a newly developed index quantifying the severity of summer drought (Drought Severity Index—DSI—based on amount of precipitation, monthly mean temperature and number of hours with sun), and (b) the amount of precipitation. Both DSI and precipitation were measured 1 or 2 years prior to emigration. We found highly significant (negative) relations between DSI (1 year before emigration) and both the total number of smolt, and the number of age 1 smolt. In addition, precipitation was (positively) related to total number of smolt, but DSI proved to be a stronger predictor compared to precipitation. In addition to drought, our results support a negative influence from older parr on the survival of age 0 trout. Our results indicate that recruitment of brown trout smolt from streams, regularly affected by summer drought, may be predicted approx. 6 months before the actual emigration the following spring. This offers an opportunity for managers to adapt protective measures in accordance with local and temporal changes in recruitment.     

pHi regulation and ultrastructural analysis in cultured gill cells from freshwater or seawater-adapted trout

Primary cultures of gill cells from freshwater and seawater-adapted trout were compared. These cultures, developed from an explant technique, exhibited a similar growth. Ultrastructural comparison between cultured and in situ cells showed that most of the cells in primary culture resembled the so called 'pavement' cells, whereas chloride cells were not observed in the cultured epithelium. Several other cells types, representing a minority of cells in primary culture, were observed (mucous cells, vesicolar cells, cells with large dense granules and cells containing lysosomes). Morphological observations of cultured pavement cells from freshwater and seawater trout gills were similar, although the density of cellular organelles in cells was less under freshwater conditions. In addition to the morphological comparison, the regulation of intracellular pH in cultured cells from freshwater and seawater gills was examined. Resting pHi was not different for freshwater or seawater gill cells. A sodium-dependent and amiloride-sensitive mechanism was found in cultured cells. Under the experimental conditions used here, this mechanism was most likely a Na⁺/H⁺ antiporter in pavement cells from freshwater and seawater-adapted trout. The comparison of pHi recovery after acidification of cells from freshwater and seawater gills showed that the activity or the number of antiporters was higher for cells from seawater trout gill.     

Interrelationships between gill chloride cell morphology and calcium uptake in freshwater teleosts

The involvement of the freshwater fish gill chloride cells (CCs) in trans-branchial calcium uptake (JinCa²⁺) was investigated. This was accomplished by assessing the interspecific relationships between the apical surface area of CCs exposed to the external environment and JinCa²⁺. Three species of freshwater teleosts, the rainbow trout (Oncorhynchus mykiss), the American eel (Anguilla rostrata) and the brown bullhead catfish (Ictalurus nebulosus), were used. Chronic (ten-day) treatment with cortisol in each species was used as a tool to evoke variations in both JinCa²⁺ and gill CC morphology in order to assess intraspecific relationships between CC surface area and JinCa²⁺. The results of quantitative morphometry, based on analysis of scanning electron micrographs, demonstrated that catfish possessed the lowest fractional area of exposed CC (CCFA) on the gill filament epithelium (12,744 ± 2248 m²/mm²) and was followed, in increasing order, by American eel (21,355 ± 981 m²/mm²) and rainbow trout (149,928 ± 26,545 m²/mm²). With the exception of catfish, chronic treatment with cortisol caused significant increases in CCFA owing to proliferation of CCs and/or enlargement of individual CCs (eel only). The rates of JinCa²⁺ closely reflected the CC fractional area in each species. The results of correlation analysis revealed significant correlations between CC fractional area and JinCa²⁺ in trout and eel. Owing to the absence of an effect of cortisol treatment, there was no significant correlation in catfish because of insufficient variation in CC fractional area in this species. CC fractional area was significantly correlated with JinCa²⁺ among the three species examined. These results suggest that CC is involved in calcium uptake in freshwater teleosts and that both intra- and interspecific differences in the rates of calcium uptake can be accounted for by variability in the surface area of exposed CCs on the gill epithelia.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

Environment-induced gill disease of cultured rainbow trout (Salmo gairdneri)

Rainbow trout from eight North German freshwater culture facilities were investigated for chronic health damage resulting from captivity. The fishes were raised under a wide variety of conditions in net cages, circulation tanks, raceways, and earthen ponds.The most obvious non-contagious health problem was gill injury. Hyperplasia of the gill filaments, leading to lamellar fusion or the development of apical caps, as well as necrotic or advanced degenerative processes were observed. In addition, the gill filaments were frequently shortened, significantly reducing the functional surface of the organ.The lesions occurred in almost the same form in all of the culture units. It is likely that they are part of a syndrome induced by the environmental conditions typical of freshwater aquaculture facilities. These are characterized by an accumulation of nitrogenous excretion and decomposition products, extreme diurnal fluctuations in the concentration of dissolved gases, and intensive feeding involving the sudden addition of large amounts of artificial products. Under such conditions even small quantities of un-ionized ammonia can be toxic for epithelial tissues and disturb the normal elimination of protein metabolites across the gills. The pathological processes may be accelerated by secondary bacterial infections.     

A specific inhibitor of mammalian kallikrein,Phe-Phe-Arg-chloromethyl ketone,inhibits the production of vasoactive substances from trout plasma by kallikrein and blocks endogenous kallikrein-like activity in trout gills

The cardiovascular effects of the kallikrein-kinin system (KKS) have not been completely characterized in lower vertebrates. In the present study, a specific, irreversible kallikrein inhibitor, Phe-Phe-Arg-chloromethyl ketone (PPACMK) was used to examine: 1. the role of the KKS in blood pressure regulation in vivo; 2. the nature of the pressor substance formed by the action of kallikrein on trout plasma in vivo and in vitro; and 3. the presence of kallikrein in trout gills and kidney. Dorsal aortic cannulated rainbow trout were used for in vivo blood pressure assays and two colorimetric serine-protease assays were used to examine tissue kallikrein activity. PPACMK alone had no effect on blood pressure in vivo. Pretreatment of porcine kallikrein with PPACMK inhibited the enzyme's pressor effect in trout by 80% and significantly attenuated the synthesis of vasopressor substance(s) from heat-treated trout plasma in vitro. Approximately 30% of gill serine protease activity was inhibited by pretreatment with PPACMK; no PPACMK-sensitive kallikrein activity was observed in the kidney. Salt water adaptation did not affect kallikrein-specific activity in the gill. These results show that the salmonid KKS does not appear to be involved in the regulation of systemic blood pressure. Further, the formation of pressor substances from trout plasma in vivo and in vitro is due to kallikrein activity and the peptides generated are similar. A kallikrein, similar to the mammalian enzyme, is found in trout gills, suggesting that the salmonid KKS may be a local mediator of gill function.     

Similarities and differences in oxynticopeptic cell ultrastructure of one marine teleost, Gadus morhua and one freshwater teleost, Oncorhynchus mykiss, during basal and histamine-stimulated phases of acid secretion

Oxynticopeptic cells (OC) from rainbow trout, Oncorhynchus mykiss, and Atlantic cod, Gadus morhua, were studied during basal acid secretion and during stimulation with histamine. An in vitro method for measuring acid secretion from isolated stomac a was used. The OC ultrastructure was studied using electron microscopy. During basal acid secretion, the OC had short apical microvilli, an extensive tubulovesicular network, large electron-dense vesicles and many mitochondria. Stimulation of acid s tion with histamine and ultrastructural changes in the OC occurred simultaneously. The acid stimulation promoted large increases in the length of microvilli, a decrease in the size of the tubulovesicular network and an increase in the diameter of the ndular lumen in cod. Large vacuoles were found to characterize the histamine-stimulated OC. These were located close to the apical membrane and sometimes opened into the lumen. During stimulation, electron-dense vesicles were often observed close to apical membrane and the large vacuoles. Sometimes these vesicles were observed inside the glandular lumen and/or the large vacuoles. It is concluded that the ultrastructure of the OC in the saltwater cod and the freshwater trout show similar characte ics as OC in other animal groups both during basal and high rates of acid secretion. In addition, vacuoles were found in both species OC during basal rates of acid secretion and they significantly increased in size during high rates of acid secretion propose that the OC vacuole have a part in the osmoregulation, since the increase was tenfold in cod but less then one-fold in trout, this difference was statistically significant.     

Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus

In this study, we established and characterized a cell line derived from the kidney of black carp (Mylopharyngodon piceus), which is an important freshwater aquaculture species. The cell line was designated as MPK and subcultured for more than 70 passages in DMEM medium containing 10% fetal bovine serum (FBS) at 28°C. MPK had a modal diploid chromosome number of 48. Moreover, a transient MPK transfection efficiency was up to 18% using a green fluorescent protein plasmid by a modified electroporation. In addition, the MPK cells showed susceptibility to spring viremia of carp virus (SVCV), as demonstrated by the presence of severe cytopathic effects (CPEs) and increased viral RNA. Unexpectedly, the MPK cells expressed pluripotency‐associated genes such as nanog, oct4 and vasa, indicating that these are possibly adult stem cells. Taken together, we have established a stable cell line from kidney that may potentially be utilized as an in vitro platform for genetic modifications and host–pathogen analysis in black carp.     

Pseudomonas putida infection in rainbow trout

A disease outbreak occurred during the Spring of 2006 among rainbow trout, Oncorhynchus mykiss, kept in growout tanks. During the outbreak 35% of the rainbow trout died. Typical clinical signs were ulceration on the dorsal surface of the fish. Based on biochemical characteristics and sequence of 16S rRNA, the causative bacteria isolated from diseased fish were identified as Pseudomonas putida. Histologically, skin ulcer initially was characterized by sloughed off epithelia and epithelial necrosis. In the advanced form of the disease, almost all layers of the skin down to the epidermis were lost. Outside of Japan, this study reports the first P. putida infection in fish.     

美国金鳟和日本鳗鲡精浆的化学组成和渗透压

本研究采用原子吸收分光光度法(AAS)和气相渗透压仪测定了人工养殖美国金鳟和日本鳗鲡精浆的化学组成和渗透压。研究结果表明,Na+、K+是精浆中的主要离子,可能在维持精浆渗透压和精子活动能力中有重要的作用。人工养殖美国金鳟与日本鳗鲡相比,精浆离子含量各项指标除K含量较高之外(P<0.01),其它各项指标均无显著性差异。本实验的两种鱼与其它种类海淡水鱼类比较,各种离子含量均较高;美国金鳟精浆渗透压值与其它鱼类相似,均保持在300 mOsm/kg的水平。     

A Comparison of the Performance of American Brook Trout (Salvelinus fontinalis Mitchell) and Rainbow Trout (Salmo gairdneri Richardson) Reared in Fish Farm Ponds and Freshwater Floating Net Cages

Following an upsurge in the interest to anglers of American brook trout (Salvelinus fontinalis) an investigation was carried out in 1978 and 1979 into their growth and survival when reared in fish farm earth ponds and freshwater floating cages. Their performance was compared with similarly reared batches of rainbow trout (Salmo gairdneri). This work shows that American brook trout require more care and attention than rainbow trout when reared artificially and that their survival to 1 year old is lower, although growth rate in the first year compares favourably.     

Importance of fatty acids in broodstock diets with emphasis on Arctic char (Salvelinus alpinus) eggs

The importance of long-chain polyunsaturated fatty acids, especially the eicosanoid precursors, is addressed in this paper. It has been generally recognized that eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are of significant importance in fish reproduction while arachidonic acid (AA, 20:4n-6) has often been overlooked. The ratio between C20 fatty acids EPA and AA might be important for many physiological functions depending on the species evolution and its requirements. Arctic char (Salvelinus alpinus) has a much more pronounced freshwater history and therefore different fatty acid requirements than the other commonly farmed salmonids such as salmon (Salmo salar), brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss). Therefore there is reason to formulate a feed that is more suitable for farming of this freshwater species. In this study, freshwater wild-origin char eggs were compared to farmed eggs of char. The ratio n-3/n-6 of total phospholipids of eggs was much lower in the wild fish, 3.5 versus 13.5, and the hatching rate of eggs from natural environment was much higher (20–70% vs. >80%). We conclude that feed based on marine raw product does not fulfill the requirements for essential fatty acids for freshwater char and we suggest that AA is supplemented to the broodstock diet and that at least linoleic acid (18:2n-6) is included in the on-growth diet formulas to lower the n-3/n-6 fatty acid ratio.     

Guanylyl cyclase stimulation by homologous and heterologous natriuretic peptides in salmonids

A range of homologous (trout ANP, trout CNP, trout VNP) and heterologous (eel ANP, eel ANP-NH₂, rat ANP, porcine CNP) NPs were tested for their effect on guanylyl cyclase in gill and kidney membrane preparations from freshwater and seawater-acclimated rainbow trout and Atlantic salmon. All NPs stimulated guanylyl cyclase at 1 μmol l⁻¹in all preparations. ANP was the most potent stimulator of kidney guanylyl cyclase and CNP the most potent stimulator of guanylyl cyclase in gills. Some differences were apparent between the potencies of homologous and heterologous peptides at 1 μmol l⁻¹: tANP was more potent than rANP in the SW trout kidney and tCNP was more potent than pCNP in FW salmon tissues. While eANP was more potent than tANP in trout gills, it was less potent than tANP in FW salmon gills. However, there was no significant difference between the potencies of eANP and eANP-NH₂ in trout or salmon gills. Salinity did not affect guanylyl cyclase activity with the exception that trout ANP at 1 μmol l⁻¹was more potent in SW trout kidneys than in FW trout kidneys. These results suggest a predomination of NPR-A in the kidney and NPR-B in the gill. It appears that salmonid NPR-A and NPR-B are relatively promiscuous in their ligand affinity, with few differences in the potencies of trout and mammalian NPs and only small differences in cGMP production where these differences do occur. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of stocking of piscivorous brown trout, Salmo trutta L., on stunted Arctic charr, Salvelinus alpinus (L.)

To study the effects on a stunted freshwater population of Arctic charr, Salvelinus alpinus (L.), two groups of large (26–45 cm) individually tagged brown trout, Salmo trutta L., were released and recaptured with gillnets after 1, 7, 11 and 63 weeks. One group of trout was trained on a fish diet before release, and the other, reared on commercial dry pellets, served as a control. Specific growth rates in both groups were negative 1 week after release and approached zero after 63 weeks. Condition factor and internal fat content decreased during the experiment. Although only 11% of the trout stomachs examined contained fish prey, charr represented 79% of the total stomach weight content. Gillnet samples of charr before and 63 weeks after the release of trout indicated a decreasing population size of charr. Individual growth and mean length of charr increased after release of trout, especially for charr at age 4 years. After the release of trout, 35% of the charr were longer than 20 cm as compared with 6% before the release.     

Differences in early developmental rate and yolk conversion efficiency in offspring of trout with alternative life histories

Partial migration, in which some individuals of a population migrate while other individuals remain resident, is generally associated with ontogenetic shifts to better feeding areas or as a response to environmental instability, but its underlying mechanisms remain relatively unknown. Brown trout (Salmo trutta) exhibit partial migration, with some individuals remaining in freshwater (freshwater resident) while others undertake an anadromous migration, where they spend time at sea before returning to breed in freshwater (migrant). We reared full‐sibling groups of offspring from freshwater‐resident and anadromous brown trout from the same catchment in the laboratory under common garden conditions to examine potential differences in their early development. Freshwater‐resident parents produced eggs that were slower to hatch than those of anadromous parents, but freshwater‐resident offspring were quicker to absorb their yolk and reach the stage of exogenous feeding. Their offspring also had a higher conversion efficiency from the egg stage to the start of exogenous feeding (so were larger by the start of the fry stage) than did offspring from anadromous parents despite no difference in standard metabolic rate, maximal metabolic rate or aerobic scope. Given these differences in early development, we discuss how the migration history of the parents might influence the migration probability of the offspring.     

Does urea reabsorption occur via the glucose pathway in the kidney of the freshwater rainbow trout?

This study tested the hypothesis that the renal reabsorption of urea occurs via the glucose transport pathway in the freshwater rainbow trout (Oncorhynchus mykiss). The relationship between glucose transport and urea transport was examined by experimentally elevating the rate of renal glucose reabsorption via infusion of the fish with exogenous glucose, and by inactivating the glucose transporters via the administration of phlorizin. Under all treatments, urea was reabsorbed against a concentration gradient, with plasma levels of urea being higher than urine levels. Glucose was almost completely reabsorbed (88%) whereas urea was reabsorbed less efficiently (47%) but to a greater extent than water (22%). Glucose and urea reabsorption were both found to be correlated with Na⁺ and Cl^– reabsorption, though the latter were 20 fold and 200–300 fold higher than glucose and urea transport rates, respectively. Glucose infusions greatly increased glucose reabsorption but urea reabsorption was unaffected. Phlorizin treatment completely blocked glucose reabsorption, but urea reabsorption was again unaffected. We conclude that there is no relationship between glucose and urea handling in the trout kidney, thus disproving the hypothesis.