Reproductive performance of cultured brown trout (Salmo trutta L.) in Chile

Data on reproductive traits in a cultured population of brown trout (Salmo trutta L.) in southern Chile were collected over three consecutive reproductive seasons: 1999, 2000 and 2001, corresponding to the first (at 3 years), second and third spawnings in females. Data were collected from individual females (n=238, 273 and 169 respectively). The reproductive season lasted mainly from June to August. The peak (July) tended to increase with each season (55.46%, 62.27% and 80.81% of total spawning fish respectively). Female body weight (470.8±102.5, 735.0±150 and 1263.9±263.4 g), total fecundity (1182±344, 1904±595 and 2744±605) and egg diameter (4.64±0.11, 4.67±0.27 and 5.24±0.12 mm) increased significantly (P<0.01) over successive reproductive seasons, particularly between the second and third spawnings. Relative fecundity, on the other hand, decreased significantly with each season (3577±471, 2591±900 and 2181±360). Following analysis of the variables over the three seasons, the correlation pattern of female body weight with total fecundity (r=0.91, n=458; P<0.001) and relative fecundity (r=?034, n=451; P<0.001) proved similar to that described in other salmonid females. Fertilization rate and survival to the eyed egg stage were also positively correlated (r=0.73, n=453; P<0.001). The systematically high values obtained for these latter variables over the seasons evaluated (consistently above 90%) are clearly greater than those we registered in other species of salmonids bred in Chile under similar conditions and suggest highly efficient biological variables that determine the gamete fertilization of this species. The brown trout is, therefore, an interesting potential aquaculture resource in Chile.     

Oxidation of glutathione during hydroperoxide metabolism in isolated hepatocytes of rainbow trout (Salmo gairdneri)

Freshly isolated rainbow trout hepatocytes were exposed to tert-butyl hydroperoxide (BuOOH), a substrate for glutathione peroxidase. BuOOH at a concentration approximately equimolar (1 mM) with intracellular reduced glutathione (GSH) caused a reversible increase in intracellular glutathione disulphide (GSSG) but did not compromise cell viability or damage membrane lipids. BuOOH at 10 mM caused a large irreversible increase in intracellular GSSG followed by efflux into the medium. Considerable leakage of lactate dehydrogenase and loss of highly unsaturated fatty acids, particularly docosahexaenoic acid also occurred. Dependence of hydroperoxide removal on flux through the hexose monophosphate pathway was suggested by the increased release of ¹⁴CO₂ from [1-¹⁴C] glucose from hepatocytes incubated with BuOOH.     

Characterization of serum and mucosal antibody responses and relative per cent survival in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization and challenge with Flavobacterium psychrophilum

Serum and mucosal antibody responses of juvenile rainbow trout, Oncorhynchus mykiss, were characterized by enzyme‐linked immunosorbent assay (ELISA) following immunization with various preparations of formalin‐killed Flavobacterium psychrophilum cells. The protective nature of these preparations was then determined by immunizing rainbow trout fry and challenging with the bacterium. Juvenile rainbow trout immunized intraperitoneally (i.p.) with formalin‐killed F. psychrophilum emulsified with Freund's complete adjuvant (FCA), and i.p. with formalin‐killed F. psychrophilum either with or without culture supernatant generated significant serum antibody responses by 6 and 9 weeks, respectively. Significant mucosal antibody responses were detected by 9 weeks only in fish immunized i.p. with killed F. psychrophilum/FCA. Following immunization and bacterial challenge of rainbow trout fry, protective immunity was conferred in F. psychrophilum/FCA and saline/FCA groups with relative per cent survival values of up to 83 and 51, respectively. Significant protection was not observed in treatment groups immunized by immersion or i.p. without adjuvant at the challenge doses tested. Results suggest that stimulation of non‐specific immune factors enhances the ability of fish to mount a protective immune response, but specific antibody appears necessary to provide near complete protection. In this study, an ELISA was developed to monitor anti‐F. psychrophilum antibody production in trout. The relationship of such responses to protective immunity suggests that future vaccination strategies against coldwater disease may require stimulation of both the innate and adaptive arms of the immune response.     

Site of acid-base relevant ion transfer in the gills of rainbow trout (Oncorhynchus mykiss) exposed to environmental hypercapnia

In order to delineate the contribution of primary vs. secondary circulatory circuits in the gill for acid-base and ionic regulation, the flow and composition of the fluids in the central venous sinus (CVS) and the systemic circuit of rainbow trout were studied by application of a previously developed microcannulation technique during normocapnic and hypercapnic conditions. The average haematocrit (Hct) of blood from dorsal aorta (DA) and sinus venosus (SV) ranged from 20.1 to 26.7%, whereas average Hct in the fluid from the branchial vein (BV), representing drainage from the central venous sinus (CVS), was in the range of 4.2 to 7.0%. Under normocapnic conditions, the largest fraction of cardiac output, 92.9%, was directed through the systemic vascular circuit, whereas the CVS circuit was perfused with 7.1 % of cardiac output. Hypercapnia did not significantly affect the blood flow distribution between the two circuits. The pattern of acid-base regulation in dorsal aortic blood reflected the characteristic response of fish exposed to environmental hypercapnia. Upon initiation of environmental hypercapnia (2% CO₂), plasma P_{CO 2} was elevated in all three flow compartments (CVS, DA, SV), inducing an extracellular respiratory acidosis of about 0.4 pH units. pH and [HCO₃ ^-] values in the DA were consistently lower, than those for both CVS and SV sites throughout the hypercapnic period. During the 8h of exposure plasma bicarbonate concentration was elevated by about 12 mM, complemented by a fall in extracellular [Cl^-] of about 10 mM in all three compartments. The amount of HCO₃ ^- gained at the CVS site during 8h of hypercapnia (3.3 mmol · kg^-1) exceeds the amount accumulated in the extracellular space (2.1 mmol·kg^-1), suggesting the CVS as the main site of ionic acid-base regulation in trout.     

The effects of elevated winter temperature and sub-lethal pollutants (low pH, elevated ammonia) on protein turnover in the gill and liver of rainbow trout (Oncorhynchus mykiss)

Appetite, growth, and protein turnover (synthesis, growth and degradation) of liver and gills were measured in juvenile rainbow trout (Oncorhynchus mykiss) fed to satiation, and exposed for 90 days to elevated winter temperatures (+2 °C above ambient) and either low pH (5.2) in softwater or 70 M total ammonia (TAmm) in hardwater. All fish increased in weight during the experiments, but those exposed to +2°C grew significantly more than those at ambient temperature due to a stimulation of appetite. During the relatively constant temperature of the first 75 days, +2 °C caused a significant increase in the rates of protein synthesis and degradation in the liver of hardwater-acclimated fish, as a result of an increase in RNA translational efficiency (KRNA). The elevated temperature also induced an increase in gill protein synthesis in softwater-acclimated fish but in this case the underlying mechanism was an increase in Cs, the capacity for protein synthesis (RNA:protein) rather than in KRNA. The addition of 70 M TAmm had no effect on protein turnover in either liver or gills of hardwater-acclimated fish. Low pH inhibited protein growth in the liver of softwater-acclimated fish at day 90 under both temperature regimes. This inhibition was effected via a decrease in protein synthesis at control temperature but via an increase in protein degradation when the fish were exposed to both low pH and +2 °C. From these results we conclude that a simulated global warming scenario has potentially beneficial rather than detrimental effects on protein turnover and growth of freshwater fish during winter.     

The effect of culture on morphology, lipid and fatty acid composition, and polyunsaturated fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells

Rainbow trout (Oncorhynchus mykiss) skin cell cultures were obtained by trypsinization of the tissue and grown in Leibovitz L-15 medium. Lipid class compositions, and fatty acid profiles of total lipids and individual phospholipid classes were determined at different times of culture. The metabolism of polyunsaturated fatty acids (PUFA) was investigated by incubating primary cultures after 7 and 14 days with [1-¹⁴C]18:2n-6 and [1-¹⁴C-]18:3n-3. The change in morphology between epithelial-like primary cultures and fibroblastic-like secondary subcultures was accompanied by alterations in the lipid composition. Polar lipids became predominant by 14 days in culture. The relative proportions of phosphatidylcholine (PC), the most abundant phospholipid, phosphatidylinositol and cholesterol increased significantly, while sphingomyelin decreased. Saturated fatty acids, 18:1n-9, n-6 and n-9PUFA were more abundant in total lipid in cultures at 14 days and 4 months than in cells initially isolated which contained higher percentages of longer chain monoenes and n-3PUFA. The changes in fatty acid composition with time in culture were observed in all the major phospholipid classes. Rainbow trout skin cells in culture desaturated and elongated both 18:2n-6 and 18:3n-3, with 20:4n-6 and 20:5n-3 being the most abundant products, respectively. PC presented the highest incorporation of radioactivity, especially following incubation with 18:3n-3. Lipid metabolism in general increased with the age of primary cultures, with both the amount of C18 PUFA incorporated and metabolized by desaturation/elongation significantly increased in 14 day cultures compared to 7 day cultures. Product/precursor ratios calculated for both n-6 and n-3 fatty acids showed that, while 6 desaturase activity was increased significantly with cell age, 5 desaturase activity was more affected by the fatty acid series, with 18:3n-3 being more readily transformed to 20:5n-3 than 18:2n-6 to 20:4n-6. Further desaturation of 20:5n-3 to hexaenes was low. Overall, the data suggested that the trout skin cell cultures were more similar to mammalian skin fibroblasts than mammalian epidermal/keratinocyte cultures.     

Effects of prolonged epinephrine infusion on blood respiratory and acid-base states in the rainbow trout: Alpha and beta effects

Rainbow trout were infused continuously for 12h with epinephrine in the presence or absence of alpha-and/or beta-adrenergic blockade to characterize the specific adrenergic mechanisms involved in the control of blood acid-base status and oxygen transport capacity. Infusion of epinephrine, alone, produced a transient respiratory acidosis, as indicated by an increase in carbon dioxide tension and a decrease in whole blood pH, yet arterial oxygen tension was elevated. Red blood cell pH increased by approximately 0.2 pH units during epinephrine infusion and this increase as well as the increase in oxygen tension were prevented by pretreatment with propranolol (a beta-adrenergic antagonist). Epinephrine infusion during alpha-adrenergic blockade caused a prolonged elevation of blood carbon dioxide tension and abolished the increases in hematocrit and hemoglobin concentrations observed during epinephrine infusion alone. Infusion of the alpha-adrenergic agonists phenylephrine (an alpha₁ agonist) or clonidine (an alpha₂ agonist) caused respiratory acidosis (decreased pH, increased CO₂ tension) and a reduction in oxygen tension. Infusion of isoprenaline (a non-specific beta agonist) caused delayed increases in carbon dioxide and oxygen tensions. We speculate that the increased carbon dioxide tension observed during epinephrine infusion is a result of beta-adrenoceptor mediated inhibition of red blood cell bicarbonate dehydration and not branchial convective or diffusive adjustments. The effects of epinephrine on blood O₂ tension, content and carrying capacity are discussed with reference to the participation of alpha- and beta-adrenergic mechanisms at the gill, spleen and red blood cell.     

Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured rainbow trout (Salmo gairdneri) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids supplemented to the culture medium was investigated in the rainbow trout cell line, RTG-2. The distribution, and the occurrence and relative extent of further desaturation and elongation of the incorporated acids was determined in individual phospholipid classes by analysis of the fatty acid compositions. RTG-2 cells exhibited Δ6 and Δ5 desaturase activities whereas Δ4 desaturase activity was almost totally absent. The percentage of precursor acids was greatest in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for possibly PA in the initial incorporation of these acids into the phospholipid pool. The compositional data indicated that individual intermediates and products of the desaturation pathways were associated with specific phospholipid classes probably via mechanisms depending upon the specificities of the acylating enzymes. The composition of phosphatidylinositol (PI) and the tightly controlled mechanisms for generating/maintaining it are consistent with a role for this phospholipid in providing precursor fatty acid for eicosanoid synthesis.     

Temporal changes in plasma thyroid hormone,growth hormone and free fatty acid concentrations,and hepatic 5′-monodeiodinase activity,lipid and protein content during chronic fasting and re-feeding in rainbow trout (Oncorhynchus mykiss)

Temporal changes in growth, plasma thyroid hormone, cortisol, growth hormone (GH) and non-esterified fatty acid (NEFA) concentrations, hepatic T₃ content and hepatic 5-monodeiodinase activity were measured in rainbow trout (Oncorhynchus mykiss) subjected to a sustained fast for up to eight weeks, and during a four-week re-feeding period. The purpose of the study was to examine aspects of the endocrine control of energy partitioning processes characteristic of short-term (acute; fasting) and long-term (chronic; starvation) food-deprivation states in fish, and to explore the role of the thyroid hormones, cortisol and GH in the energy repartitioning that takes place during an acute anabolic (re-feeding) state following chronic food deprivation.Differences in growth rate between fed and fasted groups were evident after two weeks, but significant weight loss by the fasted groups was not evident until between four and six weeks into the fast. Hepatosomatic indices (HSIs) were significantly reduced in the fasted fish within seven days, and as early as two days in one study; recovery of the HSI in fasted fish was evident within three days of re-feeding. Liver protein content (expressed as % wet weight) was consistently depressed in the fasted fish in only one of the three studies. Liver total lipid content (expressed as % wet weight) was depressed in the fasted fish within two days of food deprivation. Because of the rapid and sustained decrease in the HSI of fasted fish, the hepatic total protein and lipid reserves, when considered on a body weight basis, were markedly lowered within the first few days of the fast. Plasma GH concentrations exhibited a bi-modal pattern of change, with a transient fall in levels, followed by a sustained increase in fasted fish. The indicators of interrenal activity were suggestive of a depressed pituitary-interrenal axis in fasted animals; plasma cortisol levels were elevated to levels of fed animals within one day of re-feeding. The indicators of thyroid hormone economy (plasma thyroid hormone levels, liver triiodothyronine content, hepatic 5-monodeiodinase (MD) activity, thyroid epithelial cell height) were similarly indicative of a depressed pituitary-thyroid axis in fasted animals, with recovery to levels of the fed animals within one week. Despite the compensatory changes in accumulation of reserves (as indicated by a compensatory increase in HSI), there were no apparent compensatory changes in any of the endocrine parameters evident during the re-feeding period.     

Investigations of physiological and pathological levels of total plasma protein in rainbow trout, Oncorhynchus mykiss (Walbaum)

This study was aimed at construction of reference (physiological) value intervals determined by lower 2.5% and upper 97.5% quantiles for total plasma protein (TPP) in farmed rainbow trout, Oncorhychus mykiss (Kamloops strain), aged 10–12 months (n=755). Total plasma protein in male trout from raceway culture was found to be significantly greater (38–58.3 g L^?1) relative to females (31–51.5 g L^?1). Total plasma protein in trout from cage culture was significantly greater (32–54 g L^?1) compared with the raceway culture (31–49 g L^?1). Moderate product–moment correlations between TPP and cholesterol (r=0.8730), TPP and total calcium (r=0.8188), TPP and alanine aminotransferase (r=0.6200) and between TPP and aspartate aminotransferase (r=0.6430) were estimated. Proteinaemia was explained as a metabolic response to feed content, infections of bacterial or viral aetiology or to intoxications.