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.     

Cardiac activity,ventilation rate and acid-base regulation in rainbow trout exposed to hypoxia and combined hypoxia and hypercapnia

A computerised system for non-invasive monitoring of heart and ventilation rates and the time intervals between heart beats and between breaths was developed and used to investigate cardio-respiratory changes in rainbow trout exposed to hypoxia and to combined hypoxia and hypercapnia. Upon exposure to hypoxia and hypoxia-hypercapnia the arterial O₂ tension decreased from about 90 mmHg to about 30 mmHg. Acid-base changes were small in hypoxia whereas exposure to combined hypoxia-hypercapnia caused a large extracellular respiratory acidosis. This acidosis was completely compensated within 24h by accumulation of bicarbonate in plasma to concentrations twice the normoxic values. The ventilation rate was increased to higher values in hypoxic-hypercapnic trout than in hypoxic trout. In contrast to previous reports, the heart rate increased in hypoxia. On top of the tachycardia response to hypoxia, the heart rate was governed by circadian rhythms, with higher heart rates during the day than during the night. The time interval between heart beats varied considerably in normoxic fish. Hypoxia strongly reduced this variability, which may originate in a reduced cholinergic tone to the heart. The width of the frequency distribution of the time intervals between breaths was not affected by hypoxia. The degree of cardio-respiratory synchronization was low in both normoxic and in hypoxic and hypoxic-hypercapnic trout.     

Effects of growth hormone on plasma ionic regulation,respiration and extracellular acid-base status in trout (Oncorhynchus mykiss) transferred to seawater

The effects of trout recombinant growth hormone (rtGH) treatment (0.25 g g^–1 by intraperitoneal implant) on plasma ionic regulation, extracellular acid-base status and respiration were investigated in freshwater rainbow trout and during a 4-day period after direct transfer into seawater (35 g 1^–1).In freshwater, rtGH treatment resulted in a significant increase in gill (Na⁺, K⁺) ATPase activity and in standard metabolism (MO₂). The latter would mainly result from a higher rate of protein synthesis. Direct transfer from freshwater to seawater induced a decrease in arterial blood pH, far more pronounced in controls than in treated fish. This effect could be regarded in both groups mainly as a metabolic acidosis resulting from extracellular ion composition changes (i.e., an increase higher in chloride than in sodium, more marked in controls than in treated fish). As the rise in PaCO₂, in spite of an increase in ventilatory activity, is more significant in controls than in treated fish, it can be assumed that rtGH treatment lightened the decrease in the gas diffusing capacity of gills induced by transfer to seawater. The initial increase in MO₂ in both controls and treated fish could be the consequence of an increase in energetic cost of ventilation and osmoregulation. Then, in treated fish, the persistent high level of M may indicate a stimulation of intermediary metabolism by rtGH. In addition, the absence in treated fish of an increase in plasma lactate concentration, as observed in controls, would indicate that rtGH attenuated the decrease in O₂ affinity of haemoglobin foreseeable from the metabolic acidosis.This article is dedicated to Professor Claude Peyraud, whose recent death has deeply saddened us. We respectfully pay a tribute to his memory.     

Intracellular pH regulation of rainbow trout and carp thrombocytes

The pH regulation of rainbow trout and carp thrombocytes was investigated using the pH-sensitive fluorescent dye BCECF and a PTI Imagescan image analysis system. The cells were experimentally acidified either using the ammonium chloride prepulse method or perfusing the cells with a saline containing propionate ion. The recovery from the acidification was followed. By using ion substitutions (N-methyl-D-glucamine for sodium; TRIS vs carbon dioxide/bicarbonate buffer) and transport inhibitors (amiloride to inhibit sodium/proton exchange and DIDS to inhibit chloride and bicarbonate dependent acid extrusion mechanisms) the transport pathways were identified. The pH regulatory mechanisms of thrombocytes in both species were similar. The pH recovery after experimental acidification was fully inhibited by removing sodium from the medium. The sodium-dependent acid extrusion had both amiloride-sensitive and DIDS-sensitive components, indicating that both sodium/proton exchange and sodium-dependent bicarbonate transport were involved. The role of sodium-dependent bicarbonate movements in pH regulation was ascertained by showing that pH recovery occurred faster at a high than at a low bicarbonate concentration. Adrenergic stimulation did not affect the intracellular pH.     

Blood chemistry and acid-base balance in rainbow trout Oncorhynchus mykiss with experimentally-induced acute bacterial gill disease

Rainbow trout were experimentally infected with the causative agent of bacterial gill disease (BGD) (Flavobacterium branchiophilum) via bath challenge. All fish were cannulated with dorsal aortic catheters, had nasogastric tubes sutured in place for feeding, and were maintained individually, in plexiglass boxes with a flow-through water system. Fish were either fed, or unfed during the trial. Acute changes in blood gas, serum biochemistry and clinical parameters were monitored. By 24h post-challenge, BGD-infected trout that had been fed had significant hypoxemia, hypercapnia, increased blood ammonia, hypoosmolality, hyponatremia, hypochloremia, and increased cough and respiratory rates when compared to control levels. Unfed BGD-infected trout had similar, but less severe blood gas and clinical changes, and no electrolyte disturbances. The BGD-induced hypoxemia is likely exacerbated by increased oxygen demands brought on by feeding. It is not known what association feeding has with the development of low serum ion levels in BGD-infected trout. This is the first study to report the use of fed fish, as opposed to unfed or starved trout, in obtaining blood chemistry values from indisturbed and cannulated animals.     

Blood electrolyte and prolactin regulation in rainbow trout, Oncorhynchus mykiss (Walbaum), exposed to nitrite

Abstract. Rainbow trout, Oncorhynchus mykiss (Walbaum), exposed to environmental nitrite for 3h showed significantly reduced chloride concentrations from 120·9 ± 0·5 to 113·8 ± 2-4 mmol/1, the result of nitrite uptake by the gills with a compensatory loss of blood chloride. This change could be prevented by injection of 10 mg/kg sulpiride, which stimulates prolactin secretion through antagonism of dopamine D₂ receptors, thus increasing circulating prolactin levels. Prolactin is known to decrease membrane permeability and the increased levels of the hormone probably account for the relatively unchanged (compared to controls) plasma chloride and potassium levels in nitrite-exposed, sulpiride-treated fish.     

Edwardsiellosis in farmed rainbow trout (Oncorhynchus mykiss)

Edwardsiella tarda was isolated from naturally infected rainbow trout (Oncorhynchus mykiss) in raceway culture on a commercial fish farm where the fish were kept at a density of 110 kg m^?3 and at a water temperature of 14°C and with a photoperiod of 13 h light:11 h dark. The clinical signs of diseased fish (150 ± 20 mm standard length) were anorexia and lethargy. The most striking lesions in the fish were in the liver. There were hyperaemia and haemorrhages; on histopathological examination, the liver displayed inflammatory infiltrate in portal area, focal necrosis, dilatation of blood sinus and activation of sinusoidal cells. Infection experiments, performed 2 years after isolation of the original culture of E. tarda, were carried out under laboratory conditions at water temperatures of 15, 18 or 24°C. All experimental fish (common carp, Prussian carp, tench), intraperitoneally injected with 8 × 10⁶ cells, demonstrated a total resistance to E. tarda.     

Morphological responses of the rainbow trout (Oncorhynchus mykiss) gill to hyperoxia,base (NaHCO3) and acid (HCl) infusions

Marked morphological responses occur in the gills of freshwater rainbow trout in response to experimental acid-base disturbance and these responses play an important role in acid-base correction. Compensated respiratory acidosis induced by 70h exposure to environmental hyperoxia (elevated water PO₂) caused a 33% decrease in branchial chloride cell fractional surface area (CCFA). Metabolic alkalosis induced by normoxic recovery (6h) from hyperoxia (72h) caused a 50% increase in CCFA, whereas metabolic alkalosis induced by infusion (19h) of NaHCO₃ caused a 70% rise. However, the largest increase (135%) in CCFA was seen in response to infusion (19h) of HCl. NaCl infusion had no effect. A particular goal was to assess the relative importance of changes in CCFA vs. changes in internal substrate (HCO₃ ^–) availability in regulating the activity of the branchial Cl^–/HCO₃ ^– exchange system. For each of the experimental treatments, the accompanying blood acid-base status and branchial transport kinetics (K_m, J_max) for Cl^– uptake had been determined in earlier studies. In the present study, a positive linear relationship was established between CCFA and J^Cl– _max in individual control fish in the absence of an acid-base disturbance. By reference to this relationship, observed changes in J^Cl– _max during metabolic acid-base disturbances were clearly due to changes in both CCFA and internal substrate levels (plasma [HCO₃ ^–]) with the two factors having approximately equal influence.     

Relationship between otolith and somatic growth:consequence of starvation on acid-base balance inplasma and endolymph in the rainbow trout Oncorhynchus mykiss

The effect of a short period of starvation (5–6 weeks) on thechemistry of plasma and inner ear endolymph (Na⁺,K⁺, Cl^-, total Ca, protein, totalCO₂ concentrations and pH) was studied in trout (Oncorhynchusmykiss). In plasma, only total Ca significantly decreased, by 0.17 mM, instarved trout. A slight drop (10%) in the plasma protein content wasalso observed. Starvation induced a metabolic plasma alkalosis characterizedby a non-signifiacant increase in pH and a significant drop of 22% oftotal CO₂. In endolymph, the Na⁺,K⁺, Cl^- , total Ca and protein concentrationswere not significantly different in control and starved trout, but in thelatter, the pH of the endolymph was less alkaline (significant decrease of0.3 UpH) and the total CO₂ concentration was significantlyreduced by 50%. Experimental starvation was selected as aphysiological situation characterized by a slowing down of both somatic andotolith growth. We propose that the reduced rate of otolith growth isdirectly related to an overall reduction in the alkalinity of theendolymph.     

Effects of calcium channel blockers on pharmacologically induced contractions of rainbow trout (Oncorhynchus mykiss) intestine

Calcium depletion/replacement studies were carried out to examine the role of calcium in contraction of trout intestinal smoot muscle in vitro. Three chemically distinct calcium channel blockers were used to determine whether voltage operated calcium channels (VOCs) were involved in calcium entry with either agonist or depolarization-induced contractions. Contractions induced by depolarizing intestinal smooth muscle with potassium were totally dependent on extracellular calcium, whereas receptor-mediated responses to 5-hydroxytryptamine (5-HT) and carbachol also relied on calcium derived from intracellular stores. The calcium channel blockers, verapamil, nitrendipine, and diltiazem, all shifted the calcium-response curve for potassium to the right, supporting the existence of VOCs in trout intestinal smooth muscle. The calcium-response curve for 5-HT was also shifted to the right, suggesting that 5-HT can induce calcium uptake into the smooth muscle via VOCs, in addition to mobilizing intracellular calcium. Verapamil also appeared to block 5-HT receptors directly. Carbachol-induced contractions were only reduced by diltiazem at low concentrations of calcium (0.1–1 mM), suggesting that diltiazem has some other mechanisms of action than the other calcium channel blockers. Activation of muscarinic receptors may induce calcium entry through channels other than the VOCs, in addition to mobilizing intracellular calcium.     

Metabolism of exogenous histamine in rainbow trout (Oncorhynchus mykiss)

Information on the metabolism of exogenous histamine in fish is of much concern regarding the effect of dietary histamine on fish. Histamine catabolic enzymes, diamine oxidase and histamine N-methyl transferase were measured in the tissues of rainbow trout. Diamine oxidase was detected in the stomach, pylorus caeca and intestine. Histamine N-methyl transferase was detected only in the liver.A change in the contents of histamine and its metabolites was observed in the tissues of rainbow trout after oral administration of histamine. A large amount of imidazole acetic acid was observed in the serum, kidney, liver and muscle. On the other hand, 1-methyl histamine was detected only in the liver. Histamine and its metabolites, imidazole acetic acid and 1-methyl histamine were metabolized and diminished within 48 hr in all tissues.These results showed that histamine was metabolized by two metabolic routes in rainbow trout. One is the main pathway producing imidazole acetic acid by intestinal diamine oxidase and the other is the complementary pathway producing 1-methyl histamine by liver N-methyl-transferase.     

Nutritional regulation of somatostatin expression in rainbow trout, Oncorhynchus mykiss

Previously we characterized three DNAs from the endocrine pancreas (Brockmann body) of rainbow trout (Oncorhynchus mykiss) that encode for distinct preprosomatostatin (PPSS) molecules: one containing somatostatin-14 and its C-terminus (PPSS I) and two containing [Tyr⁷, Gly¹⁰]-somatostatin-14 at their C-termini (PPSS II′ and PPSS II″). In the present study, the regulation of PPSS expression was studied in rainbow trout placed on varying nutritional regimes (fed continuously, fasted, fasted then refed). Fish that were fasted for one week displayed reduced growth compared to their fed counterparts, but no alteration in pancreatic PPSS expression was noted between the two groups. Fish fasted for 4 and 6 weeks also were growth retarded and displayed increased levels of PPSS I mRNA and PPSS II″ mRNA compared to fed animals; PPSS II′ mRNA levels were not affected by food deprivation. Refeeding fish for two weeks following 4 weeks of food deprivation restored growth and reduced PPSS I and PPSS II″ mRNA expression to levels similar to those displayed by continuously fed fish. Changes in PPSS expression were correlated with increases in plasma SS. These results indicate that nutritional state modulates differential expression of PPSSs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Arginine effects on biochemical composition of sperm in rainbow trout,Oncorhynchus mykiss

The present study investigated the effect of arginine on seminal plasma composition in rainbow trout. Male rainbow trout broodstocks (2500 ± 200 g) were fed five practical diets (each consisting of three triplicates) supplemented with Arginine at 0.50%, 1.50% and 2.00%. The control group were fed without arginine. Broodstock feeding lasted for 90 days, and then fish semen was sampled. Results indicated no significant differences in LDH, ALP, Fe²⁺ and phosphorous content among the different treatments. The lowest levels of AST and ALT and the highest levels of Ca²⁺ and Mg²⁺ ions were observed in the treatment fed with 1.50% arginine, which showed significant differences from other treatments (P < 0.05). Moreover, the amount of Cl^?, Na⁺ and K⁺ ions was significantly increased in the seminal plasma in fish fed diets containing arginine in comparison with the control. As the amount of arginine was increased, the levels of uric acid became significantly greater in contrast to urea and glucose levels. The highest amounts of cholesterol, fructose and total protein were observed in treatments fed on 2.00%, 0.50% and 1.00% arginine, respectively, showing significant differences from other treatments (P < 0.05). The highest pH value was assayed in the 1.50% arginine treatment. Results indicated that arginine had a potential efficacy on semen quality in rainbow trout broodstocks.     

Dietary formic acid enhances apparent digestibility of minerals in rainbow trout, Oncorhynchus mykiss (Walbaum)

The effect of dietary formic acid on the availability of phosphorus (P) from a fishmeal-based diet by rainbow trout, Oncorhynchus mykiss (Walbaum), reared in brackish water (5–6 g L⁻¹) was determined. Diets containing a low amount of P (6.0 mg P g⁻¹ dry matter) were acidified with 0, 4 and 10 mL kg⁻¹ formic acid and fed to trout (average weight, 520 g) for 4 weeks. The measured pH of the diets were 6.3, 5.8 and 5.3, respectively. The intestinal solubility of P and the digestibility of P were measured by stripping the faecal matter from the fish. The apparent digestibility coefficient (ADC) of P significantly ( P < 0.05) increased from 69.5% to 75.0% of the basal diet in fish fed diets containing 10 mL kg⁻¹ formic acid. The solubility of P in the intestine was highly variable within each treatment, and the differences were not significant. The pH of intestinal content increased with the increase in dietary formic acid concentration. The ADC of magnesium and calcium also showed a significant ( P < 0.05) increase with the acidification of diet by formic acid.     

Embryonic expression of UCP2 in rainbow trout (Oncorhynchus mykiss)

Uncoupling proteins are mitochondrial anions transporters that dissociate respiration from ATP synthesis through proton leaks. Uncoupling protein 2 reportedly plays a role in several physiological processes such as energy partitioning, nutrition, and fatty acid metabolism. The mRNA expression of rainbow trout UCP2 genes (UCP2A and UCP2B) was monitored during embryogenesis and early larval development. Both genes were recruited early and displayed similar steadily decreasing patterns from fertilization until hatching. The expression of UCP2A and UCP2B appeared significantly differentiated after hatching and during the yolk sac absorption, with UCP2A displaying higher expression. We suggest that UCP2 expression profiles in the rainbow trout embryo could be associated with the utilization of lipids as a source of energy during development.     

Effects of seasonal temperature acclimatization on muscle metabolism in rainbow trout, Oncorhynchus mykiss

Maximal enzyme activities in cardiac and swimming muscles of rainbow trout (Oncorhynchus mykiss) were estimated during the natural progression of environmental temperature, between seasonal extremes of 4 and 18°C. Specific activity of marker enzymes were measured at a common temperature of 11°C to determine the potential for metabolic acclimatization. The results confirm an increased oxidative capacity in the cold, with little or no expansion in glycolytic capacity of slow muscle: citrate synthase (CS) activity was 23, 15 and 3 µmoles min^-1 g wet wt^-1 and pyruvate kinase (PK) 15, 23 and 17 µmoles min^-1 g wet wt^-1 from 4, 11 and 18°C acclimatized groups, respectively. Expansion of lipid metabolism was limited, with increased carnitine palmitoleoyltrasferase (CPT) seen only in the coldest group. Cardiac and fast muscle showed a similar response for CS but not PK or CPT which were greatest at 11 and 18°C, respectively. When compared with previous laboratory acclimation studies, these data suggest that environmental factors other than temperature are likely to influence the nature of metabolic acclimatization.     

Effect of dietary Spirulina platensis on fatty acid composition of rainbow trout (Oncorhynchus mykiss) fillet

This paper reports on inclusion of Spirulina platensis (SP) in rainbow trout (Oncorhynchus mykiss) fish diet and its effect on the fish tissue fatty acids (FAs) composition. Fish in triplicate groups with average initial body weight of 75 ± 0.23 g and age of about 7 months were fed a control diet with no supplements and with three experimental diets supplemented with 2.5, 5 and 7.5 weight percent (wt%) of SP for 12 weeks. At the end of experiment, the fillet FA compositions were evaluated. It was found that the composition of FAs in fish fillet was significantly (P < 0.05) affected by inclusion of SP in diets. As compared to the control sample, feeding the fish supplemented particularly with low levels of SP improved the quality of FAs by decreasing undesirable saturated FAs (SFAs) content from 20.03 to 17.93 % and increasing the level of some beneficial long-chain highly unsaturated FAs namely eicosapentaenoic acid (C20:5 n-3) from 6.47 to 7.27 %, docosapentaenoic acid (C22:5 n-3) from 1.42 to 1.56 % and docosahexaenoic acid (C22:6 n-3) from 1.26 to 1.35 %. Further increase in the SP inclusion level generally did not lead to desirable results. The SP inclusion in the fish diet had no significant impact on overall contents of n-3 and n-6 polyunsaturated FAs in fish fillet. However, the total highly unsaturated FAs (HUFAs) and n-3 HUFAs levels, UFAs/SFAs ratio and n-3/n-6 index were increased from 11.25, 10.65, 3.98 and 0.57 to 12.13, 11.56, 4.40 and 0.62 %, respectively, as a result of 5 wt% SP loading. In general, it can be concluded that SP inclusion up to 5 wt% of loading in rainbow trout culture can be beneficial in terms of FAs compositions of the fish fillet.     

Effect of dietary avoparcin on performances and carcass composition in rainbow trout, Oncorhynchus mykiss (Walbaum)

Two levels (40 or 80 mg kg^?1) of avoparcin were tested as a growth promoter in rainbow trout, Oncorhynchus mykiss (Walbaum), fed for 14 weeks. At 80 mg kg-¹ the weight gain was higher (P <0.05) than in the unsupplemented control or at the 40 mg kg-¹ dose. At either dose, the feed conversion rate and the protein efficiency ratio were more favourable (P <0.05) than in the control. Inclusion of the antibiotic significantly improved (P < 0.05) the apparent digestibility of lipids. The lipid content of flesh was significantly increased by inclusion of avoparcin in the diet; moreover, at the high level of inclusion the content of some unsaturated fatty acids (18:3ω3 and 20:5ω3) appeared to be significantly increased. No avoparcin residues were detected in the flesh.