Non-linear release of Bohr protons with haemoglobin-oxygenation in the blood of two teleost fishes; carp (Cyprinus carpio) and tambaqui (Colossoma macropomum)

Some teleost fishes exhibit a non-linear release of H⁺ with haemoglobin oxygen saturation (SO₂) in whole blood that may be related to the Root effect and a low apparent cooperativity in oxygen binding. To further investigate this correlation, the relationship between red cell pH (pH_i) and SO₂ was evaluated in two teleost fishes, the carp (Cyprinus carpio) and tambaqui (Colossoma macropomum). Carp possess a relatively small Root effect within the physiological pH range, while that in tambaqui is relatively large. It was therefore hypothesized that both species would possess a non-linear release of H⁺ with SO₂ under in vivo conditions, but that the degree of non-linearity would be less pronounced in carp than in tambaqui. Red cell suspensions of tambaqui showed a marked non-linear relationship between pH_i and SO₂ at in vivo pH values (pH_e=7.66), where the majority of Bohr protons were released between 50 and 100% SO₂. In whole blood of carp, the relationship between pH_i and SO₂ was almost linear at normal resting extracellular pH values (pH_e=8.10) where Hills n ₅₀ (cooperativity) was 1.3. Under acidic conditions (pH_e=7.11), n ₅₀ decreased to 0.9 and the release of H⁺ with SO₂ became slightly non-linear. The reduction in pH in carp blood was associated with the onset of the Root effect, and the oxygen tension at half saturation (P₅₀) increased from 3.8 to 38.2 mmHg. The relationship between total CO₂ and SO₂ in carp whole blood changed from being almost linear at pH_e 8.1 to being non-linear at pH_e 7.1, consistent with the relationship between pH_i and SO₂. Thus, possession of a Root effect could be a prerequisite for a non-linear release of Bohr protons with oxygenation, but the expression in whole blood may depend upon a species-specific cooperativity threshold. The non-linear release of Bohr protons with oxygenation may be a general phenomenon in teleosts with important implications for gas transport and acid-base homeostasis.     

Biochemical parameters of blood plasma and content of microcystins in tissues of common carp (Cyprinus carpio L.) from a hypertrophic pond with cyanobacterial water bloom

The aim of this study was to evaluate the dynamics of the blood plasma parameters and the content of microcystins in the tissues of common carp ( Cyprinus carpio L.) in relation to the toxic cyanobacterial water bloom. Fish (average body mass 2176±697 g) in the hypertrophic pond were exposed to natural water bloom (dominated by Planktothrix agardhii, Pseudanabaena limnetica and Limnothrix redekei ), which contained microcystins (concentration in biomass 20–181 μg g⁻¹ dry wt, concentration in water 0.3–9.5 μg L⁻¹). Biochemical parameters in fish blood plasma were analysed in 89 fish at 14-day intervals during the whole season (nine sampling periods). Our results demonstrated high variability and fluctuations in the investigated parameters. The content of microcystins and density of cyanobacterial cells correlated with some haematological indices as lipase, alanine–aminotransferase, albumin, magnesium and chlorides. The concentrations of microcystins in the muscle and liver of the fish (determined by high-performance liquid chromatography with mass spectrometer) were below the limit of detection during the monitored period [0.31 ng g⁻¹ fresh weight (f.w.) for the liver and 0.13 ng g⁻¹ f.w. for muscle]. Our study demonstrates that although known cyanobacterial toxin microcystins were not detected in the fish tissues, several biochemical parameters important for the fish physiology were modulated by natural cyanobacterial bloom.     

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.     

Species differences in the adrenergic responses of fish red cells: studies on whitefish,pikeperch, trout and carp

The occurrence and pH dependence (pHe 7–8) of the adrenergic red cell responses of two salmonids, trout and whitefish, and a percinid, pikeperch were studied. These are all species that live in well-oxygenated waters. The responses were compared to those of carp, which tolerates oxygen-deficient waters.The adrenergic responses of trout and whitefish red cells were pronounced. In these species red cell swelling, the accumulation of sodium and chloride in the cell, and the increase in red cell oxygen content at atmospheric oxygen tension were maximal at pH 7.3. In contrast, pikeperch red cells responded to -adrenergic stimulation only at extracellular pH 7.1. In carp, the adrenergic response, occurring below extracellular pH 7.5, was small as compared to the two salmonids. In each case the onset of the adrenergic response coincided with the onset of the Root effect.The differences in the adrenergic responses between the two salmonids and pikeperch suggest that the occurrence of the adrenergic response is not directly related to the environmental oxygen requirements of the species, but may be linked to the activity pattern.     

鲫血髓过氧化物酶的表达及其与血药浓度的关联性

为了研究在吡喹酮代谢过程中,鲫血髓过氧化物酶(myeloperoxidase,MPO)m RNA表达与血药浓度的相关性,以体重为(80±10.5)g的鲫鱼(Carassius auratus)为试验动物,单剂量(10 mg/kg)口灌吡喹酮(praziquantel,PZQ)后,在鲫血液中选取β-actin为内参基因,通过设计特异性引物,利用荧光定量PCR,分析了不同时间点鲫血MPO m RNA水平的相对表达量的变化;利用高效液相色谱法(high performance liquid chromatography,HPLC)测定了吡喹酮在鲫鱼体内的血药浓度,分析两者之间的相关性。结果显示,吡喹酮能够迅速进入血液,并被快速消除,药时数据符合二室开放模型。在1 h时,血液中吡喹酮的浓度达到最大值,为2.85?g/m L,96 h后血液中检测不到吡喹酮;灌药后,MPO基因表达量随时间呈先升后降趋势,在1 h时髓过氧化物酶表达量最高。此外,0.25 h、0.5 h、1 h、3 h与6 h组与对照组差异性极显著(P0.01),12 h组与对照组差异显著(P0.05)。48 h与96 h组与对照组差异不显著(P0.05)。相关性分析发现,MPO m RNA相对表达量与血液中吡喹酮的浓度之间相关系数r=0.96,为高度相关,并且推测MPO可能参与吡喹酮的氧化代谢。结论认为:(1)MPO m RNA相对表达量的升高与外源性药物吡喹酮的摄入有关。(2)血液中吡喹酮的残留量与MPO m RNA相对表达量线性相关。本研究旨在提供一种从分子水平评价水产动物体内药物残留的新思路。     

Effects of dietary vitamin K levels on growth performance,enzyme activities and antioxidant status in the hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian)

A total of 1400 juvenile Jian carp (Cyprinus carpio var. Jian) (8.93 ± 0.03 g) were fed diets containing graded levels of vitamin K at 0.027 (basal diet), 1.52, 3.02, 4.51, 6.02 and 7.52 mg kg^?1 diet for 60 days to investigate the effects of vitamin K on growth, enzyme activities and antioxidant capacity in the hepatopancreas and intestine. Percentage weight gain (PWG), feed intake and feed efficiency of fish were improved by vitamin K. Activities of trypsin, chymotrypsin, amylase and lipase in the intestine and hepatopancreas and Na⁺, K⁺‐ATPase, creatine kinase, alkaline phosphatase and gamma‐glutamyl transpeptidase in the intestine were increased by vitamin K. Malondialdehyde and protein carbonyl contents in the hepatopancreas and intestine were decreased with vitamin K supplements. Certain level of vitamin K increased antihydroxyl radical, antisuperoxide anion, superoxide dismutase, catalase, glutathione‐S‐transferase, glutathione peroxidase and glutathione reductase activities and glutathione contents in the hepatopancreas and intestine. Intestinal Lactobacillus, E. coli and Aeromonas were changed with vitamin K supplements. Together, these results indicate that vitamin K improved fish growth, digestive and absorptive ability, and anti‐oxidant capacity. The dietary vitamin K requirement of juvenile Jian carp (8.93–73.7 g) based on PWG was 3.13 mg kg^?1 diet.     

An analysis of carbon dioxide transport in arterial and venous blood of the rainbow trout,Oncorhynchus mykiss,following exhaustive exercise

Arterial and venous cannulations were used to examine the characteristics of CO₂ transport in pre and post branchial blood both at rest and during recovery from exercise. As in previous studies, exercise caused a marked decrease in the extracellular pH (pH_e) in both arterial and venous blood. Except for a transient increase in venous blood immediately following exercise, plasma total CO₂ ([C_{CO 2}]_pl) and whole blood total CO₂ ([C_{CO 2}]_wb) decreased in both arterial and venous blood during recovery. Exercise also resulted in an increase in red blood cell total CO₂ concentration ([C_{CO 2}]_i) and in the partial pressure of CO₂ (P_{CO 2}) in both arterial and venous blood. Activation of the adrenergic mechanism at the level of the red blood cell likely contributed to the increases observed in ([C_{CO 2}]_i) following exercise. At rest, the majority of the total [C_{CO 2}] carried in arterial and venous blood could be attributed to the plasma, with 2 and 9% carried in the red blood cells, respectively. However, exercise resulted in an increase in the percentage of C0₂ carried within the red blood cell to 13.5 and 20% in arterial and venous blood, respectively. The total CO₂ difference between pre and post branchial blood also increased following exercise suggesting an increase in CO₂ excretion.     

Artificial spawning of carp (Cyprinus carpio L.); differences between the effects of reproduction in females of Hungarian, Polish and French origin treated with carp pituitary homogenate or [D-Tle6, ProNHEt9] GnRH (Lecirelin)

The investigation concerned the reproduction effects in carp females of the Hungarian strain 8, Polish strain 2 and French strain F of ovulation stimulation with carp pituitary homogenate (0.3 mg kg^?1 and after 12 h 2.7 mg kg^?1) or [D‐Tle⁶, ProNHEt⁹]GnRH (Lecirelin) with metoclopramide (15 μg kg^?1 and 10 mg kg^?1 respectively). The ovulation stimulators did not affect the weight of eggs (expressed in g and as the percentage of female body weight). However, in the group treated with pituitary homogenate (I) the least‐square means estimated for these parameters were higher than in the group treated with Lecirelin (II). A statistically significant (P≤0.05) difference in the mean percentage of living embryos after 48‐h incubation was observed between groups I and II, with the eggs obtained from females of group I being of better quality. The origin of females significantly (P≤0.05) affected the weight of eggs. The weight of eggs from females of Hungarian strain 8 was higher (P≤0.05) than the weight of eggs of the other two lines. With respect to parameters for egg quality (percentage of fertilized eggs and percentage of living embryos after 24‐, 36‐ and 48‐h incubation), no effect of the origin of females was observed. The interaction between the spawning agent and the origin of the females was not statistically significant with respect to the two parameters for the weight of the eggs. The least‐square means test for the investigated interaction showed that after the application of pituitary homogenate, the weight of eggs obtained from strains 8 and F was similar (respectively 711.2 g and 665.0 g). However, after the application of Lecirelin the females of strain 8 yielded eggs of a high weight (934.3 g) and those of strain F of a very low weight (373.2 g). A statistically significant (P≤0.05) interaction between ovulation stimulator and origin of females was recorded for the percentage of living embryos after 24‐ and 36‐h incubation. A dependence was found between the latency time and the reproduction effects.     

The conversion of plasma HCO 3 − to CO2 by rainbow trout red blood cells in vitro: adrenergic inhibition and the influence of oxygenation status

This study employed a recently developed radioisotopic assay (Wood and Perry 1991) to examine the inhibition, induced by catecholamines, of the conversion of plasma HCO ₃ ^– to CO₂ in acidotic trout blood, and the influence of oxygenation status on the response. Blood was incubated in vitro at P_{CO 2}= 2 torr, and 10^–6 M noradrenaline was employed as the adrenergic stimulus. In particular we investigated whether the inhibition of plasma HCO ₃ ^– conversion could be explained by a limited supply of H⁺s for the intracellular HCO ₃ ^– dehydration reaction because of competition by the adrenergically activated Na /H⁺ exchanger. Hypoxia (P_{O 2}= 15 torr) was employed as a tool to intensify this competition. Hypoxia raised RBC pHi, pHe, and plasma total CO₂ concentration (C_{CO 2}) by the Haldane effect, and increased the magnitude of Na⁺/H⁺ activation, expressed as the change in the transmembrane pH gradient (pHe-pHi). However hypoxia did not alter the inhibition of the conversion of plasma HCO ₃ ^– to CO₂ caused by noradrenaline. Hypoxia itself stimulated the RBC-mediated conversion of plasma HCO ₃ ^– to CO₂ by about 20% in the presence or absence of noradrenaline. The conversion rate was strongly correlated with pHe, pHe-pHi, and plasma C_{CO 2} in these experiments, but not with pHi. We conclude that adrenergically mediated inhibition in the conversion of plasma HCO ₃ ^– to CO₂ by trout RBCs is not due to competitive limitation on intracellular H⁺s, but rather to changes in the electrochemical gradient for HCO ₃ ^– entry and/or to CO₂ recycling from plasma to RBC. The deoxygenated condition helps to promote CO₂ excretion at the level of the RBC.