Benefits of using the probiotic Efinol®L during transportation of cardinal tetra, Paracheirodon axelrodi (Schultz), in the Amazon

The objective of this experiment was to test the probiotic Efinol^®L during transportation of cardinal tetra, Paracheirodon axelrodi (Schultz). For the transportation, fish were distributed in 18 plastic tanks, of which nine contained the Efinol^®L (10 mg L⁻¹; probiotic treatment) and the remaining had no probiotic (control treatment). Transport lasted 24 h and three different boxes of each treatment were sampled at 3, 12 and 24 h. Up to the 12-h sampling period, no significant difference in the survival was observed; on the other hand, survival was higher at the end of the transport (24 h) in the probiotic treatment. No significant difference was found in dissolved oxygen and temperature between treatments. Conductivity, pH and alkalinity increased along the transport, but without a difference between treatments. Ammonia increased in all treatments, although it was significantly lower in the probiotic group at 24 h. Cortisol levels were significantly higher in all transported fish when compared with the basal values. At 24 h, cortisol levels in control fish were significantly higher than those in the probiotic treatment. With the observed results, we are able to conclude that the probiotic Efinol^®L is efficient during cardinal transport, lowering the mortality and helping maintain water quality possibly by lowering metabolic wastes.     

Effects of environmental Ca2+ levels on branchial chloride cell morphology in freshwater-adapted killifish Fundulus heteroclitus

ABSTRACT: To examine the involvement of chloride cells in the uptake of Ca²⁺ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca²⁺ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na⁺, K⁺-ATPase. Chloride cells, located mostly on the afferent–vascular edge of the filaments, were larger at lower Ca²⁺ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca²⁺ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca²⁺ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca²⁺ groups. Plasma osmolality decreased with decreasing ambient Ca²⁺ concentration, suggesting that environmental Ca²⁺ affects the permeability of the body surfaces. Gill Na⁺, K⁺-ATPase activity in the 0.1 mM and 0.5 mM Ca²⁺ groups were significantly higher than that in the 2.5 mM Ca²⁺ group, implying the involvement of the Na⁺–Ca²⁺ exchanger in Ca²⁺ uptake in the gills. These findings suggest that chloride cells function as the site for Ca²⁺ uptake in killifish acclimated to low Ca²⁺ environments.     

Metabolic costs of changing the cation–anion difference in the diet of juvenile African catfish Clarias gariepinus (Burchell)

The influence of dietary cation–anion difference (CAD, Na⁺ + K⁺– Cl^–, mEq kg^–1) on energy metabolism and nitrogen losses in juvenile African catfish Clarias gariepinus (Burchell) was examined in fish exposed to different dietary CAD levels (–146, 116, 497, 713 and 828 mEq kg^–1 diet). The experiment was conducted in open circuit balance respiration chambers over a 3-week period. Five 24-h monitoring periods were carried out at 3-day intervals during the experimental period with O₂ consumption, ammonia and nitrate + nitrite (NO_x) and CO₂ production being measured at 5-min intervals for each chamber. The negative dietary CAD (–146 mEq kg^–1) resulted in the highest energy expenditures (83 kJ kg^–0.8· d^–1). With increasing dietary CAD levels, heat loss gradually decreased to minimum values of 56 kJ kg^–0.8 day^–1 at a dietary CAD level of 713 mEq kg^–1. Consequently, metabolizable energy utilization efficiency (MEU, percentage of retained energy over metabolizable energy) quadratically ( P  < 0.05) increased and reached a maximum at a dietary CAD of 713 mEq kg^–1.     

Immunolocalization of Na+, K+-ATPase-rich cells in the gill and urinary system of Persian sturgeon, Acipenser persicus, fry

Localization of Na⁺, K⁺-ATPase-rich cells in the gill and urinary system of Acipenser persicus fry was performed through immunofluorescence light microscopy using a mouse monoclonal antibody IgGα₅ raised against the α-subunit of chicken Na⁺, K⁺-ATPase. Different types of epithelia were clearly identified in the gill epithelium: epithelia of branchial arch, interbranchial septum, filament and lamellar epithelium. The Na⁺, K⁺-ATPase-rich cells were found in the epithelia of branchial arch, interbranchial septum, filament, interlamellar region and also in the lamellae. Histologically, the urinary system is divided into head kidney, trunk kidney and short caudal kidney. The head kidney is composed of the pronephric tubules and the haemopoietic tissues, while the trunk kidney is composed of a large number of glomeruli and convoluted nephrons. Each nephron consisted of a large glomerulus and tubules (neck, proximal, distal and collecting tubules) which connected to ureters. Posteriorly, ureters extended and joined together to form a small urinary bladder. In the urinary system, no specific fluorescence staining was observed in the glomerulus, neck segment and proximal tubules. The distal tubule cells and collecting tubule cells showed a strong immunostaining of Na⁺, K⁺-ATPase. Epithelia of ureters and urinary bladder also showed several isolated immunofluorescent cells. Immunofluorescent cells were rich in Na⁺, K⁺-ATPase enzyme which is very important for osmoregulation.     

Comparison of physiological smolt status in descending and nondescending wild brown trout (Salmo trutta) in a Danish stream

Abstract –  Downstream movement of a wild population of brown trout was examined in a small Danish stream in relation to morphological and physiological smolt status from March to May. Downstream movement was monitored in a Wolf-type trap covering all possible passage routes in the stream. Trout caught in the trap were classified as parr, pre-smolt or smolt based on morphological criteria and compared with trout randomly caught by electrofishing upstream of the trap. Representative gill samples from trap-caught and electrofished trout were analysed for gill Na⁺, K⁺-ATPase activity and used as a measure of physiological smolt status. Only a few parr occurred in the trap. Few pre-smolts occurred in the trap evenly in March and early April. In late April, pre-smolt movement peaked. By comparison, the main downstream movement of smolts occurred in distinct peaks through late March and April. The majority of fish caught in the trap were judged as pre-smolts or smolts based on morphological criteria's and they were characterised by relatively high gill Na⁺, K⁺-ATPase activity compared with trout judged as parr. Trout caught by electrofishing upstream the trap, were classified as parr, pre-smolts and smolts early in the season (March). During and after the main smolt-run in April the distribution of the remaining trout in the brook became skewed in favour of pre-smolt and parr. The study suggests that smolting trout initiate downstream movement once having reached a certain physiological smolt condition (judged by increased gill Na⁺, K⁺-ATPase activity).     

Purification and characterization of the amylase from a small abalone <Emphasis Type="Italic">Haliotis sieboldii</Emphasis>

ABSTRACT:   Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, K⁺, Ag⁺, Na⁺ and Li⁺, but completely or partially inhibited by Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Zn²⁺. EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase.     

Changes in Plasma Cortisol, Glucose, and Selected Blood Properties in the Summer Flounder Paralichthys dentatus Associated with Sequential Movement to Three Experimental Conditions

To determine the changes in blood chemistry associated with sequential transfer of summer flounder Paralichthys dentatus (320–480 g), 300 hatchery-reared fish were moved to three different environmental conditions during a 20-d period. Fish were transferred in progression from a recirculating seawater system (22 ppt, 22.5 C) to a flow-thru seawater system (31 ppt, 20.0 C), to three small coastal net pens (33 ppt, 15.5 C), and finally to a large open ocean net pen (33 ppt, 16.0 C). For this study, eight random fish were captured at each progressive step (environmental condition), anesthetized (MS222), and bled from the caudal vein (2 mL). Transferred flounder were bled every 12 h for 48 h to collect plasma cortisol and glucose samples. Fish were bled 24 h after transport and every 3 d thereafter for osmolarity, hematocrit, hemoglobin concentration, mean corpuscular hemoglobin content, glucose, cortisol, and the electrolytes Cl^- Na⁺, K⁺ and Ca⁺. The most significant perturbations to blood chemistry (P < 0.05) occurred within 24 h of initial transfer from the recirculating to flow-thru seawater systems, suggesting an osmoregulatory rather than handling or transfer related stress. Osmolarity, electrolyte, and hematological parameters fluctuated and then recovered to stable levels by day 8 in the flow-thru seawater system. However, unlike the initial transfer, successive movement to the coastal and then the open ocean net pens produced transient increases in both plasma cortisol and glucose levels, suggesting a high level of stress associated with extended flounder handling and transfer.     

Effect of dietary pantothenic acid supplement on growth, body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var. Jian)

A 9-week feeding trial was carried out with juvenile Jian carp to study the effect of dietary pantothenic acid (PA) on growth, body composition and intestinal enzyme activities. Semi-purified diets with seven levels (4.0, 15.5, 25.6, 36.1, 45.9, 56.1 and 65.9 mg PA kg⁻¹) of supplemental calcium d -pantothenate were fed to Jian carp (13.0 ± 0.0 g). PA improved specific growth rate (SGR), protein productive value (PPV), protein efficiency ratio (PER) and lipid production value (LPV) ( P <  0.05). Fish fed the control diet had significantly lower feed efficiency (FE) than that in any other group ( P <  0.05). Body protein content increased with increasing PA levels ( P <  0.05), but moisture, lipid and ash of fish carcasses were negatively related to the graded PA levels ( P <  0.05). Intestine protein content (IPC), hepatopancreas protein content (HPC) and activity of α-amylase, lipase, trypsin, Na⁺,K⁺-ATPase, alkaline phosphatase (AKP) and gamma-glutamyl transpeptidase (γ-GT) were all positively affected by the dietary PA levels ( P <  0.05), while intestine index (ISI) and hepatopancreas index (HSI) decreased with the increment of supplemental levels of PA ( P <  0.05). These results suggested that PA could enhance fish growth and intestinal enzyme activities. The dietary PA requirement of juvenile Jian carp, Cyprinus carpio var . Jian (13.0–73.0 g), for optimal growth estimated by the broken-line analysis was 23.0 mg PA kg⁻¹ diet.     

Osmoregulatory changes in wedge sole (Dicologoglossa cuneata Moreau, 1881) after acclimation to different environmental salinities

The osmoregulatory responses of 20 days of acclimation to environmental salinities of 5‰, 15‰, 25‰, 35‰ and 55‰ were assessed in juveniles of wedge sole ( Dicologoglossa cuneata Moreau, 1881). This sole shows a good capacity to adapt to this range of environmental salinities. A direct linear relationship between environmental salinity and plasma osmolality was observed, with a calculated isosmotic point of 10.4‰ (284 mOsm kg⁻¹). Na⁺, K⁺-ATPase activity in the gills followed a 'U-shaped' relationship with environmental salinity, and a direct linear relationship in kidney tissue. Plasma cortisol levels were elevated in fish held in extreme salinities, and glucose levels were higher only in the group maintained at the highest environmental salinity. In the liver, a decrease in glycogen, lactate and amino acid contents was observed in specimens acclimated to extreme salinities (5‰ and 55‰), suggesting mobilization of liver metabolites. Metabolite levels in white muscle showed a pattern similar to the liver, with lower values in specimens acclimated to extreme salinities. We conclude that wedge sole is strongly euryhaline, but acclimation to extreme salinities comes with an energetic cost.     

Dietary pyridoxine requirement of juvenile Jian carp (Cyprinus carpio var. Jian)

In a 80-day feeding trial, a total of 1050 juvenile Jian carp ( Cyprinus carpio var. Jian) with an average initial weight of 10.71 ± 0.05 g were fed semi-purified diets containing seven graded levels of pyridoxine (0.20, 1.71, 3.23, 4.96, 6.32, 8.58 and 12.39 mg pyridoxine kg⁻¹ diet). Results indicated that with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet, percent weight gain (PWG) and specific growth rate (SGR) were improved, and no differences were found with further increase of pyridoxine levels. Feed intake also followed the similar pattern to that observed with PWG and SGR when dietary pyridoxine levels were ≤6.32 mg kg⁻¹ diet. But feed efficiency and protein efficiency ratio were not affected by pyridoxine levels. Crude protein of carcass, productive protein value and plasma ammonia concentration were improved with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet. Amylase activities in the intestine were improved with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet, but protease and lipase activities in the intestine were not affected by pyridoxine levels. Na⁺, K⁺-ATPase and Gamma-glutamyl transpeptidase activities in proximal intestine, mid intestine (MI) and distal intestine (DI) were lowest when fed the diet containing 1.71 mg pyridoxine kg⁻¹ diet. The alkaline phosphatase activities in MI and DI followed the same pattern. The dietary pyridoxine requirement of juvenile Jian carp based on PWG estimated by broken line model was 6.07 mg pyridoxine kg⁻¹ diet.     

Rearing conditions to develop seawater tolerance in underyearling sockeye salmon smolt

ABSTRACT:   In order to confirm the conditions required to produce underyearling smolts, juvenile sockeye salmon Oncorhynchus nerka were reared under different photoperiod and temperature conditions. The level of smoltification was assessed by plasma sodium concentration 24 h after transfer to sea water (plasma-Na), gill Na⁺,K⁺-ATPase activity (gill-ATPase), and plasma thyroxine (plasma-T4) and growth hormone (plasma-GH) concentrations. Mean fork length of all groups reached more than approximately 8.5 cm at the end of the experiment. The fish that experienced an alteration in photoperiod from short day (8:16 h light–dark cycle) to long day (16:8 h light–dark cycle) showed significant elevation in gill-ATPase from 9 to 16 µmol inorganic phosphate per milligram protein per hour (µmol Pi/mg pro/h) and reduction in plasma-Na from 194 to 154 mM. This group displayed distinct pigmentation on their fin margins. However, in groups reared under constant short day and constant long day, changes in gill-ATPase and plasma-Na occurred vaguely at each different time and pigmentation on fin margins were not observed. The observed photoperiod-induced changes were greater at 7°C than at 11°C. Further, the alteration of the photoperiod induced two–threefold increases in plasma-GH and plasma-T4. These results suggest that lengthening of photoperiod from short to long daylight at 7°C causes smoltification accompanied by active secretion of growth hormone and thyroxine in underyearling sockeye salmon when the fish are larger than 8.5 cm in fork length.     

Effects of the probiotic, Lactobacillus acidophilus, on the growth performance, haematology parameters and immunoglobulin concentration in African Catfish (Clarias gariepinus, Burchell 1822) fingerling

This experiment was carried out to evaluate the effects of the probiotic, Lactobacillus acidophilus , on the growth performance, haematology parameters and immunoglobulin concentration in African catfish Clarias gariepinus fingerling. Two experimental diets were formulated to contain 35 g kg⁻¹ crude protein and 10 g kg⁻¹ lipids accordingly and fed three times daily for 12 weeks to 25 C. gariepinus fingerlings per fibreglass tank in 12 replicates each. The control diet was prepared with no probiotic supplementation whereas the second diet was prepared supplemented with a probiotic, L. acidophilus , containing about 3.01 × 10⁷ colonies/g of diet. The results show that growth performance [specific growth rate (SGR) and relative growth rate (RGR)], nutrient utilization [protein efficiency ratio (PER) and feed conversion ratio (FCR)] and survival were significantly ( P <0.05) higher in fish maintained on the probiotic-supplemented diet compared with those on the control diet. Haematology parameters (packed cell volume, haemoglobin, erythrocyte sedimentation rate, red blood cell and white blood cell, total serum protein, Ca²⁺, Mg²⁺, Cl⁻, glucose and cholesterol) and total immunoglobulin concentrations were also significantly better in fish fed the probiotic-supplemented diet than in the control. Although the water quality parameters monitored were better in the fish fed the probiotic-supplemented diet than in the control, the parameters were not significantly different ( P >0.05). From the results of this experiment, we conclude that L. acidophilus can be used as a probiotic agent in African catfish culture, to enhance fish health, survival and better feed efficiency and growth performance.     

A rapid and inexpensive method to assay transport of short chain peptides across intestinal brush-border membrane vesicles from the European eel (Anguilla anguilla)

Membrane potential depolarization due to electrogenic peptide transport activity was examined in eel ( Anguilla anguilla ) intestinal brush-border membrane vesicles (BBMV) by monitoring the fluorescence quenching of the voltage-sensitive dye 3,3'-diethylthiadicarbocyanine iodide. Our experimental approach consisted of generating an internal negative membrane potential mimicking in vivo conditions and measuring membrane potential depolarization due to different extravesicular dipeptides. Peptide-dependent membrane potential depolarization was observed in both the presence and absence of extravesicular Na⁺ and was inhibited by diethylpyrocarbonate, which is consistent with the involvement of electrogenic, Na⁺-independent, H⁺-dependent peptide transport activity. Kinetic analysis indicated that peptide-dependent membrane potential depolarization is a saturable process ( K _m,app ∼ 1.5 mmol L⁻¹) and that within the 0.1–10 mmol L⁻¹ peptide range a single carrier system is involved in the transport process. Our results suggest that a peptide transport activity, kinetically resembling the PepT1(Slc15A1)-type-mediated H⁺/peptide cotransport action, can be monitored in eel intestinal BBMV using an easy and inexpensive fluorescence assay.     

Potential benefit of clove oil sedation on animal welfare during salmon smolt, Salmo salar L. transport and transfer to sea

The purpose of this study was to determine the effect of clove oil (4.0 mg L⁻¹) sedation, compared with non-sedation, on the primary (plasma cortisol), secondary (osmoregulation) and tertiary (mortality) stress responses in Atlantic salmon smolts during transport and transfer to sea. Clove oil sedation during on- and off-loading sufficiently reduced the primary stress response to lower mortality (2.1%) during transfer to sea compared with unsedated fish, which experienced a mortality rate above 12.2%. The unsedated fish experienced an acute mortality that stabilized only 6 days after the transport. None of the secondary stress responses measured in this experiment could contribute towards explaining this phenomenon, with the possible exception of plasma magnesium (Mg²⁺). Plasma Mg²⁺ differed between the groups; while plasma Mg²⁺ in the clove oil sedated group returned to pre-stress levels 72 h after transport, the unsedated group showed no such recovery even 1 week after transport, which may indicate a disturbance in the hydromineral balance, and provides a plausible explanation for the delayed mortality in this group. Eugenol-based anaesthetics appear to be promising as a stress-reducing sedative for Atlantic salmon smolts, and, if used properly, this chemical could improve animal welfare and survivability during and after common aquaculture-related incidents.     

Effects of Low Salinities on Oxygen Consumption of Selected Euryhaline and Stenohaline Freshwater Fish

The amount of energy required for osmoregulation depends on the difference between internal and external concentrations of ions (Rao 1968; Farmer and Beamish 1969), changes in corticosteroid hormone levels (Morgan and Iwama 1996), glomerular filtration rates (Furspan et al. 1984), gill and kidney Na⁺, K⁺-ATPase activity (McCormick et al. 1989; Morgan and Iwama 1998), tissue permeability to water and ions, and gill ventilation, perfusion, and functional surface area (Rankin and Bolis 1984). Differences in the energetic cost of osmoregulation play a significant role in the difference in growth rate between seawater-and freshwater-adapted fish (Morgan and Iwama 1991; Ron et al. 1995; Wang et al. 1997). Oxygen consumption is an indirect indicator of metabolic rate in fish (Cech 1990) and can be used to determine effects of salinity changes on energy costs.     

Seasonal variation in osmoregulatory and metabolic parameters in earthen pond-cultured gilthead sea bream Sparus auratus

Seasonal variations in osmoregulatory and metabolic parameters were assessed in juvenile gilthead sea bream ( Sparus auratus ) cultured in earthen ponds under a natural photoperiod and temperature. Specimens were sampled, and the plasma, gill, kidney and liver were collected during winter 2005 and 2006 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Plasma osmoregulatory parameters showed higher values in summer, while metabolic parameters presented different patterns of variations. Gill Na⁺,K⁺-ATPase activity decreased significantly in winter, while gill metabolite levels showed different patterns of variations among seasons. The enzymatic activities tested did not present a clear pattern of variation [(glutamate dehydrogenase (EC 1.4.1.2) (GDH) and hexokinase (EC 2.7.1.11) (HK)] or significant differences along seasons [glucose 6-phosphate dehydrogenase (EC 1.1.1.49)]. Kidney Na⁺,K⁺-ATPase activity decreased during summer and autumn. Different patterns of variation were observed in kidney metabolite levels while all the enzymatic activities assessed [lactate dehydrogenase-oxidase (EC 1.1.1.27) (LDH-O), HK and GDH] presented the highest values during summer. In the liver, metabolite levels and enzymatic activities did not show significant variations or present clear patterns of variation along different seasons. These results indicated seasonal variations in the osmoregulatory and metabolic parameters of different organs (blood, gill, kidney and liver) in earthen pond-cultured gilthead sea bream ( S. auratus ), which could be mainly attributed to seasonal changes in temperature.     

Localization of Zn-binding protein in the digestive tract tissue of common carp

ABSTRACT: The concentrations of Zn and sulfhydryl (SH) groups in the digestive tract tissue of common carp and some aquatic animals were studied. It was found that Zn and bound SH groups could be used as indicators for detecting the Zn-binding protein in the digestive tract tissue of common carp. The digestive tract tissue of the fish underwent subcellular fractionation, and it was found that the nuclei/cell debris fraction contained most of the DNA (85%), Na⁺/K⁺-ATPase (82%), organic phosphate (90%) and the Zn-binding protein (79%), but only part of the 5'-nucletidase and alkaline phosphatase (<23%). The nuclei/cell debris fraction of the digestive tract tissue of common carp was treated with either collagenase type I or type IV, and subfractionated by sucrose density centrifugation. It was found that treatment with collagenase type IV could release more than 50% of the Zn-binding protein, Na⁺/K⁺-ATPase and organic phosphate from collagen. Sections of digestive tract tissue of common carp were stained for Zn. It was observed that Zn can be found mainly on the edge of the epithelial layer, and everywhere in the 'membrane-like' portion of the submucosal and muscular layers. It is proposed that most of the Zn-binding protein in the digestive tract tissue of common carp is located on the basolateral plasma membranes of the epithelial cells and on the surrounding muscle cells that are attached to the collagen type IV of basal laminae.     

Effects of digestible protein levels in isonitrogenous diets on growth performance and tissue composition of juvenile Atractoscion nobilis

A study was undertaken to estimate the effects of isonitrogenous diets (ca. 604 g kg⁻¹ crude protein) containing formaldehyde-treated (FT) fish meal and graded levels of digestible protein (DP) (541, 491, 372, 347 and 247 g kg⁻¹) on growth performance and tissue composition of juveniles white seabass. Five diets were formulated to contain increasing levels of FT fish meal (from 0 to 384 g kg⁻¹) and decreasing levels of non-treated fish meal (from 480 to 96 g kg⁻¹). Each dietary treatment was fed in triplicate to apparent satiation to groups of 25 fish for 50 days. Significantly higher growth performance and feed conversion ratio were obtained in fish-fed diets containing 491 or 541 g kg⁻¹ DP, compared with all other treatments. Apparent digestibility coefficient of protein in the diets was not significantly affected by the inclusion of treated fish meal in the diets. Estimation of protein requirements using a broken-line regression analysis indicated that maximum weight gain would be obtained with a diet containing 503 ± 23 g kg⁻¹ DP. The results from this study suggest that a single-diet formulation using protein treated with formaldehyde as filler might be useful to estimate the requirement of DP for fish.