Diel changes in plasma cortisol and effects of size and stress duration on the cortisol response in European sea bass (Dicentrarchus labrax)

European sea bass (Dicentrarchus labrax), one of the most economically important fish in Mediterranean mariculture, shows high basal cortisol concentrations compared with other teleosts. The present study aims (a) to identify cortisol diel variation in fish held under a 12L:12D cycle and minimum handling stress, and (b) to establish the effect of fish size and stressor duration on the cortisol response. The results indicate high intrapopulation variability in plasma cortisol and a significant diel fluctuation with a peak value at dusk (18 h). Stressors of different intensity and/or duration affected the cortisol stress response in a differential manner according to fish size (and/or age). Maximum cortisol values in small-size fish were found at 1 and 2 h post-stress, depending on the duration of the stressor, while at 0.5 h post-stress in large fish regardless stress duration.     

Effects of dietary inclusions of red beet and betaine on the acute stress response and muscle lipid peroxidation in rainbow trout

This study evaluates the effects of red beet (RB) and betaine on rainbow trout submitted to an acute stress challenge. A control diet was compared with four experimental diets in which red beet (14 and 28%) and betaine (0.9 and 1.63%) were incorporated in different concentrations according to a factorial design. Cortisol in plasma and fin, glucose and lactate plasma levels, and malondialdehide (MDA) in muscle were all measured before the stress challenge and 30 min and 6 and 12 h after the stress challenge as parameters to determine the diet effects. RB and betaine had no effect on cortisol, glucose, and MDA basal levels. However, lactate basal levels were significantly lower on fish fed with RB and betaine. Thirty minutes after the stress challenge, there was a significant increase in plasma and fin cortisol, glucose and lactate concentrations, although fish fed with diets containing RB and betaine showed significantly higher plasma cortisol values. MDA values of fish fed with 14% RB and 0.9% betaine were significantly higher than MDA values from fish fed with 28% RB and 1.63% betaine. After 6 and 12 h, plasma and fin cortisol and lactate levels recovered in a similar trend. Glucose plasma levels recovered in almost all groups 12 h after the stress. Also, MDA values recovered basal levels after 6 and 12 h. RB and betaine did not enhance the tolerance to the stress challenge compared to the control group, although the presence of these ingredients had no negative effect on any of the stress indicators.     

Effect of blood glucose level on acute stress response of grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

Stress has a considerable impact on welfare and productivity of fish, and blood glucose level of fish may be a factor modulating stress response. This study evaluated the effect of blood glucose level and handling on acute stress response of grass carp Ctenopharyngodon idella. Fish were intraperitoneally injected with glucose at 0, 0.2, 0.5, and 1.0 mg g^?1 body mass (BM) and then were exposed to handling for 5 min. Glucose injection resulted in increase of plasma glucose level and liver glycogen content and decrease of plasma lactate level. Handling resulted in increase of plasma levels of cortisol, glucose, and lactate and plasma lactic dehydrogenase (LDH) activity and decrease of liver glycogen content. At 1 h post-stress, the plasma cortisol level was lower in the stressed fish injected with glucose at 0.5 mg g^?1 BM than the stressed fish injected with glucose at 0, 0.2, and 1.0 mg g^?1 BM. No significant differences were found in the activities of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase (PK) in the liver between the stressed and unstressed fish, regardless of the dose of glucose injection. At 1 h post-stress, the liver glucose-6-phosphatase (G6Pase) activity was higher in the fish without glucose injection than in the fish injected with glucose. This study reveals that blood glucose level can affect stress response of grass carp by modulating cortisol release and glucose homeostasis through glycogen metabolism and gluconeogenesis in the liver.     

Validation of a whole-body cortisol extraction procedure for channel catfish (<Emphasis Type="Italic">Ictalurus punctatus</Emphasis>) fry

We validated a whole-body cortisol extraction technique for channel catfish, Ictalurus punctatus, fry. Three volume enhancement methods were tested: CAL method (zero calibrator A diluent added to lipid extract), PBS method (phosphate buffered saline added to lipid extract), and VO method (food grade vegetable oil added to lipid extract). The volume enhancement extracts were evaluated using a commercial radioimmunoassay kit. Sensitivity, accuracy, precision, reproducibility, and parallelism could not be determined for the PBS method as cortisol levels were not detected in any of the extracted samples. Intra-assay coefficient of variation (CV) for the CAL and VO methods were 7.3 and 8.3%, respectively, while inter-assay CV were 9.6 and 10.6%, respectively. Based on the sensitivity, accuracy, precision, reproducibility, and parallelism results, we conclude that the CAL method is the most appropriate method for volume enhancement of catfish fry lipid extract. Using the CAL method to detect cortisol in catfish fry, fish were stressed daily for 2 weeks. Fry weights were similar throughout the study while whole-body cortisol levels were higher (P < 0.01) in stressed fish after 1 day of stress. These data show the CAL method can effectively measure whole-body cortisol in catfish fry.     

Development of a label-free immunosensor system for detecting plasma cortisol levels in fish

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. In the present study, we describe a novel label-free immunosensor for detecting plasma cortisol levels. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry. For the immobilization of the antibody on the surface of sensing electrode, we used a self-assembled monolayer of thiol-containing compounds. Using this electrode, we detect the CV signal change caused by the generation of antigen–antibody complex. The immunosensor showed a response to cortisol levels, and the anodic peak value linearly decreased with a correlation coefficient of 0.990 in diluted plasma. The specificity of the label-free immunosensor system was investigated using other steroid hormones, such as 17α, 20β-dihydroxy-4-pregnen-3-one, progesterone, estriol, estradiol, and testosterone. The specific detection of cortisol was suggested by a minimal change from ?0.32 to 0.51 μA in the anodic peak value of the other steroid hormones. The sensor system was used to determine the plasma cortisol levels in Nile tilapia (Oreochromis niloticus), and the results were compared with those of the same samples determined using the conventional method (ELISA). A good correlation was obtained between values determined using both methods (correlation coefficient 0.993). These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish plasma samples.     

Effects of hormonal manipulation on stress responses in male and female broodstocks of pikeperch Sander lucioperca

This study was designed to determine the effects of hormonal manipulation on stress responses in female and male pikeperch. Two-year-old cultured female and male broodstocks with an average weight of 337.4 ± 20.1 (mean ± SE; n = 16) and 318.7 ± 15.1 g (n = 16), respectively, were randomly allocated into four hormonal treatments each containing 4 fish. Two sexual groups of 16 fish for each gender were considered. Sexually mature male and female pikeperch were injected with either physiological saline solution (as control group), common carp pituitary extract (CPE), human chorionic gonadotropin (hCG) and or luteinizing hormone-releasing hormone analog (LHRHa₂). The blood samples were taken before hormonal injection and after ovulation and spermiation. Then the plasma levels of stress indices (cortisol, glucose, and lactate) were determined. The results showed that all CPE-, HCG-, and LHRHa_2- injected males produced sperm. In females treated with CPE and hCG, three of four ovulated, but none of LHRHa_2- and saline-injected fish spawned. Significant changes in cortisol, glucose, and lactate levels were observed among the females injected with different hormones. Plasma cortisol and glucose levels increased significantly in males injected with CPE and females injected with hCG, but no significant change was observed in lactate levels before and after hormonal induction. Comparison of two sexes revealed significant differences in glucose levels for females in some groups before injection, while CPE-injected sexes showed significant changes in cortisol and lactate concentrations. The results indicated that the induction of ovulation or spermiation stimulated stress responses especially in female pikeperch, and therefore, all the procedures should be made to minimize the disturbance during the artificial spawning.     

Endocrine (plasma cortisol and glucose) and behavioral (locomotor and self-feeding activity) circadian rhythms in Senegalese sole (Solea senegalensis Kaup 1858) exposed to light/dark cycles or constant light

The existence of daily rhythms under light/dark (LD) cycles in plasma cortisol, blood glucose and locomotor and self-feeding activities, as well as their persistence (circadian nature) under constant light (LL), was investigated in Senegalese sole (Solea senegalensis). For the cortisol and glucose rhythms study, 48 soles were equally distributed in 8 tanks and exposed to a 12:12 LD cycle and natural water temperature (experiment 1). After an acclimation period, blood was sampled every 3 h until a 24-h cycle was completed. Blood glucose levels were measured immediately after sampling, while plasma cortisol was measured later by ELISA. In experiment 2, the fish were exposed to LL for 11 days, and after this period, the same sampling procedure was repeated. For the study of locomotor and self-feeding rhythms (experiment 3), two groups of sole were used: one exposed to LD and the other to LL. Each group was distributed within 3 tanks equipped with infrared photocells for the record of locomotor activity, and self-feeders for feeding behavior characterization. The results revealed a marked oscillation in cortisol concentrations during the daily cycle under LD, with a peak (35.65 ± 3.14 ng/ml) in the afternoon (15:00 h) and very low levels during the night (5.30 ± 1.09 ng/ml). This cortisol rhythm persisted under LL conditions, with lower values (mean cortisol concentration = 7.12 ± 1.11 ng/ml) and with the peak shifted by 3 h. Both rhythms were confirmed by COSINOR analysis (p < 0.05). The synchronizing role of temperature and feeding schedule, in addition to light, is also discussed. Diel rhythms of glucose were not evident in LD or LL. As to locomotor and self-feeding activity, a very marked rhythm was observed under LD, with higher activity observed during the night, with acrophases located at 2:14 and 3:37 h, respectively. The statistical significance of daily rhythms was confirmed by COSINOR analysis. Under LL, both feeding and locomotor rhythms persisted, with an endogenous period (τ) around 22.5 h. In short, our findings described for the first time the existence of circadian cortisol and behavioral circadian rhythms in flat fish. Such results revealed the importance of taking into account the time of day when assessing stress responses and evaluating physiological indicators of stress in fish.     

Ontogeny of the cortisol stress response in yellow perch (Perca flavescens)

The ontogeny of the cortisol stress response in yellow perch (Perca flavescens) was documented by measuring resting and stress-induced whole-body cortisol levels in fish at several early development stages. In newly fertilized eggs resting cortisol levels averaged 41.0 ± 3.0 pg/embryo, and decreased to 8.0 ± 3.0 pg/embryo by day 6 where they remained constant until hatch on day 10. Acute stress challenge tests (30 sec cold shock) conducted on embryonic (days 6 and 8 post-fertilization) and larval (hatch and 2 days post-hatch) yellow perch revealed no differences in whole-body cortisol levels between stressed and unstressed individuals. In acute stress challenge tests (30 sec net confinement) conducted on post-hatch yellow perch, differences in cortisol levels between stressed (30 min post-stress) and unstressed fish were detected one week after hatching (12.3 ± 1.6 ng/g and 7.0 ± 1.3 ng/g, respectively). The magnitude of the stress response was greater in 2- to 5-week-old fish than in one-week-old fish. Changes in post-stress cortisol levels were closely associated with changes in interrenal morphology. Cumulatively, these results indicate that cortisol biosynthesis, integration and maturation of the hypothalamic-pituitary-interrenal (HPI) axis can be observed in yellow perch as early as one week after hatching. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neuropeptides isotocin and arginine vasotocin in urophysis of three fish species

In this study, for the first time, both neuropeptides isotocin (IT) and arginine vasotocin (AVT) have been identified and measured in urophysis, the neurohaemal organ of the caudal neurosecretory system of teleost fish. So far, AVT, but not IT, was quantified by radioimmunoassay (RIA) in urophysis of several fish species. We have used high-performance liquid chromatographic assay with fluorescence detection (HPLC-FL) preceded by solid-phase extraction (SPE) and liquid chromatography-electrospray ionization triple-quadrupole tandem mass spectrometry (LC-ESI MS/MS) technique to determine both neuropeptides in urophysis of three fish species. The efficiency of peptide’s SPE extraction was 79–85 %. In HPLC-FL method, the limits of detection (LOD) and quantification (LOQ) were estimated as 1.0 and 3.4 pmol/mL for IT and 0.25 and 2.20 pmol/mL for AVT. In LC–MS/MS method, LOD and LOQ were estimated as 0.4 and 1.2 pmol/mL for IT and 0.06 and 0.2 pmol/mL for AVT. The chromatographic methods are good alternative for RIA, because enable to measure both nonapeptides simultaneously in one sample. In round goby (Neogobius melanostomus), three-spined stickleback (Gasterosteus aculeatus) and sea bream (Sparus aurata), urophysial IT concentrations ranged between 0.056 and 0.678 pmol/mg tissue and AVT concentrations ranged between 0.0008 (or even below detection threshold) and 0.084 pmol/mg tissue.     

Effects of surgically implanted dummy ultrasonic transmitters on physiological response of bighead carp Hypophthalmichthys nobilis

The study assessed the effects of surgically implanted dummy ultrasonic transmitters on physiological response of bighead carp Hypophthalmichthys nobilis in April 2011. Before the surgery, 15 blood samples were extracted randomly from 195 bighead carp samples, and then the rest of the fish were divided into three groups: (1) control group, handing but no tagging, (2) sham group, surgical procedure without implantation of transmitter and (3) surgery group, surgical implantation of transmitters. In 3 h, 24 h, 7 days and 14 days after surgery, 15 fish were extracted randomly from the three groups, respectively, for sampling. Then the plasma samples were analyzed, and physiological measures of stress response (cortisol, glucose), tissue damage [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] and nutritional status [total protein, globulin, albumin, triglyceride, cholesterol, and alkaline phosphatase (ALP)] were compared. The result showed that there was no significant difference between sham and surgery groups in 3 h, 24 h, 7 days and 14 days after surgery. When compared to the control group, there were significant increases in concentrations of plasma cortisol, glucose, ALT, AST, total protein and globulin of sham and surgery groups in 3 h after surgery. After 24 h, the levels of plasma cortisol, ALT, AST, total protein, globulin and ALP were elevated in both sham and surgery groups, whereas the levels of plasma glucose had declined to normal level and plasma albumin, cholesterol and triglyceride were significantly decreased in both sham and surgery groups. After 7 days, the levels of plasma glucose, albumin and cholesterol continued to decline, while the level of plasma ALT, globulin and ALP had declined but still remained higher for sham and surgery groups than control group; however, the plasma total protein level had returned to normal. After 14 days, there was no significant difference between the three groups. The above results showed that surgical implantation of ultrasonic transmitters had indeed caused significantly negative effects on the physiological response of bighead carp. However, all the negative influences on stress response, tissue damage and nutritional status had disappeared in 14 days after surgery, and the presence of transmitters had no significant negative impact. Therefore, the bighead carp should be temporary cultured at least 14 days for the recovery of physiological response and then released into the free water for the ultrasonic telemetry research.     

The effect of allostatic load on hypothalamic–pituitary–interrenal (HPI) axis before and after secondary vaccination in Atlantic salmon postsmolts (Salmo salar L.)

The experiment consisted of three experimental groups: (1) “vaccine and stress”, (2) “stress and vaccine” and (3) control. All groups have previously been vaccinated 6 months prior to the start of the experiment. At the start of the experiment, the “vaccine and stress” group was vaccinated with Pentium Forte Plus for the second time (25.02.2008) and then given a daily stressor (confinement stressor 267 kg m-3 in 15 min) for a period of 4 weeks. The “stress and vaccine” group was given a similar daily stressor for 4 weeks and then vaccinated for the second time. The control group was neither stressed nor vaccinated a second time. The results indicates that fish in the “stress and vaccine” group may have entered an allostatic overload type 2 due to oversensitivity to ACTH, a reduced efficient negative feedback system with elevated baseline levels of plasma cortisol and reduced immune response with pronounced effects on the well-being of the animal. The “vaccine and stress” group may likewise have entered an allostatic overload type 1 response, with oversensitivity to ACTH and transient reduced efficient negative feedback system. This study shows that if plasma cortisol becomes elevated prior to vaccination, it could perhaps instigate an allostatic overload type 2 with dire consequences on animal welfare. To reduce the risk of compromising the animal welfare during commercial vaccination of salmon, one propose to grade the fish minimum a week prior to vaccination or grade simultaneously with vaccination. This could reduce the overall allostatic load during handling and vaccination and secure a healthy fish with intact immune response and improved animal welfare.     

Stress responses and disease resistance in salmonid fish: Effects of chronic elevation of plasma cortisol

Basal levels of plasma cortisol in unstressed salmonid fish are normally in the range 0–5 ng ml⁻¹. An acute stress such as handling or 1 h confinement caused a temporary elevation of the plasma cortisol levels of both brown trout,Salmo trutta L., and rainbow trout,Salmo gairdneri Richardson, in the range 40–200 ng ml⁻¹ with a return to basal levels within 24–48 h. The extent of the cortisol elevation in response to an acute stress was dependent upon both the species and strain of trout. Chronic stresses, such as prolonged confinement or crowding, resulted in an elevation of plasma cortisol levels to approximately 10 ng ml⁻¹. Under these circumstances, blood cortisol levels remained elevated for periods of up to 4 weeks before acclimation finally occurred. It is shown, by means of intraperitoneal implantation of cortisol, that chronic elevation of plasma cortisol levels in the brown trout results in a dose-dependent increase in mortality due to common bacterial and fungal diseases. This effect is apparent at plasma cortisol levels as low as 10 ng ml⁻¹, levels below those often reported as being representative of ‘unstressed’ fish. These findings are discussed in relation to the known immunosuppressive effects of corticosteroids in teleost fish.     

Physiological response,blood chemistry profile and mucus secretion of red sea bream (<Emphasis Type="Italic">Pagrus major</Emphasis>) fed diets supplemented with <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> under low salinity stress

Environmental stressors caused by inadequate aquaculture management strategies suppress the immune response of fish and make them more susceptible to diseases. Therefore, efforts have been made to relieve stress in fish by using various functional feed additives in the diet, including probiotics. The present work evaluates the effects of Lactobacillus rhamnosus (LR) on physiological stress response, blood chemistry and mucus secretion of red sea bream (Pagrus major) under low salinity stress. Fish were fed four diets supplemented with LR at [0 (LR0), 1 × 10² (LR1), 1 × 10⁴ (LR2) and 1 × 10⁶ (LR3) cells g^?1] for 56 days. Before stress, blood cortisol, urea nitrogen (BUN) and total bilirubin (T-BIL) showed no significant difference (P > 0.05), whereas plasma glucose and triglyceride (TG) of fish-fed LR2 and LR3 diets were significantly lower (P < 0.05) than those of the other groups. Plasma total cholesterol (T-CHO) of fish-fed LR3 diet was significantly (P < 0.05) lower than that of the other groups. Furthermore, total plasma protein, mucus myeloperoxidase activity and the amount of mucus secretion were significantly enhanced in LR-supplemented groups when compared with the control group (P < 0.05). After the application of the low salinity stress test, plasma cortisol, glucose, T-CHO and TG contents in all groups showed an increased trend significantly (P < 0.01) compared to the fish before the stress challenge. However, plasma total protein and the amount of secreted mucus showed a decreased trend in all groups. On the other hand, BUN, T-BIL and mucus myeloperoxidase activity showed no significant difference after exposure to the low salinity stress (P > 0.05). In addition, the fish that received LR-supplemented diets showed significantly higher tolerance against low salinity stress than the fish-fed LR-free diet (P < 0.05). The physiological status and the detected immune responses, including total plasma protein and mucus myeloperoxidase activity in red sea bream, will provide a more comprehensive outlook of the effects of probiotics to relieve stress in fish.     

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R ²: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R ²: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.     

Effects of acidic water,aluminum, and manganese on testicular steroidogenesis in <Emphasis Type="Italic">Astyanax altiparanae</Emphasis>

Metals can influence the gonadal steroidogenesis and endocrine systems of fish, thereby affecting their reproduction. The effects of aluminum and manganese in acidic water were investigated on steroidogenesis in sexually mature male Astyanax altiparanae. Whether mature male fish recover from the effects of metals in metal-free water was also assessed. The fish were exposed to 0.5 mg L^?1 of isolated or combined aluminum and manganese in acidic pH (5.5) to keep the metals bioavailable. The fish underwent 96 h of acute exposure, and samples were taken 24 and 96 h after the beginning of the experiment. The fish were then maintained in metal-free water for 96 h. Plasma levels of testosterone, 11-ketotestosterone, 17β-estradiol, and cortisol were measured. Acidic water increased the plasma concentration of testosterone and 11-ketotestosterone. Aluminum increased the testosterone levels after 96 h of exposure. Manganese increased the 17β-estradiol levels after 24 h of exposure and maintained at high levels until the end of the experiment. With the exception of acidic pH, which increased cortisol levels after 24 h of exposure, no changes were observed in this corticosteroid during the acute experiment. Aluminum and manganese together also altered steroid levels but without a standard variation. The fish recovered from the effects of most exposure conditions after 96 h in metal-free water. A. altiparanae could use reproductive tactics to trigger changes in testicular steroidogenesis by accelerating spermatogenesis and spermiogenesis, which may interfere with their reproductive dynamics.     

Stress in African catfish (Clarias gariepinus) following overland transportation

Of the many stressors in aquaculture, transportation of fish has remained poorly studied. The objective of this study was therefore to assess the effects of a (simulated) commercial transportation on stress physiology of market-size African catfish (Clarias gariepinus). Catfish weighing approximately 1.25 kg were returned to the farm after 3 h of truck-transportation, and stress-related parameters were measured for up to 72 h following return. Recovery from transportation was assessed through blood samples measuring plasma cortisol, glucose and non-esterified fatty acids (NEFA) and gill histology. Also, the number of skin lesions was compared before and after transport. Pre-transport handling and sorting elevated plasma cortisol levels compared to unhandled animals (before fasting). Plasma cortisol levels were further increased due to transportation. In control fish, plasma cortisol levels returned to baseline values within 6 h, whereas it took 48 h to reach baseline values in transported catfish. Plasma glucose and NEFA levels remained stable and were similar across all groups. Transported catfish did not, on average, have more skin lesions than the handling group, but the number of skin lesions had increased compared to unhandled animals. The macroscopic condition of the gills was similar in control, transported and unhandled catfish; however, light microscopy and immunohistochemistry revealed atypical morphology and chloride cell migration normally associated with adverse water conditions. From our data, we conclude that transportation may be considered a strong stressor to catfish that may add to other stressors and thus inflict upon the welfare of the fish.     

Effects of tank color on the growth,stress responses,and skin color of snakeskin gourami (<Emphasis Type="Italic">Trichogaster pectoralis</Emphasis>)

The effects of tank color on the growth, stress responses, and skin color of snakeskin gourami (Trichogaster pectoralis) were investigated in this study. Fish with initial body weights of 5.03±0.00 g were reared in five experimental tank colors (white, red, green, blue, and black) for 8 weeks. Each tank color was tested in triplicate with an initial stocking density of 15 fish per tank. Fish were fed with commercial sinking pellets at 4% of the average body weight per day. Growth performance, feed utilization efficiency, stress indicators (hematocrit, blood glucose, plasma cortisol levels), and skin color parameters were investigated. The fish reared in blue tanks had a significantly higher average final body weight (9.73?±?0.14 g) and significantly lower average feed conversion ratio (3.42?±?0.12) than the fish reared in black tanks (P?<?0.05). The fish reared in black tanks exhibited higher average hematocrit (36.63?±?1.11%), blood glucose (48.33?±?1.45 mg dL^?1), and plasma cortisol (9.00?±?0.56 μg dL^?1) levels than those reared in the other tank colors. However, the blood glucose levels in only the fish reared in black tanks were significantly higher than those in the fish reared in the other tank colors. The fish skin color ranged from very pale (high skin lightness) in the white tanks to very dark (low skin lightness) in the black tanks, and 80% of the variation in skin lightness were explained by the tank lightness. The use of a blue tank resulted in normal skin color; hence, blue tanks will not affect the customer acceptance of the fish. Our study revealed that blue is the most appropriate tank color for culturing snakeskin gourami.     

Physiological responses associated with capture and crowding stress in matrinxãBrycon cephalus (Gunther, 1869)

The dynamics of plasma cortisol, blood glucose, plasma chloride and liver glycogen were investigated in matrinxã (Brycon cephalus) submitted to capture and various periods of crowding. A total of 400 fish (700±22 g weight) were distributed in four ponds divided into four 50‐m² squares (25 fish/square, 350 g L^?1), where they were acclimated for 30 days. On the sampling day, after 24 h without food, all fish from three squares were transferred to the fourth square. Six fish were sampled before the procedure (control group, zero time) and 1, 3, 6 and 24 h after the capture and crowding. Each sampling was performed in a different pond to prevent additional stress. Fish were anaesthetized and blood and liver collected for biochemical analysis. Water temperature, pH, dissolved oxygen, alkalinity, ammonia and nitrite levels were within acceptable levels for matrinxã rearing. Slight but not significant increases were verified in plasma cortisol and blood glucose levels, as were decreases in plasma chloride and liver glycogen levels. The results suggest that matrinxã is highly tolerant to the procedures of capture and short‐term crowding.     

Vessel noise pollution as a human threat to fish: assessment of the stress response in gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>, Linnaeus 1758)

This study examined the effects of boat noise pollution on the stress indices of gilthead sea bream (Sparus aurata, Linnaeus 1758). To assess the stress response in these fish, biometric values and plasma parameters such as ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, total protein, cholesterol, triglycerides and osmolarity were analysed. After acclimatization of the animals, the experiment was carried out in a tank fitted with underwater speakers where the fish were exposed to sound treatments (in duplicate) consisting of: 10 days of no sound (control treatment; the animals were only exposed to the experimental tank’s background noise) and 10 days of noise derived from original recordings of motor boats, including recreational boats, hydrofoil, fishing boat and ferry boat (vessel noise treatment). The exposure to noise produced significant variations in almost all the plasma parameters assessed, but no differences were observed in weights and fork lengths. A PERMANOVA analysis highlighted significantly increased values (p < 0.05) of ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, cholesterol, triglycerides and osmolarity in the fish exposed to vessel noise for 10 days. This study clearly highlights that anthropogenic noise negatively affects fish, and they are valuable targets for detailed investigations into the effects of this global pollutant. Finally, these experimental studies could represent part of the science that is able to improve the quality of the policies related to management plans for maritime spaces (Marine Strategy Framework Directive 56/2008 CE) that are aimed at stemming this pollutant phenomenon.     

Influence of immunostimulant polysaccharides,nucleic acids,and <Emphasis Type="Italic">Bacillus</Emphasis> strains on the innate immune and acute stress response in turbots (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>) fed soy bean- and wheat-based diets

Immunostimulants are widely applied in aquaculture practice and may have beneficial effects on the immune system and physical functions allowing higher tolerance to stress. In the current study, the impact of four (i–iv) dietary active ingredients on the immune and stress response of turbot was examined in two experiments (I and II). A basal low fish meal (FM; 32%) diet was formulated and supplemented with (i) yeast β-glucan and mannan oligosaccharide (GM), (ii) alginic acid (AC), (iii) yeast nucleotides and RNA (NR), or (iv) Bacillus strains (BS). The basal diet (C-LF) and a high FM (59%) control (C-HF) were maintained. All six diets were fed to juvenile turbots for 84 days in experiment I and for additional 28 days prior to experiment II. Immunological and hematological parameters were determined in experiment I. In experiment II, physical stress response to a typical short-term (<1 day) aquaculture handling procedure (combination of capture, netting/transfer, and crowding) was investigated. For this, turbot blood was sampled before and at 0.5, 1, 4, and 24 h post stress. Plasma lysozyme activity, neutrophil reactive oxygen species (ROS) production, and total plasma protein levels did not significantly differ between treatment groups; however, plasma cholesterol increased significantly in fish fed GM, AC, NR, and C-HF compared to C-LF (I). A significant increase in plasma glucose and triglyceride was observed in GM and NR treatments, while glucose levels were significantly higher in C-HF compared to C-LF. Moreover, the immunostimulant-supplemented diets exhibited significantly lower cortisol levels compared to controls C-LF (at 0.5 h) and C-HF (at 1 h) post stress, respectively (II). According to our findings, FM substitution did not modulate the innate immune response but was associated with reduced levels of cholesterol. Dietary immunostimulants were not effective enough to boost the immune response, but we believe they might be helpful to trigger metabolic advantages during stressful handling events on fish farms.