Unstressed Fish Research Articles

Effects of filleting method, stress, storage and season on the quality of farmed Atlantic cod (Gadus morhuaL.)

The effects of filleting method, pre-slaughter stress, storage and season on drip loss, water content, water-holding capacity, rigour contraction and colour in Atlantic cod, Gadus morhua L., were examined. A total of 182 fish were sampled under commercial conditions. To test the effects of filleting method and stress, stressed (S) and unstressed (C) fish were filleted either pre (Pr) or post rigour (Po) at 1 and 48 h post mortem, receptively. The muscle pH significantly decreased from 0 to 144 h of storage in all groups. The onset of the rigour contraction was more pronounced in the Pr-S group as compared with the Pr-C group, but after 144 h of storage, no difference in degree of contraction was observed. Filleting method, stress and storage time influenced the colour of the fillets. Post-rigour filleting caused a significantly increased in measured Lightness (L*) during storage. Stress caused a significant increase in measured redness (a*). No significant difference was found for water-holding capacity. The water content was influenced by filleting method, where the Pr-S group had a significantly lower water content compared with the Pr-C groups; water content changed also according to season. The findings of this study show that stress, filleting method, time of storage and season had a significant effect on the drip loss, where a combination of all factors will determine the total loss.

Blue mussel meal as feed attractant in rapeseed protein-based diets for turbot (Psetta maximaL.)

Antinutritional factors in rapeseed products have been identified to reduce feed palatability and growth performance of turbot. Therefore, we evaluated the potential of blue mussel (Mytilus edulis L.) meal as feed attractant in rapeseed protein-based diets for turbot. Triplicate fish groups received isonitrogenous and isocaloric diets with fish meal (FM) protein replacements of 50% or 75% by rapeseed protein concentrate (RPC 50, RPC 75). These diets were supplemented with 0%, 2%, 4% or 8% of blue mussel meal. In contrast with RPC 50 diets, fish fed with RPC 75/0 showed significantly reduced daily feed intake (DFI) and specific growth rate (SGR). With increasing mussel meal inclusion, RPC 75 diets resulted in increased DFI and SGR, suggesting mussel meal as attractant in rapeseed proteinbased diets for turbot. Feed conversion was unaffected by any treatment. Protein productive value and apparent digestibility coefficients were unaffected by either RPC or mussel meal inclusion. With regard to the whole body composition, no differences in crude protein, crude lipid and ash content could be determined. Haematological characteristics were unaffected among the treatments indicating good nourished and unstressed fish. In conclusion, we demonstrated that the utilization of blue mussel meal improved the palatability of rapeseed protein-based diets for turbot.

Retention of a transgenerational marker (137Barium) in tissues of adult female anemonefish and assessment of physiological stress

Recently it was shown that female fish injected with enriched stable isotopes maternally transmit a chemical signature to larval otoliths. Validation of this larval marking technique requires laboratory experiments to determine appropriate injection concentrations and to assess any negative effects on larval and adult condition. This study investigated the temporal profile of 137barium assimilation and retention in tissues of adult female anemonefish Amphiprion melanopus (Pomacentridae) following intraperitoneal injection with either 2 or 4 μg 137Ba g−1 body mass. Mean barium isotope ratios (138Ba:137Ba) in the two groups of treated fish were not significantly different from each other, but were significantly different from those in the control group up to 56 days post-injection. This pattern of 137Ba retention was consistent across gonad, muscle, liver and bone tissues. Mean plasma cortisol concentration (an indicator of non-specific physiological stress) was not significantly different among groups and was considered to be representative of unstressed fish. Together, these results indicate that (1) A. melanopus suffer minimal physiological stress and cope well after treatment with 137Ba, (2) 137Ba is retained in female A. melanopus for a prolonged period (at least 56 days), such that multiple clutches of offspring are likely to be marked with an isotopic signature, and (3) a lower dosage of 2 μg 137Ba g−1 appears sufficient for transgenerational marking. It is concluded that 137Ba is suitable for use as a transgenerational marker and is a powerful tool to resolve long-standing enigmas such as larval dispersal distances and the fishery benefits of marine reserves.

Expression analysis of heat shock genes in the skin, spleen and blood of common carp(Cyprinus carpio)after cadmium exposure and hypothermia

Heat shock proteins are chaperones that play a pivotal role in controling multiple regulatory pathways such as stress defense, hormone signaling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, the expression patterns of four well-known heat shock genes (hsp70, hsc70-1, hsc70-2 and hsp90α) were characterized in the skin, spleen and blood cells of the common carp, under unstressed conditions and after Cd2+ treatment or hypothermia. The examined genes were expressed in a tissue-specific manner: hsc70-2 was expressed constitutively, and was at best only slightly inducible; hsp90α exhibited a high basic expression in all three tissues, whereas hsc70-1 did so only in the blood cells, the expression of hsp70 proved to be below the level of detection in unstressed fish. Cold shock induced the expression of hsp genes in the spleen (hsp90α) and blood cells (hsp70, hsc70-1 and hsp90α), while Cd2+ treatment has no effect on the expression pattern. The highest inducibilities were detected in the skin: for hsp70 an induction of at least 20-fold after cadmium exposure, for hsc70-1 of at least 30-fold and for hsp90α of 3-fold after hypothermia.

Antagonistic effect of indigenous skin bacteria of brook charr (Salvelinus fontinalis) against Flavobacterium columnare and F. psychrophilum

Industrial fish production exposes fish to potentially stressful conditions, which in turn may induce infections by opportunistic pathogens. Probiotics appear to be a promising way to prevent opportunistic infections in aquaculture. In this study, we tested the inhibitory potential of endogenous bacterial communities found in the mucus of brook charr (Salvelinus fontinalis) against two major pathogens Flavobacterium columnare and Flavobacterium psychrophilum. Nine bacterial strains were isolated from brook charr skin mucus and tested for potential antagonistic activity. Results from both agar diffusion assays and broth co-culture assays showed the presence of antagonism. We identified seven bacterial strains, collected from unstressed fish, which exerted strong antagonism against F. psychrophilum and/or F. columnare. These strains were mixed and used to treat columnaris disease in an in vivo experiment in which four distinct fish families were tested. This treatment resulted in a decrease of mortality (54–86%) across fish families indicating that candidates from the host microbiota are potentially suitable for probiotic development. This would allow for the efficient (ability to adhere and colonize the host mucus) and durable management (antagonistic effect against pathogens which would be harmless for the host and safe for its environment) of opportunistic diseases in aquaculture.

Production Performance and Stress Tolerance of Sunshine Bass Raised on Reduced Fish Meal Feeds Containing Ethanol Yeast

AbstractEthanol yeast (EY) has been shown to be an effective alternative to fish meal (FM) in diets of sunshine bass (female white bass Morone chrysops × male striped bass M. saxatilis). Diets containing blends of FM and EY yield production performance similar or superior to that of diets containing no EY. Complete replacement of FM with EY, however, reduces feed palatability, impairs production performance, and may affect other aspects of livestock vigor. Accordingly, the objectives of this study were to determine the effects of dietary EY inclusion (with or without a palatant) on the feed intake, growth performance, and physiological robustness of sunshine bass after stressor exposure. Formulations were developed containing 15, 7.5, and 0% FM with EY as the primary alternative protein source. Experimental diets were prepared according to these formulations with and without the addition of 1% soluble canola protein concentrate as a palatant. Replicate tanks of juvenile sunshine bass (∼4.4 g) were fed the experimental feeds twice daily to apparent satiation for 9 weeks. Production performance was assessed, and then the fish were subjected to a 2‐min net‐chasing stress challenge protocol. Tissue samples were collected to allow for the determination of organosomatic indices and pre‐ and postchallenge hematology. Complete replacement of FM with EY resulted in reduced growth performance and efficiency and significantly altered whole‐body proximate composition, reducing carcass protein and increasing carcass lipid. Palatant inclusion had no effect on diet acceptance or production performance. Plasma glucose and lysozyme activity were significantly affected by stress; however, dietary treatment did not affect these parameters. While cortisol levels were marginally higher among stressed fish, they were higher than expected in unstressed fish. Our results confirm previous research suggesting that the optimal level of FM in EY‐based sunshine bass feed is between 7.5% and 15%. Further research is needed to fully understand the utilization of these feeds and their effects on the physiological status of sunshine bass.Received July 13, 2010; accepted October 1, 2010

Effects of Dietary Lipid Concentration and a Dairy–Yeast Prebiotic on Growth, Body Composition, and Survival of Stressed Goldfish Challenged with Flavobacterium columnare

AbstractPreliminary feeding trials with goldfish Carassius auratus were conducted by using four practical diets with 4% or 10% supplemental poultry fat (PF) and 0% or 2% dairy–yeast prebiotic. Total lipid in the diets ranged from 9.7% to 16.4%, which is higher than the lipid level in most commercial baitfish diets. Aquarium and pool trials were conducted to compare diet effects in the presence or absence of natural foods. In aquaria, weight gain, survival, and feed conversion were similar among diets, but whole‐body lipid and dry matter percentages were higher in goldfish that were fed 10% PF diets than in fish receiving 4% PF diets. In pools, weight gain was higher in goldfish that received the 10% PF diets or the 4% PF diet with prebiotic than in fish that were fed the 4% PF diet without prebiotic. Lipid and dry matter were higher in goldfish that were given 10% PF diets than in fish that were given 4% PF diets. The presence of natural foods in pools may partly explain the differences in results between aquaria and pools. Subsequent bacterial challenges with Flavobacterium columnare were conducted separately for fish in pools that received the 4% PF and 10% PF diets. Some groups of fish were stressed prior to exposure. Stressed goldfish that were fed the 4% PF diet with prebiotic had a survival rate similar to that of unstressed fish that were fed the basal or prebiotic diets, but stressed fish that were given the 4% PF diet without prebiotic had lower survival. Unstressed goldfish that received the 10% PF diets had higher survival than stressed fish, but there was no prebiotic effect. Preliminary results indicate that the dairy–yeast prebiotic has some potential to protect stressed goldfish against F. columnare infection, but more studies are needed to determine whether dietary lipid concentration influences the results.Received February 16, 2010; accepted January 20, 2011

Thermal impairment of reproduction is differentially expressed in maiden and repeat spawning Atlantic salmon

Groups of maiden or repeat spawning Atlantic salmon were maintained during vitellogenesis in austral autumn at either 14 °C or 22 °C through until April when all fish were transferred to a spawning temperature of 8 °C. There was no difference in body weight within groups for maidens and repeats, with repeats being consistently larger than maidens, no difference in condition factor amongst groups, but consistently higher gonad weight in repeats than maidens. Gonadosomatic index (GSI) and follicle diameter were suppressed in both maidens and repeats at 22 °C, with the effect being most marked in repeat spawners. Relative fecundity (egg kg −1) determined from ovarian tissue samples also showed depression in repeats at 22 °C. Fish from both age classes held at 22 °C had a higher proportion of atretic follicles. Plasma levels of estradiol-17β (E 2) were strongly depressed in both maidens and repeats exposed to 22 °C throughout autumn but there was some evidence of recovery amongst maiden fish by late April. A similar effect was seen on plasma testosterone (T) levels. Plasma cortisol levels were generally low and typical of levels in unstressed fish indicating that stress did not account for the inhibitory effects observed. Hepatic zona pellucida protein gene expression was significantly inhibited in both maiden and repeat spawning fish reared at 22 °C, but with some evidence of recovery after temperature reduction to 8 °C. Hepatic vitellogenin (Vtg) gene expression was also lower in both maiden and repeat spawning fish exposed to 22 °C and this was accompanied by reduced plasma Vtg levels in maidens, but not repeats at 22 °C. Maidens at 14 °C began ovulating first followed by repeats at 14 °C, then repeats at 22 °C followed by maidens at 22 °C. There was reduced fertility in maidens at 22 °C relative to both maidens and repeats at 14 °C, whereas repeats at 22 °C showed intermediate fertility between 14 °C fish and 22 °C maidens. Survival to the eyed egg stage was highest in maidens at 14 °C, significantly suppressed at 22 °C in maidens, and at intermediate levels in repeats at both temperatures. This suggests that repeat spawning Atlantic salmon may be more robust in the face of thermal insult which combined with their larger size and egg production, could make their use desirable under production situations where there was any threat of exposure to higher than normal temperature.

Non-Specific Immune Parameters in Some New Candidate Species for Mediterranean Aquaculture: Results of First Studies

The aim of this paper was to describe the distribution of non-specific immune parameters in two fish species considered as new candidates for aquaculture diversification, greater amberjack and blackspot seabream. In particular, haemolysins, haemagglutinins and lysozyme were investigated to monitor their changes induced by two different stressors: 1) an experimental challenge of greater amberjack with the bacterial pathogen Listonella (Vibrio) anguillarum, which simulated an acute stress condition, and 2) a 14-day starvation period in blackspot seabream, which simulated a chronic stress condition. For each experiment, unstressed fish were kept and sacrificed at the same time intervals to be used as controls. In greater amberjack an increase in lysozyme was detected in challenged fish compared to control ones, while haemolytic activity and haemagglutinating titres showed a depressive effect following to the challenge. In blackspot seabream, starvation resulted in a reduction of lysozyme content in the mucus and plasma, as well as of haemolytic activity. The variations measured in non-specific immune parameters suggest their use as indirect markers of stress or altered health conditions.

Effect of acute crowding stress on subsequent osmotic challenge and recovery in juvenile common carp Cyprinus carpio (Linnaeus)

Effect of acute crowding stress (density of 25 g/L for 2 and 4 h) on subsequent salinity (8 g/L) tolerance was investigated in juvenile common carp (Cyprinus carpio). To assess osmoregulatory conditions, blood cortisol, glucose, Na+ and Cl− concentrations were measured at 0, 6, 24 and 72 h after osmotic challenge in three groups: control, and groups stressed for 2 and 4 h. Results indicated typical stress responses after crowding stress. In both stressed groups, cortisol was significantly higher than the control group, but no significant difference was observed within the stressed groups. Additionally, glucose levels increased after crowding stress, and there was a significant increase in the in 4-h group compared with the 2-h group. Ion concentration was not significantly affected by crowding stress. Osmotic challenge led to an increase in cortisol and glucose concentrations in unstressed fish, after 6 h, but in stressed fish, no change occurred in cortisol and glucose levels at this point compared to the pre-challenge values. There was no significant change in cortisol and glucose values at 6, 24 and 72 h after the osmotic challenge between groups. Cortisol and glucose returned to pre-stress values by 72 h following the challenge. In all groups, Na+ concentration significantly increased at 6 h after the osmotic challenge and stayed elevated until 72 h; however, there was no significant difference between groups at any time point. There was no significant difference in Cl− concentrations between groups or sampling time points. These results indicate that the common carp is a hyperosmotic regulator after exposure to saline medium, and acute crowding stress does not impair osmotic stress response.

Effect of fasting on body composition and responses to stress in sunshine bass

The integrated responses of the hormonal regulation of growth and stress in sunshine bass ( Morone chrysops X Morone saxatilis) as regulated by feed deprivation were investigated. Groups of fish were fed 1.5% of the body weight per day or offered no feed for 4 weeks. Another group of fish was not fed for 3 weeks and feed was offered during the fourth week. Fish in each group were sampled immediately before or after a 15-min low water confinement stressor after each week of the experiment. Liver mass and liver glycogen content were decreased after one week of fasting and remained low until the end of the study. However, both recovered after a week of refeeding. Intraperitoneal fat was significantly lower after two weeks of fasting and did not recover after a week of refeeding. None of these components were affected by confinement stress. Plasma glucose in unstressed fish was generally unaffected by fasting or refeeding; however, plasma glucose increased after confinement stress in fed but not in fasted fish. The cortisol stress response was unaltered by fasting and remained robust. Plasma IGF-I generally decreased in fasted fish but was not significantly lower than fed fish until the fourth week. A week of refeeding did not restore plasma IGF-I concentrations. Plasma IGF-I concentrations were higher in confinement stressed fed fish after two and four weeks but were unchanged in the fourth week. There was no change in the plasma IGF-I concentrations in fasted or refed fish due to the stress. Liver weight and liver glycogen were essentially depleted after 2 weeks of fasting. The reduction of liver glycogen greatly reduced the glucose response to stress; however, the cortisol stress response was maintained for at least four weeks of fasting. Intraperitoneal fat was decreased very little after 4 weeks of fasting. Plasma IGF-I concentrations were reduced only after 3 weeks of fasting.

Evaluation of a Dietary Dairy–Yeast Prebiotic in Juvenile Golden Shiners in Ponds

AbstractWe evaluated the performance in ponds of juvenile golden shiners Notemigonus crysoleucas fed a control diet or a diet with 2% dairy–yeast prebiotic. Fish weighing 0.1 ± 0.001 g (mean ± SD) were initially stocked into five 0.04‐ha earthen ponds per diet at 21.9 kg/ha and fed extruded 35%‐protein pellets to satiation twice daily. Subsamples of fish were weighed at 2‐week intervals for 6 weeks; the fish were harvested at 7 weeks to avoid reproduction. For golden shiners fed the control and prebiotic diets, the mean individual weight gain (2.93 and 2.89 g, respectively), net yield (214.0 and 198.8 kg/ha), feed conversion ratio (1.34 and 1.47), and survival (61.5% and 64.2%) did not differ. Condition index (Fulton's K) was higher in fish fed the control (1.15) rather than the prebiotic diet (1.08). After harvest, 100 fish per pond were acclimated to tanks before being subjected to a bacterial challenge with Flavobacterium columnare. Each pond replicate received one of three experimental treatments: confinement stress for 30 min before F. columnare exposure (stressed), no stress before F. columnare exposure (unstressed), or no stress or exposure to F. columnare (control). Whole‐body cortisol increased in stressed fish regardless of diet. After release, fish were exposed to a virulent strain (PB02) of F. columnare for 18 h. Survival over 14 d ranged from 98.3% to 100% among fish in the control and unstressed treatments fed either the control or prebiotic diets. The stressed golden shiners fed the prebiotic diet had mortality rates similar to those of the unstressed fish fed the control or prebiotic diet, but the stressed fish fed the control diet had lower mean survival (73.3 ± 4.4%) than those fed the prebiotic diet. Preliminary economic analysis indicates that the higher cost of feed containing a 2% prebiotic may be justified based on the increased survival of golden shiners in tanks exposed to stressors and pathogens.

Effect of nutritional status and sampling intensity on recovery after dorsal aorta cannulation in free-swimming Atlantic salmon (Salmo salar L.)

Recovery from implantation of a cannula in the dorsal aorta (DA) of Atlantic salmon (Salmo salar) was studied in relation to nutritional status and sampling intensity. The incentive for the study was the inconsistency between published reports and our own experience of recovery and longevity of fish exposed to this protocol. In two studies using starved fish, blood (0.3 ml) was sampled 0, 1 and 24 h after DA-cannulation, and thereafter at 48 and 72 h and thereafter once weekly for four weeks. In a third study using fed fish, four consecutive samples (0, 3, 6 and 12 h after a meal) were obtained twice a week over a four-week period. All fish displayed a sharp increase in pCO(2) and haematocrit (Hct) during surgery, followed by a marked raise in cortisol, glucose, sodium and potassium (1 h). pCO(2), pH and Hct approached baseline levels as early as the 1 h post-surgery sample, while this was not the case for cortisol and electrolytes before the 24 h post-surgery sample. Glucose did not display any significant changes post surgery. From then on, all variables displayed minor but non-significant (P > 0.05) changes indicating a steady state close to baseline values for unstressed fish. This pattern was independent of sampling procedure, i.e. repeated single or multiple samples and thus volume of blood removed. Nutritional status (fed vs. starved) did not affect post-surgical recovery pattern. Only K(+) and Hct displayed consistent and significant post-prandial patterns. We found marked differences between baseline level of cannulated fish and uncannulated control fish, in pH, K(+) and Hct indicating that cannulation may be the preferred method to obtain representative resting values in fish.

Physiological stress responses in spotted wolffish (Anarhichas minor) subjected to acute disturbance and progressive hypoxia

The spotted wolffish (Anarhichas minor) is considered a promising species for coldwater aquaculture. It is a sedentary, bottom-dwelling fish which exhibits a calm and “non-stressed” behaviour in captivity. There are, however, no reports on the physiological responses to stressors in this species. In the present study we investigated primary (cortisol secretion) and secondary (glucose mobilization) responses to common aquaculture stressors like disturbance and hypoxia. Pre-stress plasma cortisol levels were within those generally considered representative for unstressed fish (~10ng ml−1), whereas basal glucose levels were unusually low (0.3–0.4mM). After exposure to a short, but severe disturbance challenge (emptying the tank of water for 10min), the increase in plasma cortisol level was slow and relatively weak, reaching a peak level of 25ng ml−1 4 to 8h after disturbance. When the fish were exposed to a gradually decreasing oxygen level in the tank, a significantly elevated plasma cortisol level (35ng ml−1) was seen in the fish that remained in the tank until oxygen saturation had decreased to 20% oxygen saturation after 2.5h. However, a two-fold higher plasma cortisol level (~70ng ml−1) was seen in all fish exposed to reduced oxygen levels (60, 40 and 20% oxygen saturation) after 3.5h recovery in normoxic water. Plasma glucose levels showed only moderate increases (~70%) following disturbance and hypoxia challenges. An in vivo injection of ACTH caused a strong elevation of plasma cortisol (peak level ~170ng ml−1), demonstrating a high capacity for interrenal steroidogenesis in the spotted wolffish. The slow and relatively weak cortisol response to stressors, and low plasma glucose levels, may relate to the sedentary lifestyle of the spotted wolffish. The stress-response is characterised by a passive (reactive) coping style, which is considered adaptive for farming of this species.

Haematological and Immunological Responses in Juvenile Sea Bass (Dicentrarchus labrax L.) After Short-Term Acute Stress

The physiological effects of acute stress (induced by an intraperitoneal injection) on the haematocrit, haemo- globin, serum cortisol and glucose, haemagglutinins and lysozyme were investigated in juvenile sea bass Dicentrarchus labrax. A total of 40 fish were subjected to an intraperitoneal injection. Eight fish were sampled at 0, 2, 4, 24 and 48 h af- ter acute stress and compared to unstressed fish (controls) sacrificed at the same times. Intraperitoneal injection resulted in significant changes in cortisol and glucose 4 and 2 hours after exposure to stress, respectively, but not in haematocrit and haemoglobin levels. No significant differences between stressed and control fish were recorded for lysozyme content and haemagglutinating activity throughout the experiment. In juvenile sea bass acute stress caused by intraperitoneal injection was mostly associated with the increase in common stress indicators (cortisol and glucose), while no clear response in terms of non-specific immune parameters was evidenced.

Cortisol Response to a Crowding Stress: Heritability and Association with Disease Resistance to Yersinia ruckeri in Rainbow Trout

AbstractThe U.S. Department of Agriculture's National Center for Cool and Cold Water Aquaculture is conducting a breeding program for rainbow trout Oncorhynchus mykiss to develop improved germplasm for the U.S. aquaculture industry. Current selection efforts are focused on growth and disease resistance, but stress response is also a concern. Using plasma cortisol concentrations after a 3‐h crowding stress as a measure of stress responsiveness, we have previously shown family variation in cortisol responsiveness and a positive phenotypic correlation between poststressor cortisol level and growth performance within our broodstock. Selection for improved growth performance may therefore increase cortisol responsiveness to stress. In the present studies, narrow‐sense heritability (h 2) for cortisol responsiveness was estimated to be moderately high by means of midparent–offspring regression and nested family design analyses (h 2 ≥ 0.40 and 0.56, respectively). In addition, fish from the same families were used to determine associations between cortisol responsiveness to crowding and resistance to Yersinia ruckeri, the pathogenic agent of enteric redmouth disease, in an immersion challenge. Phenotypic and breeding values for cortisol responsiveness were not correlated with survivability with respect to Y. ruckeri whether or not the fish experienced repeated daily exposure to a crowding stress before the disease challenge. Furthermore, phenotypic and breeding values for poststressor plasma glucose and chloride levels and lysozyme activity did not correlate with disease survivability in stressed and unstressed fish. Finally, there was no correlation between the survival values of families when they were stressed before the disease challenge and when they were not, suggesting that there is variability among families in the effect of stress on resistance to Y. ruckeri.

Effects of Suspended Sediment on Whole-Body Cortisol Stress Response of Two Southern Appalachian Minnows, Erimonax Monachus and Cyprinella Galactura

Abstract Total immunoreactive corticosteroid (IRC) levels (adjusted for fish mass; ng/g) were measured in whole-body homogenates of 2- and 8-months old Whitetail Shiners (Cyprinella galactura) and 4-months old federally threatened Spotfin Chubs (Erimonax monachus) exposed for 48 hours to varying suspended sediment concentrations (SSC; 0, 25, 50, 100, and 500 mg/L). Hydrophobic fractions were extracted from individual frozen fish after sonication and centrifugation of tissues. Extracts were resuspended in a buffer compatible with a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Serially diluted concentrations of cortisol and extracts collected from unstressed fish were used to standardize the assay. Two-months old C. galactura had the highest resting level of whole-body IRC at 0 mg/L SSC. They also elicited the greatest response (three- to four-fold increase) when exposed to SSCs greater than 25 mg/L. Resting whole-body IRC levels were lowest in 8-months old C. galactura. For these f...

Cortisol response and immune-related effects of Atlantic salmon ( Salmo salar Linnaeus) subjected to short- and long-term stress

It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon ( Salmo salar Linnaeus) the effects of both short- (a single 15 s out of water) and long-term (4 weeks of daily handling 15 s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1β (IL-1β) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24 h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3 h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6 h post stress. Constitutive expression of IL-1β in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3 h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1β in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1 week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1β expression in macrophages isolated from stressed fish over the first 2 weeks. At weeks 1, 2 and 3 the magnitude of IL-1β response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4 weeks there was no significant difference in inducible IL-1β expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1β, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased leukocyte survival when exposed to A. salmonicida. The implications of these changes in the immune system will be discussed with respect to the use of classical indicators of stress to predict possible effects on the immune system of fish.

Stress does not inhibit induced vitellogenesis in juvenile rainbow trout

Vitellogenin (Vtg) is a widely used biomarker for xenoestrogen exposure in male fishes. In female fishes Vtg can be negatively affected by stress independent of declines in estrogen. However, few data are available on the effect of stress in male fish abnormally producing Vtg, such as when exposed to xenoestrogens. The objective for these studies was to determine the effects of stress on fish forced to produce Vtg. Three weeks prior to the experiment immature juvenile rainbow trout, Oncorhynchus mykiss, were acclimated to the experimental tanks and fed a maintenance ration. We induced Vtg synthesis by injecting 17β-estradiol (E2) 7 days prior to experimentation. Treatments in duplicate tanks were: (1) no stressor; (2) stressor; (3) E2; (4) E2 and stressor. Plasma was collected at time = 0 for baseline measurements from eight fish per tank and Vtg was significantly elevated in treated fish compared to uninjected controls. Water was drained from the stressor tanks then refilled to a level that just covered the backs of the fish. Eight fish were sampled again at 4 and 9 h, and 1, 7, and 14 days of continuous stress. Stressor tanks were refilled with water to pre-stress levels and the fish were sampled after another 2 weeks. Cortisol was significantly elevated from the unstressed fish at 4 h; however, plasma Vtg in the E2-stimulated fish was not affected by the stressor at any timepoint. These results indicate that fish capture procedures employed in the field or caging experiments likely do not lead to false negative results when plasma Vtg is used as a biomarker for xenoestrogen exposure. It also suggests that the energetic load induced by stress is insufficient to cause a reduction in Vtg, during a continuous E2 administration, at least within the timepoints examined in this study.

Experimental handling stress as infection-facilitating factor for the goldfish ulcerative disease

Experimental handling stress (EHS) was applied to clinically asymptomatic farmed goldfish ( Carassius auratus L.). EHS affected the gills and skin integrity of the fish and was accompanied by increased levels of plasma glucose, cortisol and interleukin-10 (IL-10). EHS application was followed by highly significant enhancement of the rate of infection with a virulent Aeromonas salmonicida isolate. Cumulative ulceration at the initial phase of the ensuing goldfish ulcerative disease (GUD) evidenced a facilitating role of EHS in the onset of GUD. Host susceptibility to the pathogen increased from 40% in unstressed fish to 90% in the stressed fish. A. salmonicida could be reisolated from the early-stage skin lesions only, whereas opportunistic strains, other than A. salmonicida ( A. sobria and A. hydrophila), were recovered from progressive-stage ulcers. The implication of these findings in fish aquaculture is discussed.