Stress responsiveness affects dominant-subordinate relationships in rainbow trout.

The magnitude by which plasma cortisol levels increase following exposure to a stressor is a heritable trait in rainbow trout. The relative growth in coculture of F1 lines selected for high responsiveness (HR) and low responsiveness (LR) to a confinement stressor suggested that behavioral characteristics related to food acquisition, aggression, or competitive ability might differ between the two lines. This hypothesis was tested using the F2 generation of the selected lines. The F2 lines clearly exhibited the characteristics of the F1 parents, displaying significantly divergent plasma cortisol responses to a 1-h confinement stressor and a high heritability for the trait. Behavioral differences between the lines were assessed by observing the outcome of staged fights for dominance in size-matched pairs of HR and LR fish. The identification of dominant and subordinate fish within each pair on the basis of their behavior was supported by the levels of blood cortisol in the fish attributed to each group (dominant < subordinate). Fish from the LR line were identified as dominant in significantly more trials than were HR individuals. The results suggest that behavioral attributes that affect the outcome of rank-order fights are closely linked to the magnitude of the plasma cortisol response to stress in rainbow trout. Whether the link is causal or circumstantial is not yet evident.     

Poor water quality suppresses the cortisol response of salmonid fish to handling and confinement

Confinement of brown trout in small troughs of static water for 1 h at a density of six fish 251⁻¹ stimulated the hypothalamic-pituitary-interrenal axis and resulted in an elevation of plasma cortisol from basal levels (less than 2 ng m1⁻¹) to about 100 ng m1⁻¹, the degree of stimulation being dependent upon water temperature. Confinement at a density of 30 fish 251⁻¹ resulted in a 50% suppression of this response. It is demonstrated that this effect is mediated by changes in water chemistry and not by crowding per se . Experimental manipulation of the water chemistry showed that reduced pH (7.1 → 6.3), elevated free CO₂ (63 → 520 μmoll⁻¹) or elevated ammonia (8 → 1300 μg 1⁻¹ as total ammonia nitrogen) had no individual effects on the interrenal response to acute confinement. Elevated ammonia in combination with reduced pH significantly increased the plasma cortisol levels in response to acute confinement, whereas a combination of reduced oxygen (100 → 20% saturation), elevated free CO₂ and elevated ammonia markedly suppressed (∼ 50%) the cortisol response of both brown trout and rainbow trout to acute confinement in a manner similar to that observed with trout at high densities. A compensatory increase in plasma cortisol levels was observed during the subsequent recovery of fish which had been confined for 1 h in water of poor quality. These findings are discussed in relation to the exposure of fish to multiple stresses and to the role of corticosteroids in the stress response.     

The effect of elevated blood cortisol levels on the extinction of a conditioned stress response in rainbow trout

Following previously published observations that a conditioned response (CR) was lost more quickly by rainbow trout (Oncorhynchus mykiss) exhibiting a high responsiveness to stressors than by low responding individuals this study was designed to investigate the effects of exogenous cortisol on the retention of a CR in unselected rainbow trout. Fish held in isolation were conditioned over a 10-day period by pairing an innocuous signal (conditioned stimulus, CS: a water jet played on the surface of the tank water) with a mild stressor (unconditioned stimulus, US: 30 min of confinement). This resulted in a brief elevation of plasma cortisol levels (the CR) when the fish was exposed to the CS only. The effect of exogenous cortisol on the retention of the CR was evaluated by comparing the performance of fish that received cortisol-containing slow-release intraperitoneal implants, with fish receiving vehicle-only implants. Retention of the CR was assessed at intervals up to 35 days after conditioning ceased. The CR was considered to be evident when 30 min following presentation of the CS, mean plasma cortisol levels were significantly higher in conditioned than untrained fish. On day 1 both cortisol-implanted and vehicle-implanted conditioned fish exhibited a CR. However, from day 5 onwards the CR was observed only in the vehicle-implanted and conditioned group. This finding indicates that administration of cortisol accelerated the extinction of the CR in the cortisol-implanted fish, suggesting that elevated plasma cortisol levels can impair memory processes in rainbow trout.     

Stress-induced changes in concentrations of plasma sex steroids in black bream

Cortisol levels of black bream Acanthopagrus butcheri at capture did not change with time of day, gonadal stage or season and were 1·9±0·2 and 2·8±0·4 ng ml⁻¹ for male and female fish, respectively. Confinement resulted in significantly elevated cortisol levels at all time periods; however, levels after 24 h of confinement were significantly lower than peak cortisol levels (15 min for males and 1 h for females). Confinement stress resulted in reduced levels of 17β-oestradiol (E2) and testosterone (T) within 1 h in sexually mature females. In mature males, suppression of T and 11-ketotestosterone (11KT) occurred after 30 min and 6 h of confinement, respectively. The relationship between confinement stress and levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20β P) was more complex, with levels in males being elevated after 15 min and 24 h and suppressed after 6 h of confinement. In contrast, 17, 20β P levels in females were elevated after 1 h of confinement. In regressed females, plasma E₂ and T concentrations were low at capture and were not affected by confinement stress whereas plasma 17, 20β P was elevated within 1 h. This study indicates that stress exerts a rapid inhibitory effect on gonadal steroidogenesis.     

皮质醇的周年变化与雌性虹鳟性腺成熟的关系

利用放射免疫分析法对饲养于恒定水温和自然光照下的雌性虹鳟血浆中皮质醇和性激素含量的周年变化进行了测定．结果表明：１）根据性腺结构指数和性激素分泌量判断,三龄时,雌性虹鳟达到性成熟;２）在血浆中不仅性激素而且皮质醇的变化水平与性腺结构指数的变化高度相关．排卵前性激素的水平都较高,伴随着排卵的进行性激素水平下降．而且在产卵季节虹鳟血浆中皮质醇水平也较高,三龄时皮质醇水平与性腺结构指数的相关系数为０．８６．这些结果提示,皮质醇在虹鳟的繁殖过程中可能发挥某种作用．     

Plasma cortisol concentrations before and after social stress in rainbow trout and brown trout

Two related experiments examined the relationship between plasma cortisol concentrations and the development of social hierarchies in fish. In the first, rainbow trout, Oncorhynchus mykiss, and brown trout, Salmo trutta, were observed for dominance interactions when confined within single-species pairs for 4, 48, or 168 h. Subordinate members of a pair exhibited significantly higher cortisol concentrations than dominant and single fish, but the pattern of cortisol elevation differed between the two species, being quicker to rise and increasing to a higher level in rainbow trout. Cortisol concentrations were correlated with behavioural measurements; the more subordinate the behaviour exhibited by a fish, the higher its cortisol concentration. Social stress was a chronic stressor, and no acclimation to social status occurred during the week. In the second experiment, measurements of plasma cortisol were made before pairing of rainbow trout and then after 48 h of confinement in pairs. Subordinate fish demonstrated significantly higher concentrations of plasma cortisol both before and after social stress. It therefore appears that in addition to cortisol being elevated during periods of social stress, an association may exist between initial cortisol levels and the likelihood of a fish becoming subordinate.     

Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness

The aim for this study was to examine whether the F4 generation of two strains of rainbow trout divergent in their plasma cortisol response to confinement stress (HR: high responder or LR: low responder) would also differ in stress-induced effects on forebrain concentrations of mRNA for corticotropin-releasing factor (CRF), arginine vasotocin (AVT), CRF receptor type 1 (CRF-R1), CRF receptor type 2 (CRF-R2) and AVT receptor (AVT-R). In addition, plasma cortisol concentrations, brainstem levels of monoamines and monoamine metabolites, and behaviour during confinement were monitored. The results confirm that HR and LR trout differ in their cortisol response to confinement and show that fish of these strains also differ in their behavioural response to confinement. The HR trout displayed significantly higher locomotor activity while in confinement than LR trout. Moreover, following 180 min of confinement HR fish showed significantly higher forebrain concentrations of CRF mRNA than LR fish. Also, when subjected to 30 min of confinement HR fish showed significantly lower CRF-R2 mRNA concentrations than LR fish, whereas there were no differences in CRF-R1, AVT or AVT-R mRNA expression between LR and HR fish either at 30 or 180 min of confinement. Differences in the expression of CRF and CRF-R2 mRNA may be related to the divergence in stress coping displayed by these rainbow trout strains.     

Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Krøyer) influences the physiological response to an acute stressor

The influence of infection with the juvenile stages of the sea louse, Lepeophtheirus salmonis (Kr?yer) on the response of rainbow trout Oncorhynchus mykiss (Walbaum) to a net confinement protocol was investigated. The experiment consisted of two groups of seawater-adapted rainbow trout, one which was exposed to a total of 4000 nauplii/copepodid stages of L. salmonis 30, 25 and 14 days prior to confinement. Confinement elicited a greater stress response in the lice-exposed fish, than in the controls, as seen by higher plasma cortisol and glucose levels. A reduced spleen somatic index in exposed fish following 6 h confinement coincided with increased erythrocyte and lymphocyte numbers in the blood. Circulating lymphocyte numbers were significantly reduced in both groups 24 h post-confinement, when a lower alternative complement activity was recorded in control fish. Prior to confinement, lice-exposed fish had an elevated serum lysozyme activity and reduced oxygen radical production by blood leukocytes. Following confinement, lysozyme activity was gradually reduced in lice-exposed trout. During confinement, oxygen radical production decreased in control fish and increased in infested fish. Overall, transient exposure to juvenile lice altered the response to a second stressor, which has implications for management procedures of L. salmonis exposed fish.     

Crowding causes prolonged leucopenia in salmonid fish, despite interrenal acclimation

Crowding for 3 weeks significantly reduced the coefficient of condition of both brown trout and rainbow trout. However, acclimation of the hypothalamic-pituitary-interrenal (HPI) axis, as assessed by changes in plasma cortisol levels, occurred within 6 days for brown trout and within 10 days for rainbow trout. Blood lactate levels were significantly reduced in the crowded fish of both species throughout the experiment. Sexual maturation of the male fish significantly elevated the number of circulating red blood cells in both species, reduced the lactate levels in brown trout and elevated cortisol levels in the rainbow trout. Despite the relatively rapid interrenal acclimation, the numbers of thrombocytes and lymphocytes in the blood of both species were significantly reduced during the period of crowding and it is concluded that changes in the composition of circulating blood cells are more reliable indicators of chronic crowding stress than are plasma cortisol levels. These findings are discussed in relation to the role of the HPI axis in suppressing the defence systems of salmonid fish during periods of chronic stress.     

ACTH does not mediate divergent stress responsiveness in rainbow trout.

Two lines of rainbow trout selected for high (HR) and low (LR) responsiveness to a standardised confinement stressor displayed a sustained divergence in plasma cortisol levels during a 3-h period of confinement (max.: HR: 167+/-13 ng ml(-1); LR: 103+/-8 ng ml(-1); P<0.001). However, no significant difference in plasma ACTH levels was evident (max: HR: 153+/-9 pg ml(-1); LR: 142+/-7 pg ml(-1)). Dexamethasone (DEX) was administered to HR and LR fish to block endogenous adrenocorticotropin (ACTH) release. Administration of a weight-adjusted dose of ACTH to the DEX-blocked fish elevated plasma cortisol levels to a significantly greater extent in HR (233+/-24 ng ml(-1)) than LR (122+/-14 ng ml(-1)) fish (P<0.001). Plasma cortisol levels in DEX-blocked HR and LR fish after sham injection were low but also significantly different (HR: 6.7+/-1 ng ml(-1); LR: 2.2+/-0.2 ng ml(-1); P<0.001). These results indicate that modulation of cortisol responsiveness to stressors in HR and LR fish resides, at least in part, downstream of the hypothalamic-pituitary axis.     

Primary and secondary indices of stress in the progeny of rainbow trout (Oncorhynchus mykiss) selected for high and low responsiveness to stress

A series of pooled gamete matings was carried out employing eggs and milt from mature male and female rainbow trout selected for a consistently high- or low-responsiveness to stress, as indicated by post-stress plasma cortisol elevation. Development of the progeny was closely monitored and the responsiveness to stress of the progeny of high-responding parents and the progeny of low-responding parents was assessed by two methods. For a period of 14 months, at approximately monthly intervals, the plasma cortisol elevation evoked by a standardized confinement stress was determined in fish from each group, and secondly, on one occasion, the time-course of the plasma cortisol response to a 24-h period of confinement was monitored. Progeny of high-responding parents snowed a significantly greater cortisol response to stress than the progeny of low-responding parents during both testing procedures. However, when the effect of a 14-day confinement stress was examined, high-responding progeny showed a more rapid recovery of plasma cortisol levels, while levels in the low-responding progeny, although initially lower, showed a more sustained elevation. To assess the possible functional implications of these observations, circulating lymphocyte numbers, an immunologically important cortisol-sensitive component of the blood cell complement, were determined. The duration of the lymphocytopenia observed following the onset of confinement was found to be related to the initial, not the sustained, cortisol response. These data suggest that manipulation of the sensitivity to stress of fish is feasible by selective breeding, but that careful.choice of the indices employed to identify traits considered desirable is necessary.     

Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss)

Eurasian perch (Perca fluviatilis) is a promising aquaculture candidate, but the growth performance of this non-domesticated species may be negatively affected by its stress responsiveness to intensive culture conditions. To evaluate this potential problem, juvenile Eurasian perch were exposed to a standardized handling stressor twice a week for an 8-week period. A similar study was conducted on domesticated rainbow trout (Oncorhynchus mykiss) for comparison of intra- and inter-specific differences. The stressed fish of both species showed lower body growth than the non-stressed control fish, however, the final mean body mass was 35.4% lower in the stressed Eurasian perch than in the non-stressed controls, compared to 22.8% difference between the two groups in rainbow trout. The stress responsiveness was examined by comparing the post-stress cortisol and glucose levels in repeatedly stressed fish and fish exposed to the stressor only once. The cortisol stress response in both species strongly indicated a habituation to the repeated stressor. Thus, repeatedly stressed Eurasian perch reached maximum cortisol levels of 130 ng/mL after 0.5 h compared to 200 ng/mL in the fish stressed once, while considerably smaller differences in cortisol levels were shown between the repeatedly and single stressed rainbow trout. Rainbow trout also showed lower post-stress glucose levels in the repeatedly stressed fish compared to the single stressed fish. In contrast, the glucose levels in both groups of Eurasian perch increased abruptly after stress treatment and remained elevated at approximately 19 mM for 6 h; levels were three times as high as the peak levels 3 h post-stress in rainbow trout. Together, the habituation of the stress response shown in both species did not eliminate the growth difference found in the repeatedly stressed fish versus the control fish. Further, the lower growth performance of Eurasian perch compared to rainbow trout could partly be due to the increased energy consumption in the more stress responsive Eurasian perch.     

Does socially induced stress in rainbow trout cause chloride cell proliferation?

At the end of a 2-week confinement period, subordinate rainbow trout Oncorhynchus mykiss had significantly higher circulating concentrations of plasma cortisol than did the dominant fish with which they were paired. Physiological effects linked to elevated plasma cortisol concentrations in subordinate fish included loss of weight and a lowering of condition factor. However, there were no significant differences in gill epithelium chloride cell numbers or blood plasma ion concentrations between dominant and subordinate fish. It is concluded that elevated plasma cortisol concentrations elicited by the social stressors of the present study did not cause proliferation of chloride cells.     

Relationship between stress, feeding and plasma ghrelin levels in rainbow trout, Oncorhynchus mykiss

Sexually immature rainbow trout were acclimated to small-volume (1 m³) holding tanks and then exposed to short-term stress to examine the relationship between feeding, stress, plasma ghrelin levels and other plasma stress parameters. Plasma ghrelin levels showed an increase 24 h after a single feed, plasma lactate and glucose levels decreased over the same period and plasma cortisol levels were low and constant. One hour of confinement stress resulted in elevations of plasma cortisol, glucose and lactate and depression of plasma ghrelin levels. In a separate experiment, 2 h of confinement stress also depressed feeding immediately after stress, concomitant with increases in plasma cortisol, lactate and glucose; however, in this case there was no change in plasma ghrelin concentrations. A repeat of the 2-h confinement experiment using fish that had not been acclimated to small-volume holding tanks produced a more marked elevation in plasma cortisol and a stronger suppression of feeding post-stress but in this case also, there was no change in plasma ghrelin levels. The results of this study confirm that feeding in rainbow trout is suppressed by confinement stress although the effect is transitory in this domesticated stock. Similar to that in other fishes, plasma ghrelin levels appear to be modulated by feeding status and may be influenced by stress, suggesting an orexigenic role for ghrelin in rainbow trout.     

The effect of short term confinement stress on binding characteristics of sex steroid binding protein (SBP) in female black bream (Acanthopagrus butcheri) and rainbow trout (Oncorhynchus mykiss)

The effect of short term confinement stress on sex steroid binding protein (SBP) binding characteristics was examined in female black bream (Acanthopagrus butcheri), and rainbow trout (Oncorhynchus mykiss). Black bream were sampled immediately after capture from the wild and again after 1, 6 or 24 h confinement. Rainbow trout were sampled before and after 5 h confinement. Confinement of black bream for 6 h after capture significantly reduced the binding capacity of SBP. Binding affinity also tended to be lower after confinement. There were no differences in binding affinity or capacity of black bream SBP after 1 or 24 h confinement, or rainbow trout SBP after 5 h confinement. Plasma from rainbow trout at 3 and 6 h after treatment with cortisol was compared to plasma from saline-injected controls. No significant differences in binding characteristics were detected, but there was a trend of decreased binding capacity in cortisol-injected fish compared to controls at 6 h post-injection. Relative binding studies indicated that plasma cortisol at concentrations 100x or more greater than plasma estradiol (E(2)) may displace E(2) from SBP in black bream, and act to reduce circulating levels of E(2) through increased clearance of free steroid. Physiological levels of cortisol did not displace E(2) from SBP in trout. The observed changes in SBP and the competition of physiological concentrations of cortisol for SBP binding sites may generate a component of the stress-induced falls in plasma levels of E(2) reported across a range of species.     

Extinction of a conditioned response in rainbow trout selected for high or low responsiveness to stress

Two lines of rainbow trout (Oncorhynchus mykiss) that exhibit divergent endocrine responsiveness to stressors also display disparate behavioral traits. To investigate whether the high-responding (HR) and low-responding (LR) fish also differ in cognitive function, the rate of extinction of a conditioned response was compared between the two lines. Groups of HR and LR fish were exposed to a paired conditioned stimulus (CS; water off) and unconditioned stimulus (US; confinement stressor). After exposure to 18 CS-US pairings, at least 70% of individuals of both lines acquired a conditioned response (CR) manifested as an elevation of blood cortisol levels on presentation of the CS only. Post-conditioning, the fish were tested by presentation of the CS at weekly intervals, for 4 weeks, with no further reinforcement, and the extinction of the CR in the two lines was compared. The decline in mean plasma cortisol levels after exposure to the CS over successive tests suggested that the CR was retained for a shorter period among the HR (<14 days) than LR fish (<21 days). The frequency of individuals within each line whose plasma cortisol levels indicated a stress response when exposed to the CS was significantly greater among the LR than HR fish at 14 and 21 days with no HR fish falling into this category at 21 days. At 28 days post-conditioning, there were no HR fish and only three LR fish were categorized as "stressed". These results suggest that there are differences in cognitive function between the two lines. Possible mechanisms underlying these differences are discussed.     

Effects of copper on the acute cortisol response and associated physiology in rainbow trout

The aim of this study was to determine the effects of chronic waterborne copper (Cu) exposure on the acute stress-induced cortisol response and associated physiological consequences in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 30 μg Cu/L in moderately hard water (120 mg/L as CaCO(3)) for 40 days, following which time the acute cortisol response was examined with a series of stressors. At 40 days, a 65% increase in Cu was observed in the gill, but no accumulation was observed in the liver, brain or head kidney. Stressors such as air exposure or confinement did not elicit an increase in circulating cortisol levels for Cu-exposed fish, in contrast to controls. However, this inhibitory effect on the acute cortisol response appeared to have few implications on the ability of Cu-exposed fish to maintain ion and carbohydrate homeostasis. For example, plasma Na(+), Ca(2+) and glucose levels as well as hepatic glycogen levels were the same post-stress in control and Cu-exposed fish. Trout were also challenged with exposure to 50% seawater for 48 h, where Cu-exposed trout maintained plasma Na(+), glucose and hepatic glycogen levels. However, Cu-exposed fish experienced decreased plasma K(+) levels throughout the Cu exposure and stress tests. In conclusion, chronic Cu exposure resulted in the abolition of an acute cortisol response post-stress. There was no Cu accumulation in the hypothalamus-pituitary-interrenal axis (HPI axis) suggesting this was not a direct toxic effect of Cu on the cortisol regulatory pathway. However, the lack of an acute cortisol response in Cu-exposed fish did not impair the ability of the fish to maintain ion and carbohydrate homeostasis. This effect on cortisol may be a strategy to reduce costs during the chronic stress of Cu exposure, and not endocrine disruption as a result of toxic injury.     

Behavioral and Neuroendocrine Correlates of Selection for Stress Responsiveness in Rainbow Trout--a Review

In rainbow trout the magnitude of the cortisol response to stressshows both consistency over time and a moderate to high degreeof heritability, and high responding (HR) and low responding(LR) lines of rainbow trout have been generated by individualselection for consistently high or low post-stress cortisolvalues. Using 2nd and 3rd generation fish, we tested the hypothesisthat differential stress responsiveness is associated with behavioralalterations in the HR-LR trout model. LR fish showed a tendencyto become socially dominant, a rapid recovery of food intakeafter transfer to a novel environment, and a reduced locomotorresponse in a territorial intrusion test. Furthermore, stressinduced elevation of brain stem and optic tectum concentrationsof the monoamine neurotransmitters serotonin, dopamine, andnorepinephrine and their metabolites suggests that both synthesisand metabolism of these transmitters were elevated after stressto a larger degree in HR than in LR trout. A divergent patternwas seen in the hypothalamus, where LR fish displayed elevatedlevels of 5-hydroxyindoleacetic acid (a serotonin metabolite)and 3-methoxy-4-hydroxyphenylglycol (a norepinephrine metabolite).Thus, selection for a single trait, cortisol responsiveness,in rainbow trout is associated with concurrent changes in bothbehavior and central signaling systems. The apparent parallelto genetically determined stress coping styles in mammals, andthe existence of similar trait associations in unselected populationsof rainbow trout, suggests an evolutionarily conserved correlationbetween multiple traits. Continuing studies on the HR and LRtrout lines are aimed at providing the physiological and geneticbasis for new marker-assisted selection strategies in the rapidlydeveloping finfish aquaculture industry, as well as increasedknowledge of the function and evolution of central neuroendocrinesignaling systems.     

Differences in the sensitivity of brown trout, Salmo trutta L., and rainbow trout, Salmo gairdneri Richardson, to physiological doses of cortisol

Interspecific differences in the stress response of fish may be due, in part, to differences in the sensitivity of target tissues to cortisol. The relative response of brown and rainbow trout to a standardized dose of cortisol was assessed by monitoring condition (K factor), the number of circulating lymphocytes and mortality due to disease, following cortisol treatment. Cortisol implantation resulted in a significant decline in K factor and number of circulating lymphocytes in immature brown trout, but not in immature rainbow trout, despite plasma cortisol levels being similar in both cases. Cortisol implantation in mature brown and rainbow trout significantly increased the mortality rate due to bacterial and fungal infection compared with control fish. Furthermore, the mortality rate due to disease was significantly greater in brown trout than rainbow trout, despite both groups receiving similar doses of steroid.     

Administration of cortisol to brown trout, Salmo trutta L., and its effects on the susceptibility to Saprolegnia infection and furunculosis

Oral administration and intraperitoneal implantation can be used to administer cortisol to the brown trout. Both techniques produce a dose-dependent increase in plasma cortisol concentration and the experimental procedures reduce the stress response in control fish to a minimum level. The plasma levels of the steroid are within the physiological range for the fish and the time of the response to acute oral administration is similar to that for such stresses as crowding, pollution and confinement. The evidence strongly indicates that elevated cortisol levels can significantly increase the susceptibility of salmonid fish to a wide range of infectious diseases including furunculosis and fungal infection. The present study has shown that this can occur at cortisol levels within the normal physiological range of the fish.