A comparison of plasma glucose and plasma cortisol as selection markers for high and low stress-responsiveness in female rainbow trout

Two groups of female rainbow trout displaying consistently divergent plasma cortisol responses to a 3 h period of confinement were identified following five separate confinement episodes at monthly intervals. High-responding (HRC) and low-responding individuals (LRC) continued to display divergent cortisol responses to confinement up to 21 months after the start of the study (342 ± 34 ng ml-1 and 208 ± 21 ng ml-1 respectively at the final sample; p<0.01). HRC fish were significantly larger than LRC fish throughout the study period (533 ± 13 g and 422 ± 10 g respectively overall; p<0.001), although significant differences in specific growth rate (SGR) were apparent only at the start of the study. Individual fish were also selected from the same population on the basis of their plasma glucose levels following confinement (HRG: 189 ± 6; LRG 121 ± 3 mg dl-1; p<0.001). However, the two selection traits (cortisol and glucose) identified separate subsets of the experimental population. HRG fish were also significantly larger than LRG fish although this difference was not so pronounced as for the cortisol-selected fish. There was no reciprocal relationship between body weight and stress responsiveness; fish selected from the population on the basis of high or low body weight displayed no divergence in either cortisol or glucose responses to confinement. Differences in size and SGR may indicate that HR fish adapted more rapidly to changes in environmental and social factors at the start of the study than LR fish did

Modification of the plasma cortisol response to stress in rainbow trout by selective breeding

Male and female rainbow trout were segregated into high- and low-responding individuals (HR, LR) on the basis of their plasma cortisol response to a 3 h period of confinement imposed at five, monthly, intervals. Consistent divergence was obtained in the responsiveness of the two groups, although the difference between LR and HR groups was greater in female fish (56 c.f. 116 ng ml-1) than in males (45 c.f. 69 ng ml-1). Progeny groups (full-sib families) were obtained from the pairing of HR males and females and LR males and females. A third progeny group (US) was obtained by random pairing of parents which were not selected as HR or LR. Post-stress plasma cortisol levels in the progeny were first tested at 6 months after hatch and were significantly correlated with the response of the corresponding parental groups, HR>US>LR (178, 126, 81 ng ml-1 respectively). The difference in responsiveness between LR and HR groups was demonstrated in all four subsequent tests over a 12 month period. There were no significant differences in baseline plasma cortisol levels in LR and HR groups prior to confinement. During a 4h period of confinement, the differences in plasma cortisol levels between LR and HR fish were sustained throughout, indicating that the trait upon which the fish were selected was related to absolute maximum levels of circulating cortisol, not the rate of change of cortisol levels during exposure to a stressor. A moderately high heritability (h2) for confinement-induced plasma cortisol of 0.41 was obtained by a parent-progeny regression. Manipulation of stress-responsiveness in fish by selective breeding offers scope for optimising performance under intensive rearing conditions but also provides a useful research tool for investigating the operation of the endocrine stress response

Contrasting seasonal modulation of the stress response in male and female rainbow trout

Between June and September the magnitude of the plasma cortisol response of maturing male and female rainbow trout to confinement was indistinguishable. A progressive increase in confinement-induced cortisol levels in both sexes occurred during the period May to September, associated with the seasonal rise in water temperature. Between September and January there was a more than 50% reduction in the magnitude of the cortisol response to confinement in male fish which coincided with declining water temperature and significant increases in plasma 11-ketotestosterone and elevated plasma testosterone levels. In contrast, female trout showed no evidence of a decline in cortisol responsiveness to confinement during the period September to January. Plasma estradiol-17β levels were significantly greater in females than males throughout the study period and this difference was maximal between September and January. However, plasma testosterone was also elevated in females during this period and levels overall were higher than those in male fish. Previous studies have shown estradiol-17β and testosterone to have diametrically opposed effects on stress responsiveness in trout, with the former enhancing, and the latter suppressing, the cortisol response to a stressor. The relative roles of androgens, estrogen and water temperature in modulating the stress responsiveness of rainbow trout is discussed

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 co-culture 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 1h 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

Indicators of reproductive performance in rainbow trout Oncorhynchus mykiss (Walbaum) selected for high and low responsiveness to stress

A number of indicators of reproductive performance were assessed in rainbow trout divergently selected on the basis of their plasma cortisol response to a standardised stressor. For both male and female rainbow trout, body weight was significantly related to stress responsiveness. High-responding (HR) females were significantly heavier than low-responding (LR) females overall and HR males were significantly heavier than LR males overall. There was no significant difference in the SGR of HR and LR female fish during the experimental period while in contrast the mean SGR of HR males was significantly greater than that of LR males. The divergent confinement-induced levels of blood cortisol in HR and LR groups were stable for more than one year after selection. There was no significant difference in the extent to which confinement stress reduced blood estradiol-17 levels in HR and LR females despite the large difference in relative blood cortisol levels. The onset and rate of ovulation was similar within the HR and LR groups. Differences in fecundity, relative fecundity and egg volume and weight were wholly attributable to the different mean body weights of the HR and LR fish. There was no difference between the sperm counts of HR and LR males. There was significantly higher mortality among eggs derived from HR parents overall between fertilization and hatch but maximum mean mortality did not exceed 12%. Mean time to eyeing in the HR groups was 224 degree days and in the LR groups was 244 degree days. Hatching commenced at 344 and 347 degree days in the HR and LR groups respectively and was complete within 412 and 416 degree days respectively. Overall, selection for stress responsiveness was associated with no significant benefits or costs in reproductive performance

Plasma cortisol and 17β-estradiol levels in roach exposed to acute and chronic stress

Plasma cortisol levels were measured as an indicator of physiological stress in roach subjected to brief handling, or to a 14 day period of confinement, and in undisturbed control fish, during winter (water temperature 5oC) and summer (16oC), at which time plasma 17β-estradiol levels were also determined. Cortisol levels in undisturbed roach were low (mean 8.1 ng ml-1 at 5oC; 1.4 ng ml-1 at 16oC) and both handling and handling + confinement significantly elevated blood cortisol levels to 400 and 140 ng ml-1 respectively (at 5oC) and 700 and 600 ng ml-1 respectively (at 16oC). Blood cortisol levels had almost returned to baseline within 4 hours following handling alone but in fish subjected to handling and prolonged confinement cortisol levels remained elevated for up to 168 hours. Differences in baseline and post-stress levels of cortisol, and in the rate of recovery from acute stress, were observed at the two different temperatures and the possible factors underlying these differences are discussed. Circulating levels of 17β-estradiol were significantly reduced within 24 hours of exposure to either acute handling or chronic confinement indicating that the reproductive endocrine system in roach is sensitive to disruption by stressors

Divergence of endocrine and metabolic responses to stress in two rainbow trout lines selected for differing cortisol responsiveness to stress

Rainbow trout (Oncorhynchus mykiss) of two lines selected for low (LR) and high (HR) cortisol stress-responsiveness were subjected to confinement for a period of 336 hours. Endocrine (plasma cortisol, hepatic cortisol binding) and metabolic (plasma glucose, lactate, amino acids; hepatic glycogen and alanine aminotransferase levels) indices of stress were measured at intervals in confined and unconfined fish of both lines. During confinement plasma cortisol concentration reached maximum values earlier in HR fish (2 hours) than in LR fish (6 hours) returning to control values within 336 hours in both lines. Paradoxically, although both HR and LR lines displayed a characteristic metabolic stress response, these changes were more pronounced in LR fish. Plasma glucose and lactate levels increased during confinement in both lines but to a significantly greater extent in LR fish. Confinement significantly elevated plasma amino acids to a greater extent in LR fish than in HR fish. Liver glycogen concentration was depleted most rapidly in LR fish but was significantly higher in confined fish of both lines than controls at the end of the experiment. No significant changes were observed in hepatic alanine aminotransferase activity during confinement. Confined fish of both lines displayed a decrease in hepatic cortisol receptor abundance within 24 h and this was more sustained in HR fish. The more pronounced disturbance of a broad range of indicators of stress in confined LR fish, compared to HR fish, throws doubt on the magnitude of the cortisol response being the primary driver of these differences

The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices

Indicators of a generalised stress response (changes in cortisol, glucose, RNA:DNA ratio and total protein) when measured in whole-body preparations of individual sticklebacks display significant alterations in response to acute (hours) and chronic (days) disturbances and food withdrawal. In addition, changes in alkali-labile phosphorous, a specific biomarker of exposure to estrogenic contaminants, can be detected in whole-body preparations of estrogen-exposed sticklebacks confirming that the measurement of biomarkers normally assessed in a specific tissue can be equally possible in whole fish

High blood cortisol levels and low cortisol receptor affinity: is the chub, Leuciscus cephalus, a cortisol-resistant teleost?

In contrast to the relatively minor intra- and inter-species differences in blood cortisol levels reported for salmonid species, there is a more pronounced distinction between cortisol levels among the Salmonidae and Cyprinidae, with both basal and stress-induced cortisol levels markedly higher in the latter. This study shows that in the chub, Leuciscus cephalus, a widely distributed European cyprinid, mean blood cortisol levels during stress (1500 ng ml-1) exceeded those reported for most other species of fish and even in unstressed chub, cortisol levels (50 – 100 ng ml-1) were within the range known to cause immunosuppression, growth retardation and reproductive dysfunction in salmonid fish. The chub appears to be atypical only with respect to plasma cortisol levels; the levels of plasma glucose and plasma lactate in unstressed and stressed chub are similar to those reported for other species. Plasma levels of 11-ketotestosterone in males and 17beta-estradiol in females are lower than reported for salmonids but similar to other cyprinid species and display clear stress-induced reduction. Comparative analysis of the binding characteristics of the trout and chub gill cortisol receptor revealed that the total number of binding sites in gill tissue for each species was similar (Bmax; approx 50 – 100 fmol mg-1 protein). However, the affinity of the binding site for cortisol displayed an 8-fold difference between the species (rainbow trout: Kd approx. 6 nM; chub: Kd approx. 50 nM). Therefore, the potentially adverse effects of high circulating levels of cortisol found both at rest and under conditions of stress in chub may be offset by the lower affinity of the cortisol receptor, rather than the abundance of target-tissue receptor sites. This strategy is similar to that reported for some glucocorticoid-resistant rodent species and New World primates

Evaluation of biochemical methods for the non-destructive identification of sex in upstream migrating salmon and sea trout

Female-specific markers of reproductive activity (plasma 17β-estradiol, E2; vitellogenin, VTG; alkali-labile phosphoprotein phosphorous, ALP) were measured over 12 months in a captive population of brown trout. During the early months of the reproductive season (February – May) and using the concentration of plasma E2 or plasma ALP as a marker for females the proportion of fish in which sex was misidentified was high (15% - 50%). The misidentification rate was considerably lower (1 – 8%) using plasma VTG. Preliminary evaluation of a commercial immunochromatographic VTG test system as a screen for the presence or absence of VTG in plasma from brown trout provided results that were consistent with those obtained from direct measurement of plasma VTG levels by ELISA. These preliminary conclusions were verified by sampling upstream-migrating sea trout and salmon trapped over a 6 month period. Plasma E2 levels did not satisfactorily discriminate between male and female sea trout and salmon. However, plasma VTG levels in both species were bimodally distributed and it was assumed that this divergence corresponded to male (plasma VTG levels 800 μg ml-1) fish. Plasma ALP provided a more accurate indication of sex in the wild salmon and sea trout than was suggested by the pilot study on captive brown trout. The commercial immunochromatographic VTG test system provided results that were wholly consistent with the data obtained from the trapped fish by direct measurement of plasma VTG