Mean Corticosterone Research Articles

Effects of Midazolam on Corticosterone and Blood Gases in Spectacled Eiders Prior to Transmitter Implantation

ABSTRACTStress and physical exertion may affect the physiology and behavior of wildlife during and after capture, and consequently, survival following release. Such effects may reduce the quality and quantity of the data obtained from captured wildlife. We captured spectacled eiders (Somateria fischeri), a species listed as threatened under the United States Endangered Species Act, in western Alaska, USA, during spring 2018 for surgical implantation of satellite transmitters. We evaluated the efficacy of midazolam, a benzodiazepine sedative given at capture, to reduce stress and physical exertion. We dosed spectacled eiders with either midazolam (5 mg/ml, = 2.2 mg/kg intramuscular; n = 20) or saline (0.7 ml intramuscular; n = 20) at the point of capture. We assessed sedation level and collected blood samples upon arrival to the field surgery site and at anesthetic induction. We found that midazolam reduced mean corticosterone concentration by 29% and median lactate concentration by 30.3% at the mean arrival time (42 min post‐dosing) relative to the control group. These effects had abated by the mean induction time (99 min post‐dosing). Unexpectedly, blood pH was reduced in the midazolam treatment relative to controls at both arrival and induction, which likely resulted from sedative‐induced respiratory depression that was easily treated with intubation and mechanical ventilation, and administration of the reversal drug, flumazenil. Low blood pH was not associated with negative post‐surgical outcomes, as had been found in spectacled eiders with acidosis caused by anaerobic metabolism typical of physical exertion. Intramuscular injection of midazolam in the field effectively reduced stress and physical exertion in spectacled eiders prior to surgical implantation of transmitters. © 2021 The Wildlife Society.

Opium Effect in Pregnancy on the Dynamics of Maternal Behavior: Testing a Neurochemical Model

Background: Investigations into neurochemical mechanisms of opioid addiction are difficult due to the complexity of behavior and multiplicity of involved neurotransmitter and hormonal systems. The aim of this study was to examine the benefits of structured analysis of these mechanisms using the framework of the neurochemical model Functional Ensemble of Temperament (FET) and the example of maternal behavior under the condition of opium consumption in pregnancy. The FET differentiates between (a) endurance, (b) speed of integration, and (c) emotionality aspects of behavior suggesting that these systems are differentially regulated by (a) serotonin-neuropeptides-brain-derived neurotrophic factor (BDNF), (b) dopamine-GABA, and (c) opioid receptor systems, correspondingly. The FET also suggests that mu-opioid receptors (MORs) binding the endorphines (including opium’s ingredient morphine) have a stronger association with regulation endurance, whereas delta-OR have a stronger association with integration of behavior and kappa-OR – with the perceptual mobilization seen in anxiety. To test the predictions of this model, we compared the impact of massive MOR dysregulation on 3 behavioral aspects of behavior and on serotonin, BDNF, and corticosterone levels. Methods: The study used 24 female white Wistar rats which were randomly divided into (1) control group: pregnant rats without any intervention; (2) opium-exposed group: animals that were exposed to opium during pregnancy and after the delivery until the end of the study. At the end of the study, the levels of BDNF, serotonin (5-HT) in the hippocampus of the mother’s brain, and serum corticosterone, as well as 12 aspects of the maternal behavior were evaluated. The differences between control and experimental groups were assessed using the t test for independent samples. Results: The BDNF and serotonin concentrations in the hippocampus of the mother rats which were exposed to opium were lower than in the control group; the mean corticosterone in exposed mothers was higher than in the control group. Behaviorally, opium-consuming mothers showed lower endurance in 4 distinct behavioral categories (nesting, feeding, grooming, and retrieval) than the mothers in the control group. Ease of integration of behavior was affected to a lesser degree, showing a significant effect only in 1 out of 5 applied measures. Self-grooming, seen as an emotionality-related aspect of behavior, was not affected. Conclusion: Opium exposure during pregnancy in our experiment primarily reduced the endurance of rat’s maternal behavior, but the speed of integration of behavioral acts was less affected. This negative impact of opium on endurance was associated with a decrease of BDNF and serotonin levels in the hippocampus and an increase in corticosterone level in opium-consuming mothers. There is no effect of opium exposure on self-grooming behavior. This pattern supports the FET hypothesis about the role of 5-HT and BDNF in endurance, differential regulation of endurance, integrative and emotionality aspects of behavior, and differential association of the MOR system with endurance aspects, in comparison with kappa- and delta opioid receptors.

Validation of an Alternative Feather Sampling Method to Measure Corticosterone.

Simple SummaryResearch projects on birds’ welfare or their stress physiology are often complemented by measurements of corticosterone level in feathers. Until now, the common standard for this method is to collect the feathers by plucking, a procedure which on living birds is presumed to be painful and to cause stress. Therefore, in most European countries an animal experiment application is required. The aim of this study was to validate an alternative, possibly less stressful sampling method: cutting the feathers close to the skin. The examined species were geese and ducks from a conventional poultry husbandry. There was no relevant difference between the two methods assessed according to statistical analysis. In conclusion, it is reasonable to assume that feather cutting could be established as an alternative sampling method for measuring corticosterone. Nevertheless, we recommend further research on other species to confirm these results.The most common feather sampling method for feather corticosterone measurement is by plucking the feathers from the bird’s skin. This procedure performed on living, restrained birds is qualified as an animal experiment according to German/European legislation, which has to be applied for from the competent authorities. The Directive 2010/63/EU requires the full implementation of the 3-R Principle of Russel and Burch in animal experiments, which means not only to replace the use of animals, but also to reduce the number of animals used and to refine procedures whenever possible. In response to this issue, the aim of this study was to validate an alternative, less invasive sampling method by cutting feathers close to the skin in comparison to the gold standard of plucking them. For this proof-of-principle study, a conventional poultry husbandry with trial groups of geese (Anser anser domesticus) and ducks (Anas sterilis) was selected. All birds were kept under the same living conditions to standardize the influencing factors regarding husbandry, and thus, their stress levels. Feather samples were collected between the shoulders from 46 geese and 51 ducks, both by cutting as well as by plucking, directly after slaughter for meat production. Feather corticosterone levels were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Results were compared using Bland–Altman plots and concordance correlation coefficients (CCC). It could be seen that concordance between corticosterone levels in cut and plucked feathers was rather poor: 0.38 for Anser, and 0.57 for Anas. However, comparing the mean corticosterone values in pg/mm of each species with their respective standard deviations, the differences between the methods were negligible. As the results showed that the differences between the individuals were markedly greater than the differences between the methods, the determination of corticosterone levels in cut feathers is valid compared to using plucked feathers. The validation tests of ELISA showed only acceptable repeatability and reliability. Hence, the results should be verified in further studies. In conclusion, it is recommended for future research to use cut instead of plucked feathers for corticosterone measurement.

Corticosterone in central North Pacific male humpback whales (Megaptera novaeangliae): Pairing sighting histories with endocrine markers to assess stress

Developing a better understanding of the stress response is critical to ensuring the health and sustainability of marine mammal populations. However, accurately measuring and interpreting a stress response in free-ranging, large cetaceans is a nascent field. Here, an enzyme immunoassay for corticosterone was validated for use in biopsy samples from male humpback whales (Megaptera novaeangliae). Analyses were conducted on 247 male North Pacific humpback whale blubber samples, including 238 non-calves and 9 calves that were collected on the Hawaiian breeding and Southeast Alaskan feeding grounds from 2004 to 2006. Significant relationships were found when corticosterone concentrations were examined by year, age class and distribution between locations. When examined by year, corticosterone concentrations for male humpback whales were higher in Hawaii in 2004 than in 2005 and 2006 (p < 0.05). Corticosterone concentration also varied by age class with initially high concentrations at birth which subsequently tapered off and remained relatively low until sexual maturity was reached around age 8–10 years. Corticosterone concentrations appeared to peak in male humpback whales around 15–25 years of age. Blubber biopsies from Alaska and Hawaii had similar mean corticosterone concentrations, yet the variability in these samples was much greater for whales located in Hawaii. It is clear that much work remains to be done in order to accurately define or monitor a stress response in male humpback whales and that specific attention is required when looking at age, sex, and yearly trends. Our results suggest that a stress response may be most impacted by age and yearly oceanographic conditions and needs to be initially examined at the individual level.

Melanization, α-melanocyte stimulating hormone and steroid hormones in male western fence lizards from nine populations

Hormones can mediate suites of correlated traits. Melanocortins regulate melanin synthesis and elements of the melanocortin system can directly, and indirectly, affect a number of other traits, such as stress reactivity. Trait correlations within the melanocortin system have been studied mainly in birds and mammals but less so in reptiles. We examined adult male western fence lizards (Sceloporus occidentalis) and if melanization was correlated with plasma levels of three hormones, including peptide hormone α-melanocyte stimulating hormone (α-MSH), testosterone and corticosterone, and ectoparasite loads. This lizard is darker at higher elevations in California, and we compared five high-elevation and four low-elevation populations during comparable periods of the breeding season at each site. We first validated use of an α-MSH assay kit with lizard plasma. Since Anolis carolinensis is one of the few species with published values for α-MSH plasma levels, we assayed both Anolis and Sceloporus plasma and compared hormone values to those we generated for Anolis to the publish values. We also evaluated effects of different methods of storing spiked plasma pools on resulting α-MSH concentrations. Plasma levels of α-MSH did not differ significantly, but some populations differed significantly in mean corticosterone and mean testosterone. Combining all individuals from the nine populations, we found that individual variation in α-MSH was not associated with individual variation in melanization, but levels of α-MSH were positively associated with plasma testosterone and negatively associated with corticosterone. The lack of association between individual levels of melanization and expression of most other traits differs from a growing number of within-population studies of melanization, and we discuss what differences in physiological mechanisms could produce different hypothetical patterns. Circulating levels of -MSH are only one element of the melanocortin system; in situ synthesis of α-MSH by the skin and the diversity of melanocortin receptors could also contribute to variation in traits mediated by the melanocortin system and should be examined.

An Analysis of the Hypothalamic-Pituitary-Adrenal Axis Functions in Cirrhotic Rats in Response to Surgical Stress

Introduction The activation of hypothalamic-pituitary-adrenal (HPA) axis through severe diseases and stress courses leads to a rise in circulatory cortisol for an adequate response to stress. This axis is one of the important systems that involve in neuroendocrine response to the surgical stress. Hepatoadrenal syndrome that is a manifestation of adrenal insufficiency (AI) in the course of liver disease is described as insufficient production of steroid hormones mainly cortisol due to primary dysfunction of the adrenal gland or secondary malfunction of the HPA axis to provoke the adrenal gland leading to severe illness and increased mortality. Through this evidence, we presented this question as to whether cirrhotic patients have a greater mortality rate than other patients after surgery and if the HPA axis is partly responsible for this phenomenon. Also how the adrenal gland functions during surgery in cirrhotic rats. We conducted this study to assess the effect of cirrhosis on the HPA axis through surgery in cirrhotic rats by evaluating the changes in serum corticosterone level and blood sugar before, immediately, and 30 minutes after surgery. Method This study was performed in the animal lab approved by the Ethics Committee of Tehran University of Medical Sciences in 2014, on 25 male Wistar rats. Thioacetamide was used for induction of cirrhosis in rats with new method of monitoring weekly changes of rats' weight which had 100% success in procedure and reduction in mortality rate. Laparotomy was performed on all of the rats during 9–12 in the morning within 10–15 minutes. Laparotomy was chosen as surgical stress because of its simplicity and feasibility. Three blood samples were obtained from each rat immediately after inducing anesthesia, immediately after the conclusion of surgery, and 30 minutes after surgery. The plasma concentration of corticosterone was measured with enzyme-linked immunosorbent assay test. P value of 0.05 or less was considered as statistical significance. Result Cirrhotic rat group consisted of 15 rats and control group consisted of 10 rats. There was a significant difference in the mean level of corticosterone and blood sugar between the cirrhotic rat group and control group in the 3 time levels (P=0.044/P < 0.001). Pairwise comparison of mean corticosterone and blood sugar levels between case (mean: 249.359 ± 3.90) and control (mean: 262.40 ± 4.69) showed a significant difference (P=0.04, 95% CI = 0.30–25.79/P < 0.001, 95% CI = 129.62–233.96). Unlike the control group, the level of serum corticosterone was compared in the cirrhotic rat group (group 1) before, immediately, and 30 minutes after surgery, which showed a significant difference in our study (P value = 0.005). However, this result was also significant in comparing the blood sugar in 3 time levels of surgery in the control group (P value < 0.001) but not in the cirrhotic rat group (P value = 0.233). Conclusion There was a significant rise in corticosterone levels during 3 time levels of surgery in cirrhotic rats; nevertheless, this elevation was significantly lower than the control group. Also the mean level of blood sugar was higher in the control group than in cirrhotic rats. However, this difference was significant in comparison with the same times of surgery between the two groups. These results approximately can substantiate our hypothesis that AI in the field of cirrhosis would also affect the response of HPA axis to stress during and after surgery that can be concomitant with higher rate of cardiovascular unsteadiness incidences, deteriorating the severity of illness and rise in mortality rate.

EFFECT OF ROUTINE HANDLING AND TRANSPORTATION ON BLOOD LEUKOCYTE CONCENTRATIONS AND PLASMA CORTICOSTERONE IN CAPTIVE HISPANIOLAN AMAZON PARROTS ( AMAZONA VENTRALIS).

Increased glucocorticoids cause a characteristic stress leukogram in mammalian taxa. It is assumed that avians exhibit a similar response, but to date, there have been no controlled studies to correlate serial endogenous corticosterone levels to hematologic values. An established flock of 18 Hispaniolan Amazon parrots ( Amazona ventralis) was used as a model in a crossover study. The treatment group was subjected to the stress of transport, restraint, and common clinical procedures with serial blood samples collected at 20-min intervals for hematology and corticosterone levels; the control group was sampled at the same intervals. Longitudinal data analysis was performed with linear mixed modeling. For all hematologic analytes, the baseline value had a significant positive effect on subsequent values (all P < 0.001). The white blood cell, heterophil, and eosinophil counts and heterophil to lymphocyte ratio increased over time in the treatment group, whereas it remained stable in the control group ( P = 0.016, P < 0.001, P < 0.001, P = 0.02, respectively, for the time*treatment effect). Lymphocyte absolute counts decreased over time, although not significantly; the decrease was significant for the relative lymphocyte count in the treatment group. Monocytes and basophils were not significantly altered. The treatment group had a higher mean corticosterone level overall than the control group by approximately 60% ( P = 0.008). The mean corticosterone level also increased over time in both groups by three- to fourfold ( P < 0.001) by 20 min then plateaued. These results demonstrate that some significant hematologic changes may arise with routine handling and transportation of birds and should be accounted for in hematologic interpretation of cell counts.

P.2.020 - Blunted basal corticosterone pulsatility predicts post-exposure susceptibility to post-traumatic stress disorder phenotype in rats

Introduction Glucocorticoid (GC) hormones play a major role in orchestrating the physiological and behavioral reactions essential for homeostasis. As such, these hormones enable the organism to prepare for, respond to, and cope with the acute demands of physical and emotional stressors [1]. The secretion of GC hormones is a dynamic process which displays, in addition to a circadian pattern of release, an ultradian rhythmicity in the blood and in target tissues [2] Inadequate GC release following stress not only delays recovery but can also interfere with the processing or interpretation of stressful information, resulting in long-term disruptions of memory integration processes [3]. An example of such an impaired process is post-traumatic stress disorder (PTSD) [4]. In the rat, corticosterone ultradian pulses have a near-hourly frequency and changes in the amplitude of these pulses throughout the 24 h cycle determine the circadian variation in hormone secretion [2]. Aims We used an animal model of PTSD [5] in order to assess whether stress-induced impairment of behavioral responses is mediated by an aberrant secretion of corticosterone. Methods Serial blood samples were collected manually via the jugular vein cannula during the inactive phase in conscious male rats at 20-min intervals for a period of 5 h before and 6.5 h after exposure to predator scent stress (PSS). The outcome measures included behavior in an elevated plus-maze and acoustic startle response 7 days after exposure. Individual animals were retrospectively classified as having extreme, partial, and minimal behavioral responses according to pre-set cut-off criteria for behavioral response patterns [5] and blood samples were analyzed retrospectively for corticosterone secretion patterns. For the behavioral results and for serum corticosterone levels, the statistical analyses were performed using one-way analysis of variance (ANOVA), bonferroni tests were used to examine differences between individual groups. To gain additional understanding about the relationship between behavioral measures and corticosterone pulsatility parameters, two sets of regression analysis were conducted: Pearson's correlation analysis was used to describe the relationship between each behavioral variable and corticosterone pulsatility parameters across the entire sample. The contribution of each variable that exhibited a significant correlation (or trend) was then evaluated using a multiple stepwise-regression analysis. Results Under basal conditions, the amplitude of ultradian oscillations of corticosterone levels, rather than the mean corticosterone level or the frequency of corticosterone pulsatility, was significantly reduced in individuals who displayed PTSD-phenotype 8 days later. In addition, extreme disruption of behavior on day 8 post-exposure was also characterized by a blunting of corticosterone response to the stressor. Animals with behavior that was only partially affected or unaffected displayed none of the above changes. Conclusions Blunted basal corticosterone pulse amplitude is a pre-existing susceptibility or risk factor for PTSD, which originates from prior (life) experiences and may therefore predict post-exposure PTSD-phenotype in rats. Further studies confirming these findings in animals and in human subjects are required to ascertain whether dysregulation in pulsatility indeed predisposes individuals to, or in fact underlies, faults in the HPA-axis response to stress and renders them vulnerable to PTSD.

Blunted basal corticosterone pulsatility predicts post-exposure susceptibility to PTSD phenotype in rats

The basal activity of the hypothalamic–pituitary–adrenal axis is highly dynamic and is characterized by both circadian and ultradian (pulsatile) patterns of hormone secretion. Pulsatility of glucocorticoids has been determined to be critical for optimal transcriptional, neuroendocrine, and behavioral responses. We used an animal model of post-traumatic stress disorder (PTSD) to assess whether stress-induced impairment of behavioral responses is correlated with aberrant secretion of corticosterone. Serial blood samples were collected manually via the jugular vein cannula during the light-(inactive)-phase in conscious male rats at 20-min intervals for a period of 5 h before and 6.5 h after exposure to predator scent stress. The outcome measures included behavior in an elevated plus-maze and acoustic startle response 7 days after exposure. Individual animals were retrospectively classified as having “extreme”, “partial”, or “minimal” behavioral responses according to pre-set cut-off criteria for behavioral response patterns. Corticosterone secretion patterns were analyzed retrospectively. Under basal conditions, the amplitude of ultradian oscillations of corticosterone levels, rather than the mean corticosterone level or the frequency of corticosterone pulsatility, was significantly reduced in individuals who displayed PTSD-phenotype 8 days later. In addition, extreme disruption of behavior on day 8 post-exposure was also characterized by a blunting of corticosterone response to the stressor. Animals with behavior that was only partially affected or unaffected displayed none of the above changes.Blunted basal corticosterone pulse amplitude is a pre-existing susceptibility or risk factor for PTSD, which originates from prior (life) experiences and may therefore predict post-exposure PTSD-phenotype in rats.

Corticosterone responses to capture and restraint in Australasian Gannets, Morus serrator, at Cape Kidnappers, New Zealand

Birds can respond to threats or potential threats in their immediate environment with increased secretion of the glucocorticoid hormone corticosterone. The size of a corticosterone response to capture reflects the sensitivity of a bird to stimuli from its immediate environment, and there is marked variation between individual birds in their corticosterone responses. Whilst corticosterone responses to capture have been described in many species of birds, there are few reports of corticosterone responses in Australasian seabirds, and the aim of the present study was to describe individual and mean corticosterone responses to capture and restraint in Australasian Gannets (Morus serrator) at Cape Kidnappers, New Zealand. Corticosterone concentrations were low initially, then increased after capture in all gannets, with mean concentrations 55.77 ± 4.72 ng mL−1 after 30 min of restraint. This is the first report of corticosterone responses in gannets. Mean corticosterone concentrations at 30 min were within the range of responses reported for the blue-footed booby, another species within the family Sulidae, suggesting that different species of Sulidae have similar sensitivities to their immediate environment. Further studies of corticosterone in gannets and other Australasian seabirds will be worthwhile to provide information about how these seabirds can respond to changes in their environment.

Individual variation and repeatability of corticosterone responses of little penguins (Eudyptula minor) sampled in two successive years at Oamaru, New Zealand

Plasma corticosterone concentrations increase when birds experience a stressor, and plasma corticosterone responses to a capture and handling stressor have been measured in many species of birds. Whilst it is assumed that the reported corticosterone responses reflect the inherent sensitivity of each bird to the stressor, responses of the same birds have rarely been measured at intervals of one or more years. The current study was conducted to measure the repeatability in two successive years of corticosterone responses of little penguins (Eudyptula minor) at Oamaru, New Zealand. There was a wide range of individual corticosterone responses to capture and restraint in 96 little penguins in 2012 and 50 penguins sampled at the same time of year in 2013. There were significant repeatabilities for the ranks of corticosterone at 15, 30 and 60min (r=0.416±0.160, r=0.636±0.115 and r=0.380±0.166 respectively) and for the ranks of integrated corticosterone responses (r=0.594±0.126) for 23 birds sampled in both years. There were no significant relationships between the size of corticosterone responses and age, body weight or condition index. Mean corticosterone concentrations at 60min were 114.22±6.65ng/ml in 2012 and 116.94±6.42ng/ml in 2013. Mean corticosterone responses did not differ between two successive years and were greater than responses of other penguin species. Penguins are well suited to long term studies in which corticosterone responses are measured annually as potential measures of changing marine environmental conditions.

Koncentrace kortikosteronu v krevní plazmě nosnic (Gallus domesticus) v průběhu snáškového cyklu

The aim of this work was to study corticosterone concentration in the plasma of laying hens (gallus domesticus) during the laying period. The experiment included 12 ISA Brown laying hens, which were caged in enriched cage system according to EU directive 1999/74. They were fed by complete diet for laying hens. Blood samples were taken between 15th and 75th week of age. Mean corticosterone concentration during the whole period was 2.86 ± 0.086 nmol.l-1and ranged from 1.76 ± 0.135 to 3.98 ± 0.0357 nmol.l-1. Plasma corticosterone concentration increased significantly (P≤0.01) at the beginning of the laying period. Then followed slight variation until 47th week of age. Corticosterone concentration increased significantly (P≤0.01) again at 52nd week of age to 3.98 ± 0.236 nmol.l-1. From 58th week of age to end of laying period plasma corticosterone concentration decreased and reached the value of 2.22 ± 0.210 nmol.l-1.

Revisiting the “visible burrow system”: The impact of the group, social rank, and gender on voles under owl attack

In the present study, corticosterone levels and behavior were compared between voles (Microtus socialis) that were attacked by a barn owl (Tyto alba) and voles that did not experience such attack. Both female and male voles were exposed to the owl either together with their groupmates or when socially isolated. As hypothesized, corticosterone levels were higher in voles after the owl attack, and were higher in females than in males. However, blood corticosterone was higher in voles that experienced the attack in groups compared with the socially-isolated voles. The latter result seems enigmatic, since group members usually benefit from the “social buffering” conferred by their group-mates. It is suggested that contagious vigilance among group members accounts for the higher mean corticosterone level in grouped compared to socially-isolated voles, over-shadowing the possible impact of social buffering. We also found a negative correlation between body mass and corticosterone level, with more high-mass voles showing low corticosterone levels compared with low-mass voles. This finding accords with a previous study in which the behavior of high-mass voles was less affected by owl attack compared to low-mass voles. The novelty of the present results therefore lies in supporting, at the hormonal level, past behavioral findings in rats and voles, and in demonstrating that high-mass voles, by virtue of their physical strength and perhaps also their life experience, are less stressed by the owl attack and become the leaders and stabilizers of their groups.

Molt-associated Changes in Hematologic and Plasma Biochemical Values and Stress Hormone Levels in African Penguins (Spheniscus demersus)

Handling, including blood collection, has often been discouraged in molting penguins because it is considered an additional stress imposed on birds already experiencing major physiologic stress associated with molting. To evaluate the degree of physiologic stress posed by molting, we compared the hematologic and plasma biochemical values and hormone levels of molting and nonmolting African penguins, Spheniscus demersus. Five male and 5 female penguins randomly chosen were given complete physical examinations, were weighed, and blood samples were taken at 7 time points before, during, and after the molt. Data were analyzed by linear mixed-model analysis of variance. Throughout the study, behavior and appetite remained normal. Catecholamine levels were highly variable within and among subjects, whereas mean corticosterone levels were significantly different between baseline, molt, and postmolt values. Significant differences from baseline values were observed in many of the hematologic analytes; however, only decreases in hematocrit and red blood cell count values were considered clinically significant. Anemia due to experimentally induced blood loss as a possible cause of the significant hematologic changes was ruled out based on results of a follow-up control study during the nonmolt season, which showed no significant changes in hematocrit level or total red blood cell counts when using similar sampling protocols, which indicates that these changes were associated with molt.

High corticosterone and sex reversal in common carp (Cyprinus carpio L.) with adrenal hyperplasia caused by P450c17a2 deficiency

The aim of this study was to investigate the inheritance of adrenal hyperplasia, caused by 17α-hydroxylase deficiency, and its association with female to male sex reversal in common carp. Inbred strains used in this experiment were E4 (XX, female, normal), E5 (XX, male, adrenal hyperplasia) and E7 (XX, male, adrenal hyperplasia). F1 females (E4E5: XX, normal) were crossed to E5 and E7 males to produce backcross progeny (BC5 and BC7), or reproduced by gynogenesis to produce doubled haploid progeny (DH). Fish (age 6months) were subjected to a net confinement stressor and sacrificed to collect blood plasma for analysis of cortisol and corticosterone, and to determine sex. Values for plasma corticosterone showed a clear segregation pattern, consistent with a single recessive gene model. There was a highly significant difference between high and low corticosterone responders in BC and DH progeny groups. Mean corticosterone values for high corticosterone responders in the three groups BC5, BC7 and DH were, respectively: 1706, 1760 and 1366ng/ml. For low corticosterone responders values were 1.5, 1.1 and 0.6ng/ml. High corticosterone responders had on average low levels of cortisol: 21.3, 24 and 17.3ng/ml for BC5, BC7 and DH. Low corticosterone responders had normal levels (66.7, 105.1 and 65.2ng/ml for BC5, BC7 and DH, respectively). Sex ratios were significantly different between high and low corticosterone responders. High corticosterone responders were predominantly male, while low corticosterone responders were female or intersex. High corticosterone responders had well developed testis and were fertile indicating that in the gonads, 17α-hydroxylase is not impaired. The observed effect on sex shows that 17α-hydroxylase deficiency most likely augments sex reversal in common carp, either directly through high levels of plasma corticosterone, or through paracrine effects of elevated ACTH production.

Inter-island differences in the corticosterone responses of North Island Saddlebacks (Philesturnus rufusater) in New Zealand do not suggest selective effects of translocation

North Island Saddlebacks (Philesturnus rufusater) have been the subject of a well-documented sequence of translocations to isolated islands around New Zealand. We measured corticosterone stress responses in North Island Saddlebacks derived from the ancestral population and from a series of three sequential translocations. Each translocation might impose a selective pressure on the founder populations of North Island Saddleback. This selective filter might favour a particular corticosterone response linked through an associated coping ability. Evidence of selection would be reflected in a progressive shift in the mean corticosterone response related to the number of sequential translocations. Mean corticosterone responses to a standardised stressor were highest in birds descended from a population subject to two sequential translocations. However, responses measured at localities representing the original source population and populations derived from one and three sequential translocations were not significantly different from each other. Accordingly, our data suggest that translocations do not necessarily cause directional selection on the stress responses of these vulnerable populations of birds.

Response of restraint stress-selected lines of Japanese quail to heat stress and Escherichia coli challenge

Japanese quail selected for divergent corticosterone response to restraint stress were evaluated for their susceptibility to heat stress and challenge with Escherichia coli. These quail lines are designated as high stress (HS), low stress (LS), and the random-bred control (CS) lines. Heat stress (35°C, 8 h/d) began at 24 d until the end of the study at 39 d. Birds were challenged with an aerosol spray containing 2 × 10(9) cfu of E. coli at 25 and 32 d. At 38 d, the surviving birds were necropsied and the intestinal tract was screened for both Salmonella and Campylobacter. Body weights of the CS birds were higher than both HS and LS at 17, 25, and 32 d. At 32 d, there was no difference in mortality between males and females and the CS line had higher mortality compared with the LS line with the HS line being intermediate. At 38 d, females of the CS line that were both heat stressed and challenged had a mortality incidence of 25%, which was significantly higher than male birds of the same line and treatment (5.3%). There was an increased incidence in Salmonella enterica serotype Agona isolation after heat stress, with the LS birds having less isolation than the HS birds. Mean corticosterone levels of male birds were not significantly affected by line, heat stress, or E. coli challenge; however, the LS line subjected to heat stress had one-third the level of the HS line, a difference identical to that seen in the original selection for response to restraint stress.

Inverse urinary corticosterone and testosterone metabolite responses to different durations of restraint in the cane toad (Rhinella marina)

Non-invasive measurement of urinary corticosterone and testosterone metabolites in amphibians provides opportunities for endocrine studies of responses to physiological and psychological stressors. Typically, corticosterone metabolite concentrations increase in frog urine within 1–2h of a mild capture and handling stress protocol. However, no study has investigated the effect of duration of manual restraint on the changes in corticosterone and reproductive hormones in amphibians. We quantified urinary corticosterone and testosterone metabolite responses for 8h following various durations of manual restraint (control, 5, 15 or 30min) in adult male cane toads (Rhinella marina) under controlled laboratory conditions. All toads had a corticosterone stress response over 8h to our standard capture and handling stressor. The mean corticosterone stress response was significantly higher after 15 or 30min restraint in comparison to the control (no restraint) or to 5min restraint. Manual restraint for 5, 15 or 30min caused a significant reduction in urinary testosterone concentrations over 8h. We also provide a novel method of quantifying plasticity in corticosterone stress responses in amphibians with respect to restraint duration using the concept of a “reaction norm”. The reaction norm, which was calculated as slope of the regression line of integrated corticosterone response against restraint duration, was 9.69 (pg corticosterone/μg creatinineh)/min for male toads. In summary, corticosterone and testosterone responses to restraint are affected by restraint duration in male toads. Glucocorticoid reaction norms can be applied to study the change in physiological stress hormonal response with respect to restraint duration in other amphibian species.

Maternal corticosterone regulates nutrient allocation to fetal growth in mice

Stresses during pregnancy that increase maternal glucocorticoids reduce birth weight in several species. However, the role of natural glucocorticoids in the mother in fetal acquisition of nutrients for growth remains unknown. This study aimed to determine whether fetal growth was reduced as a consequence of altered amino acid supply when mice were given corticosterone in their drinking water for 5 day periods in mid to late pregnancy (day, D, 11-16 or D14-19). Compared to controls drinking tap water, fetal weight was always reduced by corticosterone. At D16, corticosterone had no effect on materno-fetal transfer of [(14)C]methylaminoisobutyric acid (MeAIB), although placental MeAIB accumulation and expression of the Slc38a1 and Slc38a2 transporters were increased. However, at D19, 3 days after treatment ended, materno-fetal transfer of MeAIB was increased by 37% (P < 0.04). During treatment at D19, placental accumulation and materno-fetal transfer of MeAIB were reduced by 40% (P < 0.01), although expression of Slc38a1 was again elevated. Permanent reductions in placental vascularity occurred during the earlier but not the later period of treatment. Placental Hsd11b2 expression, which regulates feto-placental glucocorticoid bioavailability, was also affected by treatment at D19 only. Maternal corticosterone concentrations inversely correlated with materno-fetal MeAIB clearance and fetal weight at D19 but not D16. On D19, weight gain of the maternal carcass was normal during corticosterone treatment but reduced in those mice treated from D11 to D16, in which corticosterone levels were lowest. Maternal corticosterone is, therefore, a physiological regulator of the amino acid supply for fetal growth via actions on placental phenotype.

CORTICOSTERONE AND THYROXINE IN COLD-STUNNED KEMP'S RIDLEY SEA TURTLES (LEPIDOCHELYS KEMPII)

Kemp's ridley sea turtles (Lepidochelys kempii), a critically endangered species, frequently strand on the shores of Cape Cod (Massachusetts, USA) in late autumn in a state of "cold-stunning" exhibiting low body temperature and related clinical issues. Stranded turtles are transported to the New England Aquarium (Boston, Massachusetts, USA) for treatment and rehabilitation. This study tested the hypothesis that cold-stunned sea turtles might exhibit high corticosterone ("stress hormone") or low thyroxine (which is often affected by temperature), or both, and that monitoring of both hormones may be useful for assessing recovery. In a retrospective analysis, 87 archived plasma samples were assayed from 56 cold-stunned juvenile Kemp's ridley sea turtles for corticosterone and free thyroxine (fT4). Upon admission, mean corticosterone was the highest yet reported for a population of sea turtles (39.3 +/- 2.5 ng/ml; mean +/- standard error of the mean [SEM]) and fT4 was usually undetectable. On admission, corticosterone was negatively correlated with white blood cell count but was not correlated with blood glucose. There were no differences in either hormone between survivors and nonsurvivors on admission. After 18+ days in recovery, surviving turtles' corticosterone dropped significantly to levels typical of baseline in other species (0.9 +/- 1.0 ng/ml) while fT4 increased significantly (1.3 +/- 1.5 pg/ml). During recovery, corticosterone was positively correlated with blood glucose and was not correlated with white blood cell count. Turtles that showed persistent deficits in feeding, activity, or both during recovery had significantly lower fT4 than did turtles with no such deficits. The "high corticosterone, low fT4" endocrine profile seen on admission may be a useful marker of cold-stunning in this and other species. Further studies are necessary to determine whether low thyroid hormones play a causal role in deficits in feeding and activity during recovery. Monitoring of both hormones may be useful for triage, monitoring of recovery, and assessing readiness for release.