Effect of neonatal isolation on responses to subsequent exposure to isolation stress in young chickens.

The aim of the present study was to investigate effects of isolation at an early age on behavioral and physiological responses of chickens to an isolation challenge at two weeks of age. Birds were assigned to a control group or to one of three treatments where chicks were isolated for 5 min per day. The groups were 1) no isolation (control); 2) early isolation (EI; 2 to 4 days of age); 3) late isolation (LI; 5 to 7 days of age); or 4) full isolation (FI; 2 to 7 days of age). All groups of chicks were challenged with isolation for 5 min at two weeks of age, with distress vocalizations (DV), stepping and jumping behavior measured. Hypothalamic and blood samples were collected at the end of isolation challenges. There were no significant differences between groups in body weight gain at 2 weeks of age. Latency of jump was lower in the LI group compared with the control group, but DV and number of steps were not affected by isolation treatment during the neonatal period. There were no significant differences among groups in plasma glucose or FFA concentrations. Gene expression for hypothalamic corticotropin-releasing hormone, was lower in the EI than the control group, with no differences in expression between control and LI or control and FI groups. There were no significant differences among groups in the expression of arginine vasotocin, thyrotropin-releasing hormone, neuropeptide Y, proopiomelanocortin, and orexin genes. These results suggest that isolation in the first week of life may affect responses to isolation of chicks when they are older, and that there may be a critical period of several days for this effect to occur.

Av-UCP single nucleotide polymorphism affects heat production during cold exposure in chicks.

OBJECTIVE: Uncoupling protein one (UCP1) is involved in thermogenesis, especially in non-shivering heat production. In chickens, a single nucleotide polymorphism (SNP) of the av-UCP (avian UCP) gene has been reported to be associated with body weight gain and increased abdominal fat. The purpose of this study was to examine the relationship between the av-UCP gene SNP and heat production in chicks. METHODS: C/C and T/T male chicks (Rhode Island Red) of av-UCP gene SNP (g. 1270, C > T) were exposed to a low temperature environment (16 °C for 15 min) and their physiological responses were compared. RESULTS: After cold exposure, mean rectal temperatures of C/C chicks were higher than those of T/T chicks. In pectoral muscle, genes expression of av-UCP and carnitine palmitoyltransferase-1 were higher in C/C chicks than T/T chicks. Hypothalamic expression levels of thyrotropin-releasing hormone and proopiomelanocortin genes were higher in C/C chicks than T/T chicks. Expression of hypothalamic corticotropin-releasing hormone, arginine vasotocin, brain-derived neurotrophic factor and neuropeptide Y genes did not differ between C/C and T/T chicks. In addition, plasma free fatty acid levels in C/C chicks were lower than those of T/T chicks. CONCLUSION: These results suggest that the av-UCP gene SNP affects non-shivering heat production via the hypothalamo-pituitary-thyroid axis and fatty acid metabolism in the chicken.

Repeated thermal conditioning during the neonatal period affects behavioral and physiological responses to acute heat stress in chicks.

OBJECTIVE: The aim of the present study was to investigate the effects of repeated thermal conditioning (RTC) at an early age on physiological and behavioral responses in chicks. METHODS: Birds were assigned to one of the four treatments in which the RTC was exposure to 40 °C for 15 min daily. The treatments were 1) no thermal conditioning (control); 2) early exposure group (EE; RTC from 2 to 4 days of age); 3) later exposure group (LE; RTC from 5 to 7 days of age); or 4) both early and later exposure (BE; RTC from 2 to 7 days of age). All groups of chicks were challenged with high ambient temperature (40 °C for 15 min) at two weeks of age. RESULTS: During heat challenge, initiation times of dissipation behaviors (panting and wing-drooping) were measured. Rectal temperature and respiration rate were measured after and before heat challenge. Hypothalamic samples and blood were collected at the end of heat challenges. Initiation times of dissipation behaviors and rectal temperature were not affected by the treatments. Increases in respiration rate in response to heat challenge were suppressed by early RTC treatment. There was no clear pattern of glucose levels in relation to thermal conditioning, whereas plasma corticosterone levels were decreased by early treatment (EE and BE groups). Hypothalamic thyrotropin releasing hormone gene expression was suppressed by early and later thermal conditioning and suppressed further by both early and later exposure. Neuropeptide Y gene expression in the BE group was lower than in the other groups, with a similar trend for corticotropin releasing hormone expression. CONCLUSION: Our results suggest that the effect of repeated thermal conditioning on the central thermoregulatory system depends on the number of times that chicks experienced conditioning. In addition, repeated thermal conditioning has greater effects on the acquisition of thermotolerance when conditioning occurs in chicks of two to four days of age in comparison with chicks of five to seven days of age.

Cortisol responses of goldfish (Carassius auratus) to air exposure, chasing, and increased water temperature.

Fish can respond to stimuli from the internal or external environment with activation of the hypothalamo-pituitary-interrenal (HPI) axis and the secretion of cortisol. Stimuli that activate the HPI axis of fish include short term air exposure and increases in water temperature. The present study was conducted to determine how quickly cortisol concentrations increase in goldfish subjected to an increase in water temperature, and to compare the response to an increase in water temperature with responses to other stimuli. Plasma cortisol concentrations varied widely between individual goldfish, with concentrations ranging from 9.1 to 516.0 ng/mL in goldfish on the day of arrival from the supplier. Mean cortisol concentrations in undisturbed goldfish were low (4.5 ±â€¯1.0 ng/mL). Mean cortisol concentrations in fish exposed to air for 3 min and in fish that experienced chasing for 10 min were markedly elevated 15 min after the beginning of the stimuli (132.6 ±â€¯31.0 and 121.1 ±â€¯23.9 ng/mL respectively). Mean cortisol concentrations in fish that experienced an increase in water temperature rose to 22.2 ±â€¯7.6 ng/mL after 15 min, declined to <10 ng/mL at 30 and 60 min then increased and were elevated (79.0 ±â€¯10.8 ng/mL) at 240 min. Cortisol measurements can be used to indicate the responsiveness of fish to changes in water temperature and goldfish will be a convenient study species for the development of studies of plasticity in responses of fish to increases in water temperature that are happening due to climate change.

An acute increase in water temperature can increase free amino acid concentrations in the blood, brain, liver, and muscle in goldfish (Carassius auratus).

Water temperature directly affects the body temperature in fish, so increasing water temperatures in oceans and rivers will lead to increases in fish body temperatures. Whilst a range of responses of fish to increases in water temperature have been measured, amino acid metabolism in a fish under high water temperature (HT) conditions has not been investigated. The aim of this study was to determine the effects of an acute increase in water temperature on oxygen consumption, plasma cortisol concentrations, and free amino acid concentrations in plasma and several tissues in goldfish (Carassius auratus). Oxygen consumption and plasma cortisol concentrations were increased in goldfish exposed to HT (30 ± 1 °C) for 200 min compared with goldfish at a control water temperature (CT 17 ± 1 °C). Oxygen consumption and plasma cortisol concentrations in both groups of fish combined were positively correlated. When goldfish were exposed to HT for 300 min oxygen consumption and plasma concentrations of 15 free amino acids were increased compared with goldish at CT. Concentrations of several free amino acids were increased to varying extents in the brain, liver, and muscle tissues. In conclusion, an acute increase in water temperature affected amino acid metabolism differently in the brain, liver, and muscle tissues. Goldfish will be a useful species for further studies of the possible roles of various amino acids in the brain, muscle, and liver during acute increases in water temperature in fish.

Serum prolactin and testosterone levels in captive and wild brown kiwi (Apteryx mantelli) during the prebreeding, breeding, and incubation periods.

In brown kiwi (Apteryx mantelli), the male is the primary incubator, a trait that is relatively rare among birds. The maintenance of avian incubation behavior is controlled by the protein hormone prolactin (PRL). Although steroid hormone concentrations in both wild and captive kiwi have previously been reported, this study is the first to report levels of PRL in captive and wild male and female kiwi through the prebreeding and breeding seasons, and to directly compare testosterone (T) concentrations between captive and wild males during the breeding and incubation periods. Female PRL concentrations increased at the time of oviposition, whereas male PRL concentrations rose gradually between the prebreeding and incubation periods. Although males are considered the main incubator, an increase in PRL levels could help females maintain behaviors such as nest guarding, or to take over incubation the event of mate loss. A gradual increase in PRL allows the male to be ready for incubation during the long breeding season. Interestingly, T concentrations in captive males did not decrease during incubation and was significantly higher than in wild males. Continual elevated T could have an impact on sperm production through negative feedback, thereby contributing to the low egg fertility seen in captive kiwi. Therefore, determining the underlying reason for the differences in hormone levels could be significant, if not vital, for improving the success of captive kiwi breeding programs.

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.

Oxidative damage and brain concentrations of free amino acid in chicks exposed to high ambient temperature.

High ambient temperatures (HT) reduce food intake and body weight in young chickens, and HT can cause increased expression of hypothalamic neuropeptides. The mechanisms by which HT act, and the effects of HT on cellular homeostasis in the brain, are however not well understood. In the current study lipid peroxidation and amino acid metabolism were measured in the brains of 14 d old chicks exposed to HT (35 °C for 24- or 48-h) or to control thermoneutral temperature (CT; 30 °C). Malondialdehyde (MDA) was measured in the brain to determine the degree of oxidative damage. HT increased body temperature and reduced food intake and body weight gain. HT also increased diencephalic oxidative damage after 48 h, and altered some free amino acid concentrations in the diencephalon. Diencephalic MDA concentrations were increased by HT and time, with the effect of HT more prominent with increasing time. HT altered cystathionine, serine, tyrosine and isoleucine concentrations. Cystathionine was lower in HT birds compared with CT birds at 24h, whilst serine, tyrosine and isoleucine were higher at 48 h in HT birds. An increase in oxidative damage and alterations in amino acid concentrations in the diencephalon may contribute to the physiological, behavioral and thermoregulatory responses of heat-exposed chicks.

Expression and localization of the neuropeptide Y-Y4 receptor in the chick spleen: mRNA upregulation by high ambient temperature.

High ambient temperatures (HT) can increase diencephalic neuropeptide Y (NPY) expression, and central injection of NPY attenuates heat stress responses while inducing an antioxidative state in the chick spleen. However, there is a lack of knowledge about NPY receptor expression, and its regulation by HT, in the chick spleen. In the current study, male chicks were used to measure the expression of NPY receptors in the spleen and other immune organs under acute (30 vs. 40 ± 1°C for 3 h) or chronic (30 vs. 40 ± 1°C for 3 h/day for 3 days) exposure to HT and in response to central injection of NPY (47 pmol, 188 pmol, or 1 nmol). We found that NPY-Y4 receptor mRNA was expressed in the spleen, but not in other immune organs studied. Immunofluorescence staining revealed that NPY-Y4 receptors were localized in the splenic pulp. Furthermore, NPY-Y4 receptor mRNA increased in the chick spleen under both acute and chronic exposure to HT. Central NPY at two dose levels (47 and 188 pmol) and a higher dose (1 nmol) did not increase splenic NPY-Y4 receptor mRNA expression or splenic epinephrine under HT (35 ± 1°C), and significantly increased 3-methoxy-4-hydroxyphenylglycol (MHPG) concentrations under HT (40 ± 1°C). In conclusion, increased expression of NPY-Y4 receptor mRNA in the spleen under HT suggest that Y4 receptor may play physiological roles in response to HT in male chicks.

Individual variation in glucocorticoid stress responses in animals.

When stimuli from the environment are perceived to be a threat or potential threat then animals initiate stress responses, with activation of the hypothalamo-pituitary-adrenal axis and secretion of glucocorticoid hormones (cortisol and corticosterone). Whilst standard deviation or standard error values are always reported, it is only when graphs of individual responses are shown that the extensive variation between animals is apparent. Some animals have little or no response to a stressor that evokes a relatively large response in others. Glucocorticoid responses of fish, amphibian, reptiles, birds, and mammals are considered in this review. Comparisons of responses between animals and groups of animals focused on responses to restraint or confinement as relatively standard stressors. Individual graphs could not be found in the literature for glucocorticoid responses to capture or restraint in fish or reptiles, with just one graph in mammals with the first sample was collected when animals were initially restrained. Coefficients of variation (CVs) calculated for parameters of glucocorticoid stress responses showed that the relative magnitudes of variation were similar in different vertebrate groups. The overall mean CV for glucocorticoid concentrations in initial (0 min) samples was 74.5%, and CVs for samples collected over various times up to 4 h were consistently between 50% and 60%. The factors that lead to the observed individual variation and the extent to which this variation is adaptive or non-adaptive are little known in most animals, and future studies of glucocorticoid responses in animals can focus on individual responses and their origins and significance.

Corticosterone responses and personality in birds: Individual variation and the ability to cope with environmental changes due to climate change.

Birds can respond to an internal or external stimulus with activation of the HPA axis and secretion of corticosterone. There is considerable individual variation in corticosterone responses, and individual responses can be very different from the mean response for a group of birds. Corticosterone responses and behavioural responses to environmental stimuli are determined by individual characteristics called personality. It is proposed that birds with low corticosterone responses and proactive personalities are likely to be more successful (have greater fitness) in constant or predictable conditions, whilst birds with reactive personalities and high corticosterone responses will be more successful in changing or unpredictable conditions. The relationship between corticosterone responses and fitness thus depends on the prevailing environmental conditions, so birds with either low or high corticosterone responses can have the greatest fitness and be most successful, but in different situations. It is also proposed that birds with reactive personalities and high corticosterone responses will be better able to cope with environmental changes due to climate change than birds with proactive personalities and relatively low corticosterone responses. Phenotypic plasticity in corticosterone responses can be quantified using a reaction norm approach, and reaction norms can be used to determine the degree of plasticity in corticosterone responses of individual birds, and mean levels of plasticity in responses of species of birds. Individual corticosterone responses and personality, and reaction norms for corticosterone responses, can in future be used to predict the ability of birds to cope with environmental changes due to climate change.

Are baseline and short-term corticosterone stress responses in free-living amphibians repeatable?

Amphibians respond to environmental stressors by secreting corticosterone, a stress hormone which promotes physiological and behavioral responses. Capture handling can be used to stimulate physiological stress response in amphibians. The use of single blood sampling and presentation of mean data often limits the quantification of within and between individual variation in baseline and short-term corticosterone stress responses in amphibians. It is important for studies of amphibian physiological ecology to determine whether baseline and short-term corticosterone stress responses are consistent or not. We quantified repeatability (r), a statistical measure of consistency, in baseline and short-term corticosterone stress responses to a standard capture and handling stress protocol in free-living adult male cane toads (Rhinella marina). Corticosterone metabolite concentrations were measured entirely non-invasively in male toad urine samples via an enzyme-immunoassay. During the first sampling occasion, urine samples were collected manually from individual male toads (n=20) immediately upon field capture. Toads were handled for 5min then transferred to plastic bags (constituting a mild stressor), and urine samples were collected hourly over 8h in the field. The toads were resampled for baseline (0h) urine corticosterone with hourly urine sampling over 8h (for quantification of the stress induced corticosterone) at 14 day intervals on three consecutive occasions. Within and between sample variations in urinary corticosterone metabolite concentrations were also quantified. All toads expressed a corticosterone stress response over 8h to our standard capture and handling stress protocol. Variations both within and between toads was higher for corrected integrated corticosterone concentrations than corticosterone concentrations at baseline, 3 or 6h. Baseline urinary corticosterone metabolite concentration of the male toads was highly repeatable (r=0.877) together with high statistical repeatabilities for 3h (r=0.695), 6h (r=0.428) and 8h (r=0.775) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r=0.807; r=0.743 respectively). This study highlights that baseline and short-term corticosterone stress responses are repeatable in free-living amphibians. Future studies should utilize this non-invasive tool to explore repeatability among seasons and across years, and determine its functional significance in relation to behavioral ecology and reproduction in amphibians generally.

Repeatability of baseline corticosterone and short-term corticosterone stress responses, and their correlation with testosterone and body condition in a terrestrial breeding anuran (Platymantis vitiana).

Repeatability of physiological response variables, such as the stress hormone corticosterone, across numerous sampling occasions is an important assumption for their use as predictors of behaviour, reproduction and fitness in animals. Very few studies have actually tested this assumption in free-living animals under uncontrolled natural conditions. Non-invasive urine sampling and standard capture handling protocol have enabled the rapid quantification of baseline corticosterone and short-term corticosterone stress responses in anuran amphibians. In this study, established non-invasive methods were used to monitor physiological stress and urinary testosterone levels in male individuals of the terrestrial breeding Fijian ground frog (Platymantis vitiana). Adult male frogs (n = 20) were sampled at nighttime on three repeated occasions at intervals of 14 days during their annual breeding season on Viwa Island, Fiji. All frogs expressed urinary corticosterone metabolite responses to the capture and handling stressor, with some frogs showing consistently higher urinary corticosterone responses than others. Ranks of corticosterone values at 0, 4 and 8 h, and the corrected rank were highly significant (r = 0.75-0.99) between the three repeated sampling occasions. Statistical repeatabilities were high for baseline corticosterone (r = 0.973) and for corticosterone values at 2 h (r = 0.862), 4 h (r = 0.861), 6 h (r = 0.820) and 8 h (r = 0.926), and also for the total (inclusive of baseline corticosterone values) and the corrected integrated responses (index of the acute response) [r = 0.867 and r = 0.870]. Urinary testosterone levels also showed high statistical repeatability (r = 0.78). Furthermore, variation in baseline and short-term corticosterone stress responses was greater between individuals than within individuals. Baseline urinary corticosterone was significantly negatively correlated with the corrected integrated corticosterone response (r = -0.3, p < 0.001) but non-significantly with body-condition (r = -0.04) and baseline urinary testosterone (r = -0.07). In contrast, the corrected integrated corticosterone response was positively correlated (non-significantly) with baseline urinary testosterone (r = +0.04) and body-condition (r = +0.08). Urinary testosterone levels and body-condition were significantly negatively correlated (r = -0.23, p < 0.001). The results suggest that male frogs with higher levels of testosterone could have depleted energy reserve during the breeding period. The acute corticosterone responses help in replenishing energy that is needed for breeding and survival. The results also provide some support to the 'cort-fitness' hypothesis as highlighted by the negative correlation between baseline corticosterone and body-condition. It is most likely that the acute corticosterone response is adaptive and linked positively with reproductive fitness and survival in male anurans.

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.

Urinary corticosterone metabolite responses to capture and handling in two closely related species of free-living Fijian frogs.

Studies of baseline (unstressed) and short-term corticosterone stress responses in free-living amphibians can provide crucial information on the physiological responses of different populations to environmental change. In this study, we compared baseline and urinary corticosterone metabolite responses of free-living adult males and females of two closely related Fijian frogs of the Platymantis genus (Family: Ceratobatrachidae). Fijian ground frogs (Platymantis vitiana) live on the ground while Fijian tree frogs (Platymantis vitiensis) are arboreal. We captured free-living frogs and applied our moderate stress protocol (5 min handling during urine sampling at hourly intervals), with urinary corticosterone metabolite concentrations measured by enzyme-immunoassay. Mean urinary corticosterone metabolite concentrations in male and female Fijian ground frogs increased from 0 to 2 h and continued to increase to peak concentrations 5-6 h after capture. Mean baseline corticosterone concentration was significantly different between sexes (higher in males than females) only for Fijian ground frogs. There was no significant difference between sexes in the integrated corticosterone responses for both species. Mean baseline and urinary corticosterone metabolite responses of Fijian tree frogs were lower than those of Fijian ground frogs. Corticosterone levels increased for 4-5 h in both species and began to decrease again 7 h after initial capture. Corticosterone responses were consistently higher for Fijian ground frogs than Fijian tree frogs. Individuals in both species showed markedly variable corticosterone responses over the 8h duration of the stressor, with some individuals showing low stress responses and others showing high stress responses. The magnitude of the corrected integrated response of the ground frogs was almost twice that of the tree frogs. These differences in baseline and short-term corticosterone stress responses between these two species could be a consequence of ecological differences including micro-habitat, predator interactions and/or competitive interactions with the introduced cane toad (Rhinella marina). Comparisons of corticosterone responses between populations and species provide a valuable tool for measuring the physiological responses of the amphibians to environmental change.

Effects of temperature on urinary corticosterone metabolite responses to short-term capture and handling stress in the cane toad (Rhinella marina).

Extreme temperature can cause metabolic, immune and behavioural changes in amphibians. Short-term stress hormonal response via increased secretion of corticosterone enables amphibians to make necessary physiological and behavioural adjustments for coping with stressors. The effect of temperature on short-term corticosterone responses has not been studied in amphibians. In this study, this relationship was evaluated in adult male cane toads (Rhinella marina). We acclimated male toads (n=24 toads per group) at low, medium and high temperature (15, 25 or 35°C) under controlled laboratory conditions for a 14 day period. After thermal acclimation, short-term corticosterone responses were evaluated in the toads subjected to a standard capture and handling stress protocol over a 24h period. Corticosterone metabolites in toad urine were measured via enzyme-immunoassay. During acclimation, mean baseline urinary corticosterone level increased after transfer of the toads from wild into captivity and returned to baseline on day 14 of acclimation for each of the three temperatures. At the end of the 14 days of thermal acclimation period, baseline corticosterone level were highest for toad group at 35°C and lowest at 15°C. All toads generated urinary corticosterone responses to the standard capture and handling stressor for each temperature. Both individual and mean short-term corticosterone responses of the toads were highest at 35°C and lowest at 15°C. Furthermore, Q(10) values (the factor by which the reaction rate increases when the temperature is raised by 10°) were calculated for mean corrected integrated corticosterone responses as follows; (15-35°C) Q(10)=1.51, (15-25°C) Q(10)=1.60; (25-35°C) Q(10)=1.43. Both total and corrected integrated corticosterone responses were highest for toads at 35°C followed by 25°C and lowest for the 15°C toad group. Overall, the results have demonstrated the thermodynamic response of corticosterone secretion to short-term capture and handling stress in an amphibian species.

Individual variation and repeatability in urinary corticosterone metabolite responses to capture in the cane toad (Rhinella marina).

Urinary corticosterone metabolite enzyme-immunoassay (EIA) can be used for the non-invasive assessment of baseline levels and corticosterone responses in amphibians. In this study, urinary corticosterone responses of wild male cane toads (Rhinella marina) to confinement and repeated handling were measured to quantify individual variation in corticosterone responses for the first time in an amphibian species. Urine samples were collected at 0 h in the wild, hourly from 2 to 8 h after transfer into captivity, and again at 12 and 24 h in captivity. Toads were then held in captivity and subjected to the same sampling protocol on three occasions at 14 days intervals to quantify variation in corticosterone metabolite responses within and between toads. Baseline and individual corticosterone metabolite responses in male cane toads were generally consistent, with high statistical repeatabilities for 0 h (r=0.630), 6 h (r=0.793), 12 h (r=0.652) and 24 h (r=0.721) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r=0.567, p=0.033; r=0.728, p=0.014 respectively). Urinary corticosterone responses appear to be a stable, repeatable trait within individuals. Corticosterone responses in amphibians can be more readily measured when urine rather than plasma samples are collected, and the protocol established in the current study can now be applied to the study of variation in corticosterone responses in other amphibians.

Corticosterone responses differ between lines of great tits (Parus major) selected for divergent personalities.

Animal 'personality' describes consistent individual differences in suites of behaviors, a phenomenon exhibited in diverse animal taxa and shown to be under natural and sexual selection. It has been suggested that variation in personality reflects underlying physiological variation; however there is limited empirical evidence to test this hypothesis in wild animals. The hypothalamic-pituitary-adrenal axis is hypothesized to play a central role in personality variation. Here we tested whether in great tits Parus major variation in personality traits is related to plasma concentrations of corticosterone (CORT). Using a capture-restraint protocol we examined baseline and stress-induced CORT levels in two captive experimental groups: (1) birds selected for divergent personalities ('fast-bold' and 'slow-shy' explorers); and (2) non-selected offspring of wild parents. We first tested for differences in CORT between selection lines, and second examined the relationship between responses in a canonical personality test and CORT concentrations in non-selected birds. We found support for our prediction that the slow-shy line would exhibit a higher acute stress response than the fast-bold line, indicating a genetic correlation between exploratory behavior and stress physiology. We did not, however, find that continuous variation in exploratory behavior co-varies with CORT concentrations in non-selected birds. While our results provide support for the idea that personality emerges as a result of correlated selection on behavior and underlying physiological mechanisms, they also indicate that this link may be particularly evident when composite personality traits are the target of selection.

Hypothalamic gonadotropin-inhibitory hormone precursor mRNA is increased during depressed food intake in heat-exposed chicks.

The regulation of food intake in chickens (Gallus gallus domesticus) represents a complex homeostatic mechanism involving multiple levels of control, and regulation during high ambient temperatures (HT) is poorly understood. In this study, we examined hypothalamic mRNA expression of gonadotropin-inhibitory hormone (GnIH) to understand the effect of HT on an orexigenic neuropeptide. We examined the effects of HT (35 °C ambient temperature for 1, 24 or 48 h) on 14-day old chicks. HT significantly increased rectal temperature and suppressed food intake, and also influenced plasma metabolites. The expression of GnIH precursor mRNA in the diencephalon was significantly increased in chicks at 24-and 48 h of HT when food intake was suppressed significantly, whilst no change was observed for GnIH precursor mRNA and food intake at 1h of HT. In situ hybridization and immunocytochemistry further revealed the cellular localization of chicken GnIH precursor mRNA and its peptide in the paraventricular nucleus (PVN) in the chick hypothalamus. We examined plasma metabolites in chicks exposed to HT for 1 or 48 h and found that triacylglycerol concentration was significantly higher in HT than control chicks at 1h. Total protein, uric acid and calcium were significantly lower in HT chicks than control chicks at 48h. These results indicate that not only a reduction in food intake and alteration in plasma metabolites but also the PVN-specific expression of GnIH, an orexigenic agent, may be induced by HT. The reduced food intake at the same time as GnIH expression was increased during HT suggests that HT-induced GnIH expression may oppose HT-induced feeding suppression, rather than promote it. We suggest that the increased GnIH expression could be a consequence of the reduced food intake, and would not be a direct response to HT.

Stress-induced activation of ovarian heat shock protein 90 in a rat model of polycystic ovary syndrome.

AIM: Polycystic ovarian syndrome is the most common endocrine disorder affecting infertile women of reproductive age. This study evaluated the activation of heat shock protein 90 (Hsp 90) during the formation of stress-induced polycystic ovaries. MATERIAL & METHODS: Female Sprague-Dawley rats (180-200 g) were subjected to one of two stress-inducing conditions; animals were either treated with adrenocorticotropic hormone daily for 18 days or were exposed to daily cold stress for three weeks. Non-treated rats sampled during proestrus or diestrous served as controls. Blood samples were collected from the left ventricles of anesthetized rats and concentrations of follicle-stimulating hormone, luteinizing hormone, estradiol, testosterone and corticosterone were measured in all rats. The expression of messenger RNA for androgen receptor, estrogen receptor-α and -ß, nerve growth factor receptor, and glucocorticoid receptor, and protein expression for Hsp 90 was also assessed in the rat ovaries. RESULTS: Stress increased glucocorticoid receptor and androgen receptor expression, and decreased estrogen expression. Nerve growth factor receptor expression was greater in treated than diestrous rats and less in treated than proestrous rats. Ovarian Hsp 90 protein expression was increased in rats treated with adrenocorticotropic hormone or cold stress. Serum follicle-stimulating hormone levels were reduced and testosterone and corticosterone levels increased by stress, whilst luteinizing hormone and estradiol levels were similar to levels in diestrous and proestrus control rats respectively. CONCLUSIONS: The results indicate that stress, via the activation of ovarian Hsp 90 and changes in steroid hormone receptor expression and serum reproductive hormone levels, may be involved in the induction of polycystic ovaries in rats.