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Abstract: Based on plasma cortisol concentrations it is widely accepted that transport is stressful to horses. So far, cortisol release during transport has not been evaluated in depth by non-invasive techniques such as analysis of salivary cortisol and faecal cortisol metabolites. Transport also causes changes in heart rate and heart rate variability (HRV). In this study, salivary cortisol, faecal cortisol metabolites, heart rate and HRV in horses transported by road for short (one and 3.5 h) and medium duration (8 h) were determined. With the onset of transport, salivary cortisol increased immediately but highest concentrations were measured towards the end of transport (4.1±1.6, 4.5±2.6, 6.5±1.8 ng/ml in horses transported for one, 3.5 and 8 h, respectively). Faecal cortisol metabolite concentrations did not change during transport, but 1 day after transport for 3.5 and 8 h had increased significantly (p <0.01), reflecting intestinal passage time. Compared to salivary cortisol, changes in faecal cortisol metabolites were less pronounced. Heart rate increased and beat-to-beat (RR) interval decreased (p <0.05) with the onset of transport. Standard deviation of heart rate increased while root mean square of successive RR differences (RMSSD) decreased in horses transported for 3.5 (from 74±5 to 45±6 ms) and 8 h (from 89.7±7 to 59±7 ms), indicating a reduction in vagal tone. In conclusion, transport of horses over short and medium distances leads to increased cortisol release and changes in heart rate and HRV indicative of stress. The degree of these changes is related to the duration of transport. Salivary cortisol is a sensitive parameter to detect transient changes in cortisol release. [Copyright &y& Elsevier]

Glucocorticoid (GC) hormones rise in response to stressors, including natural events including weather or predator presence, and human activities, such as hunting, scientific research or recreational visits. However, because blood sampling itself causes stress and is dangerous or even impossible in some wildlife species, feedback-free methods for GC determination are needed to assess stress in these animals. Faecal GC analyses have thus gained interest. Here, we validate a non-invasive method to estimate the physiological stress in the Upland goose Chloephaga picta. An adrenocorticotropin hormone (ACTH) challenge was conducted in captive adults (female and male), and droppings were collected before, during and after the experiment. Corticosterone metabolite (CM) secretion in response to the ACTH challenge was measured with several enzyme immunoassays (EIA) to find the most appropriate test. We used CM levels during the periods before and after the experiment as control data. An EIA for 11-oxoetiocholanolone achieved the highest response to the ACTH challenge and also reflected a stress response to unfamiliar environment. Furthermore, CM concentrations of dry samples were highly correlated with the corresponding non-dried (frozen) samples. The data suggest that this method is appropriate to measure the stress in Upland geese, and that samples can be stored either frozen or dry form. [ABSTRACT FROM AUTHOR]

Abstract: Stressful events may contribute to low reproductive efficiency due to glucocorticoid-mediated inhibition of hormone secretion in a variety of species. We therefore investigated effects of stress related to management of mares around artificial insemination on secretion of cortisol and fertility parameters. To avoid further disturbance of mares by frequent blood sampling, faecal cortisol metabolites (fCM) were determined instead (sample collection at 8-h intervals). A total of 50 mares (16 maiden, 17 barren, 12 foaling, 5 teaching mares) were included in the study. Mares were brought to the AI centre in vans or trailers (driving time between 30min and 5h). Teaching mares were housed in the clinic and had therefore not to be transported. Mares were inseminated either with fresh/cooled-shipped or frozen semen. Rectal palpations and ultrasound examinations were performed at 24- to 48-h intervals, in animals inseminated with frozen semen at 6-h intervals during the last 48h before ovulation. In maiden, barren and foaling mares, fCM concentrations in faeces tended to be higher than in teaching mares at all times after arrival at the AI centre. At 24 and 48h after arrival, fCM concentrations in maiden mares were significantly higher than in teaching mares (24h: maiden mares 12.3±3.1ng/g, barren mares 8.5±1.2ng/g, foaling mares 11.0±2.4ng/g, teaching mares 3.8±0.6ng/g, p <0.05). The time from arrival at the AI centre to detection of ovulation did not differ among the different groups of mares and was 4.5±0.4, 5.0±0.5, 3.8±0.5 and 5.6±0.9 days in maiden, barren, foaling and teaching mares, respectively (n.s.). Pregnancy rates were 53, 53, 55 and 60%, respectively (n.s.). The time from arrival at the AI centre to detection of ovulation was 4.4±0.3 days and 4.9±0.3 days in mares inseminated with fresh/shipped (n =39) or frozen semen (n =11; n.s.), respectively. The frequency of follicular checks influenced fCM secretion and was statistically significant at 16h before ovulation (fresh/shipped semen: fCM 6.9±0.7ng/g faeces, frozen semen: fCM 16.9±5.2ng/g faeces, p <0.01). In the mare, gynaecological examinations seem to act as stressors and may increase cortisol secretion. However, this does not negatively influence fertility and in animals familiar with that procedure fCM concentrations are not elevated. [Copyright &y& Elsevier]

We studied mechanisms responsible for reproductive skew in female Alpine marmots. In each social group, only dominant females produced weaned young although subordinate females had similar body condition, were fertile as indicated by high levels of oestradiol during the mating season, and occasionally started pregnancies. During the period of gestation, dominant females initiated significantly more agonistic interactions against subordinate females, resulting in significantly increased levels of glucocorticoids and decreased levels of progesterone in subordinates. Results suggested that reproductive suppression in female Alpine marmots is mediated by the negative effects of stress (glucocorticoids) on the activity of the hypothalamic-pituitary-gonadal axis. The strength of competition between subordinate and dominant females was affected by their relatedness. Dominant females attacked unrelated subordinate females more, whereas amicable behaviour was observed mainly between dominant females and their daughters. These differences could be explained by differences in indirect fitness: related subordinate females benefited from warming the offspring of the dominant female during hibernation. Reproductive suppression was apparently costly for dominant females, because their reproductive success decreased as the number of adult subordinate females in a group increased. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour. [Copyright &y& Elsevier]

Reports on a study which estimated physiological stress in a small percellariiform seabird, the Wilson's storm-petrel Oceanites oceanicus, as a means of understanding how limited resources are partitioned between provisioning parents and their chicks. Analysis of adrenocortical activity of Wilson's storm-petrels during the breeding season by measuring tetrahydrocorticosterone concentrations in extracts of feces and urine of chicks and adults; Indication that tetrahydrocorticosterone levels can be used to assess adrenal activity for signs of physiological stress; Results which indicate that the petrels respond to unfavorable conditions by maintaining their own body condition and reducing provision of food to their chicks.

Traditionally androgen concentrations are measured invasively in blood plasma. However, non-invasive methods to detect androgens are desirable, as this reduces interference with the natural behavior of the study species and multiple samples can be obtained relatively easy. The aim of this study was to validate a method to measure androgens non-invasively in excrements of male European stonechats (Saxicola torquata rubicola). Extracts of excrements of a male stonechat injected with [3H]testosterone ([3H]T) were chromatographically separated using high performance liquid chromatography (HPLC). The resulting HPLC fractions were then analyzed with a radioimmunoassay against testosterone (T-RIA). The results showed that the assay picked up major metabolites of [3H]T. The physiological relevance of excreted androgen metabolites was further validated by showing that injection of exogenous GnRH to seven males led to a significant increase in excreted androgen metabolites. In contrast, androgen metabolite levels of six saline-injected control males did not increase. Furthermore, excrements from nine males were collected from January until April to see whether the typical seasonal increase in testosterone levels can also be traced when measuring excreted androgen metabolites. As expected, there was a significant seasonal increase in androgen metabolite concentrations. Thus, the T-RIA measures androgen metabolites in droppings of male European stonechats and to our knowledge this study represents the first validation of a non-invasive androgen assay in a passerine bird. [Copyright &y& Elsevier]

Although greylag geese Anser anser establish long-term monogamous pairbonds, some of the existing pairs do split up (divorce) and new pairs are formed during the annual spring mating period. In this study, male greylag geese which were involved in the challenge of an existing pairbond (challenged males and challengers) were regarded as ‘natural experimental’ groups and compared with males in stable pairbonds (unchallenged males and male-paired males, a common male strategy when the availability of females is low). In total, 37 males were investigated. The analysis included a description of the seasonal patterns of hormone levels, aggression and courtship. We tested whether hormone levels correlated with aggressive and courtship behaviours. Finally, we compared hormonal and behavioural patterns amongst the four groups. Immunoreactive testosterone (T) and corticosterone (B) equivalents were measured in faecal samples. Individual hormone levels were correlated with frequencies of agonistic male-male interactions and with frequencies of male-female courtship. During early mating and pre-laying phases, T was at its seasonal maximum, which may have masked hormone-behaviour correlations. During egg-laying, at the onset of seasonally decreased T, agonistic male-male interactions and the frequencies of courtship behaviour were significantly correlated with T. Unchallenged males had higher rates of agonistic interactions than any other males. However, unchallenged and challenged males tended to excrete T at higher levels than challengers. The high rates of being attacked and elevated levels of faecal B were indicative of the social conflict experienced by challengers. No hormonal differences were observed between heterosexually paired males and male-paired males. In summary, pairbond status and situations of social conflict had a modulating effect on T and B; however, in this study, the two hormones seem to be affected independently of one another. [ABSTRACT FROM AUTHOR]

To monitor adrenocortical activity in Adélie penguins (Pygoscelis adeliae), we validated an enzyme immunoassay (EIA) for faecal glucocorticoid (GC) metabolites. An adrenocorticotropin hormone (ACTH) challenge was conducted on a paired female and male. The EIA for tetrahydrocorticosterone showed a clear response to the ACTH challenge in both birds. After high-performance liquid chromatography using pooled samples generated from the ACTH challenge, and analysing each individual fraction, three immunoreactive peaks were detected. Both biological and chemical validations strongly suggest that the EIA can be a useful tool for non-invasively measuring GC metabolites in faeces of Adélie penguins. [ABSTRACT FROM AUTHOR]

Although it is known that hormone concentrations vary considerably between individuals within a population, how they change across time and how they relate to an individual’s reproductive effort remains poorly quantified in wild animals. Using faecal samples collected from wild red deer stags, we examined sources of variation in faecal cortisol and androgen metabolites, and the potential relationship that these might have with an index of reproductive effort. We also biologically validated an assay for measuring androgen metabolites in red deer faeces. We show that variation in hormone concentrations between samples can be accounted for by the age of the individual and the season when the sample was collected. Faecal cortisol (but not androgen) metabolites also showed significant among-individual variation across the 10-year sampling time period, which accounted for 20% of the trait’s phenotypic variance after correcting for the age and season effects. Finally, we show that an index of male reproductive effort (cumulative harem size) during the mating season (rut) was positively correlated with male cortisol concentrations, both among and within individuals. We suggest that the highest ranking males have the largest cumulative harem sizes (i.e. invest the greatest reproductive effort), and that this social dominance may have associated behaviours such as increased frequency of agonistic interactions which are associated with corresponding high levels of faecal cortisol metabolites (FCM). [ABSTRACT FROM AUTHOR]

Ambient temperature and air pressure are relevant stimuli that can elicit hormonal responses in alignment with adjusting individuals’ physiology and behaviour. This study investigated possible changes in corticosterone (C) and testosterone (T) and contingencies with behaviour in response to ambient temperature and air pressure, and it evaluated the temporal response dynamics of these hormones in 12 individual Greylag geese ( Anser anser ) over 26 and 12 individual Northern bald ibis ( Geronticus eremita ) over 27 days, during late winter. Immunoreactive metabolites of C and T were analysed non-invasively from 626 fecal samples by means of group-specific antibodies and correlated to behaviour and weather factors. In both species, high C levels correlated with low temperatures 24 h before sampling, but low C levels correlated with high air pressure 6–12 h before sampling. In both species, C levels and behavioural activity were negatively correlated. In addition, temperature had a positive influence on T levels in both species 12–24 h before sampling. The fact that weather conditions influenced changes in levels of C, while social interactions did not, is indicative of a general mechanism of graduated physiological adjustment to environmental variations affecting metabolism, stress responses and behaviour. [ABSTRACT FROM AUTHOR]

Background The clinical signs of hyperadrenocorticism (hypercortisolism) in dogs are known to be caused by chronic overexposure to glucocorticoids. The quantification of cortisol in serum, saliva or urine reflects the cortisol concentration at the time of sample collection, but in suspected hyperadrenocorticism it may be preferable to examine a long-term parameter of cortisol production. Hypothesis/Objectives There is a need for a noninvasive method to monitor the long-term production of cortisol in dogs. It seems possible that measuring cortisol levels in hair could represent such a method. Animals Hair was collected from 12 dogs with hyperadrenocorticism and from 10 healthy control dogs. Methods Immunoreactive cortisol, cortisone and corticosterone concentrations were determined by enzyme immunoassay. High-performance liquid chromatography was performed to test the validity of the cortisol assay. Results Levels of immunoreactive cortisol, cortisone and corticosterone were significantly higher in dogs with hyperadrenocorticism than in control dogs. The difference was most pronounced for the cortisol level. Conclusions and clinical importance The determination of cortisol in hair offers the advantage that sampling is easier and less invasive than taking blood, urine, faeces or saliva. Measuring cortisol in hair may represent a valuable tool for the diagnosis of hyperadrenocorticism in dogs. [ABSTRACT FROM AUTHOR]

Abstract: Salivary alpha-Amylase (sAA) is a starch digesting enzyme. In addition to its function in the context of nutrition, sAA has also turned out to be useful for monitoring sympathetic nervous system activity. Recent studies on humans have found a relationship between intra-individual changes in sAA activity and physical and psychological stress. In studies on primates and other vertebrates, non-invasive monitoring of short-term stress responses is usually based on measurements of cortisol levels, which are indicative of hypothalamic–pituitary–adrenal activity. The few studies that have used both cortisol levels and sAA activity indicate that these two markers may respond differently and independently to different types of stress such that variation in the degree of the activation of different stress response systems might reflect alternative coping mechanisms or individual traits. Here, we present the first data on intra- and inter-individual variation of sAA activity in captive bonobos and compare the results with information from other ape species and humans. Our results indicate that sAA activity in the bonobo samples was significantly lower than in the human samples but within the range of other great ape species. In addition, sAA activity was significantly higher in samples collected at times when subjects had been exposed to stressors (judged by changes in behavioral patterns and cortisol levels) than in samples collected at other times. Our results indicate that bonobos possess functioning sAA and, as in other species, sAA activity is influenced by autonomic nervous system activity. Monitoring sAA activity could therefore be a useful tool for evaluating stress in bonobos. [Copyright &y& Elsevier]

Abstract: Based on cortisol release, a variety of situations to which domestic horses are exposed have been classified as stressors but studies on the stress during equestrian training are limited. In the present study, Warmblood stallions (n =9) and mares (n =7) were followed through a 9 respective 12-week initial training program in order to determine potentially stressful training steps. Salivary cortisol concentrations, beat-to-beat (RR) interval and heart rate variability (HRV) were determined. The HRV variables standard deviation of the RR interval (SDRR), RMSSD (root mean square of successive RR differences) and the geometric means standard deviation 1 (SD1) and 2 (SD2) were calculated. Nearly each training unit was associated with an increase in salivary cortisol concentrations (p <0.01). Cortisol release varied between training units and occasionally was more pronounced in mares than in stallions (p <0.05). The RR interval decreased slightly in response to lunging before mounting of the rider. A pronounced decrease occurred when the rider was mounting, but before the horse showed physical activity (p <0.001). The HRV variables SDRR, RMSSD and SD1 decreased in response to training and lowest values were reached during mounting of a rider (p <0.001). Thereafter RR interval and HRV variables increased again. In contrast, SD2 increased with the beginning of lunging (p <0.05) and no changes in response to mounting were detectable. In conclusion, initial training is a stressor for horses. The most pronounced reaction occurred in response to mounting by a rider, a situation resembling a potentially lethal threat under natural conditions. [ABSTRACT FROM AUTHOR]

Abstract: Public health agencies utilize aerial insecticides to interrupt an active West Nile virus (WNV) transmission cycle, which may expose WNV-infected birds to these agents. Although resmethrin has been considered benign to birds, no studies have evaluated whether the environmentally employed form of resmethrin with PBO synergist (synergized resmethrin (SR)) can suppress avian immunity to WNV infection and enhance a bird''s host competence. Recognizing that wild birds confront toxicological stressors in the context of various physiological states, we exposed four groups (n =9–11) of 9-week-old chickens (Gallus domesticus) to drinking water with either SR (three alternate days at 50μg/l resmethrin+150μg/l piperonyl butoxide), CORT (10 days at 20mg/l to induce subacute stress), the combination of SR and CORT, or 0.10% ethanol vehicle coincident with WNV infection. Compared to controls, SR treatment did not magnify but extended viremia by 1 day, and depressed IgG; CORT treatment elevated (mean, 4.26log10 PFU/ml) and extended viremia by 2 days, enhanced IgM and IgG, and increased oral virus. The combination of SR and CORT increased the number of chickens that shed oral virus compared to those treated with CORT alone. None of the chickens developed a readily infectious viremia to mosquitoes (none ≥5log10 PFU/ml), but viremia in a CORT-exposed chicken was up to 4.95log10 PFU/ml. Given that SR is utilized during WNV outbreaks, continued work toward a complete risk assessment of the potential immunotoxic effects of SR is warranted. This would include parameterization of SR exposures with immunological consequences in wild birds using both replicating (in the laboratory) and non-replicating (in the field) antigens. As a start, this study indicates that SR can alter some immunological parameters, but with limited consequences to primary WNV infection outcome, and that elevated CORT mildly enhances SRs immunotoxicity in chickens. [Copyright &y& Elsevier]

In long-term monogamous birds reproductive success varies considerably among pairs. Determinants of reproductive success may be individual as well as pair-specific parameters, including the degree of compatibility and coordination among pair partners. However, little is known about the consistency of partner compatibility with regards to social contexts and life-history changes. In the long-term monogamous, biparental greylag geese, reproductive success was previously found to correlate positively with the degree of hormonal compatibility within pairs. In the present study, we analyzed the degree of within-pair testosterone covariation (TC) in relation to individual and pair-specific life history and social instability. We found that greylag goose pairs facing active social challenge had lower degrees of TC than those in unchallenged pair-bonds, whereas the permanent attachment of a third individual to an existing pair or the number of previous partners did not correspond with changed TC. Furthermore, TC decreased with increasing pair-bond duration and increased with female age but was not related with age of the male partner or other life-history parameters. Hence, our data suggest that hormonal partner compatibility in greylag geese is not a stable trait, but rather reflects a pair's status quo, which may be particularly affected by the stability of the social environment. [ABSTRACT FROM PUBLISHER]

Abstract: Prenatal learning plays an important role in the ontogeny of behavior and birds provide a useful model to explore whether and how prenatal exposure to hormones of maternal origin can influence prenatal learning and the development of behavior. In this study we assessed if prenatal exposure to yolk testosterone can influence auditory learning in embryos of Northern bobwhite quail (Colinus virginianus). We experimentally enhanced testosterone concentrations in bobwhite quail eggs prior to incubation. The embryos from these T-treated eggs as well as control embryos that had received the vehicle-only or were non-treated were exposed to an individual bobwhite hen''s maternal call for 120 min over the course of the day prior to hatching. All chicks were tested at 24 h following hatching for their auditory preference between the familiar bobwhite maternal call versus an unfamiliar bobwhite maternal call. T-treated chicks spent significantly more time in proximity to the familiar call compared to the unfamiliar call and also showed shorter latencies to approach the familiar call than control birds. Increased emotional reactivity, i.e. propensity to express fear responses, was also found in T-treated chicks. Baseline heart rates recorded in a second group of T-treated embryos and control embryos did not differ, which suggests no effect of yolk testosterone on baseline arousal level. To our knowledge this is the first demonstration of the influence of prenatal exposure to testosterone on auditory learning. [Copyright &y& Elsevier]

Abstract: Urinary metabolites of androgens and cortisol were measured in free-living male and female bonobos. Sex differences and correlations between adrenal and gonadal steroid excretion were investigated. The immunoreactive concentrations of androgens were measured with two different androgen assays. One assay used a testosterone (T) antibody raised with a 17β-hydroxy group, and the other employed an antibody raised against a reduced form, 5α-androstane-17α-ol-3-one-CM (17α) with cross reactivity for epitestosterone and 5α-androstanedione. Both assays have been used in bonobo and chimpanzee studies where non-invasive techniques were employed. The levels of 17α-androgen metabolites were 1.7- and 3-fold higher than those of T-metabolites in males and females. The two androgen assay results correlated in males but not females. There was a sex difference in the T-metabolites measured. Male levels were significantly higher. Levels of 17α in the two sexes were similar. Cortisol metabolite levels (CORT) were similar between the sexes. The T-metabolites were significantly correlated with CORT in males but not in females. In females, the 17α-androgen metabolites correlated with CORT. This suggests that either androgen secretion or metabolism differs between the sexes. A parsimonious interpretation of the androgen assay cortisol/androgen correlation differences would be that larger components of dehydroepiandrosterone (DHEA), androstenedione or epitestosterone from the adrenal androgens were being excreted and measured in the females. The CORT/T metabolite interactions in males may reflect male-specific social or metabolic endocrine conditions. [Copyright &y& Elsevier]

Abstract: Studies have demonstrated an effect of yolk testosterone levels on the physiology and behavior of nestling birds. In order to investigate this phenomenon experimentally in a precocial bird, we enhanced yolk testosterone, but within the physiological range, by injecting 50 ng testosterone in ethanol into Japanese quail Coturnix japonica eggs prior to incubation. The chicks hatching from these as well as from control eggs that had received the carrier-only or were left unmanipulated were subject to a number of behavioral tests from hatching to the age of 3 weeks. In addition, fecal samples were taken during a 90-min isolation period to determine a physiological response to a stressor. Experimental chicks performed a detour task faster and approached novel objects sooner than did the controls. Chicks from treated eggs took a longer time to start distress vocalizing and also produced less distress calls during open-field trials, took on average a larger number of trials for them to show tonic immobility and also excreted lower levels of corticosterone metabolites (BM) than did the controls. In response to a stressor, excreted BM was initially higher in the control chicks, as compared to the experimental birds. Induced behavioral effects were independent of sex with no sex treatment interactions found. In sum, experimentally enhanced testosterone levels in the eggs shifted individual behavioral phenotype towards “bold” or “proactive”, irrespective of sex. We conclude that testosterone in the yolk influences the coping style of hatchlings and may be a potential means of maternal influence on offspring phenotype. [Copyright &y& Elsevier]

Testosterone modulates male vertebrates’ sexual and social behaviour. We experimentally investigated the testosterone-sensitive behaviours in male greylag geese (Anser anser) by implanting silastic tubes containing crystalline testosterone during the mating season (February; 5 implanted and 5 control males) and in the early winter (November; 7 and 7). Focal animals were part of a semi-tame, unrestrained flock with fully intact social relationships. Excreted testosterone and corticosterone immunoreactive metabolites (TM, BM) were determined by enzyme immunoassay. Individual faecal samples and behavioural protocols were collected daily over a period of 5 weeks, including 1 control week before implantation. In February, no significant behavioural effects of the supplemental testosterone were observed, which may be due to the naturally occurring high systemic androgen levels in spring. In November, however, implanted males had higher TM excretion rates and performed status signalling behaviour (“beak up”) more frequently than control males. No differences between implanted and control males were found with respect to BM, agonistic interactions or vigilance behaviour. Furthermore, during the second week after implantation, TM positively correlated with the frequency of “beak up” of implanted males, whilst their female partners were attacked with lower latency by other members of the flock than the females of control males. Hence, status signalling in greylag ganders seems to be testosterone-sensitive year-long and “inappropriate” status signalling of males may draw attacks towards their females. [ABSTRACT FROM AUTHOR]

In many species, seasonal activities such as reproduction or migration need to be fine-tuned with weather conditions. Air pressure and temperature changes are the best parameters for such conditions. Adapting to climatic changes invariably involves physiological and behavioral reactions associated with the adrenals. In the present study, we investigated the effects of ambient temperature and air pressure on excreted immuno-reactive metabolites of corticosterone (BM) and androgens (AM). Focal individuals were 14 paired male greylag geese (Anser anser) from a semi-tame, unrestrained flock. BM and AM were measured in individual fecal samples over 25 days in November and December. Two different ACTH-validated assays were used for the assessment of BM: the first one cross-reacting with 11β,21-diol-20-one structures (“old assay”) and the second one with 5β,3α,11β-diol structures (“new assay”). With the “new assay,” BM correlated negatively with the minimum ambient temperature of the night before, which may reflect corticosterone involvement in thermoregulation. BM also correlated positively with the minimum air pressure of the previous afternoon, which supports the value of air pressure for predicting weather conditions. Together, these reactions suggest a role of the adrenals in responding behaviorally and physiologically to changes in weather. Preliminary analysis indicated a higher sensitivity to the excreted glucocorticosteroid metabolites in the “new assay.” As expected for outside the mating season, no relationships were found between excreted AM and the weather parameters considered. The gradual changes in BM excretion in parallel with weather conditions may be part of the fine-tuning of physiology and behavior by environmental clues. [Copyright &y& Elsevier]

Haematology and plasma biochemistry values are useful tools for ecological research, providing information on the physiological state and adaptation of individuals to their habitat, changes in nutritional state of birds, body condition, the level of parasite infestation, etc. We studied the effect of stress factors on haematological and plasma biochemistry values in adult and nestling Wilson’s storm petrels Oceanites oceanicus (Aves, Procellariiformes). We measured packed cell volume, triglyceride levels, plasma protein levels, plasma hue and plasma corticosterone of nestlings and breeding adults at King George Island, South Shetland Islands. We used a snow storm as a natural experiment to test what effect starvation has on the stress response of nestlings. In particular, we predicted that: (1) plasma protein concentrations and plasma hue reflect ectoparasite load, (2) triglycerides and hue reflect the nutritional state, and (3) corticosterone levels increase with ectoparasite load and starvation. In line with our predictions, plasma triglycerides were higher in nestlings than adults, indicating a resorptive nutritional state in nestlings, during which dietary fat is deposited in adipose tissues. In adults, plasma triglycerides were positively correlated with body mass. Corticosterone levels increased in response to handling in adults, while we did not find a stress response in nestlings in good condition. However, nestlings sampled after their nests had been blocked by a snow storm showed a stress response in excess of that of adults. In nestlings sampled after the snow storm, corticosterone peak levels were positively correlated with the infestation intensity of the ectoparasite Philoceanus robertsi (Phthiraptera: Ischnocera), suggesting that the stress response is increased when more stressors act at the same time. In adults, plasma hue and proteins decreased with increasing ectoparasite load. [ABSTRACT FROM AUTHOR]

Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2 h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10 h postinjection in experiment 1 and about 4 h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5α-pregnane-3β,11β,21-triol-20-one (measuring corticosterone metabolites with a 5α-3β,11β-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals’ sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology. [Copyright &y& Elsevier]

Presents a study which looked at the impact of social stress and dominance in greylag geese. Detailed information on the methodology used to conduct the study; Discussion based on the results of the study.

Simple Summary: Testosterone is the main reproductive hormone in male vertebrates. Conventional methods to assess testosterone rely on invasive blood sampling procedures, which can induce pain and distress to the animals. Here we successfully validated a non-invasive method to determine testosterone levels by assessing testosterone metabolites (TMs) in excreta of mice. We investigated the effects of sex and daytime on the metabolism and the excretion of TMs and validated the applied EIA to measure TMs. Further, we assessed diurnal fluctuations in TM excretions in both sexes and across strains. We found that males excreted more radiolabeled TMs via their feces compared to females. TM excretion patterns did not differ between sexes but TM excretion occurred faster in urinary than fecal samples. Animals excreted TM faster during the night than during the day. Daytime had no effect on the formed TMs; however, males and females formed different TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences between mouse strains. Our non-invasive method to assess fecal TMs can be widely used and will benefit various research disciplines. Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a non-invasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the applied EIA to measure TMs and assessed diurnal fluctuations in TM excretions in male and female mice and across strains. We found that males excreted significantly more radiolabeled TMs via the feces (59%) compared to females (49.5%). TM excretion patterns differed significantly between urinary and fecal samples and were affected by the daytime of ³H-testosterone injection. Overall, TM excretion occurred faster in urinary than fecal samples. Peak excretion of fecal TMs occurred after 8 h when animals received the 3H-testosterone in the morning, or after 4 h when they received the 3H-testosterone injection in the evening. Daytime had no effect on the formed TMs; however, males and females formed different types of TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences in the TM levels of C57BL/6J and B6D2F1 hybrid males. Finally, we successfully validated our applied EIA (measuring 17β-hydroxyandrostane) by showing that hCG (human chorionic gonadotropin) administration increased TM levels, whereas castration reduced them. In conclusion, our EIA proved suitable for measuring fecal TMs in mice. Our non-invasive method to assess fecal TMs can be widely used in various research disciplines like animal behavior, reproduction, animal welfare, ecology, conservation, and biomedicine. [ABSTRACT FROM AUTHOR]

Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans , which contains the human estrogen receptor alpha and a reporter construct, in which β-galactosidase gene expression is controlled by an estrogen-responsive-element. The estrogen response of the fungal biosensor was validated with blood, urine, feces, milk and saliva. All matrices were screened for estrogenic activity prior to and after chemical extraction and the results were compared to an enzyme immunoassay (EIA). The biosensor showed consistent results in milk, urine and feces, which were comparable to those of the EIA. In contrast to the EIA, no sample pre-treatment by chemical extraction was needed. For 17β-estradiol, the biosensor showed a limit of detection of 1 ng/L. The validation of the biosensor for pregnancy diagnosis revealed a specificity of 100% and a sensitivity of more than 97%. In conclusion, we developed and validated a highly robust fungal biosensor for detection of estrogen activity, which is highly sensitive and economic as it allows analyzing in high-throughput formats without the necessity for organic solvents. [ABSTRACT FROM AUTHOR]

The welfare of animals in captivity is of considerable societal concern. A major source of stress, especially for wild animals, is the lack of control over their environment, which includes not being able to avoid contact with human beings. Paradoxically, some studies have shown that interactions with human beings may improve the welfare of wild animals in captivity. Here, we investigated the behavioural (behaviours indicative of cooperation or stress) and physiological (variations in salivary cortisol concentrations) effects of the increasingly used practice of training wild animals as a way to facilitate handling and/or as behavioural enrichment. We evaluated the effects of indoor training sessions with familiar caretakers on nine human-socialised individuals of a wild species, the wolf (Canis lupus), in comparison to nine individuals of its domesticated form, the dog (Canis lupus familiaris). All animals were raised and kept in intraspecific packs under identical conditions—in accordance with the social structure of the species—in order to control for socialisation with human beings and familiarity with training. We also collected saliva samples of trainers to measure GC and testosterone concentrations, to control for the effects of trainers’ stress levels on the responses of the animals. During the training sessions, separated from pack members, the animals stayed voluntarily close to the trainers and mostly adequately performed requested behaviours, indicating concentration to the task. Similarly to dogs, the salivary cortisol level of wolves–used as an index of stress—dropped during these sessions, pointing to a similar stress-reducing effect of the training interaction in both subspecies. The responses to the requested behaviours and the reduction in salivary cortisol level of wolves and dogs varied across trainers, which indicates that the relaxing effect of training has a social component. This points to another factor affecting the welfare of animals during the sessions, beside the rewarding effect of getting food and control over the situation by successfully completing a task. As all responses performed by the animals corresponded to cues already familiar to them, the reported effects were likely due to the above cited factors rather than to a learning process. Our results support previous findings suggesting that training is a potentially powerful tool for improving welfare in some wild social canids by creating structured and positive interactions between these animals and their human caretakers. [ABSTRACT FROM AUTHOR]

Mothers may affect the future success of their offspring by varying allocation to eggs and embryos. Allocation may be adaptive based on the environmental conditions perceived during early breeding. We investigated the effects of food supplementation and predation risk on yolk hormone transfer in the pied flycatcher Ficedula hypoleuca. In a food supplementation experiment, females were food-supplemented prior to and during egg-laying and androgen concentrations were measured throughout the laying order. Predation risk was investigated in three different studies combining both correlative data, where flycatchers bred in close proximity to two different predator species that prey upon adult flycatchers (either Tengmalm's owl Aegolius funereus or pygmy owl Glaucidium passerinum), and an experimental manipulation, where flycatchers were exposed to cues of a nest predator (least weasel Mustela nivalis). Females receiving food supplementation laid eggs with lower concentrations of androstenedione (A4) than females not receiving food supplements. Yolk testosterone (T) concentration showed the same pattern but the difference was not statically significant. Testosterone (but not A4) concentration increased within clutches, from the first to the last egg, independently of the food supplementation. Females breeding under high predation risk did not differ from control females in their yolk androgen levels (A4, T or progesterone). However, concentrations of A4 tended to be lower in the proximity of pygmy owls, which could indirectly increase offspring survival after fledging. Food supplementation during egg-laying seems to have a stronger impact on maternal transfer of androgens than predation risk. Food availability and predation risk could differentially affect the trade-offs of androgen allocation for the offspring when raised in good vs. dangerous environments. [ABSTRACT FROM AUTHOR]

Estrogens are important for the bone development and health. Exposure to endocrine disrupting chemicals during the early development has been shown to affect the bone phenotype later in life. Several studies have been performed in rodents, while in larger animals that are important to bridge the gap to humans there is a paucity of data. To this end, the pig as large animal model was used in the present study to assess the influence of gestational estradiol-17β (E2) exposure on the bone development of the prepubertal and adult offspring. Two low doses (0.05 and 10 μg E2/kg body weight) referring to the ‘acceptable daily intake’ (ADI) and the ‘no observed effect level’ (NOEL) as stated for humans, and a high-dose (1000 μg E2/kg body weight), respectively, were fed to the sows every day from insemination until delivery. In the male prepubertal offspring, the ADI dose group had a lower strength strain index (p = 0.002) at the proximal tibia compared to controls, which was determined by peripheral quantitative computed tomography. Prepubertal females were not significantly affected. However, there was a higher cortical cross-sectional area (CSA) (p = 0.03) and total CSA (p = 0.02) at the femur midpoint in the adult female offspring of the NOEL dose group as measured by computed tomography. These effects were independent from plasma hormone concentrations (leptin, IGF1, estrogens), which remained unaltered. Overall, sex-specific effects on bone development and non-monotonic dose responses were observed. These results substantiate the high sensitivity of developing organisms to exogenous estrogens. [ABSTRACT FROM AUTHOR]

Measuring hair cortisol has attracted interest as a long term parameter for chronic stress evaluation. However, some studies support the hypothesis that locally produced cortisol, originating from the hair follicle or skin cells, affects concentrations in the hair. In an animal model the influence of different treatments (extensive brushing, administration of a hyperemising fluid that enhances blood circulation or a synthetic glucocorticoid) on the local cortisol production of hair was evaluated. Therefore eight sheep were sheared and the area of the skin surface of the back was quartered, with three quarters being daily subjected to a certain treatment and one quarter remaining untreated. The skin areas were sheared again after three weeks and cortisol concentrations of all wool samples were determined by immunoassay. Systemic cortisol concentrations were additionally monitored with faecal samples, indicating a significant decline in concentrations of glucocorticoid metabolites between week 1 and 2 or 3, respectively. We found no significant difference in hair cortisol concentrations between fields before treatment ( p = 0.310). Comparing matched fields before and after treatment, we found no significant differences in wool cortisol concentrations for fields treated with hyperemising fluid as well as for the control fields ( p = 0.329, p = 0.097). Hairs exposed to either extensive brushing or dexamethasone fluid had significantly higher immunoreactive cortisol concentrations after three weeks of treatment ( p = 0.016, p = 0.01). We therefore advise cautious interpretation when measuring hair cortisol concentrations as a parameter for chronic stress, because external factors may have a significant influence on the results. [ABSTRACT FROM AUTHOR]

Blood levels of corticosterone have been traditionally analyzed to assess stress levels in birds; however, measuring steroid hormone metabolites in feces and droppings has gained much interest as a noninvasive technique successfully used for such purposed in vertebrates. Diet may affect these fecal metabolite levels (e.g., due to nutritional stress), however, this variable has not been taken into account in studies with chicks despite the great dietary flexibility of many avian species. In this study, we addressed for the first time this key issue and validated the technique in wild gull-billed tern chicks ( Gelochelidon nilotica ). Several enzyme immunoassays were used to determine the most appropriate test to measure the stress response. Subsequently, we performed an experiment in captivity to assess adrenocortical activity in gull-billed tern chicks fed with two diets: piscivorous vs. insectivorous. Finally, the relation between the chicks’ growth rate and excreted immunoreactive glucocorticoid metabolites (EGMs) was also evaluated. We found the immunoreactive cortisone metabolites to be a good index of stress (as being an index of adrenocortical reactivity) in chicks of this species. Fish-fed chicks had higher levels of cortisone metabolites when comparing both concentration and total daily excreted metabolites. Within each treatment diet, cortisone metabolite levels and growth rates were negatively correlated. These findings suggest that the diet should be considered when using this technique for comparative purposes and highlight the trade-off between stress levels and chicks growth rates. [ABSTRACT FROM AUTHOR]

The management of captive animals has been improved by the establishment of positive reinforcement training as a tool to facilitate interactions between caretakers and animals. In great apes, positive reinforcement training has also been used to train individuals to participate in simple medical procedures to monitor physical health. One aim of positive reinforcement training is to establish a relaxed atmosphere for situations that, without training, might be very stressful. This is especially true for simple medical procedures that can require animals to engage in behaviours that are unusual or use unfamiliar medical devices that can be upsetting. Therefore, one cannot exclude the possibility that the training itself is a source of stress. In this study, we explored the effects of medical positive reinforcement training on salivary cortisol in two groups of captive ape species, orangutans and bonobos, which were familiar to this procedure. Furthermore, we successfully biologically validated the salivary cortisol assay, which had already been validated for bonobos, for orangutans. For the biological validation, we found that cortisol levels in orangutan saliva collected during baseline conditions were lower than in samples collected during three periods that were potentially stressful for the animals. However, we did not find significant changes in salivary cortisol during medical positive reinforcement training for either bonobos or orangutans. Therefore, for bonobos and orangutans with previous exposure to medical PRT, the procedure is not stressful. Thus, medical PRT provides a helpful tool for the captive management of the two species. [ABSTRACT FROM AUTHOR]

Abstract: External and internal stressors prolong parturition in different species. At parturition, sympathoadrenal activation should be avoided because an increased sympathetic tone may cause uterine atonia via β2-receptors. We hypothesized that at physiological parturition, horses are under parasympathetic dominance, and stress-response mechanisms are not activated during delivery of the foal. To evaluate stress responses, heart rate, heart rate variability, catecholamines, and cortisol were analyzed in mares (n = 17) throughout foaling. Heart rate decreased from 2 hours before (51 ± 1 beats/minute) to 2 hours after delivery (41 ± 2 beats/minute; P < 0.05). Heart rate variability variables, standard deviation of the beat-to-beat interval, and root mean square of successive beat-to-beat differences, changed over time (P < 0.05) with the highest values within 15 minutes after delivery. The number of mares with atrioventricular blocks and the number of atrioventricular blocks per mare increased over time (P < 0.01) and were significantly elevated from 15 minutes before to 45 minutes after birth of the foal. Salivary cortisol concentrations increased to a maximum at 30 minutes after delivery (25.0 ± 3.4 ng/mL; P < 0.01). Plasma epinephrine and norepinephrine concentrations showed significant fluctuations from rupture of the allantochorion to expulsion of the fetal membranes (P < 0.01) but were not markedly elevated at any time. In conclusion, mares give birth under high parasympathetic tone. Cortisol release during and after foaling is most likely part of the endocrine pathways regulating parturition and not a labor-associated stress response. [Copyright &y& Elsevier]

Abstract: The maintenance and development of conservation areas by grazing of large herbivores, such as Przewalski's horses, is common practice. Several nature conservation areas house male bachelor groups of this species. When males are needed for breeding they are removed from the groups, often without considering group compositions and individual social positions. However, alpha animals are needed for ensuring group stability and decision making in potentially dangerous situations in several species. To investigate the role of the alpha male in a bachelor group, we observed the behaviour of five Przewalski's horse males during the enlargement of their enclosure. We analyzed the group's social structure and movement orders, as well as the animals' connectedness, activity budgets, and whether they moved with preferred group members and how factors such as social rank influenced the horses' behaviour. We also investigated the excretion of glucocorticoid metabolites (GCM) via faeces of the horses while exploring a new area as a parameter of glucocorticoid production. Our results show that the alpha male is important for a bachelor group in changing environmental conditions. The alpha male had the highest level of connectedness within the group. When exploring the new environment, its position in the group changed from previously being the last to being the first. Furthermore the whole group behaviour changed when exploring the new area. The stallions showed reduced resting behavior, increased feeding and did not stay close to each other. We found that the excretion of glucocorticoid metabolites of most horses rose only marginally during the first days on the new area while only the alpha male showed a significant increased amount of glucocorticoid production during the first day of the enclosure enlargement. [Copyright &y& Elsevier]

Although some information exists on the stress response of horses in equestrian sports, the horse-rider team is much less well understood. In this study, salivary cortisol concentrations, heart rate (HR) and heart rate variability (HRV), SDRR (standard deviation of beat-to-beat interval) and RMSSD (root mean square of successive beat-to-beat intervals) were analysed in horses and their riders (n = 6 each) at a public performance and an identical rehearsal that was not open to the public. Cortisol concentrations increased in both horses and riders (P < 0.001) but did not differ between performance and rehearsal. HR in horses and riders increased during the rehearsal and the public performance (P < 0.001) but the increase in HR was more pronounced (P < 0.01) in riders than in their horses during the public performance (from 91 ± 10 to 150 ± 15 beats/min) compared to the rehearsal (from 94 ± 10 to 118 ± 12 beats/min). The SDRR decreased significantly during the equestrian tasks in riders (P < 0.001), but not in their horses. The RMSSD decreased in horses and riders (P < 0.001) during rehearsal and performance, indicating a decrease in parasympathetic tone. The decrease in RMSSD in the riders was more pronounced (P < 0.05) during the performance (from 32.6 ± 6.6 to 3.8 ± 0.3 ms) than during the rehearsal (from 27.5 ± 4.2 to 6.6 ± 0.6 ms). The study has shown that the presence of spectators caused more pronounced changes in cardiac activity in the riders than it did in their horses. [ABSTRACT FROM AUTHOR]

In general, avian species produce clutches of more than one egg. Differential investment in egg weight and hormone levels is possible and their effects on development and behaviour can be measured. Here we investigated changes in three hormones (testosterone, T; androstenedione, A4 and progesterone, P4) in the outer layer of yolks of infertile domestic goose eggs over the course of a laying season. There was a significant change in the concentration of all three hormones: T and P4 increased in a linear manner across the season whilst A4 increased until mid season and then steadily decreased. The correlation between T and P4 (r = 0.613, P<0.001), and between T and A4 (r = 0.746, P<0.001) were both positive, but the relationship between A4 and P4, while positive was not strong (r = 0.379, P<0.001), although still significant. The increase in P4 over the laying season is consistent with the transition from a laying to a broody state. The increase in T is consistent with a trade-off for the seasonal decline in egg weight. [ABSTRACT FROM AUTHOR]

11-Oxoetiocholanolone and related substances are important metabolites of cortisol and are excreted via feces in ruminants. To investigate whether 11-ketotestosterone (11-KT) or its immunoreactive metabolites are formed and excreted in ruminant feces, an enzyme immunoassay (EIA) was developed and validated. The antibody was raised in rabbits against 11-KT-3-CMO:bovine serum albumin with biotinylated 11-KT as a label. The assay showed a sensitivity of 0.3 pg/well. To validate the assay biologically, 6 rams were injected with a synthetic analogue of the adrenocorticotropic hormone (Synacthen, 2 µg/kg body wt). An aliquot was collected of each fecal portion spontaneously defecated 8 h before Synacthen injection to 24 h after injection and stored at −20 °C until analysis. Samples (0.5 g) were extracted using 80% methanol and immunoreactive metabolites measured using the 11-KT EIA and an already established 11,17-dioxoandrostane (11,17-DOA) EIA. High-performance liquid chromatography separation revealed no peak in the same elution position as authentic 11-KT; therefore, reacting substances were referred to as 11-KT equivalents. In the case of 11-KT immunoreactive substances, the values increased from baseline (median, 136 ng/g feces) to a peak concentration (median, 424 ng/g) 10 to 14 h after Synacthen injection and declined afterwards. Concentrations of 11,17-DOA showed the same pattern, but the values were 2 to 4 times higher. From this data, the authors conclude that 11-KT-like substances, specifically C19O3-androgens with a 17ß-hydroxy group, were present in the feces. These substances originate from the adrenals and are most likely cortisol metabolites. Environ Toxicol Chem 2013;32:1332-1336. © 2013 SETAC [ABSTRACT FROM AUTHOR]

Salivary alpha amylase (sAA) is the most abundant enzyme in saliva. Studies in humans found variation in enzymatic activity of sAA across populations that could be linked to the copy number of loci for salivary amylase (AMY1), which was seen as an adaptive response to the intake of dietary starch. In addition to diet dependent variation, differences in sAA activity have been related to social stress. In a previous study, we found evidence for stress-induced variation in sAA activity in the bonobos, a hominoid primate that is closely related to humans. In this study, we explored patterns of variation in sAA activity in bonobos and three other hominoid primates, chimpanzee, gorilla, and orangutan to (a) examine if within-species differences in sAA activity found in bonobos are characteristic for hominoids and (b) assess the extent of variation in sAA activity between different species. The results revealed species-differences in sAA activity with gorillas and orangutans having higher basal sAA activity when compared to Pan. To assess the impact of stress, sAA values were related to cortisol levels measured in the same saliva samples. Gorillas and orangutans had low salivary cortisol concentrations and the highest cortisol concentration was found in samples from male bonobos, the group that also showed the highest sAA activity. Considering published information, the differences in sAA activity correspond with differences in AMY1 copy numbers and match with general features of natural diet. Studies on sAA activity have the potential to complement molecular studies and may contribute to research on feeding ecology and nutrition. [ABSTRACT FROM AUTHOR]

Background: Environmental challenges might affect the maternal organism and indirectly affect the later ontogeny of the progeny. We investigated the cross-generation impact of a moderate heat challenge in chickens. We hypothesized that a warm temperature–within the thermotolerance range- would affect the hormonal environment provided to embryos by mothers, and in turn, affect the morphology and behavioral phenotype of offspring. Methodology/Principal Findings: Laying hens were raised under a standard thermal condition at 21°C (controls) or 30°C (experimental) for 5 consecutive weeks. A significant increase was observed in the internal temperature of hens exposed to the warm treatment; however plasma corticosterone levels remained unaffected. The laying rate was not affected, but experimental hens laid lighter eggs than the controls during the treatment. As expected, the maternal thermal environment affected yolk hormone contents. Eggs laid by the experimental hens showed significantly higher concentrations of yolk progesterone, testosterone, and estradiol. All chicks were raised under standard thermal conditions. The quality of hatchlings, growth, feeding behavior and emotional reactivity of chicks were analyzed. Offspring of experimental hens (C30 chicks) were lighter but obtained better morphological quality scores at hatching than the controls (C21 chicks). C30 chicks expressed lesser distress calls when exposed to a novel food. Unlike C21 chicks, C30 chicks expressed no preference for energetic food. Conclusion/Significance: Our findings suggest that moderate heat challenge triggers maternal effects and modulate the developmental trajectory of offspring in a way that may be adaptive. This suggests that the impact of heat challenges on captive or wild populations might have a cross-generation effect. [ABSTRACT FROM AUTHOR]

Japanese Quail ( Coturnix japonica) are a common avian laboratory model, and measuring corticosterone metabolites (CM) non-invasively from Quail droppings would be of broad interest. Using the enzyme immunoassay suitable for measuring CM in droppings of Chicken ( Gallus domesticus) and other galliform species, we tested the CM measured in Quail droppings after stimulation with adrenocorticotropic hormone. An ACTH challenge indicated that the assay did not detect major amounts of CM in Japanese Quail excreta. Therefore, we aimed at testing whether steroid metabolism and clearance differed between closely related species. We compared the CM excretion patterns in male and female Japanese Quail and Chicken. After intravenous injection with [H]-labelled corticosterone, we collected all droppings for 24 h, including caecum excreta. Males and females of both species excreted radioactive CM with two peaks. The first radioactivity-peak presumably was from uric acid (Quail 45/55 min; Chicken 120/75 min post-injection, males/females respectively), whereas the second peak probably represented faecal excretion (180/210 min; 240/140 min, respectively). Several highly polar radioactive CM, but no corticosterone, was separated by chromatography in droppings of both species. Number and identity of CM considerably differed between males and females, as well as between urine and faecal excretion. The nature of excreted CM in Quail droppings remains unresolved. However, we show that knowing the effective time for dropping collection is essential for interpreting stimulus-specific CM responses, and mixing samples from rapid urine and slower faecal excretion should be avoided. [ABSTRACT FROM AUTHOR]

Effects of deployment of miniaturised transmitters and loggers have been studied mainly in diving seabirds such as penguins, and less so in flying seabirds. However, some studies of albatrosses and petrels recorded extended trip durations and elevated rates of nest desertion following device attachment, especially if transmitter loads exceeded 3 % of adult mass. Studies have usually compared performance parameters such as trip duration, meal mass, breeding success or rate of return in the next season between birds with devices and controls. We here examined the effects of geolocator loggers (Global Location Sensing, (GLS)) on thin-billed prions Pachyptila belcheri (130 g), by comparing performance parameters and additionally eco-physiological parameters. GLS weighed ca. 1 % of the body mass, and were fixed on leg rings, which may influence the flight efficiency by creating an asymmetric load. We found no differences in the performance parameters, either in the season of attachment or the season following recovery. Similar stable isotope ratios in adult blood and feather samples further indicated that the foraging ecology was not influenced. However, after 1 year of logger deployment, adults differed in their hormonal response to stress: while baseline corticosterone levels were not influenced, corticosterone levels in response to handling were elevated. Moreover, increased heterophil/lymphocyte ratios and a decreased tail growth in winter suggest that carrying the GLS was energetically costly, and adults adapted physiologically to the higher work load, while keeping up a normal breeding performance. [ABSTRACT FROM AUTHOR]

Abstract: Tertiary wastewater treatment plant effluent before and after ozonation (0.6–1.1gO3/g DOC) was tested for aquatic ecotoxicity in a battery of standardised microbioassays with green algae, daphnids, and zebrafish eggs. In addition, unconjugated estrogen and 17β-hydroxyandrogen immunoreactive substances were quantified by means of enzyme immunoassays, and endocrine effects were analysed in a 21-day fish screening assay with adult male and female medaka (Oryzias latipes). Ozonation decreased estrogen-immunoreactivity by 97.7±1.2% and, to a lesser extent, androgen-immunoreactivity by 56.3±16.5%. None of the short-term exposure ecotoxicity tests revealed any adverse effects of the tertiary effluent, neither before nor after the ozonation step. Similarly in the fish screening assay, reproductive fitness parameters showed no effects attributed to micropollutants, and no detrimental effects of the effluents were observed. Based on the presented screening, ozonation effectively reduced steroid hormone levels in the wastewater treatment plant effluent without increasing the effluent''s ecotoxicity. [Copyright &y& Elsevier]

In this study, effects of weaning on behavioral and physiological stress parameters in young horses (foals) were determined. Foals were weaned either simultaneously without the presence of adult horses (group A, n == 6), or in the presence of two adult females familiar but unrelated to the foals (group B, n == 5), or weaned consecutively by removing two mother horses per day (group C, n == 6). Behavior, locomotion, salivary cortisol concentration, beat-to-beat (RR) interval, heart rate variability (HRV) and weight were determined. Group A foals lost weight for 2 days (mean ± SEM) − 8.3 ± 1.6 kg, p < 0.05. Weaning was followed by increased vocalization which was least pronounced in foals of group B ( p < 0.05). Locomotion was most pronounced on weaning day in foals of group A and lowest in group B ( p < 0.05). Weaning increased salivary cortisol concentration on the day of weaning in groups A and B and for 2 days in group C ( p < 0.05). The RR interval decreased most pronouncedly in group A foals ( p < 0.05). There were no consistent changes in HRV. Based on cortisol release and behavior, weaning is associated with stress but this was least pronounced in foals weaned in the presence of two familiar but unrelated adult female horses. [ABSTRACT FROM AUTHOR]

Abstract: Parents, and particularly mothers, can influence their offspring''s development in non-genetic ways. Maternal effects can occur during the mothering phase as well as during the embryonic phase. Prenatal maternal effects in birds can be mediated by yolk steroid hormones that influence subsequent offspring development. Studies have focused mainly on the influence of laying females'' living conditions on yolk hormonal contents, and rarely on the effects of individual characteristics. Here, we investigated prenatal influence of parent age on yolk steroid levels and on offspring phenotype. We compared Japanese quail at two different ages: at the beginning of their reproductive cycle (11weeks old: age 1) and six months later, after egg production peak (37weeks old: age 2). Egg composition, reproductive outcomes, and offspring growth, sexual development and behaviour were studied at both ages. We found that laying rate, fertility and chick survival rates declined between age 1 and age 2. Age 2 eggs had relatively lighter shells and higher yolk plus albumen contents; they also had lower testosterone contents. Age 2 offspring weighed more at hatching than did age 1 offspring; subsequently their growth patterns differed and their sexual development was more precocious. Age 2 offspring were less emotional than age 1 offspring when encountering a novel environment, and they appeared more sensitive to social separation. Our study shows, for the first time in a bird species, a strong impact of parental age on offspring phenotype, and especially on behaviour, an impact that is possibly mediated via modulation of yolk testosterone content. [Copyright &y& Elsevier]

An animal's emotional responses are the result of its cognitive appraisal of a situation. This appraisal is notably influenced by the possibility of an individual to exert control over an aversive event. Although the fact that environment controllability decreases emotional responses in animals is well established, far less is known about its potential trans-generational effects. As the levels of avian yolk hormones can vary according to the mother's environment, we hypothesized that housing environment of mothers would modulate the quality of her eggs and in turn her offspring's behaviour. Two groups of female Japanese quail were constituted: a group that had access to a place to hide in their home-cage (Hd, n = 20) and a group that had nowhere to hide (NoHd, n = 20) when stressed. Both groups were submitted to daily human disturbances for a twenty-day-period. Hd females produced eggs with both less testosterone and androstenedione than did NoHd females. The emotional and social reactivity of Hd females' offspring were lower and their growth was slower than those of NoHd females' offspring. Our results show that a minor difference in housing environment had substantial effects on eggs and offspring. The presence of a shelter probably helped quail to cope with daily human disturbances, producing less reactive offspring. This transgenerational effect caused by an opportunity to hide could lead to applications in care of laboratory animals, conservation biology and animal welfare. [ABSTRACT FROM AUTHOR]

Abstract: Large inter-species differences have been found in yolk corticosterone amounts in avian eggs. While some studies have failed to detect significant amounts of corticosterone, in other species high amounts have been recorded, such as in a recent study of southern rockhopper penguins Eudyptes chrysocome chrysocome. However, attention has been drawn recently to the fact that many antibodies for corticosterone measurement cross-react with other steroids present in the yolk. In particular, progesterone and related substances can occur in yolk in high concentrations, such that also low cross-reactions of corticosterone assays may lead to measurement errors. We thus performed high-performance liquid chromatographic (HPLC) analyses of yolk extracts and determined the concentration of immunoreactive corticosterone, as well as cross-reacting progesterone and cortisol in egg yolks of southern rockhopper penguins and imperial shags Phalacrocorax atriceps albiventer. We found that high gestagen concentrations in the yolk result in large measurement errors for yolk corticosterone, even when the cross-reactivity seems small. This was observed for both species. We further found species-specific differences in the actual corticosterone amounts present in the egg yolks. [Copyright &y& Elsevier]

Abstract: Maternal stress effects on offspring development have been studied largely in rodents and primates, and to a lesser extent in farm animals. Potential lack of knowledge concerning prenatal stress on farm animals is regrettable because they are frequently subjected to a variety of husbandry stressors. Above all, effects of maternal stress on poultry offspring have been neglected. Prenatal effects in birds are known to involve maternal steroids present in eggs. In this study, we investigated the effects of daily unpredictable stressors applied to laying Japanese quail on their offspring''s morphological and behavioural development. We also investigated the effects of our procedure on female reproductive output and on egg characteristics (weight, composition, yolk sex steroid levels). Our procedure induced only a mild stress: laying and egg fertilization rates of stressed females were not impaired; they remained similar to those of control females. Nonetheless, our stress procedure had an impact on some egg characteristics: stressed females’ eggs were heavier, contained more albumen and tended to have higher yolk testosterone levels than control females’ eggs. Stressed females’ offspring hatched earlier, were heavier at hatching and had a different growth pattern than did control females’ offspring. They also appeared to have a higher emotional reactivity than control chicks when encountering a novel environment and they reacted more strongly following social separation. Our study revealed that mild stressors applied to laying Japanese quail can increase the emotional reactivity of their chicks and suggested that maternal stress effects on offspring are mediated by changes in egg composition and yolk testosterone levels. We stress the particular relevance to the poultry industry of our findings highlighting the importance of taking into account the environment of laying females, as this environment can influence their progeny''s behaviour and therefore their subsequent adaptation to husbandry conditions and ultimately their welfare. [Copyright &y& Elsevier]

Abstract: In nestlings, glucocorticoid (GC) secretion has short-term and long-term f itness consequences. For example, short-time elevations trigger begging activity, whereas chronically elevated GC levels impair body condition, growth and cognitive abilities. Despite a growing body of literature on personality traits, the effects of selection for fast and slow exploration on GC secretion have received little attention. We compared baseline and stress-induced hypothalamic–pituitary–adrenal (HPA) axis activity of hand-reared great tit nestlings of lines selected for fast and slow exploration. Nestling droppings were collected under three conditions: control, test (following handling stress, day 14 after hatching) and the following day. The concentrations of excreted immunoreactive corticosterone metabolites (CM) were determined via an enzyme immunoassay. We also observed nestlings'' begging behaviour. CM differed significantly between the lines. Nestlings of the fast line excreted lower CM than slow-line birds. In response to handling stress, nestlings excreted significantly higher concentrations of CM than during the control and on the day after handling. Sex and begging activity were not related to CM levels. Under the control condition, but not after handling, males begged significantly more often than females. In both lines, adults excreted significantly less CM compared to nestlings. Both nestlings and adults of the slow line produced higher baseline CM values than fast-line birds. Fast-line nestlings excreted lower baseline CM than nestlings of a wild population not selected for fast or slow exploration. Slow-line nestlings did not. Our results show that selection on the basis of exploratory behaviour affected HPA axis reactivity. [Copyright &y& Elsevier]

Individual phenotypic characteristics of many species are influenced by non-genetic maternal effects. Female birds can influence the development of their offspring before birth via the yolk steroid content of their eggs. We investigated this prenatal maternal effect by analysing the influence of laying females' social environment on their eggs' hormonal content and on their offspring's development. Social instability was applied to groups of laying Japanese quail females. We evaluated the impact of this procedure on laying females, on yolk steroid levels and on the general development of chicks. Agonistic interactions were more frequent between females kept in an unstable social environment (unstable females) than between females kept in a stable social environment (stable females). Testosterone concentrations were higher in unstable females' eggs than in those of stable females. Unstable females' chicks hatched later and developed more slowly during their first weeks of life than those of stable females. The emotional reactivity of unstable females' chicks was higher than that of stable females' chicks. In conclusion, our study showed that social instability applied to laying females affected, in a non-genetic way, their offspring's development, thus stressing the fact that females' living conditions during laying can have transgenerational effects. [ABSTRACT FROM AUTHOR]

Glucocorticoids promote the mobilization of energy stores and they may facilitate the expression of energetically expensive functions. Early arrival on the breeding grounds in migratory species and territorial competition are energetically demanding activities that may be supported by elevated baseline glucocorticoid levels. Here, we evaluated the associations between the baseline levels of excreted corticosterone (CORT) metabolites of male Pied Flycatchers ( Ficedula hypoleuca) just after arriving on their breeding area and timing of arrival, considering ornamental traits indicative of social status, like forehead patch size and black plumage coloration, as well as heat shock protein levels (HSP60). We observed a positive association of CORT metabolites with HSP60 levels, which are synthesized under several environmental challenges affecting cell homeostasis. Our data showed a negative association between arrival date and CORT metabolite levels, possibly as a result of the higher energetic demands imposed by the hard environmental conditions experienced at the time of an early arrival after migration. We observed a negative relationship of forehead patch dimensions and CORT metabolite levels, suggesting that dominance is associated with low baseline CORT metabolites. Also, males that expressed a higher degree of territorial behaviour when exposed to a playback song of a conspecific at their nest-box showed higher CORT metabolites upon arrival than males that expressed a lower degree of territorial behavior. This may indicate that elevated baseline CORT metabolite levels may facilitate an intense territorial competition in males. Thus, male–male competition may be a factor affecting observed baseline glucocorticoid levels in migratory birds. [ABSTRACT FROM AUTHOR]

Glucocorticoid metabolites enter the aquatic environment via mammalian excrements. Molecular structures of their C19O3 metabolites strongly resemble the major fish androgen 11-ketotestosterone. Therefore, we tested the hypothesis that the cortisol metabolite 5α-androstan-3,11,17-trione acts similarly to 11-ketotestosterone by employing a fish screening assay for endocrine-active substances. After 21 d, both 11-oxygenated compounds had masculinized sex characteristics of the anal fin in female medaka in a dose-dependent manner. Environ. Toxicol. Chem. 2010;29:1613–1620. © 2010 SETAC [ABSTRACT FROM AUTHOR]