Fecal Corticoid Research Articles

7 Validation of a commercial ELISA kit for non-invasive measurement of equine cortisol concentration

The measurement of fecal corticosteroid metabolites (FCM) is a method for quantifying potential stress. The sampling method is relatively non-invasive, especially when compared with blood cortisol. Fecal collection reduces concern for a rise in cortisol from the sampling method itself and has been shown to be a more consistent measure of cortisol in horses without the daily diurnal rhythm observed in blood cortisol. Commercial ELISA kits offer many benefits over previously validated RIA methods but lack validation. Further, time point of fecal collection has not been established. The objective of this study was to provide both analytical and biologic validation of fecal and plasma samples from horses using a commercial cortisol ELISA Kit (Arbor Assays DetectX Cortisol ELISA kit, K003-H1). In October, horses (4 M, 4 F, 8 ± 7 yr) were transported in pairs for a 15-min transportation session using a livestock trailer that included a divider to prevent physical contact and limit visual contact. Blood samples were collected via jugular venipuncture before, immediately after, and 60 min post-transportation. Fecal samples were collected via drop or grab sampling before transportation and 20, 24, and 28 h post-transportation. Data were analyzed using PROC MIXED in SAS. Inter-assay and intra-assay coefficients of variation for FCM were 9.0 and 10.8%, respectively and for plasma cortisol 11.0 and 7.2%, respectively. Dilutionalparallelism was observed between dilutions 1:20 and 1:40 for fecal samples and 1:100 and 1:250 for plasma samples. The recovery of a known spike for plasma cortisol and FCM were 98.2 and 100.0%, respectively. Plasma cortisol concentrations increased in response to trailering (73.4 ± 7.6 ng/ml, 0 min post-transportation, P = 0.02) compared with pre-transportation (41.2 ± 7.6 ng/ml) and began to decline 60 min post-transportation (51.0 ± 7.6 ng/ml) when compared with immediately post-trailering (P = 0.09). Plasma cortisol concentrations pre-trailering were not different from 60 min post-trailering. FCM concentrations increased 24 h post-trailering (10.8 ± 1.7 ng/g, P = 0.04) when compared with pre-transportation (7.4 ± 1.7 ng/g). FCM concentrations pre-trailering were not different from 20 h (8.68 ± 1.7 ng/g, P = 0.64) or 28 h post-trailering (10.0 ± 1.7 ng/g, P = 0.11). These data support that 15 min of transport with limited visual/physical contact between conspecifics is adequate to elicit a stress response in horses and that changes in FCM are observed 24 h post-stressor. In conclusion, the Arbor Assay Cortisol ELISA kit is a reliable, economic option for the measurement of cortisol in equine plasma and fecal samples.

Instability of fecal glucocorticoid metabolites at 4°C: Time to freeze matters.

The use of fecal corticosteroid metabolites (FCMs) has proven to be well suited to evaluate adrenocortical activity, a major component of the stress response, particularly in wildlife. As with any tools, confounding factors and drawbacks must be carefully considered. Among them, sample preservation and storage are of particular importance, as they can affect stability of FCMs and lead to biased results and interpretations. Arguably, immediate freezing of fecal samples upon collection is the best practice to preserve FCM integrity, however, for logistical reasons, this condition is rarely feasible in the field. It is generally argued that temporary storage of samples at low above-zero temperature is an acceptable way of preserving samples in the field before freezing them for long-term storage. However, to our knowledge, there is no empirical study that demonstrates the stability of fecal metabolites in samples stored at +4°C. In this study, we collected a fresh fecal sample from 20 captive roe deer, each of which was homogenized and split into three subsamples (60 subsamples in total) to investigate the effects on FCMs levels of temporary storage at +4°C for 24 h and 48 h before freezing versus immediate freezing at -20°C after feces collection. Compared to immediate freezing, mean FCMs levels decreased by 25% every 24 h when feces were stored at +4°C before freezing. The variance of FCMs levels followed the same pattern, leading to a clear reduction in the ability to detect biological effects. Minimizing the storage time at +4°C before freezing should therefore be seriously considered when establishing sampling and storage protocols for feces in the field for adequate hormonal profiling.

Experimenter familiarization is a crucial prerequisite for assessing behavioral outcomes and reduces stress in mice not only under chronic pain conditions

Rodent behavior is affected by different environmental conditions. These do not only comprise experimental and housing conditions but also familiarization with the experimenter. However, specific effects on pain-related behavior and chronic pain conditions have not been examined. Therefore, we aimed to investigate the impact of different housing conditions, using individually ventilated and standard open top cages, inverted day-night cycles, and experimenter familiarization on male mice following peripheral neuropathy using the spared nerve injury (SNI) model. Using a multimodal approach, we evaluated evoked pain-related- using von Frey hair filaments, measured gait pattern with the CatWalk system, assessed anxiety- and depression-like behavior with the Elevated plus maze and tail suspension test, measured corticosterone metabolite levels in feces and utilized an integrative approach for relative-severity-assessment. Mechanical sensitivity differed between the cage systems and experimenter familiarization and was affected in both sham and SNI mice. Experimenter familiarization and an inverted day-night cycle reduced mechanical hypersensitivity in SNI and sham mice. SNI mice of the inverted day-night group displayed the slightest pronounced alterations in gait pattern in the Catwalk test. Anxiety-related behavior was only found in SNI mice of experimenter-familiarized mice compared to the sham controls. In addition, familiarization reduced the stress level measured by fecal corticosteroid metabolites caused by the pain and the behavioral tests. Although no environmental condition significantly modulated the severity in SNI mice, it influenced pain-affected phenotypes and is, therefore, crucial for designing and interpreting preclinical pain studies. Moreover, environmental conditions should be considered more in the reporting guidelines, described in more detail, and discussed as a potential influence on pain phenotypes.

Comparative assessment of plasma cortisol and fecal corticoid metabolites (FCM) of Atlantic salmon (Salmo salar L.) subjected to acute- and long-term stress

Farmed Atlantic salmon (Salmo salar L.) are subject to a variety of stressors throughout production. Elevations of cortisol level in the blood are one of the major endocrine primary stress responses in vertebrates and are widely used as stress indicator. However, blood sampling is invasive and stressful procedures. Cortisol instantly released into the blood at sampling can easily interfere with the initial stress response of interest. Fecal corticoid metabolites (FCM) have been suggested for a less invasive assessment of stress in fish. In the present study, we evaluated stress responses by using plasma cortisol and FCM as stress indicators in Atlantic salmon. Atlantic salmon (with average weight 42 g) were exposed to 3 different stressors: 1) parr-smolt transformation; 2) infestation with salmon lice (Lepeophtheirus salmonis) for two-weeks; 3) infection with infectious salmon anaemia virus (ISAV) for four weeks. The results demonstrated that FCM levels correlated well with the plasma cortisol levels at single-point sampling during long-term stress (p < 0.05). Significant increases of both plasma cortisol and FCM were found two weeks after initiated 24 h light. Each tank was sampled twice, with around 40-min interval. The chasing during the first sampling was used as an acute stressor. The effects of sampling procedures on plasma cortisol and FCM levels are compared. Blood and feces were analyzed by an Enzyme-Linked Immunosorbent Assay (ELISA). Plasma cortisol and FCM levels increased within 40 min after fish perceived an acute stressor when the average weight of fish was below 100 g. Attenuated cortisol stress responses to acute stress were found after fish experiencing long-term stress. Collectively, FCM reflects changes in plasma cortisol and can be an alternative to analysis of plasma cortisol in Atlantic salmon, complementing evidence of using FCM as stress indicators for monitoring and improving fish welfare. The results highlight that standard sampling procedures and good experimental designs should be established to ensure robust and repeatable results in the study of stress based on the FCM measurement. Future study will investigate representative timepoints for FCM sampling, with particular attention to the types of stress and sizes of the fish.

Impact of environmental conditions on the levels of stress and breeding performance in Wistar rats: conventional environment versus environmentally controlled housing.

It has been established that providing comparable and standard environmental conditions to house experimental rats is of high importance. However, there is growing evidence that rats prefer higher temperatures owing to their thermoneutral zone that lies between 28-34°C. This experiment validates the stress levels of Wistar rats of both sexes housed in conventional conditions about 24-31°C and a relative humidity of 70-92% in a tropical animal facility when compared to rats housed in controlled temperatures of 20-24°C with humidity maintained between 30-70%. Adequate air-changes to maintain ammonia-free environment was provided with a power-exhaust system in a conventional setup and with the individually ventilated caging system in the environmentally controlled group. To assess stress, faecal corticosteroid metabolite assay was done in naïve animals and in a set of animals that underwent retro-orbital bleeding under general anaesthesia. Mothering ability, fecundity and preweaning mortality were also compared between animals housed in two different conditions. Results showed no differences in stress hormone levels between the groups. However, faecal weights differed between males and females in both naïve and orbital bled groups. Animals housed in controlled environment clearly had better breeding data with lesser preweaning mortality in comparison to the conventionally housed rats. Further studies are required to elucidate whether the results are comparable when conducted during all seasons of the year at different locations across the globe.

Circulating and Excreted Corticosteroids and Metabolites, Hematological, and Serum Chemistry Parameters in the Killer Whale (Orcinus orca) Before and After a Stress Response

Comparisons of circulating and excreted corticosteroid profiles during a biological stress response could provide more data concerning the timing and magnitude of acute hormone changes in different sample matrices. A stress test was performed in 13 zoo-based killer whales (Orcinus orca) whereby animals were elevated out of the water on a rising lift-bottom platform for 20 min. Paired blood and feces were tested for cortisol, corticosterone, aldosterone, and their metabolites and hematological and serum chemistry parameters. Circulating corticosteroids significantly increased during the stress test and returned to pre-treatment concentrations within 24 h, indicating hypothalamic-pituitary-adrenal-axis activation. Concentrations of fecal corticosteroid metabolites did not change significantly during the stress test. Significant differences between pre-test levels and the stress test stage were observed for counts of white blood cells and absolute neutrophils, consistent with a stress leukogram. For serum chemistry, significant deviations from baseline levels were seen for the majority (20/37) of measured parameters. This study provides a direct measurement of the physiologic response of clinically healthy killer whales to a stress event. However, due to the lack of significant changes in fecal hormone metabolite profiles, further investigation of the excretory dynamics of corticosteroids during acute and chronic stress is warranted.

Negligible hormonal response following dehorning in free-ranging white rhinoceros (Ceratotherium simum).

The white rhinoceros (Ceratotherium simum) is experiencing unsustainable poaching losses fuelled by a demand for horn. Increasingly, private and state reserves are dehorning their rhinoceros populations in an attempt to reduce poaching pressure. Rhinoceroses use their horns in social interactions as well as during resource access and so its partial removal as part of reserve management practices may adversely influence these behaviours. Physiological stress can correlate with animal welfare, reproductive state and health and thus acts as a useful indicator of these parameters. To establish whether dehorning causes a physiological stress response, glucocorticoid and gonadal steroid profiles of free-ranging white rhinoceroses were determined through the collection and analysis of faecal steroid metabolites before and after dehorning. Faecal corticoid profiles were not influenced by the number of occasions a rhinoceros had been dehorned or by the number of days that had elapsed since dehorning. Furthermore, there was no apparent suppression in the concentrations of testosterone or progesterone metabolites in males and females, respectively, after exposure to multiple dehorning procedures. These findings should increase wildlife managers' confidence that dehorning does not negatively impact white rhinoceros physiology as measured hormonally.

Behavioral and endocrine correlates of dominance in captive female Jackson's hartebeest (Alcelaphus buselaphus).

For species that form dominance hierarchies, such as group-living ungulates, aggressive interactions can pose a challenge to successful captive management. For example, Jackson's hartebeest (Alcelaphus buselaphus), a rare antelope of east Africa, can be difficult to maintain in captivity because aggression within female dominance hierarchies can lead to injury and death. We quantified behavioral and endocrine correlates of dominance in a captive herd of ten female hartebeest with the goal of understanding how to minimize dangerous interactions. We observed hartebeest for 18 months in a 4-ha enclosure on St. Catherines Island, Georgia. We quantified type of agonistic behavior, initiator and recipient of the behavior, who displaced whom, and age and relative size of participants. We also used fecal steroid analysis to take weekly measurements of corticoids, a hormonal correlate of stress. Hartebeest maintained a stable, linear dominance hierarchy. Only 5.9% of interactions were reversals of established rank. Most dominance interactions were low intensity, but 17.3% of interactions carried a high risk of injury (chase, horn butt, horn clash, horn clash on knees, and head push). High-ranking individuals usually initiated interactions. Most interactions were between animals of similar rank, and animals of intermediate rank participated in the highest number of interactions. The frequency of high-intensity interactions was unrelated to rank. Rank was correlated with age, but not size. There was no relationship between rank and fecal corticoid levels. Dominance interactions in hartebeest are not a function of social instability, and the frequency of dangerous interactions is not a function of rank.

The importance of individual heterogeneity for interpreting faecal glucocorticoid metabolite levels in wildlife studies

Abstract As a non‐invasive and inexpensive method, the use of faecal glucocorticoid metabolites (FGM) analysis in wildlife research is increasing. Various environmental factors have been shown to influence FGM levels, or faecal corticosteroid metabolites (FCM) levels in birds, but most studies do not account for inter‐individual variance, which we hypothesized may substantially affect results. We combined FCM analysis with genetic analysis to identify the sex and individual's identity in samples collected in three consecutive winters; with repeated samples per individual, across the entire range of an endangered population of capercaillie Tetrao urogallus in south‐western Germany. Using generalized additive mixed models, we modelled FCM levels as a function of sex, season and environmental covariates at two spatial scales: location and home range. We compared two models: one including information on the individual animal and the other excluding this information (i.e. naïve model) to assess the influence of individual heterogeneity on the results obtained. Models accounting for inter‐individual differences explained 44.0% and 45.1% (at the location and home‐range scale respectively), while only very little (4.0% and 5.1%, respectively) was explained by the environmental predictors. When ignoring individual effects, the model results changed considerably with other, previously non‐informative predictors, becoming significant. In the full models, accounting for inter‐individual variance, weather conditions had no effect at either scale. FCM levels were negatively correlated with habitat quality at the sampling location, while human recreation at the home‐range scale led to elevated FCM levels. In the naïve models, two additional predictors appeared significant: one weather variable at the local scales and two at the home‐range scale. In all models, seasonal FCM patterns differed significantly between males and females. Synthesis and applications. By combining faecal corticosteroid metabolites (FCM) analysis with genetic individual assessment, we demonstrate that individual heterogeneity can explain most of the variance in faecal corticosteroid metabolites levels and that ignoring this information can lead to erroneous conclusions when testing for environmental stressors. We therefore stress the importance of identifying individuals when studying faecal corticosteroid metabolites in wildlife and recommend combining faecal corticosteroid metabolites analyses with genetic analyses to adequately address this issue.

Exposure to chronic variable social stress during adolescence alters affect‐related behaviors and adrenocortical activity in adult male and female inbred mice

Rodent models provide valuable insight into mechanisms that underlie vulnerability to adverse effects of early-life challenges. Few studies have evaluated sex differences in anxiogenic or depressogenic effects of adolescent social stress in a rodent model. Furthermore, adolescent stress studies often use genetically heterogeneous outbred rodents which can lead to variable results. The current study evaluated the effects of adolescent social stress in male and female inbred (BALB/cJ) mice. Adolescent mice were exposed to repeat cycles of alternating social isolation and social novelty for 4 weeks. Adolescent social stress increased anxiety-related behaviors in both sexes and depression-related behavior in females. Locomotion/exploratory behavior was also decreased in both sexes by stress. Previously stressed adult mice produced less basal fecal corticosteroids than controls. Overall, the novel protocol induced sex-specific changes in anxiety- and depression-related behaviors and corticoid production in inbred mice. The chronic variable social stress protocol used here may be beneficial to systematically investigate sex-specific neurobiological mechanisms underlying adolescent stress vulnerability where genetic background can be controlled.

Long Term Physiologic and Behavioural Effects of Housing Density and Environmental Resource Provision for Adult Male and Female Sprague Dawley Rats.

Simple SummaryThe effect of housing on rodent well-being has received increasing scrutiny in recent years in North America in an attempt to define adequate standards of animal care. Typically, the adequacy of cage size is determined by species and body weight, with little consideration being given to the cage complexity and impact on animal welfare. Further, most studies evaluate different caging methods over relatively short periods of time, which may not be realistic for typical colony settings. Inappropriate housing may lead to boredom, aggression, and the development of abnormal behaviour and function, which in turn may affect animal responses during research protocols. In this study, we evaluated the impact of group housing rats by sex in double wide cages with the addition of polyvinyl chloride (PVC) tubes for shelter, a nylon chew toy, and a piece of food enrichment given three times weekly over the course of five months. These groups were compared to rats that were single or pair-housed by sex in standard cages provided only with a nylon chew toy. We found that group-housed rats were more active and continued to engaged in positive social behaviours, such as grooming, for the duration of the study, with minimal impact on other aspects of animal function.There is considerable interest in refining laboratory rodent environments to promote animal well-being, as well as research reproducibility. Few studies have evaluated the long term impact of enhancing rodent environments with resources and additional cagemates. To that end, male and female Sprague Dawley (SD) rats were housed singly (n = 8/sex), in pairs (n = 16/sex), or in groups of four (n = 16/sex) for five months. Single and paired rats were housed in standard cages with a nylon chew toy, while group-housed rats were kept in double-wide cages with two PVC shelters and a nylon chew toy and were provided with food enrichment three times weekly. Animal behaviour, tests of anxiety (open field, elevated plus maze, and thermal nociception), and aspects of animal physiology (fecal corticoid levels, body weight, weekly food consumption, organ weights, and cerebral stress signaling peptide and receptor mRNA levels) were measured. Significant differences were noted, primarily in behavioural data, with sustained positive social interactions and engagement with environmental resources noted throughout the study. These results suggest that modest enhancements in the environment of both male and female SD rats may be beneficial to their well-being, while introducing minimal variation in other aspects of behavioural or physiologic responses.

Establishment of a non-invasive method for stress evaluation in farmed salmon based on direct fecal corticoid metabolites measurement

Fish welfare is an important issue for growth of the aquaculture industry. Stress responses represent animal's natural reactions to challenging conditions and could be used as a welfare indicator. Cortisol level is relevant to fish welfare condition, and is a readily measured component of the primary stress response system. Generally, cortisol is measured by blood sampling. However, fish blood cortisol level could be instantly influenced by handling-stress at sampling. Fecal corticoid metabolites (FCM) are a mixture of several different metabolites with a wide range of polarities. Thus, feces could be promising alternative less handling-sensitive and non-invasive biological matrices for cortisol evaluation in Atlantic salmon. In this study we developed non-invasive method for determination of fecal corticoid metabolites in farmed Atlantic salmon (Salmo salar L.) using enzyme-linked immunosorbent assay (ELISA).It was demonstrated that salmon FCM extracted from salmon feces is insoluble in non-polar solvents like diethyl ether, but well soluble in polar solvents like methanol. The proper extraction ratio could be one ml 100% methanol for 100 μL of the liquid part of salmon feces or 100 mg of the solid part. The FCM directly detected in unextracted liquid part of feces correlated well with the FCM extracted from both liquid and solid part of the corresponding samples, without significant difference. Thus, it is feasible to measure FCM directly in the liquid part of salmon feces without any extraction procedure.Then, we applied this assay for FCM analysis in the group of salmon that experienced salmon pancreas disease (PD) and amoebic gill disease (AGD). We demonstrated 1) both plasma cortisol and FCM increased significantly during the outbreak of inflammatory disease (P < 0.01). Plasma cortisol level was elevated from 28 ± 40 ng/ml to 164.4 ± 62.5 ng/ml, FCM from 14.4 ± 13.2 ng/ml to 170.7 ± 89.7 ng/ml 2) Growth and starvation has no significant impact on either cortisol or FCM level. 3) FCM correlated well with plasma cortisol level (P < 0.01). Furthermore, there seems more individual variation in plasma cortisol levels than in FCM levels.These results suggest FCM could be directly analyzed in liquid part of salmon feces without extraction. This directly detected FCM level could represent the total fecal FCM level and plasma cortisol level. This simple and non-invasive method makes FCM a proper indicator for salmon welfare.

Assessing Stress in Arctic Lemmings: Fecal Metabolite Levels Reflect Plasma Free Corticosterone Levels.

Interest in the ecology of stress in wild populations has triggered the development of noninvasive methods for quantifying stress hormones. Measurement of fecal corticosteroid metabolites (FCMs) is one such method, but it is still unclear whether FCMs can be a reliable proxy of free plasma glucocorticoids. To assess the validity of this assumption, we carried out a robust assessment on brown lemmings (Lemmus trimucronatus) from Bylot Island, Nunavut, Canada, that were hand captured and anesthetized and related plasma glucocorticoid levels to fecal metabolite glucocorticoid levels. We examined endogenous factors that could explain interindividual variability. Blood corticosterone was measured from samples obtained on capture and 30 min later, and FCM levels were measured from animals kept in captivity for 72 h. Plasma free corticosterone increased 135-fold over baseline values 30 min after capture, which confirmed that initial handling was perceived as a stressor. We found that FCM levels were highly related with free (marginal [Formula: see text] = 0.53) but not with total ([Formula: see text] = 0.02) corticosterone levels, regardless of age, sex, and reproductive condition. FCM levels started increasing 2 h after capture and reached maximum levels 4 h after capture. No circadian rhythm in FCMs was found. Plasma total corticosterone levels were much higher in adult females compared with adult males, but this difference was much smaller when measuring free corticosterone levels and FCM levels. Our results suggest that FCM levels are good measures of stress by being closely related to plasma free corticosterone levels in brown lemmings.

Personality modulates proportions of CD4+ regulatory and effector T cells in response to socially induced stress in a rodent of wild origin

The way how individuals respond to chronic challenges can vary tremendously, and such differences are closely linked to personality. The few available studies on individual differences in stress-related immunosuppression in non-human mammals have been mainly carried out with laboratory strains. We conducted a study in male mound-building mice (Mus spicilegus) of wild origin. We distinguished between high (HAN) and low anxious/neophobic (LAN) personality types, quantified by subjects' consistent and associated behavioral responses in repeated elevated plus maze and novel object tests. After reaching maturity, parts of the subjects were regularly confronted to different resident pairs over a period of 5days to provoke a condition of chronic social stress, while others were used as untreated controls. We measured fecal corticosteroid metabolite (FCM) concentrations and different cellular immune parameters from blood and spleen. Socially confronted HAN showed higher increases in FCM concentrations than LAN, indicating a more pronounced physiological stress response in the former personality type. HAN of the experimental group also showed lower percentages of effector T cells (Teff) and higher regulatory T cells (Treg) in the spleen; the latter are known for their immunosuppressive activity. Considering the ratio of Teff/Treg, animals with higher increases in FCM concentrations during the late period of the experiment showed a stronger shift towards Treg cells, supporting immunosuppressive effects of chronically elevated corticosteroid levels. Summarizing, our results strongly suggest that immunomodulatory effects of socially induced stress were altered by individual differences in anxiety/neophobia, emphasizing the significance of personality in shaping physiological responses to challenge.

STEREOTYPIC BEHAVIORS IN BEARS

Confined bears are deprived of natural stimulus, such as large roaming domains, active foraging and enriched habitats, making them vulnerable to develop stereotypic behaviors. Motivation drives and neuro-pathological imbalance are the causes. Various stereotypic behavior types have been reported, including oral, head and locomotory related behaviors, and pacing are the most commonly seen. Stereotypic behavior level could be monitored using an observation-reporting system or fecal corticoid. Environmental and feeding enrichment are the proposed solutions; however, habituation could be developed. Parallel intermittent and consecutive environmental enrichment and unpredictable feeding schedule would be beneficial. Medication, especially long-term and low-dosage fluoxetine, was found to be effective in alleviating bear stereotypic behaviors.

Effects of stocking density and environmental enrichment on behavior and fecal corticosteroid levels of pigs under commercial farm conditions

Abstract In intensive pig farming of Western and Southern Europe, welfare concerns are still often related to barren environments and crowded conditions. Pig producers need to balance the requirements to improve welfare conditions at farm with practical considerations. The aim of this study was to determine the extent to which the reduction of stocking density and the provision of suspended pieces of hard wood as environmental enrichment have an influence on both behavior and fecal corticosteroid concentrations in commercially housed growing-finishing pigs. A total of 640 growing pigs were arranged in a 2 × 2 factorial design with stocking density (high, 1.0 m 2 /pig and low, 1.5 m 2 /pig) and environmental enrichment (yes or no) as factors. Ten replicate pens were allocated to each treatment. Observations of behavior (instantaneous scan sampling) were made in each pen at 2-week intervals, when pigs were from 15 to 31 weeks old. Fecal samples were collected to determine corticosteroids concentration in the 40 selected pens. The pigs housed in low density showed a higher (although not significant) level of exploration of pen furniture than crowded pigs (10.11% vs. 8.53%, respectively; P = 0.09). Similarly, social interactions were observed more frequently ( P ≤ 0.001) among the pigs in the enriched (10.27%) than in barren (6.69%) pens. The pigs housed in barren pens had greater incidences of tail biting (barren: 1.35%, enriched: 0.42%; P ≤ 0.01) and aggression (barren: 1.30%, enriched 0.61%; P ≤ 0.05). Crowded pig in barren pens spent less time moving (2.26%) compared to other treatments. Uncrowded pigs in enriched pens spent less time lying inactive (43.97%) and feeding (14.48%) compared to other treatments. Fecal corticosteroids doubled their concentration from the first (56.74 ng/g) to the last (108.10 ng/g) sampling date ( P ≤ 0.001). The crowded pigs showed higher ( P ≤ 0.001) concentration than the pigs housed in low stocking density (85.09 ng/g and 76.08 ng/g, respectively). No differences were found in corticosteroid concentrations between the pigs housed in barren and enriched pens. To conclude, the reduction of stocking density modified the pigs' behaviors and reduced the fecal corticosteroid levels, highlighting an improvement of welfare conditions. The provision of suspended pieces of hard wood modified the pigs' behaviors but did not exert relevant effects on fecal corticosteroid levels.

Chronic unpredictable stress during adolescence causes long-term anxiety

Exposure to stress during adolescence can cause long-term changes in behavior and cognition. Anxiety diagnoses rise during adolescence and are increased by adverse experiences. Currently, it is unknown how long stress during adolescence alters anxiety in adulthood. We found that rats exposed to chronic unpredictable stress during adolescence expressed altered behavior 6.5 months later; showing increased anxiety in a feeding test in a novel environment. Although behavioral changes indicative of anxiety were detected in late adulthood, the basal levels of fecal corticoid metabolites in prior-stressed rats did not differ from unstressed, control rats.

Validation of a radioimmunoassay-based fecal corticosteroid assay for Richardson’s ground squirrels Urocitellus richard-sonii and behavioural correlates of stress

Abstract We validated a radioimmunoassay-based method quantifying fecal glucocorticoid metabolites (FGMs) from captive male and female Richardson’s ground squirrels Urocitellus richardsonii. Blood samples were drawn to explore the correlation between plasma cortisol and FGM concentrations. We also injected groups of squirrels with normal saline (CTL; control), adre-nocorticotropic hormone (ACTH; stimulating adrenal activity), or dexamethasone (DEX; suppressing adrenal activity). Potential correlations between stress and behaviour were explored through quantification of fecal pellet production and the intervention necessary to elicit defecation, as well as the behaviour of subjects in the context of handling. Changes in plasma cortisol concentration between capture (baseline), and following handling (stress-induced) were also quantified for free-living squirrels. While glucocorticoid concentrations recovered from feces during our captive-animal study were not well correlated with plasma cortisol concentrations, and uncorrelated with defecation or behaviour, FGM concentrations did reflect the activation of the hypothalamic-pituitary-adrenal (HPA) axis. FGM concentrations increased significantly during initial captivity, but declined to baseline level as individuals acclimated to the novel environment. Injection of subjects with ACTH increased FGMs above baseline, confirming activation of the HPA axis. Plasma cortisol concentrations increased significantly with induced stress, indicating that capture and handling activated the glucocorticoid stress response even among previously handled, free-living subjects. Our findings validate a non-invasive tool that will afford new insight into the physiological processes underlying social, reproductive and antipredator behaviour of Richardson’s ground squirrels.

Experimental chronic noise is related to elevated fecal corticosteroid metabolites in lekking male greater Sage-Grouse (Centrocercus urophasianus).

There is increasing evidence that individuals in many species avoid areas exposed to chronic anthropogenic noise, but the impact of noise on those who remain in these habitats is unclear. One potential impact is chronic physiological stress, which can affect disease resistance, survival and reproductive success. Previous studies have found evidence of elevated stress-related hormones (glucocorticoids) in wildlife exposed to human activities, but the impacts of noise alone are difficult to separate from confounding factors. Here we used an experimental playback study to isolate the impacts of noise from industrial activity (natural gas drilling and road noise) on glucocorticoid levels in greater sage-grouse (Centrocercus urophasianus), a species of conservation concern. We non-invasively measured immunoreactive corticosterone metabolites from fecal samples (FCMs) of males on both noise-treated and control leks (display grounds) in two breeding seasons. We found strong support for an impact of noise playback on stress levels, with 16.7% higher mean FCM levels in samples from noise leks compared with samples from paired control leks. Taken together with results from a previous study finding declines in male lek attendance in response to noise playbacks, these results suggest that chronic noise pollution can cause greater sage-grouse to avoid otherwise suitable habitat, and can cause elevated stress levels in the birds who remain in noisy areas.

Adrenal activity in maned wolves is higher on farmlands and park boundaries than within protected areas

In this study we measured excreted fecal corticoid metabolites (FCM) in maned wolves (Chrysocyon brachyurus) living within a protected reserve, on farmlands or in a boundary zone between the two habitats, and determined the impacts of season and reproductive status on adrenal activity. Feces were collected within a national park (n=191 samples), a park boundary zone (n=39) and on nearby farmlands (n=27), processed and analyzed by enzyme immunoassay. FCM amounts from samples collected on farmlands were higher (P<0.05) than in those collected inside the reserve and from the boundary zone. In relation to seasonality, FCM were elevated (P<0.05) in spring (September–November) when wolf pairs were raising young. We then divided the samples collected during breeding season (March–August) into cycling females and male/non-cycling females based on fecal progesterone: fecal testosterone ratio. FCM concentrations of the former collected inside the park were higher than (P<0.05) than the latter group. However, there were no differences in FCM levels between the two groups for samples collected in the boundary zone and on farmlands. Furthermore, FCM concentrations of male/non-cycling females samples collected on farmlands were 2- to 5-fold higher (P<0.05) than in counterparts collected inside the park. The consistently high FCM concentrations in samples collected on farmlands indicate that, in addition to seasonality, gender and reproductive status, anthropogenic pressures also contribute to elevating adrenal steroid for individuals living in altered habitat.