Faecal Cortisol Metabolites Research Articles

Relationships between boldness and fecal steroid hormone levels in captive alpine musk deer (Moschus chrysogaster) in China

Animals adapt to environmental changes through behavioral and physiological regulation, and there are individual differences in their ability to adapt to the environment, which are associated with individual personality. Boldness is a fundamental axis of animal personality, characterizing an animal's response to unfamiliar environments and threats. From June 1 to July 28, 2023, a personality assessment experiment utilizing a novel stimulus test with an unfamiliar human was conducted on 39 captive alpine musk deer (Moschus chrysogaster) at the Zhuanglang Musk Farm in Gansu Province, China. We recorded avoidance reaction indicators of musk deer in the presence of an unfamiliar human, and measured the level of the musk deer's boldness, influencing factors, and the relationships with fecal steroid hormone levels. Based on cluster analysis, 39 captive musk deer could be divided into bold (boldness score, BSS = 0.994 ± 0.008, n = 3) and shy groups (BSS = 0.327 ± 0.145, n = 36) and the average BSS in 39 musk deer was 0.378 ± 0.228. Factors such as age, sex, and density of enclosures did not have a significant effect on the boldness of musk deer. There was a significant negative correlation between the musk deer's boldness and the level of fecal cortisol metabolites. However, the correlations between boldness and testosterone levels in males and estradiol levels in females were not significant. Based on the results of our study, in the practice of musk deer farming, the measurement of the boldness can help predict the individual's adaptation to environmental stress, and it can also provide a new research perspective for the study of the boldness of solitary animals that are forced to live in groups.

Hibernating or not hibernating? Brown bears' response to a mismatch between environmental natural cues and captive management, and its welfare implications.

In wild brown bears, likely factors triggering hibernation response to harsh environmental conditions are temperature, photoperiod, and food resources availability. In fact, constantly fed captive brown bears are described as skipping hibernation being active all year-round. Is the hibernation response so flexible and subordinate to contingencies, or else is an adaptation that, if dismissed, may negatively impact on bear well-being? This study investigates the potential hibernation response in captive brown bears under unvaried management conditions using an integrative approach simultaneously analyzing multiple animal-based variables together with environmental covariates. Data from a mid-latitude zoo revealed distinct behavioral, fecal glucocorticoids, and body condition score seasonal fluctuations, resembling natural hibernation cycles, despite constant food access. Environmental variables like photoperiod and visitor numbers significantly influenced activity levels. Bears exhibited behaviors indicative of hyperphagia and fall transition, such as appetitive feeding and denning behaviors. Hormonal analyses revealed high fecal cortisol metabolites levels during hyperphagia, suggesting physiological responses to seasonal changes. Findings underscore the importance of environmental cues and food availability in shaping zoo bear behavior and physiology. Considering that the hibernating vs. non-hibernating description might represent an oversimplification, management strategies should deal with captive bear potential need to freely express their adaptive predispositions by accommodating their natural behaviors, such as providing denning spots and adjusting diet composition as soon as typical hyperphagic and predenning behaviors emerge, ultimately enhancing their well-being.

Investigating the biological relevance of measuring gastrointestinal cortisol metabolite levels to assess stress responses in Atlantic salmon (Salmo salar L.) after an acute stress

Monitoring stress levels of farmed Atlantic salmon (Salmo salar) is important to ensure fish welfare and optimize farm operations. Feces could be a promising matrix for assessing stress responses in fish, based on their properties of low-invasive sampling and allowing repeated sampling over time. Meanwhile, elevated levels of cortisol metabolites (CMs) in feces indicate the increases in plasma cortisol levels (PLA) after exposure to acute stress. However, the dynamics of fecal CMs following acute stress in Atlantic salmon remain unclear. In this study, a confinement stress involving chasing and crowding was conducted to investigate the responses of gastrointestinal CMs to an acute stressor in Atlantic salmon. The post-smolts, with an average weight of 155.21 g, were sampled before and at 30 min, 1.5, 6, 12, 18, 24, 36, and 48 h after the onset of stress. Blood and gastrointestinal contents from the stomach, proximal intestine, and distal intestine of each fish were collected and subsequently analyzed, using competitive enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the pre-stress level of PLA was low (4.28 ± 6.13 ng/ml) and reached a peak within 30 min following stress. The levels of CMs in gastrointestinal contents from stomach (SCMs), proximal intestine (PCMs), and distal intestine (DCMs) in pre-stress group were 0.82 ± 0.50, 18.31 ± 6.14 and 16.04 ± 6.69 ng/g, respectively. Gastrointestinal CMs increased significantly within 30 min and the peak levels of SCMs (3.51 ± 3.75 ng/g), PCMs (68.19 ± 23.71 ng/g) and DCMs (65.67 ± 23.37 ng/g) were found at 1.5 h post-stress. The significant increases in PCMs and DCMs post-stress validate the biological relevance of measuring intestinal CMs for assessing acute stress responses in Atlantic salmon. No significant difference was noted between PCMs and DCMs across all samples, suggesting that intestinal contents can serve as a suitable matrix compared with feces when measuring the responses of CMs to acute stress. The time lag between the peak of PLA levels and their reflection in the intestinal contents exceeded 1 h, indicating that using intestinal contents as a matrix to assess stress levels in fish can extend and delay the sampling window. This study highlights valuable guidance for determining the optimal times to utilize intestinal contents for measuring stress responses, providing further insights into the dynamics of fecal CM following acute stress.

The impact of net cleaning on fecal cortisol metabolites in farmed Atlantic Salmon

Stress assessment is crucial in aquaculture to ensure and improve the health and well-being of production animals including fish. The primary objective of this study was to utilize the assessment of fecal cortisol metabolites (FCMs) as a low-invasive stress determination method for farmed Atlantic salmon (Salmo salar L.) and to suggest an appropriate sampling timeframe for this measurement.Blood, mid-intestinal contents, and feces samples were collected before, during, and at 2 and 20 h after a stress-inducing event, namely, net cleaning. Plasma cortisol (PC) and its metabolite levels in the mid-intestinal contents (MICMs) and FCMs were assessed by an enzyme-linked immunosorbent assay (ELISA). The research reveals a distinctive temporal profile, with a sequential elevation in levels of PC, MICMs and FCMs in response to the effects of net cleaning. Peak FCMs levels were observed approximately 20 h after net cleaning and were significantly higher than peak PC levels (P < 0.05). The findings suggest that FCMs are a promising indicator for acute stress assessment. To enhance the understanding and improve the applicability of FCMs as stress indicators in fish farming practices the critical challenges regarding feces sampling and FCMs analyses are further discussed. Future research aimed at developing practical protocols and industry standards will emphasize method validation and include a greater number of sampling points as well as an expanded experimental period.This study signifies a crucial step toward understanding the role of FCMs as a reliable stress indicator in fish. It underscores the importance of accounting for the delayed and extended sampling patterns when assessing stress in aquatic settings. Moreover, it offers a less invasive and more labor-efficient approach for monitoring the overall health and welfare of salmonids than PC analysis, thereby promoting sustainability in this vital industry.

Environmental enrichment during yard weaning alters the performance of calves in an attention bias and a novel object recognition test

The weaning of beef calves in yards places multiple stressors on the animals, and environmental enrichment may help mitigate some of these stressors and improve animal welfare. This trial assessed the impacts of enrichment provision to beef calves during yard weaning using measures of biological functioning, behaviour, and affective state. Overall, calves utilised the brush more than the other provided enrichments, which were a hanging rope and a ball. Enrichment influenced the behaviours of calves during both an attention bias test, with enriched calves exhibiting behaviours associated with greater anxiety, and a novel object recognition test, with enriched calves spending less time interacting with objects. In their home pens, enriched calves performed more drinking and grooming behaviours. However, no significant differences were seen between treatments for body weight, faecal cortisol metabolites, and internal body temperature. Enrichment also did not influence any longer-term measurements of body weight, flight speed, or crush score. The study design was impacted by mud, requiring the regrouping of the animals. Thus, some results should be interpreted with caution. Overall, this study demonstrates that further work is required into the assessment of affective states for these animals as it could not be confirmed whether the results seen indicate that calf welfare was improved or impaired through enrichment provision.

Management of dairy heifers: Can operant conditioning be an effective and feasible tool to decrease stress and ease animals' close contact and handling?

Beside health monitoring, a regular check of dairy heifers' growth rate is desirable, but it is rarely done since procedures that require restraint and handling can be associated with substantial stress for both animals and farmers. Inexperienced heifers, especially if they are highly responsive to humans, may find restraint and handling potentially aversive. This study investigated whether training heifers of different age and responsiveness toward humans (RTH), through operant conditioning, could reduce stress in animals, ease close contact and handling, and be feasible in terms of farmer's effort. We assessed 60 Holstein heifers of 2 age classes (Young, n = 29, 291 ± 39; Old, n = 31, 346 ± 62 d) according to the Avoidance Distance Test (ADT) and classified them as Confident (C, n = 20), Neutral, (N, n = 21) or Non-confident (NC, n = 19). Half of the heifers of each age and RTH class was trained (Tr, n = 29), whereas the other half was not (NTr, n = 31). The Tr heifers were subjected to target training for 8 sessions and positively reinforced with feed to allow being touched on the muzzle, rump and perineum. In case a heifer refused positive reinforcement, the trainer stepped back as negative reinforcement. In the last week of the experiment the effect of training on the reaction to handling was assessed in all heifers. We measured heart rate (HR), root mean square of successive inter-beat interval differences (RMSSD) and fecal cortisol metabolites (FCMs). The presence of behavioral distress signs was recorded as well. The ADT was performed a second time 24 h after the measuring session. All the trained heifers, regardless of RTH class, successfully accomplished the target training task in 6 sessions, spending on average 25.3 s per session, each. All the trained heifers allowed to be touched on the rump and perineum at the end of the 4th session. Training NC heifers required more time compared with the others. Trained heifers showed higher RMSSD (14.2 vs. 16.9), indicating a lower vagal tone, and thus, a slightly lower stress level than NTR. Training did not lead to differences in HR, FCMs or presence of stress behavioral signs. NC heifers had the highest mean baseline FCM values (38.4 vs. 30.3 vs. 29.1 ng/g) compared with N and C. NC heifers showed also the lowest value of FCMs, 12 h after the measuring session (36.7 vs. 44.6 vs. 49.7 ng/g), likely due to a decreased responsiveness of the adrenal gland to a stressor. The average avoidance distance decreased between the beginning and the end of the experiment, especially for N and NC heifers, regardless whether they were trained or not. These results show how using operant conditioning on some heifers, not only decreased their vagal tone, but reduced the responsiveness to humans of all the animals, trained and not trained; in the latter case, through non-associative learning, such as habituation.

Faecal cortisol levels in a wild Iberian red deer population are best explained by prior weather conditions

AbstractThe responsiveness of the hypothalamic–pituitary‐adrenocortical axis to stressors is crucial for wild animals to survive and adapt to environmental changes without compromising individual welfare. We analysed the influence of prior weather conditions, seasonality, the influx of ecotourism, and nutrition on stress levels in a wild population of red deer in a Mediterranean hunting reserve in the Pre‐Pyrenees, Spain. We used faecal cortisol metabolites as a proxy for physiological and psychological stress, and faecal triiodothyronine metabolites as an indicator of nutritional stress. Faecal analyses were chosen because it is a non‐invasive technique that does not alter an individual's behaviour, and it is easy to conduct in a wild population, something which presents added challenges due to the lack of a controlled environment. Our results indicate that prior weather conditions, rather than just seasonality, best explained the variations in these hormones within seasons and among years. On the contrary, the results showed that high levels of the cortisol hormone did not necessarily correspond to low levels of the triiodothyronine hormone. This could be a sign of acute nutritional stress in this population. Finally, we did not find an effect of the ecotourism influx on cortisol levels, suggesting that this deer population tolerates the presence of tourists.

How do grazing beef and dairy cattle respond to virtual fences? A review.

Virtual fencing (VF) is a modern fencing technology that requires the animal to wear a device (e.g., a collar) that emits acoustic signals to replace the visual cue of traditional physical fences (PF) and, if necessary, mild electric signals. The use of devices that provide electric signals leads to concerns regarding the welfare of virtually fenced animals. The objective of this review is to give an overview of the current state of VF research into the welfare and learning behavior of cattle. Therefore, a systematic literature search was conducted using two online databases and reference lists of relevant articles. Studies included were peer-reviewed and written in English, used beef or dairy cattle, and tested neck-mounted VF devices. Further inclusion criteria were a combination of audio and electrical signals and a setup as a pasture trial, which implied that animals grazed in groups on grassland for 4h minimum while at least one fence side was virtually fenced. The eligible studies (n = 13) were assigned to one or two of the following categories: animal welfare (n studies = 8) or learning behavior (n studies = 9). As data availability for conducting a meta-analysis was not sufficient, a comparison of the means of welfare indicators (daily weight gain, daily lying time, steps per hour, daily number of lying bouts, and fecal cortisol metabolites [FCM]) for virtually and physically fenced animals was done instead. In an additional qualitative approach, the results from the welfare-related studies were assembled and discussed. For the learning behavior, the number of acoustic and electric signals and their ratio were used in a linear regression model with duration in days as a numeric predictor to assess the learning trends over time. There were no significant differences between VF and PF for most welfare indicators (except FCM with lower values for VF; P = 0.0165). The duration in days did not have a significant effect on the number of acoustic and electric signals. However, a significant effect of trial duration on the ratio of electric-to-acoustic signals (P = 0.0014) could be detected, resulting in a decreasing trend of the ratio over time, which suggests successful learning. Overall, we conclude that the VF research done so far is promising but is not yet sufficient to ensure that the technology could not have impacts on the welfare of certain cattle types. More research is necessary to investigate especially possible long-term effects of VF.

Fecal thyroid hormone metabolites in wild ungulates: a mini-review.

This review aims to analyse the fluctuations of fecal thyroid hormone metabolites (FTMs) related to environmental and individual variables in different species of wild ungulates and provide a collection of assay methods. The great advantage of fecal sampling is being completely non-invasive. A systemic search was conducted from 2019 to 2024, using data sources PubMed, Scopus, Web of Science, and the World Wide Web, and ten studies were found on this topic. Three studies used the radioimmunoassay method for FTMs analysis, while the others used a less expensive enzyme-linked immunosorbent assay. Most of these papers validated the method for the species-specific matrix. Related to the studied variables, some authors analysed FTM fluctuations only concerning individual variables, and others in response to both. Temperature and fecal cortisol metabolites (FCMs) were the most studied environmental and individual variables, respectively. Since FTMs are an integrative measure of plasma thyroid hormones, the information obtained from a non-invasive-assay method regarding wild ungulate physiology is becoming of great interest to the scientific community.

Sounds of danger and post-traumatic stress responses in wild rodents: ecological validity of a translational model of post-traumatic stress disorder

In the wild, animals face a highly variable world full of predators. Most predator attacks are unsuccessful, and the prey survives. According to the conventional perspective, the fear responses elicited by predators are acute and transient in nature. However, the long-term, non-lethal effects of predator exposure on prey behavioral stress sequelae, such as anxiety and post-traumatic symptoms, remain poorly understood. Most experiments on animal models of anxiety-related behavior or post-traumatic stress disorder have been carried out using commercial strains of rats and mice. A fundamental question is whether laboratory rodents appropriately express the behavioral responses of wild species in their natural environment; in other words, whether behavioral responses to stress observed in the laboratory can be generalized to natural behavior. To further elucidate the relative contributions of the natural selection pressures influences, this study investigated the bio-behavioral and morphological effects of auditory predator cues (owl territorial calls) in males and females of three wild rodent species in a laboratory set-up: Acomys cahirinus; Gerbillus henleyi; and Gerbillus gerbillus. Our results indicate that owl territorial calls elicited not only “fight or flight” behavioral responses but caused PTSD-like behavioral responses in wild rodents that have never encountered owls in nature and could cause, in some individuals, enduring physiological and morphological responses that parallel those seen in laboratory rodents or traumatized people. In all rodent species, the PTSD phenotype was characterized by a blunting of fecal cortisol metabolite response early after exposure and by a lower hypothalamic orexin-A level and lower total dendritic length and number in the dentate gyrus granule cells eight days after predator exposure. Phenotypically, this refers to a significant functional impairment that could affect reproduction and survival and thus fitness and population dynamics.

Weathering the storm: Decreased activity and glucocorticoid levels in response to inclement weather in breeding Columbian ground squirrels

Inclement weather can rapidly modify the thermal conditions experienced by animals, inducing changes in their behavior, body condition, and stress physiology, and affecting their survival and breeding success. For animals living in variable environments, the extent to which they have adapted to cope with inclement weather is not established, especially for hibernating species with a short active season that are constrained temporally to breed and store energy for subsequent hibernation. We examined behavioral (foraging activity) and physiological (body mass and fecal cortisol metabolites) responses of Columbian ground squirrels (Urocitellus columbianus), small hibernating rodents inhabiting open meadows in Rocky Mountains, to 3 events of inclement weather (two snow storms in May 2021 and May 2022, one heavy rainfall in June 2022). We found that individuals adapted to inclement weather conditions by (1) reducing above-ground activity, including foraging, (2) decreasing the mobilization of stored resources as indicated by a decrease in the activity of the hypothalamo-pituitary-adrenal (HPA) axis and lower fecal cortisol metabolites in the hours/days following periods of inclement weather; and (3) compensating through increased foraging and more local activity when favorable conditions resumed. As a result, body mass and growth did not decrease following short periods of inclement weather. Columbian ground squirrels were well-adapted to short periods of inclement weather, coping via modifications of their behavior and the activity of the HPA axis.

Linear regression between fecal cortisol metabolites and circulating cortisol levels of peri-partum ewes: Suggesting a non-invasive alternative sampling method

The protection of animal rights is essential when conducting sampling, and it is crucial to develop and use noninvasive sampling methods. Therefore, this study evaluated fecal cortisol metabolites (FCMs) in ewes around parturition as a noninvasive method and an alternative to invasive blood sampling for assessing blood cortisol concentrations. An important objective of the present study was to obtain regression equations to estimate serum cortisol concentrations according to the measured FCMs. For this purpose, from 45 days before to 45 days after parturition, blood and fecal samples were taken every two days from 35 adult pregnant ewes. Serum cortisol levels and FCMs were assessed in all samples. There was a positive and significant correlation between serum cortisol and FCMs in the entire study. Furthermore, a linear regression formula was defined between these parameters, and an estimated value of each parameter can be obtained by substituting one parameter in the formula. The evaluating FCMs has several advantages over the analysing blood cortisol, including convenience and stress-free sampling in animals. The animal rights are respected due to the lack of need for blood sampling, thereby providing the possibility of multiple non-invasive sampling at different periods. Using the results of this study, the evaluation of FCMs in sheep can be considered as a reliable alternative for blood sampling.

Predicting Sex in White Rhinoceroses: A Statistical Model for Conservation Management.

Ensuring the effective management of every rhinoceros population is crucial for securing a future for the species, especially considering the escalating global threat of poaching and the challenges faced in captive breeding programs for this endangered species. Steroid hormones play pivotal roles in regulating diverse biological processes, making fecal hormonal determinations a valuable non-invasive tool for monitoring adrenal and gonadal endocrinologies and assessing reproductive status, particularly in endangered species. The purpose of this study was to develop a statistical model for predicting the sex of white rhinoceroses using hormonal determinations obtained from a single fecal sample. To achieve this, 562 fecal samples from 15 individuals of the Ceratotherium simum species were collected, and enzyme immunoassays were conducted to determine the concentrations of fecal cortisol, progesterone, estrone, and testosterone metabolites. The biological validation of the method provided an impressive accuracy rate of nearly 80% in predicting the sex of hypothetically unknown white rhinoceroses. Implementing this statistical model for sex identification in white rhinoceroses would yield significant benefits, including a better understanding of the structure and dynamics of wild populations. Additionally, it would enhance conservation management efforts aimed at protecting this endangered species. By utilizing this innovative approach, we can contribute to the preservation and long-term survival of white rhinoceros populations.

Housing relocation does not have to induce a significant stress response in captive Japanese macaques (Macaca fuscata).

Previous studies suggest that housing relocation may be stressful for captive non-human primates. Our study investigated the stress levels of Japanese macaques during a housing relocation by measuring their daily fecal cortisol metabolites, which are often used as an indicator of stress. Ten adult Japanese macaques, single-housed for research purposes, were relocated to a new facility. Fecal samples were collected daily for 7 days. Cortisol metabolite concentrations were determined via enzyme immunoassay. No significant differences in cortisol metabolite levels were found in 7 days, but concentration levels showed that the highest median was associated to the relocation day. The minimal cortisol metabolite increase suggests that there was a slight activity increase in the hypothalamic-pituitary-adrenal axis. Techniques encouraging cooperation of the monkeys, the short time duration of the relocation, and consistency in the environment may have contributed to the minimal stress levels observed.

Assessing the impact of essential oil blends as antibiotic alternatives on nutritional and metabolic parameters in non-forage-based diet-fed Nellore steers

Essential oils (EO) are antimicrobials with the potential to replace conventional ones in ruminant diets. Despite the advantage of the EO serving markets that prohibit antibiotics in animal nutrition, the results that prove their effectiveness vary with the type of EO, dose, diet, etc. This study aimed to assess the effects of a blend of essential oil, based on cinnamaldehyde and diallyl disulfide, associated or not with antibiotics on nutritional and metabolic parameters for feedlot Nellore steers fed non-forage-based diet. Five Nellore steers were distributed in a 5×5 simple Latin square design. The treatments assessed were a basal no-roughage diet with the inclusion of monensin (MON), monensin + virginiamycin (MV), monensin + blend of EO (MEO25), monensin + blend of EO (MEO35), and a blend of essential oil (EO). Feed, orts, feces, ruminal fluid, blood, and urine samples were utilized to assess the intake, digestibility, ruminal pH, ruminal NH3, nitrogen balance, microbial nitrogen synthesis, fecal cortisol metabolites, blood glucose, and D and L-lactate. No difference (P &gt; 0.05) has been observed in additives in intake, nutrient digestibility, and ingestive behavior. There was a trend (P = 0.0553) of the effect of additives on ruminal pH, and the MV presented the highest value. No difference (P &gt; 0.05) for the ruminal NH3, microbial N synthesis, and nitrogen balance concentration was also verified. The blood glucose and blood lactate concentration were not affected (P &gt; 0.05) by additives. The blend of essential oils based on cinnamaldehyde and diallyl disulfide could then be indicated in replacement or association with antibiotics in a non-forage-based diet for Nellore steers.

Welfare impacts associated with using aversive geofencing devices on captive Asian elephants

Animal-borne aversive geofencing devices (AGDs, or satellite-linked shock collars) are commercially available and used on livestock to restrict their movement within a virtual boundary. This technology has potential application as a human-wildlife conflict mitigation tool, where problem animals might be conditioned to avoid human-dominated habitats by associating an audio warning with a subsequent electric shock, which is delivered if the audio warning is ignored. Ensuring that high standards of animal welfare are maintained when implementing such tools is important for acquiring manager and community acceptance of such approaches. We conducted two pilot experiments with eight captive Asian elephants using mild electric shocks from a modified dog-training collar fitted around the neck, as part of an ongoing effort to develop AGDs suitable for mitigating human-elephant conflict. As part of these experiments, we assessed elephants' behavioural and physiological stress before, during and after the experiments. During the experiments elephants wore collars for up to nine consecutive days and received a small number of electric shocks on 1–3 consecutive days. Bootstrapped principal component analysis showed that daily activity budgets of individual elephants on experiment days were not different from the pre-experiment days. Generalised linear mixed-effects model (GLMM) showed that anxiety/stress behaviours increased on the first day of acclimatising to the collar and on testing days (i.e. days they received shocks) of the first experiment, but not during the second experiment relative to pre-experiment days. Analysis of faecal cortisol metabolite (FCM) concentrations using GLMM showed that FCM concentrations were higher in samples collected ∼24 hrs and ∼48 hrs after testing days compared to baseline levels as expected given the lag time for excretion of cortisol metabolites. These elevated anxiety/stress behaviours and FCM concentrations returned to baseline levels shortly after the experiment. Therefore, we conclude that AGDs did not produce lasting behavioural or physiological stress effects in elephants during this short-term study but recommend further studies with a larger sample of elephants to confirm the transferability of these findings.

Non-invasive assessment of fecal glucocorticoid and androgen metabolites in the pygmy hippopotamus (Choeropsis liberiensis)

The pygmy hippopotamus (Choeropsis liberiensis) is an endangered species endemic to the Upper Guinea Forest ecosystem in West Africa. We have limited information concerning the species’ reproduction and well-being under managed care. We therefore developed non-invasive methods for characterizing gonadal androgen and adrenal hormone profiles in pygmy hippos using fecal samples collected from 12 males and 12 females housed in North American zoological institutions. We aimed to: 1) identify and validate enzyme immunoassays (EIAs) for measuring metabolites of corticosteroids and testosterone in feces; and 2) test whether gonadal activity is correlated with previous breeding history, season or type of housing. For glucocorticoids, several EIAs for measuring metabolites were investigated. A group-specific EIA exhibiting cross-reactivity with 11,17-dioxoandrostane (DOA) metabolites of cortisol most clearly reflected adrenocortical activity in response to pharmocological challenge with adrenocorticotropic hormone (ACTH) in both males and females. However, day-to-day concentrations of this metabolite in the feces of pygmy hippos that did not undergo ACTH challenge were near the detection limits of the assay, making this EIA impractical for assessing glucocorticoid activity in this species. Another group-specific EIA, exhibiting cross-reactivity with 5α-pregnane-3β,11β,21-triol-20-one, produced biologically relevant data and evidence of an appropriate response to pharmacological challenge with exogenous ACTH. The testosterone metabolite assay C196 (Arbor Assays, Ann Arbor, Michigan, USA) also produced biologically coherent data: adult males exhibited the highest mean androgen metabolite concentrations (477 ng/g), followed by adult females (259 ng/g) and juvenile males (160 ng/g). Proven breeding males had higher, but not significantly different, mean concentrations (472 ng/g) to unproven males (352 ng/g; P = 0.400). Similarly, adult males housed outdoors year-round in subtropical climates exhibited higher, but not statistically different mean concentrations (554 ng/g) to males in temperate climates that were housed indoors at least part of the year (412 ng/g; P = 0.208). There were, however, significant differences in mean concentrations among seasons for adult males, with higher values in spring (546 ng/g) and summer (542 ng/g) than in autumn (426 ng/g) and winter (388 ng/g, P = 0.003). In conclusion, we identified EIAs for the measurement of fecal metabolites of androgens and glucocorticoids that can be used for further studies to monitor gonadal activity in male pygmy hippos and adrenocortical activity in both sexes. We also identified a seasonal trend in male gonadal activity in this species under managed care in North America. Finally, our findings highlight an important consideration when using non-invasive methods for evaluating fecal cortisol metabolites: ACTH used for pharmacological validation of an EIA does not necessarily equate to biological relevance.

Intraspecific Interactions Decrease Stress Affecting Welfare in Shelter Dogs: A Comparison of Four Different Housing Conditions.

Shelters are stressful environments for domestic dogs (Canis familiaris). Evaluating dogs' welfare is crucial to improve their life condition and to promote a better management of shelters. We aimed at verifying which variables improved welfare in 10 shelter dogs ((hosted in the shelter "Centro cinofilo Caerite" in Bracciano (Rome)) by analysing their behavioural responses in different environmental conditions. Furthermore, faecal samples were taken to measure cortisol metabolites (CM), a non-invasive method to evaluate adrenocortical activity in dogs. Dogs were observed for a total of 400 h in 4 different cage conditions: (i) alone in a cage; ii) alone in an enriched cage; (iii) in cage with conspecifics; (iv) in cage with regular interaction with humans outside the cage. Alone in the cage situation showed highest frequencies of displacement activities (Friedman test: χ2 = 13.32; p = 0.004). In contrast, being in the cage with conspecifics seems to reduce displacement activity frequency, as well as the level of faecal cortisol metabolites (Friedman test: χ2 = 8.04; p = 0.045). Our results suggest that conspecifics' presence is the best way to reduce stress in shelter dogs. This research could provide some useful guidelines for managing shelters and improving dogs' life condition.

Season-dependent impact of forage quality on stress in alpine chamois.

Chronically heightened stress levels in wildlife species may have detrimental effects on individual life history traits, for example, through the increased likelihood of disease, parasitic infections, and overall reduced fitness. Understanding the drivers of stress may thus have great potential for informing wildlife conservation. Although the role of climate and individual status is well studied in stress ecology, the impact of related stressors such as dietary quality is of increasing interest to wildlife research and conservation. In this study, fecal cortisol metabolites (FCMs) in Alpine chamois Rupicapra r. rupicapra used as bioindicators of stress, and their relationship with forage quality-measured as the percentage of fecal crude protein (CP)-were investigated. Data collection took place in 2011 and 2012 in the Gran Paradiso National Park (Western Italian Alps), on 22 individually marked adult males. The relationship between FCMs and CPs was analyzed through linear models and separated between winter and summer months, accounting for the effect of potentially confounding exogenous and endogenous variables. After AICc-based model selection, we found that forage quality was negatively related to FCM levels in Alpine chamois during the summer months, meaning that higher quality forage was associated with the decreased expression of stress hormones. However, during the winter months, we did not find a significant relationship, potentially as a result of forage quality being ubiquitously poor. Although the mechanisms through which dietary variations impact FCM concentrations in wildlife populations are largely unknown, the occurrence of significant relationships between forage quality and stress levels supports potentially important implications for the long-term effect of climatic changes on the fitness of wildlife populations.

Elevated glucocorticoids during the ovarian follicular phase predict conception in wild female chacma baboons

Mating related behavior during ovarian cycling can be energetically demanding and constitute a significant stressor, requiring physiological responses to mediate investment in reproduction. To better understand the proximate mechanisms underlying these responses, we examine hormonal and behavioral variation across the ovarian cycle during conceptive and nonconceptive cycles in wild female chacma baboons (Papio ursinus). We quantified immunoreactive fecal estradiol, progesterone, and cortisol metabolites for 21 adult females, and calculated activity budgets and rates of received aggression from over 5000 15-min behavioral samples. We found conception to be associated with higher concentrations of both estradiol and cortisol during the follicular phase, but no difference in progesterone between conceptive and nonconceptive cycles for either the follicular or luteal phase. While females spent less time feeding during the follicular compared to the luteal phase, we found no difference in time spent feeding, moving, or copulating between conceptive and nonconceptive cycles of the same phase. Rates of received aggression also were similar across the ovarian cycle, with no difference between conceptive and nonconceptive cycles. Finally, we found positive associations between cortisol and estradiol, indicating that glucocorticoids (GCs) do not suppress hypothalamic-pituitary-gonadal (HPG) activity and reproductive function in this context. Overall, our results suggest that elevated GCs may play an adaptive role in mobilizing energy during sexually receptive periods of ovarian cycling.