Evaluation of Fecal Glucocorticoid Metabolite Levels in Response to a Change in Social and Handling Conditions in African Lions (Panthera leo bleyenberghi)

Natural weathering conditions can influence faecal cortisol metabolite (FCM) measurements in wildlife if fresh faeces cannot be collected immediately following defaecation. In this study, we evaluated this issue in a threatened Australian marsupial, the greater bilby (Macrotis lagotis). Fresh (<12 h since defaecation) faecal samples (n = 19 pellets per bilby) were collected one morning from seven adult bilbies kept in captivity. One control faecal sample (Day 1) from each bilby was immediately frozen. The remaining faecal pellets were randomly positioned outdoors. Subsequently, we froze one faecal pellet every 24 h for 19 days. FCM levels in bilby faeces were quantified using an enzyme-immunoassay. Mean FCM levels showed variation (daily mean coefficients of variation [CV %]) of 56.83–171.65% over 19 days. Overall, FCM levels were affected by exposure time; however, multiple comparisons showed that no significant change in FCM occurred after environmental exposure (no significant difference in mean FCM between control (Day 1) with any of the exposure days (Days 2–19). Individuals and sex also affected FCM levels. We found no correlation between mean daily CVs with daily minimum–maximum temperatures or rainfall. Our results indicate that FCM in bilby faeces is fairly stable to long-term environmental exposure (19 days). In future, freshly excreted bilby faeces (where the sample maintains a distinct odour for 9–13 days) should be used to study FCM levels in wild bilbies.

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20 degrees C until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37 degrees C for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 +/- 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350% above baseline). On one occasion, we did not detect any variation. Mean (+/- SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 +/- 26.7, after: 213.7 +/- 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

Summary Non‐invasive faecal cortisol metabolite (FCM) analysis is a well‐established tool to quantify stress in captive and free‐ranging species. While the method has great potential, its suitability in field studies might be limited when faecal samples from unknown individuals are used. Possible factors affecting final results and thus jeopardizing correct data interpretation are individual and sex‐specific variation, storage conditions and uneven distribution of metabolites in the faeces. We tested these factors on a population of free‐ranging Alpine chamois Rupicapra rupicapra rupicapra in the Austrian Alps. Faecal samples (n = 183) were analysed with an established enzyme immunoassay (EIA). To further validate the assay for FCM in chamois, a high‐performance liquid chromatography (HPLC) was performed. Sex‐specific differences in metabolite excretion were evaluated. Effects of storage length and temperature on FCM were tested with two experiments. The distribution of metabolites in the faeces was determined by the analysis of subsamples of single faecal samples. Potential individual effects on FCM levels and individually variable reactions to stressful events were evaluated with a simulation experiment. Patterns of immunoreactive peaks after HPLC separation were similar for different faecal samples, except in one sample of a male. In the stability tests, storage time at ambient temperature prior to freezing and the individual were the most important variables in modulating FCM. Concentrations within single samples varied significantly between pellets. Analysis of faecal samples collected from June to October showed a highly significant seasonal trend (P < 0·001) and a considerable variance of FCM levels within the population. Simulations confirmed that individual reactions to stressors in terms of varying gradients and FCM levels can explain the observed FCM patterns. Using FCM to assess adrenocortical function requires measuring extensively metabolized products of glucocorticoids, whose excretion and detection in faeces depend on several environmental, endogenous and methodological factors. In free‐ranging wildlife, these factors and the intrinsic individual differences in FCM excretion generate systemic noise and substantially distort final results. Therefore, sampling of unknown individuals inevitably jeopardizes meaningful interpretation of data, if the above named factors are not taken into consideration.

Horses are highly sensitive to stress, which can affect their wellbeing and lead to various health issues. Effective and objective stress assessment is therefore crucial for improving their care and management. The production of the glucocorticoid cortisol is increased in response to stressful stimuli and its metabolites can be measured non-invasively in feces. Therefore, this study aimed to explore the impact of different environmental and physiological stressors on fecal cortisol metabolite (FCM) concentrations in horses, with a particular focus on the relationship between stress, health, and welfare. We hypothesized that increased FCM levels may be indicative of disease and thus improve early detection and subsequent intervention. Fresh fecal samples of N = 41 horses (20 geldings and 21 mares) from the same herd were collected once weekly for 1 year. Horses had been housed in the same stable for at least a month and were accustomed to the habitat, daily routine, and social groups. Environmental conditions, health data, and potentially stressful events were recorded. Fecal concentrations of 11,17-dioxoandrostanes were measured via 11-oxoetiocholanolone enzyme immunoassay. We showed stable baseline FCM concentrations of 6.3 ng/g feces (range 0.6-28.1 ng/g feces). During the summer months, median FCM concentrations increased significantly (p < 0.05; One Way RM ANOVA), and this increase correlated with higher ambient temperatures (p < 0.0001, adj r 2 = 0.669, Pearson Product Moment correlation). Additionally, other factors such as breed, coat color, and housing conditions influenced the FCM concentrations. Stressful events, such as riding exams and some painful conditions, also resulted in elevated FCM levels, although the magnitude of these responses varied across individual horses. However, not all diseases were associated with increased FCMs. Our findings emphasize the complexity of the hypothalamic-pituitary-adrenal axis in horses, suggesting that while high FCM levels can indicate stress, they may not be reliable biomarkers for early disease detection. Particularly in the light of climate change, the impact of heat stress in the summer months should not be neglected and measures to improve the housing conditions accordingly should become an essential part of equine health management.

Due to the increasing demand for methods to quantify adrenal activity in response to stressors in wild animals, we evaluated whether stimulation with adrenocorticopic hormone (ACTH) increases faecal cortisol metabolite levels in European badgers. We also conducted several experiments to evaluate the effect of storage time and collection methods on cortisol metabolite levels of faecal samples. Faecal samples were collected from five captive European badgers (Meles meles) and cortisol metabolites were quantified by an enzyme immunoassay. Faecal cortisol metabolite levels increased 1-2 days after ACTH injection and dropped to pre-treatment levels 3 days after ACTH stimulation. We found that cortisol metabolite levels were not affected by the 8-hour time lapse from collection in the field until freezing of faecal samples, environmental exposure (temperature 25.66 ± 2.55°C; range 21.20-32.20) or mean relative humidity 62.77 ± 9.90% (range 42.00-79.00). Cortisol metabolites were not homogenously distributed in the scats. Cortisol metabolite levels were significantly lower when faecal extracts and samples were frozen for long periods of time (14 weeks or more). Thus, the measurement of faecal cortisol metabolites is a suitable method for the non-invasive evaluation of adrenocortical activity in European badgers. However, some aspects related to the collection and storage of faecal samples should be considered in future studies conducted with this species.

Effects of birth date and natal dispersal on faecal glucocorticoid concentrations in juvenile Common hamsters

Human activities involving noise emission can affect wild animals. European mink was exposed to road noise and human voice playbacks to analyze how sound intensity level and duration of both noises altered the time that individuals were active and if their fecal cortisol metabolite (FCM) levels varied. A Hierarchical Analysis Cluster was performed to establish 2 mink groups with respect to both noise source type: short duration/low intensity (SL) and long duration/high intensity (LH). We performed general linear mixed models to evaluate the variation in locomotor activity duration (s) and FCM (nanogram per gram) levels, respectively. The results showed both road noise and human voices decreased locomotor activity duration in SL more sharply compared with LH, and human voices were the triggers that induced the most pronounced response to both exposure conditions. FCM (ng/g) levels increased in SL compared with LH during road noise while the opposite happened during human voices. Differences based on sex and age of individuals were observed. In conclusion, noise characteristics given by the sound type determined the variations in locomotor activity duration while noise exposure level determined the variations in FCM (ng/g) levels. Attention should be paid to noisy activities (e.g., recreational activities for visitors in protected natural areas) and loud groups of people to conserve wildlife, especially noise sensitive species.

Simple SummaryThe measurement of faecal cortisol metabolites (FCMs) is increasingly used to monitor physiological stress responses in different animal species. Before this method is applied in coming stress- and welfare-related studies of farmed blue foxes (Vulpes lagopus), a species-specific validation is first required. In the current study, a 5α-pregnane-3ß,11ß,21-triol-20-one enzyme immunoassay was found suited to measure FCMs and thus hypothalamus–pituitary–adrenal (HPA) axis activity in farmed blue foxes. FCMs can therefore serve as a valid indicator of stress in future welfare studies of blue foxes.Welfare studies of blue foxes would benefit from a measurement of faecal cortisol metabolites (FCMs) as a non-invasive, physiological stress parameter reflecting hypothalamus–pituitary–adrenal (HPA) axis activity. Before implementation, a species-specific validation of such a method is required. Therefore, we conducted a physiological validation of an enzyme immunoassay (EIA) to measure FCMs in blue foxes. Twenty individuals (nine males and eleven females) were injected with synthetic adrenocorticotrophic hormone (ACTH) and faecal samples were collected every third h for two days. The FCM baseline levels were assessed based on the first sampling day (control period, 144 samples), followed by the ACTH injection and the second day of sampling (treatment period, 122 samples). FCMs were analysed with a 5α-pregnane-3ß,11ß,21-triol-20-one EIA. We compared the estimated mean FCM concentrations of the treatment samples to the baseline average. All samples for the two periods were collected at the same time of the day, which enabled to test the data also with an hourly pairwise comparison. With the two statistical approaches, we tested whether a possible diurnal fluctuation in the FCM concentrations affected the interpretation of the results. Compared to the baseline levels, both approaches showed 2.4–3.2 times higher concentrations on time points sampled 8–14 h after the ACTH injection (p < 0.05). The estimated FCM concentrations also fluctuated slightly within the control period (p < 0.01). Inter-individual variations in FCM levels were marked, which highlights the importance of having a sufficient number of animals in experiments utilising FCMs. The sampling intervals of 3 h enabled forming of informative FCM curves. Taken together, this study proves that FCM analysis with a 5α-pregnane-3ß,11ß,21-triol-20-one EIA is a valid measurement of adrenocortical activity in the farmed blue foxes. Therefore, it can be utilised as a non-invasive stress indicator in future animal welfare studies of the species.

Simple SummaryMulti-unowned-cat environments can be highly stressful for cats, and infectious and parasite diseases spread quickly and are difficult to prevent. This study aimed to determine the occurrence of intestinal parasites and fecal cortisol metabolites (FCM) in cat feces collected from different multi-cat environments and assess the effect of housing conditions on intestinal parasites and FCM levels in order to develop more efficient control strategies. Cat fecal samples from rescue shelters, catteries and feline colonies were analyzed with coprological methods to detect intestinal parasite patency and determine FCM. Helminth infection was mainly detected in free-roaming cats. In confined cats, protozoa infections were more likely detected in shelter cats than in cattery cats. Exposure to dogs was associated with parasite infection and cats highly exposed to dogs with visual contact and audible barking showed higher intestinal protozoa prevalence than cats that were not exposed to dogs. FCM levels were correlated with enclosure size and protozoa infection. Reducing stress by improving housing in terms of enclosure size and avoiding exposure to dogs may have an impact on the occurrence of intestinal parasites, especially protozoa.Housing conditions were assessed in different unowned multi-cat management models in order to evaluate their impact on the occurrence of intestinal parasites and fecal cortisol metabolite (FCM) levels. Fresh stool fecal samples were collected from rescue shelters, catteries and feline colonies for coprological analyses in order to detect intestinal parasite patency and fecal cortisol metabolites. A questionnaire provided information about the facilities, management and housing conditions of cats, including information about dog exposure, enclosure size, environment enrichment and changes to group composition. Overall, intestinal parasite infection was detected in 58.2% of fecal samples collected. The occurrence of intestinal parasites detected in free-roaming cats was 82.2%, mainly due to helminth infection. The parasite infection rate was 57.3% in rescue shelters and 34.6% in catteries. In confined cats, protozoa infection was more likely detected in rescue shelters than in catteries (RR = 2.02 (1.30–3.14), p = 0.0012). Although the FCM values were very variable between cats, the enclosure size and parasite infection were correlated with the average FCM. A small enclosure size was correlated with high fecal cortisol metabolites (p = 0.016). Protozoa-positive samples showed higher FCM levels than negative samples (p = 0.0150). High dog exposure was statistically associated with protozoa infection (p = 0.0006). The results indicated that improving housing, especially in terms of floor space and avoiding dog exposure, reduces stress and can thus be applied to make control strategies in multi-unowned-cat environments more efficient, especially when cats are confined.

Simple SummaryFaecal cortisol metabolites (FCMs) are a useful, non-invasive tool for the assessment of stress in koalas. However, FCM stability after defecation is a critical issue. Therefore, we exposed faecal pellets of koalas to three different environmental conditions and measured FCMs with three recently evaluated enzyme immunoassays (EIAs). Because water loss over time exerted the greatest influence on FCMs, we strongly recommend collecting freshly defecated pellets in koalas.Faecal material can be a valuable source of information for a range of animal health aspects and can be used to measure faecal cortisol metabolites (FCMs). FCM values can relate to physiological stress responses. However, freshly defecated pellets are not always available and environmental conditions, such as temperature and humidity, might affect faecal pellet consistency and FCM levels. Therefore, the impact of environmental conditions on FCMs needs to be evaluated. We collected 107 pellets from two female and two male koalas, exposed them to three types of treatments, and analysed FCMs in these samples with three enzyme immunoassays (EIAs). After analysis, the original FCM values were mathematically corrected for water loss. Results show that the FCMs were more stable when measured using tetrahydrocorticosterone (50c) and 5α-pregnane-3β,11β,21-triol-20-one (37e) EIAs, and were less stable when measured with the cortisol EIA. With 50c, the FCM values did not vary significantly over time either before or after the adjustment with water in the environment treatment group. For samples kept under constant low (25 °C) and high (35 °C) temperatures, the 50c FCM values did not vary significantly over time, after adjustments were made for water loss. Thus, this study highlights the importance of considering the suitability of faecal field samples for FCM analysis. Because water loss was the main driver of FCM changes, we strongly recommend collecting koala pellets that are freshly defecated, despite the effort and time it might take to collect such pellets.

Assessment of stress in captive wildlife populations requires reliable biochemical indicators that can be measured through non-invasive sampling methods. This research evaluated cortisol and corticosterone metabolite concentrations in fecal and hair samples from 147 animals representing 23 species housed across four Nigerian zoological facilities. Sampling occurred over 18 months to capture seasonal variation and responses to management changes. Fecal glucocorticoid metabolite concentrations varied significantly among species, with felids showing highest baseline values and bovids demonstrating lowest levels. Hair cortisol concentrations provided integrated stress assessments over longer timeframes, revealing chronic elevation in animals with limited environmental enrichment. Strong correlations emerged between biochemical markers and behavioral indicators of stress including stereotypic movements and reduced feeding activity. Implementation of enrichment programs resulted in 34.7% reduction in fecal cortisol metabolites and 41.2% decrease in stereotypic behaviors over six-month observation periods. Species-specific reference ranges were established for common zoo animals, providing practical tools for welfare monitoring. These findings demonstrate that integrated biochemical and behavioral assessment enables objective evaluation of captive wildlife welfare, supporting evidence-based management decisions.

Fecal cortisol metabolite levels in free-ranging North American red squirrels: Assay validation and the effects of reproductive condition

Fecal cortisol metabolites under anonymized sampling: Robust estimates despite significant individual heterogeneity

Anthropogenic land use and its after-effects are potential sources of stress for European ground squirrel (&lt;em&gt;Spermophilus citellus&lt;/em&gt;) populations, which increasingly have to cope with human impact throughout the species’ range. To determine whether habitat alteration impacts the stress load of free-ranging populations in Austria, we live-trapped and faecal-sampled individuals both in a nearly unaltered steppe habitat (TD) and in a strongly altered alfalfa meadow (FB). Overall and seasonal faecal cortisol metabolite (FCM) concentrations were analysed and compared between the two study sites. FCM levels of adult males and of juveniles of both sexes were higher at FB than at TD. Adult females showed no such differences, but exhibited increased faecal progesterone metabolites (FPM) levels at both sites during June. Our results indicate that human activities affected stress load in adult males and juveniles. The altered vegetation led to highly abundant food at FB and, together with the isolation of the habitat, caused a high population density. This apparently intensified social stress in certain periods of the active season. Elevated FCM levels in both adult males and juveniles at FB coincided with the period of highest population density, when all juveniles had emerged from the natal burrows, and hibernation had not started yet. At the same time, predation pressure and human recreational activities also peaked. The highest FCM levels were found in juveniles at FB shortly after natal emergence, suggesting that this age class is most vulnerable to social stress, predation and human disturbance. The lack of a measurable stress response in adult females may be due to increased progesterone concentrations attenuating the stress-induced elevation of glucocorticoids.

Simple SummaryUrination outside the litterbox (also known as periuria) is a very frequent problem seen by veterinary behaviourists and is a common reason for the relinquishment of cats. Veterinary behaviour textbooks describe two forms of periuria (spraying and latrining), including characteristics of both, and speculations, such as spraying is more closely associated with stress. With the aim of evaluating the arousal underpinning emotional stress in cats showing periuria, we studied recorded behaviours as well as faecal cortisol metabolite (FCM) levels of 11 “sprayer” and 12 “latriner” cats along with their controls (i.e., cats that did not show periuria) from the same multi-cat homes of three to nine cats. The results indicated that households in which a cat exhibits urine spraying are generally more aroused than households with latrining cats, but “sprayers” are not more aroused than their housemates. In practical terms, such results suggest that behaviour management to control periuria in these households should be focused on all cats not just the “sprayers”.It is often suggested that both latrining and spraying in the home are associated with increased stress in cats. However, the scientific evidence for this is weak. We therefore examined faecal cortisol metabolite (FCM) levels in subjects using a case-control design. Eleven spraying and 12 problematic latrining cats (assessed as healthy after detailed medical examinations on an initial population of 18 spraying and 23 latrining cats) were assessed along with behaviourally normal and similarly healthy control subjects from the same multi-cat (n = 3–9) households. Individual faecal samples were collected by owners from both “case” and “control” cats after observing them defecate in all but one pair in each group. A total of five samples per cat (typically taken on a weekly basis) were collected and submitted to extraction procedures prior to FCM analysis via an 11-oxoaetiocholanolone enzyme immunoassay (EIA). Participant cats, both “cases” (nine “sprayers” and eight “latriners”) and controls, were also individually video recorded (together with the owner) for 5 min in a dedicated room. FCM levels were significantly higher in individuals (“sprayers” and their controls) from spraying households than from the latrining households (“latriners” and their controls), but there was no significant difference between cats from the same household. Within a video observation test, cats from spraying houses spent proportionally more time moving (as opposed to stationary), but again there was no difference between cats from the same house. These results indicate that households in which a cat exhibits urine spraying, are generally more aroused, but “sprayers” are not more aroused than their housemates. Accordingly, we suggest appropriate management needs to be applied to the whole household to help alleviate the potential stress of all the cats in the home, and not just the one expressing this through urinary spraying behaviour.