Monitoring the hypothalamic–pituitary–adrenal (HPA) axis through determination of fecal cortisol metabolite (FCM) levels is a non-invasive method useful for understanding how handling and social conditions may affect the physiological status of zoo animals. The present study used FCM analysis to evaluate whether the HPA axis activity of a lion pride was modified by a change in social and handling conditions after the death of the dominant male. Five African lions (Panthera leo bleyenberghi), two males and three females, were included in the study. Fecal samples were collected before and after the death of the dominant male. To avoid cohabitation conflicts between males before the dominant male died, subgroups were established and subjected to weekly changes between indoor and outdoor facilities. After the death of the dominant male, these management dynamics ceased, and the remaining four lions were kept together outdoors. Significant lower group FCM concentrations (p < 0.001) were detected after the decease of the dominant male, probably associated with a decrease in daily handling, together with a more stable social environment. Overall, the present study indicates the effect of different management scenarios on the HPA axis activity and differentiated physiological responses to the same situation between individuals.

In many countries, horses remain involved in traditional equestrian events such as those celebrated in Menorca (Balearic Islands, Spain) every year since at least the 14th century. The present study aimed to evaluate the variations in salivary cortisol concentrations to estimate the physiological stress response in horses at the Menorca patronal festivals. Two different editions (years 2016 and 2018) of the festivals in honor of the Virgin of Grace in Maó (Menorca, Spain) were studied. Nineteen and seventeen Pure Breed Menorca stallions were included in the study, respectively. The stallions were aged between seven and twelve years. During celebrations, samples were collected before the start of the festivals between 8–9 a.m. and during the festivals at 8–9 p.m. On the second day of celebrations, the samples were collected at 8–9 a.m. and 3–4 p.m. Finally, on the day after the festivals, one sample was collected at 8–9 p.m. Additionally, a control group was sampled at 8–9 a.m., 3–4 p.m., and 8–9 p.m. Salivary cortisol concentrations were assessed by using a commercial enzyme immunoassay kit specially validated to quantify salivary cortisol in horses. Salivary cortisol concentrations did not show significant differences between sampling hours in the control group (p > 0.05). All the samples collected during festivals were significantly higher than samples of the control group (p < 0.05). Within the twenty-four hours after the end of the celebrations, cortisol concentrations returned to baseline levels and did not differ significantly from the control group (p > 0.05). Hence, the present study describes that the participation of the horses in these particular acts generate an acute and transitory stress response. Overall, the current work provides a reasonable basis for future research on the stress physiology and well-being of horses participating in traditional celebrations or similar events.

Assessing steroid hormones through feces provides invaluable insight on the stress and reproductive physiology of wildlife, and has been broadly applied to monitor the health and welfare of wild animals managed under human care. This study utilized fecal hormone monitoring to evaluate adrenal and gonadal activity in 18 captive lions (Panthera leo) across five Spanish zoological institutions, focusing on how biological and management factors affect these metrics. We analyzed fecal glucocorticoid metabolites (FGM) concentrations and, additionally in females, fecal progesterone (FPM), estradiol (FEM), and androgen (FAM) metabolites in relation to their reproductive status. Results indicated significant variability in FGM levels among individuals, with no consistent trends influenced by sex or zoo environment, including changes in the frequency of providing environmental enrichment at two zoos. Importantly, reproductive status significantly impacted adrenal and gonadal hormone levels; ovariectomized lionesses showed lower concentrations of FGM, FPM, and FEM compared to intact and deslorelin‐implanted females, the latter of which exhibited higher and more variable FAM levels. These findings advance our understanding of hormone patterns in lions and suggest implications for their management in captivity.

Immunoglobulin A (IgA) has been investigated as a stress biomarker with the potential to complement glucocorticoid measurements in welfare assessments. This study aimed to develop the methodology and validate an enzyme immunoassay (EIA) for quantifying IgA in feces (FIgA) of lions ( Panthera leo ), investigate excretion patterns of FIgA under baseline conditions in captive lions, and explore its relationship with fecal glucocorticoid metabolites (FGM). Feces were collected from 11 lions housed in stable social groups at four Spanish zoos over a period of two to six weeks. FIgA was reliably quantified using a commercial EIA, with concentrations ranging from 0.28 to 794.17 mu g IgA/g feces, showing substantial intra- and inter-individual variability. Females had significantly higher FIgA concentrations than males (113.10 vs 54.96 mu g IgA/g feces; p < 0.01). Additionally, FIgA concentrations varied across zoos (p p < 0.001). Positive correlations were found between FIgA and FGM for all samples combined (rho = 0.43, p < 0.001) and across individual means (rho = 0.70, p < 0.05), but not consistently when examining each lion separately. This study demonstrates for the first time that IgA can be reliably quantified in lion feces, paving the way for its application in welfare studies.

Oxytocin (OT) has emerged as a central focus in stress regulation and animal welfare research. While commonly measured in blood, saliva, and urine, its presence in feces remains unexplored. Fecal OT (fOT) could be highly valuable for wildlife studies, where direct handling is impractical. This study explored OT detection in feces using lions (Panthera leo) as a model species. The objectives included developing a reliable fOT extraction protocol, describing baseline fOT patterns in captive lions, and evaluating its relationship with fecal glucocorticoid metabolites (fGM) under non-stressful conditions. Fecal samples from 16 lions in stable social groups across five Spanish zoos were collected over two to six weeks. A methanol-based extraction and commercial enzyme immunoassay were successfully validated for fOT quantification. Concentrations ranged from 3.00 to 296.64 ng OT/g of feces, with notable intra- and inter-individual variability. Age, sex, and contraceptive status had no significant influence on fOT (p > 0.05), but concentrations differed significantly between zoos (p < 0.001). No consistent relationship was found between fOT and fGM levels under baseline conditions. This study is the first to demonstrate OT detection in feces. The validated methodology provides a foundation for investigating fOT as a biomarker of stress and welfare in wild and domestic species.

The Mediterranean tortoise Testudo hermanni inhabits different regions bordering the northwestern Mediterranean. This species is vulnerable, protected by legislation, and involved in various breeding and reintroduction programs. Wild populations face numerous environmental and anthropogenic stressors that can potentially interfere with their conservation. While seasonal changes in stress-response biomarkers, such as glucocorticoids and thyroid hormones, have been widely studised in mammals and birds, there is a paucity of research in reptile species. Therefore, the present study aimed to evaluate the seasonal fluctuations in corticosterone and total triiodothyronine levels in adult and juvenile Hermann’s tortoises (Testudo hermanni) as a measure of the physiological stress response. Blood samples were collected seasonally (winter, spring, summer, and autumn) and posteriorly analyzed by using a specific and validated enzyme immunoassay for both hormones, respectively. The results showed that corticosterone levels varied seasonally and differed between sexes, whereas total triiodothyronine levels changed seasonally but did not differ between sexes. Notably, juveniles exhibited no seasonal changes in either corticosterone or total triiodothyronine levels. Additionally, no correlation between blood extraction duration and hormonal concentrations was observed. This study is pioneering in its comprehensive evaluation of corticosterone and total triiodothyronine changes across all four seasons, including winter, and its focus on juvenile Hermann’s tortoises.