loading . . . Donkey hair cortisol concentrations are associated with carrying heavy load and being beaten at work - BMC Veterinary Research Background Cortisol is often considered an indicator of stress. However, it is not a straightforward indicator of stress in animals and is more closely linked to arousal and hypothalamic-pituitary-adrenal axis (HPA) axis activation. To better understand what animals are experiencing, other stressors, e.g., donkeys carrying heavy loads and being beaten, should be considered when interpreting cortisol levels. Our research aimed to assess how carrying mounted loads and working in challenging conditions impact donkey hair cortisol concentrations (HCC). For this purpose, thirty-five working donkeys were used. Data on the percent body weight ratio (%BWR) carried, the presence of skin wounds and lameness, provision of food and water, mounted load type, whether donkeys were beaten during work, and their working area were collected. Donkey hair samples were analyzed for HCC using an enzyme-linked immunosorbent assay (ELISA) kit and a multispectral plate reader. A multiple linear regression model was fitted to the data by stepwise backward elimination of variables. Results Donkeys were used for carrying fodder (n = 18; 51.4%) and bricks (n = 17; 48.6%) on their backs. Over half of the donkey owners provided water (54.3%, n = 19), and just under half provided food access (45.7%, n = 16) during the working day. The median HCC and %BWR were 4.34 pg/mg (interquartile range [IQR] 3.46–6.27 pg/mg) and 70.77% (IQR 57.0– 85.12%), respectively. Overall, 88.57% of donkeys carried loads above 50% BWR. High %BWR (estimate = 0.04, t = 2.75, p < 0.001), being beaten while working (estimate = 1.60, t = 4.49, p < 0.001), and carrying bricks (estimate = 0.96, t = 2.14, p = 0.04) were predictors of high HCC. Conclusion Carrying heavy loads of bricks and being beaten while working are likely stressful and can lead to increased activity of the HPA, resulting in higher levels of hair cortisol. Hair cortisol measurement is a valuable tool for assessing chronic stress in donkeys. Further research is necessary to assess HCC in donkeys working in different industries, such as mining, cart pulling, field plowing, and modern farming for hides and dairy products, to determine the relationship between cortisol, chronic stress, and the lives of animals. https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-025-05021-2
