Aim: The changes of the hypothalamic pituitary adrenal (HPA) axis response to a long distance transportation results in increase of adrenocorticotropic hormone (ACTH) and cortisol levels. The purpose of the study was to quantify the level of short-term road transport stress on circulating ACTH and cortisol concentrations, related to the effect of previous handling and transport experience of horses. Materials and Methods: The study was performed on 56 healthy horses after short-term road

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... rt of 30 km. The horses were divided into four groups, Groups A, B, C, and D, with respect to the handling quality: Good (Groups A and B), bad (Group D), and minimal handling (Group C) conditions. According to the previous transport, experience horses were divided as follows: Horses of Groups A and D had been experienced long-distance transportation before; horses of Groups B and C had been limited experience of transportation. Results: One-way RM-ANOVA showed significant effects of transport on ACTH changes in Groups B and C and on cortisol changes in both Groups A and B. Groups A and B showed lower baseline ACTH and cortisol values than Groups C and D; Groups A and B showed lower post-transport ACTH values than Groups C and D. Groups A, B, and C showed lower post-transport cortisol values than Group D. Only Groups A and B horses have shown an adequate capacity of stress response to transportation. Conclusion: The previous transport experience and quality of handling could influence the HPA axis physiological responses of horses after short-term road transport.