Repeatability of the ACTH stimulation test as reflected by salivary cortisol response in healthy horses

•ACTH stimulation test for evaluation of adrenal cortex function and stress assessment.•Administration of ACTH in horses stimulates the secretion of cortisol in horses.•ACTH stimulation test is repeatable in the short and long term.

The aim of this study was to further characterize the ACTH stimulation test as reflected by salivary cortisol response and to measure the short- and long-term repeatability of it in healthy horses as a tool to assess the capacity of the adrenal cortex to secrete cortisol. Nineteen healthy horses were subjected to 3 ACTH stimulation tests. Intervals were 2 wk and 5 mo between the first and second and the second and third tests, respectively. A dose of 1-μg/kg BW synthetic ACTH was injected intravenously. Saliva samples were collected at baseline and at 30, 60, 90, 120, 150, and 180 min after administration for cortisol measurements using a competitive enzyme immunoassay. A repeated measures ANOVA was used to compare values within and among horses. Mean ± SD total increase in cortisol concentrations integrated over the entire sampling period was 34.5 ± 11.0 ng/mL. The highest measured concentration at a single time point was 9.7 ± 2.7 ng/mL and was reached after 122 ± 22 min. For the short- and long-term repeatability, intraclass correlation coefficient was 0.90 and 0.33, respectively. The 3 ACTH stimulation tests results differed significantly among (P < 0.00001) but not within (P = 0.538) individual horses. The Freiberger stallions had a higher salivary cortisol baseline concentration and a lower response to ACTH stimulation as compared with Warmblood mares and geldings. The present study confirmed that the administration of ACTH in healthy horses reliably stimulates the salivary secretion of cortisol and shows that the test is repeatable in the short- and long-term.