Behavioral and physiological responses of horses at the Costa Rican National Horse Parade: Developing welfare indicators for real-time assessment

The Costa Rican National Horse Parade is an annual cultural equestrian event involving approximately 2,000 horses. To address growing welfare concerns, this study evaluated behavioral and physiological indicators of stress in participating horses. Behavioral data were collected using an ethogram at 3 observation stations (start, middle, and end) along the 3.5-km route. During the 6.5-h event, 24 trained observers conducted one-minute evaluations of randomly selected horses (n = 557). Stress-related behaviors, including ear position, neck hyperflexion, and head tossing, were recorded, along with rider behaviors (e.g., excessive whip use and aggressive riding). Eye wrinkle depth (EWD; none, soft, deep) and eye white visibility (EWV; visible, nonvisible) were assessed in 346 horses using photographs. Physiological indicators were measured in 10 horses (5–15 years old) from the same facility, including body weight (BW), body condition score (BCS), and cortisol levels (blood and fecal). Blood cortisol (BC) levels were measured on farm at rest and 5 min post-exercise, 21 d pre-event, and immediately post-event. Fecal cortisol (FC) samples were collected at rest (21 d pre-event) and 16 h post-event. A chi-squared test was used to assess differences in behavioral stress indicators and observation stations (P < 0.05) and also to evaluate EWD and EWV across stations (P < 0.05). BC data were not normally distributed (P < 0.05), leading to the use of a Kruskal-Wallis test. FC concentrations were analyzed using a paired t-test. Statistical analyses were conducted using Jamovi (v2.3.28) and RStudio (v4.2.3). Significant differences (P < 0.05) were found among observation stations. Stress-related behaviors increased at the end station, coinciding with heightened aggressive rider behavior and excessive whip use. EDW and EWV assessments showed that deep EWD (44.23%) and EWV (40.38%) peaked at the middle station. Physiological assessments showed a 6 kg decrease in BW and a 0.02-point reduction in BCS, though these changes were not statistically significant (P > 0.05). Differences in BC levels (rest: 13.27 ng/mL, post-exercise: 20.19 ng/mL, post-event: 40.71 ng/mL; P < 0.05) were found, with post hoc Mann-Whitney U tests confirming significance for all comparisons (P ≤ 0.011). FC concentrations doubled post-event (mean rest: 11.10 ng/mL, post-event: 27.09 ng/mL, P = 0.0008). These findings indicate increased physiological and behavioral distress, highlighting the need for improved welfare protocols during the event.