Sample storage method and collection time effects on horse hair cortisol concentrations

Abstract

Serum cortisol concentration is the gold standard for evaluating stress in horses. However, it can be affected by circadian rhythms, environmental factors, and other excitatory events. Hair cortisol is less subject to these variables and is less invasive to collect, but there is a lack of published information on hair cortisol concentrations in horses. The objectives of this study were to examine (1) the impact of sample storage (room temperature vs. frozen) and (2) the effect of collection date (spring vs. fall), on cortisol concentration in equine hair. It was hypothesized that frozen samples would yield higher concentrations than room samples, and that the samples taken in the spring would yield higher concentrations than those in the fall. Tail hair samples (1 g) were collected from 32 university-owned horses in the spring and fall semesters of 2024. Each horse's samples were split, with 0.5 g maintained at room temperature (20 to 22°C) and 0.5 g being frozen (−28.89°C). Spring samples were stored for 8 mo before analysis, and fall samples were stored for 2 mo. Samples were analyzed based on previously published methods. Log-transformed hair cortisol concentrations were analyzed using a general linear model and mixed model in SAS to assess the effects of storage condition (frozen vs. room temperature) and sample date (spring vs. fall). Significance was declared at P > 0.05. Both factors significantly influenced hair cortisol concentrations, with frozen samples showing higher concentrations than room samples (P = 0.03) and fall samples exhibiting higher concentrations than spring samples (P = 0.03; Table 1). These findings highlight the importance of accounting for storage conditions and seasonal variation in cortisol analyses.