Comparing Hair Cortisol, DHEA, and Cortisol-to-DHEA Ratios from Various Body Sites in Anatolian Shelter Dogs

Hair-based biomarkers offer a non-invasive window into chronic stress physiology, yet the influence of sampling body site on hair cortisol, dehydroepiandrosterone (DHEA), and their molar ratio has not been systematically characterized in domestic dogs. This study compared these three biomarkers across three anatomical sites—shoulder (mid-scapula), neck (dorsal midline), and rump (sacroiliac region)—in 30 neutered Anatolian shelter dogs (2–3 years; 15 males, 15 females; 30 dogs total, with each dog sampled at all three body sites) housed in individual kennels over eight weeks. Hair samples were collected at week 0 and week 8, extracted by methanol pulverization, and quantified by validated ELISA protocols. Hair cortisol differed significantly by site (p < 0.001), as did DHEA (p < 0.01) and cortisol-to-DHEA ratios (p < 0.001). Cortisol decreased modestly over eight weeks (p < 0.05). DHEA was inversely correlated with cortisol (r = −0.62, p < 0.01). The shoulder site yielded the highest absolute concentrations and the greatest cortisol-to-DHEA ratio, combined with the lowest within-site coefficient of variation (CV = 11.2% vs. 13.8% at neck and 15.6% at rump), jointly supporting its designation as the preferred anatomical sampling location. The cortisol-to-DHEA ratio is identified as a nuanced welfare indicator that integrates both allostatic load and adaptive resilience capacity, with implications for standardized non-invasive stress assessment protocols in shelter medicine.