Measures of Oxidative Status Markers in Relation to Age, Sex, and Season in Sick and Healthy Captive Asian Elephants in Thailand

**Background:** Oxidative stress is a pathological condition that can adversely affect animal health, but little research has been conducted on wildlife species. The Asian elephant (Elephas maximus) is endangered, and captive populations face various diseases. This study aimed to analyze five oxidative status markers (reactive oxygen species [ROS], malondialdehyde [MDA], albumin, glutathione peroxidase [GPx], and catalase) and fecal glucocorticoid metabolites (fGCMs) in healthy and sick captive Asian elephants in Thailand, examining differences by age, sex, and season.

**Methods:** Blood was collected from 137 healthy elephants (mean age 16.8 ± 16.4 years; range 1–61; 45 males, 92 females) and 20 sick elephants (mean age 27.6 ± 20.0 years; 4 males, 16 females) from tourist camps in northern Thailand. Sick elephants presented with weakness (n=1), puncture wounds (n=2), gastrointestinal distress (n=5), eye problems (n=3), musculoskeletal conditions (n=4), and elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) (n=5). Serum oxidative markers were measured using established assays: ROS via DEPPD method, MDA via TBARS assay, albumin via automated chemistry analyzer, GPx via CUPRAC method, and catalase via spectrophotometric method. Fecal samples were analyzed for fGCMs using a corticosterone enzyme immunoassay. Data were analyzed using generalized linear models (GLM) with age group, sex, and season as main effects, plus interactions. Pairwise comparisons used Tukey post hoc tests. A subset of age-/sex-/camp-matched healthy elephants (n=30) was compared to sick groups using Kruskal-Wallis tests. Pearson correlations were calculated between biomarkers and blood parameters.

**Key Results:** ROS and albumin concentrations were highest in aged elephants (>45 years). No sex differences were found for any biomarker. Significant interactions were observed for age group and season for ROS and catalase, and for sex and season for GPx. In pairwise comparisons, ROS concentrations were higher in adult females in summer (2.35 ± 0.07 mg/L) compared to juveniles and subadults in the rainy season (p < 0.05). Catalase activity was lower in juvenile males, subadult males, adult females, and aged females in summer compared to subadult and adult elephants in winter and rainy season (p < 0.05). For health status, EEHV-HD elephants showed the most significant changes: MDA was more than double that of healthy elephants (p < 0.01), albumin was only 68% of healthy (p < 0.01), GPx was higher (p < 0.05), and catalase was almost double (p < 0.05). No differences in fGCMs were found between healthy and sick groups overall, though variability was higher in sick animals. Positive correlations were found between catalase and fGCMs (r = 0.23, p < 0.05) and between several red blood cell parameters and oxidative markers.

**Clinical Implications:** This study provides the first comprehensive analysis of multiple oxidative status markers in Asian elephants. Age and season should be considered when interpreting these biomarkers. The marked changes in EEHV-HD elephants (elevated MDA, GPx, catalase; decreased albumin) suggest oxidative stress plays a role in this fatal disease. These markers could aid in early diagnosis and monitoring of EEHV-HD and other conditions. However, sample sizes for sick elephants were small, and longitudinal studies are needed to determine temporal changes and predictive value. The lack of sex differences and minimal seasonal effects may reflect the relatively stable climate in Thailand and the protective effects of elephant skin pigmentation. These findings offer additional tools for health assessment in captive elephants, potentially improving disease management and conservation efforts.