Differentiating the estrous cycle phases using vaginal vestibule pH and cytology in Asian elephants (Elephas maximus) in human care

Abstract

Reproductive dysfunction in elephants is often confirmed through blood hormonal assays, as behavioral observations alone are insufficient. While fecal and saliva hormonal analyses offer welfare-friendly alternatives, significant resources are required, limiting practicality in field applications. To improve reproductive management and ensure animal welfare, this study aimed to investigate pH measurements and cytological analysis of the vaginal vestibule to differentiate estrous cycle phases in female elephants, both those with regular and irregular cycling. A total of 106 vaginal vestibule cytological smears from eight regularly cycling females and 39 smears from three irregularly cycling females (confirmed by progesterone and luteinizing hormone [LH] enzyme immunoassays, n = 308 serum samples) were analyzed using staining techniques and pH strip measurements. Based on pH, smears were categorized into acidic, neutral, and alkaline groups. Intermediate cells (ICs) varied significantly across the three groups. In regularly cycling females, ICs dominated during diestrus (67.5 %), whereas estrus was characterized by the absence of ICs and a predominance of superficial cells. ICs decreased in metestrus (36.3 %; P < 0.05). Mean pH levels ranged from acidic in diestrus (5.8) to alkaline in estrus (8.5). In irregularly cycling elephants, pH (6.3) differed from proestrus and estrus in irregularly cycling elephants. Moreover, pH was significantly correlated with progesterone (r = -0.25) and LH (r = 0.4) among all females. Using cytology exclusively was insufficient to differentiate between regular and irregular cyclic statuses; however, when combined with pH measurements, it effectively identified the estrous phases in regularly cycling elephants. This study offers novel findings that could serve as practical, welfare-focused metrics for reproductive assessments in elephants.