Validation of reproductive monitoring techniques in free-living female giant armadillos (Priodontes maximus)

The giant armadillo ( Priodontes maximus ) is classified as Vulnerable and remains one of the least studied neotropical mammals, with major gaps in knowledge of its reproduction limiting reproductive management strategies. This study presents the first integrated reproductive assessment of female giant armadillos, aiming to validate practical tools for reproductive monitoring under field conditions. Between May 2023 and January 2025, six females (one juvenile and five adults) were evaluated in the Pantanal and Cerrado biomes of Brazil. Assessments included external genitalia morphology, vaginal cytology, serum estradiol and progesterone concentrations, and vaginal microbiota. Camera trap records were retrospectively analyzed to determine pregnancy status at the time of capture and no females were pregnant during the study period. The mean estradiol and progesterone concentrations in adults were 422.5 ± 620.4 pg/mL and 5.8 ± 3.3 ng/mL, respectively, while the juvenile concentrations were 10.2 pg/mL and 1.8 ng/mL, respectively. Significant correlations were detected among reproductive parameters, including positive associations between superficial cells and vulvar edema ( r s = 0.72), and between large intermediate cells and progesterone concentration ( r s = 0.75). Negative correlations were observed between parabasal cells and both superficial cells ( r s = -0.82) and estradiol concentration ( r s = -0.68). Vaginal cytology revealed cyclic epithelial maturation consistent with hormonal and morphologic variations and vaginal microbiota was predominantly composed of Enterobacteriaceae. These findings demonstrate that the integration of vaginal cytology, external genitalia assessment, and hormonal profiling is a promising approach for reproductive monitoring in P. maximus , providing essential baseline data for future research and conservation efforts. • First integrated reproductive assessment of free-living female giant armadillos. • Vaginal cytology reflects hormonal and external genital changes in P. maximus . • Significant correlations among vulvar morphology, vaginal cytology and hormones. • Vaginal microbiota in wild females is predominantly composed of Enterobacteriaceae.