Fecal Hormone Metabolites Concentrations Collected Longitudinally Confirm Spontaneous Polyestry in Mt. Graham Red Squirrels (Tamiasciurus fremonti grahamensis)

The Mount Graham red squirrel (Tamiasciurus fremonti grahamensis, MGRS) is a federally endangered subspecies endemic to southeastern Arizona. Despite the establishment of ex situ conservation programs, reproduction outside the wild has not occurred. We monitored fecal hormone metabolites (FHM) to noninvasively assess estrous cycling and reproductive hormone dynamics in ex situ-managed MGRS. We compared ex situ findings with two in situ (wild) populations. We tracked longitudinal changes in estradiol and progesterone metabolites in three ex situ managed females and evaluated whether ovulation was spontaneous or induced. We detected 22 ovulation events over two years, with timing and regularity consistent with spontaneous polyestrous cycling. Comparative hormone analyses revealed appreciably lower progesterone and corticosteroid metabolite levels in ex situ individuals, whereas estradiol levels were comparable to those of wild counterparts. These findings suggest that maintaining environmental, and social conditions, e.g., lighting, temperature, and conspecific proximity, in ex situ settings is critical and could influence reproductive success. By confirming spontaneous ovulatory cycling in MGRS and demonstrating the utility of FHM as a noninvasive reproductive assessment tool, this study advances ex situ breeding efforts and provides a framework for reproductive monitoring in other endangered taxa.