Hormonal and Environmental Drivers of Spermiation in the Endangered Mountain Yellow-Legged Frog (Rana muscosa): Toward Biologically Informed Assisted Reproductive Technologies.

Amphibians are among the most threatened vertebrates, yet assisted reproductive technologies (ARTs) remain underutilised in their conservation. We developed and evaluated a biologically relevant, non-lethal sperm collection protocol for Rana muscosa, a critically endangered frog in a long-term conservation breeding program. Thirteen hormone treatments were tested across six post-injection time points, and sperm quality was assessed via concentration, motility, osmolality, and pH. Generalised linear mixed models revealed that gonadotropin-releasing hormone (GnRHa) alone significantly outperformed hCG-based regimens. The 3 {micro}g/g GnRHa dose yielded the highest sperm concentration and sustained motility from 3 to 24 hours post-injection. Motility was highest under moderately acidic (pH 6.5-7.0) and hypoosmotic (75-100 mOsm/kg) conditions. To support decision-making, we applied a refined Wildlife Sperm Index (WSI) that weights motility and concentration as primary functional-yield traits, with motility given greater relative weight given its direct role in fertilisation, and treats pH and osmolality as secondary environmental-compatibility traits. Under this revised composite score, 3 {micro}g/g GnRHa achieved the highest overall WSI score, outperforming 2 {micro}g/g and 4 {micro}g/g GnRHa, which ranked second and third, respectively, despite their more favourable pH and osmolality profiles. These findings provide the first evidence-based ART protocol for R. muscosa and offer a transferable framework for optimising gamete collection, IVF, and cryopreservation in line with other amphibian species, advancing both genetic management and species recovery goals.