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 underutilized in their conservation. We developed and evaluated a biologically optimized, 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. Generalized linear mixed models revealed that gonadotropin-releasing hormone (GnRHa) alone significantly outperformed hCG based regimens. The 3 ug per 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 to 7.0) and hypoosmotic (75 to 100 mOsm per kg) conditions. To support decision-making, we used a Wildlife Sperm Index (WSI) developed by our team that integrates sperm traits and environmental parameters into a single composite score. While 3ug per g GnRHa ranked highest by concentration alone, 4ug per g GnRHa achieved the top WSI score due to greater environmental (pH and osmolality) compatibility. These findings provide the first evidence-based ART protocol for R. muscosa and offer a transferable framework for optimizing gamete collection, IVF, and cryopreservation in line with other amphibian species, advancing both genetic management and species recovery goals.