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

Abstract 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 µg/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 used a Wildlife Sperm Index (WSI) developed by our team that integrates sperm traits and environmental parameters into a single composite score. While 3 µg/g GnRHa ranked highest by concentration alone, 4 µg/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. Competing Interest Statement The authors have declared no competing interest.