Semen cryopreservation methods were studied for North American river otters to help with genetic management and conservation. Extender composition and freezing methods were tested to see how they affected sperm quality after thawing. Results showed that using an extender with 4% glycerol and 1% Equex, and freezing with the pellet method, gave the best post-thaw sperm motility, longevity, and acrosomal integrity. The timing of glycerol addition and pre-freeze cooling rate didn't make a big difference in sperm quality. This study provides valuable information for preserving otter sperm and could be useful for other endangered otter species.