This pioneering study aimed to evaluate the cryopreservation of semen from P. falkneri (n = 4) and P. motoro (n = 4), maintained ex situ at the Sao Paulo Aquarium, Brazil. For this purpose, the animals were physically restrained, biometric data of the disc and clasper were obtained, and semen was collected through manual massage. Total motility and progressive motility parameters were evaluated using Computer-Assisted Sperm Analysis (CASA) with IVOS II equipment and Animal Breeders II software. The semen extenders INRA 96 and OptiXcell were used to assess their efficacy in sperm cryopreservation. INRA required the addition of 5% dimethyl sulfoxide (DMSO) as a cryoprotectant. The results indicated that there was no difference in semen motility values before and after freezing with INRA + DMSO (p = 0.6226). On the other hand, samples cryopreserved with OptiXcell showed a difference in semen motility post-thaw (p = 0.0156). These findings contribute to a broader study on optimizing cryopreservation protocols to ensure long-term viability and fertility of semen, enhancing genetic diversity and supporting wild population restoration. A multidisciplinary approach integrating reproductive biology, ecology, physiology, and assisted reproduction technologies, aligned with the One Conservation concept, is essential for advancing conservation and management strategies for these threatened species.