Enhancing aquaculture efficiency: Development of a reliable cryopreservation protocol for Leiarius marmoratus.

Leiarius marmoratus is a freshwater catfish widely used in Brazilian aquaculture for hybrid production with Pseudoplatystoma spp., representing an important genetic resource for sustainable aquaculture. This study aimed to establish a standardized and reproducible sperm cryopreservation protocol through the evaluation of different cryoprotectant formulations and thermal conditions to maximize post-thaw sperm quality. Sperm from 34 males were diluted in Beltsville Thawing Solution® (BTS®) at a 1:2 dilution ratio at collection and evaluated under different cryoprotectant conditions to identify the most effective formulation for preserving post-thaw sperm quality. Methanol-based treatments consistently outperformed other permeable and non-permeable cryoprotectants. The optimized protocol, BTS® (5%) with 10% methanol at a final dilution ratio of 1:10 using 0.25 mL straws, yielded superior results. Additionally, this protocol yielded a total motility of 41%, significantly higher than trehalose-based treatments. Furthermore, 10% methanol provided the highest membrane integrity, indicating superior structural preservation of sperm after thawing. In addition, the protocol demonstrated consistent performance across experimental replicates, indicating its robustness under varying handling conditions. Validation under field conditions confirmed the reproducibility of the optimized protocol and its ability to maintain post-thaw sperm quality parameters under commercial aquaculture condition. Overall, the use of 10% methanol in combination with BTS® proved to be the most efficient approach for L. marmoratus sperm cryopreservation, supporting artificial reproduction and contributing to the conservation of native catfish genetic resources.