A stepwise methodology for developing fish sperm cryopreservation protocols: application to the endangered Pogonias courbina

Wild fish populations face growing threats from anthropogenic activities, climate change, and natural disasters. Aquatic germplasm banking is a vital strategy for preserving genetic diversity and supporting sustainable aquaculture. This study proposes a sequential, decision-guided framework for developing species-specific sperm cryopreservation protocols, applied here to the endangered Pogonias courbina, listed as endangered in Brazil and vulnerable by the IUCN. A series of experiments was conducted to identify optimal conditions for thawing (26 °C for 14 s), equilibration time (10 min), extenders (HBSS, 3% glucose, 5% glucose), and both permeable (dimethyl sulfoxide, propylene glycol, dimethylformamide, ethylene glycol) and non-permeable cryoprotectants (glucose, fructose, sucrose, trehalose, skimmed powdered milk, egg yolk). Sperm quality was assessed using Computer-Assisted Sperm Analysis (CASA). Three treatments with the highest post-thaw motility (MOT) were selected to fertilize oocytes from two hormonally-induced females. No significant difference was observed between sperm from wild and farmed males. The best kinetic parameters and fertilization rates were obtained using the combination of HBSS +10% DMSO +5% egg yolk (MOT: 71.8%). Based on these findings, we propose practical recommendations regarding extender selection, equilibration time, cryoprotectant combinations, and thawing conditions that support high post-thaw viability and successful fertilization. This is the first study to report successful artificial reproduction of P. courbina using cryopreserved sperm, establishing a reproducible protocol that can be applied for captive breeding, restocking, and germplasm banking. The proposed framework offers a generalizable approach for developing cryopreservation protocols in fish species.