Optimizing Sperm Cryopreservation from Four Endangered Korean Amphibian Species: Species-Specific Effects of Cryoprotectants and Cooling Regimes on Membrane-Integrity Viability

Global amphibian populations are declining rapidly and the development of effective cryopreservation protocols for germ cells has become a critical tool in ex situ conservation programs. Post-thaw membrane-integrity viability in four endangered Korean amphibians (<i>Dryophytes suweonensis</i>, <i>Pelophylax chosenicus</i>, <i>Kaloula borealis</i>, and <i>Hynobius yangi</i>) were evaluated. Sperm were cryopreserved using dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF) at 10-30% (<i>v</i>/<i>v</i>) in combination with 0.6 M sucrose, and were frozen at two suspension heights (5 cm vs. 10 cm) above liquid nitrogen. Post-thaw membrane-integrity viability was assessed using a SYBR-14/propidium iodide membrane-integrity assay (LIVE/DEAD kit). Low concentrations of permeating cryoprotectants (CPs) improved membrane-integrity viability, whereas high concentrations led to high toxicity, particularly with DMSO. Across species, DMF produced the highest membrane-integrity viability and the most consistent performance. The cooling rate influenced membrane-integrity viability, with samples frozen at 10 cm exhibiting greater viability, reflecting the balance between intracellular ice formation during rapid cooling and solution effects during slow cooling. Optimal conditions for <i>D. suweonensis</i> were 15% DMSO at 10 cm (86.5% membrane-integrity viability); for <i>P. chosenicus,</i> 10% DMF at 10 cm (75.5%); and for <i>K. borealis</i>, 10% DMSO at 5 cm (81.6% membrane-integrity viability). <i>Hynobius yangi</i> showed modest improvement under 15% DMF at 5 cm (19.7%), although overall membrane-integrity viability was low. ED₅₀ modeling indicated species-specific thresholds requiring low CP concentrations. Sperm cryopreservation outcomes in amphibians are strongly influenced by CP type, concentration, cooling regime, and species physiology. GLM and ED₅₀ modeling provide a methodological framework for refining cryopreservation strategies for non-model, endangered species.