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

## TL;DR

This study identifies optimal cryopreservation methods for sperm from four endangered Korean amphibians, showing that cryoprotectant type, concentration, and cooling speed significantly affect sperm viability.

## Contribution

The study provides species-specific cryopreservation protocols for four endangered amphibians using empirical testing of cryoprotectants and cooling regimes.

## Key findings

- DMF at low concentrations provided better membrane-integrity viability than DMSO across species.
- Slower cooling (10 cm above liquid nitrogen) improved viability for most species.
- Optimal conditions varied per species, with the highest viability observed for Dryophytes suweonensis at 86.5%.

## Abstract

Amphibian populations are rapidly declining, and sperm cryopreservation is essential for conservation. This study tested the post-thaw membrane-integrity viability of sperm from four endangered Korean species following cryopreservation using two different cryoprotectants (dimethyl sulfoxide [DMSO] or N,N-dimethylformamide [DMF] with sucrose) and two cooling methods (freezing at 5 or 10 cm above the liquid nitrogen surface). Low cryoprotectant levels improved membrane-integrity viability, while high concentrations—especially DMSO—were toxic. DMF offered more consistent protection, and slower cooling (10 cm) enhanced viability. Optimal conditions were 15% DMSO at 10 cm for Dryophytes suweonensis (86.5%), 10% DMF at 10 cm for Pelophylax chosenicus (75.5%), and 10% DMSO at 5 cm for Kaloula borealis (81.6%). Hynobius yangi showed low membrane-integrity viability overall, peaking at 19.7% with 15% DMF at 5 cm.

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 (Dryophytes suweonensis, Pelophylax chosenicus, Kaloula borealis, and Hynobius yangi) were evaluated. Sperm were cryopreserved using dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF) at 10–30% (v/v) 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 D. suweonensis were 15% DMSO at 10 cm (86.5% membrane-integrity viability); for P. chosenicus, 10% DMF at 10 cm (75.5%); and for K. borealis, 10% DMSO at 5 cm (81.6% membrane-integrity viability). Hynobius yangi 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.