Optimizing Equilibration Time to Enhance Post-Thaw Viability of Cryopreserved Zebrafish ( Danio rerio ) Ovarian Fragments

Cryopreservation enables the long-term storage of viable biological material at ultra-low temperatures and forms the foundation for germplasm cryobanks that maintain valuable genetic lines of model organisms such as zebrafish ( Danio rerio ). However, reliable and reproducible cryopreservation protocols for fish germline stem cells remain difficult to develop, partly because key steps such as equilibration are often overlooked or assigned arbitrarily. Here, we optimized equilibration time for cryopreservation of zebrafish ovarian tissue. Ovarian fragments were equilibrated in 2 M methanol + 0.1 M glucose + 10% egg yolk for varying durations (15-120 minutes) before controlled slow-cooling and storage in liquid nitrogen. Post-thaw viability was assessed using a Trypan Blue exclusion assay. A 60-minute equilibration yielded the highest viability of ovarian cells in Experiment 1 (55.68 ± 1.74%), whereas a 30-minute equilibration yielded the highest viability in Experiment 2 (75.86 ± 2.44%), but was not significantly different from the 60-minute equilibration in Experiment 2 (75.58 ± 2.04%) ( p = 0.9983). Equilibration alone accounted for a 48.71% increase in post-thaw viability relative to controls. The framework presented here provides a reproducible method for determining species-specific equilibration optima and supports the development of effective germplasm cryobanks for both model and endangered fish species.