Optimizing equilibration time to enhance post-thaw viability of cryopreserved zebrafish (Danio rerio) ovarian fragments

Abstract

Germplasm cryobanks are a vital ex situ conservation strategy for preserving the genetic material of endangered species, as the process of cryopreservation enables living material to be stored without deteriorating almost indefinitely. However, cryopreservation protocols yielding a high post-thaw viability of germ cells are difficult to develop, as there are several interconnected steps that can significantly alter the outcome. The purpose of my study was to optimize the equilibration time for the cryopreservation of zebrafish (Danio rerio) ovarian fragments, since previous studies often neglected this step and instead utilized an arbitrary equilibration time. Using a repeated-measures design, ovarian fragments were exposed to an equilibration treatment (control, 15, 30, 45, or 60 minutes in experiment 1; 30, 60, 90, or 120 minutes in experiment 2) and cryopreserved via slow-cooling. 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 as a factor alone accounted for a 48.71% increase in viability, confirming this step should not be overlooked. The method employed in this study can be used to calculate the optimal equilibration time for any fish species, a necessary step in developing effective cryopreservation protocols to preserve the genetic diversity of endangered species.