Optimization of open-pulled straw (OPS) vitrification for isolated zebrafish blastoderms: a foundation for whole-embryo cryopreservation

BACKGROUND: Fish embryo cryopreservation is hindered by low surface-to-volume ratios, poor membrane permeability, and yolk-mediated resistance to dehydration. The massive yolk prevents uniform cryoprotectant equilibration, leading to lethal toxicity during vitrification. These biological barriers lead to lethal ice formation and chilling injury, hindering long-term germplasm preservation in teleosts. OBJECTIVE: Teleost embryos consist primarily of the blastoderm and a massive yolk sac. Temporarily modulating the composition and volume of the yolk sac prior to cryopreservation may enhance its permeability to cryoprotectants (CPAs), potentially synchronizing the equilibration kinetics of the yolk with those of the blastoderm to improve overall embryonic cryotolerance. We first sought to identify the developmental stage at which zebrafish embryos become most suitable for cryopreservation and to establish an optimized vitrification protocol for isolated blastoderms at this stage. These efforts serve as a fundamental prerequisite for the ultimate goal of successfully cryopreserving intact embryos. MATERIALS AND METHODS: Zebrafish blastoderms at various developmental stages were surgically isolated from the yolk sac. Vitrification was performed using the Open Pulled Straw (OPS) method, utilizing a combination of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as the primary cryoprotectants (CPAs). RESULTS: Optimized vitrification using an OPS-based DMSO/EG protocol yielded high viability. Following transplantation, vitrified???thawed RFP-labeled blastomeres successfully integrated into host embryos, demonstrating sustained developmental competence through the 21-somite stage. CONCLUSION: OPS vitrification effectively preserves blastoderm developmental competence, providing a technical framework to bypass yolk-mediated barriers and establishing a vital foundation for achieving cryopreservation of intact teleost embryos.