External N-2-fluorophenyl-D-gluconamide does not affect zebrafish embryogenesis

Cryopreservation of zebrafish embryos remains challenging due to their low tolerance for cryoprotectant exposure and their susceptibility to lethal ice formation. Ice recrystallization inhibitors (IRIs) such as N-2-fluorophenyl-D-gluconamide (2FA) have shown promise in improving post-thaw survival in other systems, yet their biocompatibility in zebrafish embryos has not been established. Here, we evaluated the cytotoxicity and teratogenicity of 2FA during discrete developmental stages and under continuous 72-h exposure. Embryos were submerged in embryo media containing 9 mM or 15 mM 2FA during the blastula, gastrula, segmentation, pharyngula, or hatching stages, and hatch rate and morphology were assessed at 72 h post fertilization. Regardless of concentration or exposure window, 2FA exposure did not reduce hatch rate, increase mortality, or elevate abnormal morphology relative to embryo-media controls. Thus, these results demonstrate that 2FA is well tolerated by zebrafish embryos at concentrations up to 15 mM. These results support further investigation into 2FA efficacy during cryopreservation.