C-2026: Observations of damage to stage V zebrafish oocytes in contact with extracellular ice

Abstract

The successful banking of zebrafish germplasm by embryo or oocyte cryopreservation remains elusive, due to the rapid onset of cryoinjury following extracellular ice formation. The present study used high-speed imaging to investigate the causes of damage to Stage V zebrafish oocytes following contact with extracellular ice and subsequent cooling. During cryomicroscopy experiments in the absence of cryoprotectant additives, extracellular ice formation was initiated approximately 8 mm away from the oocyte; the resulting crystals were then allowed to envelop the oocyte during an isothermal hold at −0.9 °C. When oocytes were exposed to the external ice for up to 20 min at this temperature, only 13% exhibited any evidence of injury (rupture or darkening). In contrast, our previous studies have shown that when zebrafish embryos are maintained in contact with external ice for 20 min at −0.4 °C, mechanical damage occurred in as many as 47% of embryos. Controlled-cooling experiments with oocytes were performed at rates of 0.5, 1, and 2 °C/min, all of which were preceded by a 5-min isothermal hold at −0.9 °C, to allow equilibration with the extracellular ice. In all 68 oocytes observed, cryoinjury manifested as a sudden increase in oocyte opacity (darkening) during the cooling ramp. The average darkening temperature decreased slightly with increasing cooling rate, but this effect was not statistically significant (ANOVA, p > 0.25). However, we found a statistically significant effect of oocyte age (time since collection) on the darkening temperature (ANOVA, p p