Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at −5 °C for 30 min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60 min at 25 °C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16 Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25 °C for 30 min, the maturation rate decreased in solution with 0.51 Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40 Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25 °C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at −5 °C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.