Sensitivity to chilling of medaka ( Oryzias latipes) embryos at various developmental stages

As an essential step toward cryopreservation of fish embryos, we examined the chilling sensitivity of medaka ( Oryzias latipes) embryos at various developmental stages. Embryos at the 2–4 cell, 8–16 cell, morula, blastula, and early gastrula stages were suspended in Hanks solution. They were chilled to various temperatures (usually 0 °C), kept for various periods (usually 20 min), then cultured for up to 14 d to determine survival (assessed by the ability to hatch). Embryos at the 2–4 cell stage were the most sensitive to chilling to 0 °C, but sensitivity decreased as development proceeded. The survival rate of 2–4 cell embryos was affected after 2 min of chilling at 0 °C; although the rate decreased gradually as the duration of chilling increased, 38% of them still survived after 40 min of chilling. Embryos at the 2–4 cell stage were sensitive to chilling at 0 or −5 °C, but much less sensitive at 5 or 10 °C. The survival rate of 2–4 cell embryos subjected to repeated rapid cooling and warming was similar to that of those kept chilled. When early gastrula embryos were preserved at 0 or 5 °C, the hatching rate did not decrease after 12 and 24 h of chilling, respectively, but then decreased gradually as storage was prolonged; however, 3–10% of the embryos hatched even after storage for 10 d. In conclusion, although later-stage medaka embryos would be suitable for cryopreservation (from the perspective of chilling sensitivity), chilling injury may not be serious in earlier stage embryos.