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Fig. 3 Low-temperature treatment (LT) during the early stages of embryonic development reduces off-target mutagenesis without decreasing on-target mutagenesis in medaka. A To investigate the effect of low-temperature conditions on embryo survival, embryos were cultured at 28 °C (126 eggs) and 16 °C (115 eggs). The survival rate at 16 °C dramatically decreased as development progressed. B Temporal changes in on-target and off-target mutation rates were examined. Those of DJ-1 (on-target) and Off-D (off-target) were calculated at each developmental stage (n = 10). While DJ-1 reached nearly 100% mutation rate by Stage 13, Off-D showed no clear upward trend in mutation rate beyond Stage 13. Each data point represents mean ± SE. C, D, E The effect of LT on On-Target and Off-Target mutation rates. (C)DJ-1 and Off-D (n = 8–10) (B) p4hb and Off-P (n = 9) (C) avt and Off-A (n = 8) The 28 °C group was cultured at 28 °C until hatching, while the LT group was cultured at 16 °C until Stage 13, then shifted to 28 °C until hatching. No significant difference in mutation rates was observed in the on-target regions between the LT and 28 °C groups. However, for off-target regions, LT significantly reduced them compared to 28 °C. N.S.: no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Columns and error bars represent mean ± SE