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Fig. 4 The effect of early low-temperature treatment (LT) on on-target and off-target mutation rates in zebrafish. The 28 °C group was cultured at 28 °C until hatching, while the LT group was cultured at 22 °C for 1 day, then shifted to 28 °C until hatching. No significant difference in mutation rate was observed between the 28 °C and LT groups for ywhaqa (n = 7–10). However, for Off-Y1, LT significantly reduced the mutation rate compared to 28 °C (n = 7–10). For Off-Y2, although the difference was not statistically significant, the mutation rate tended to be lower in the LT group(n = 7–10). N.S.: no significant difference, *: p < 0.05. Columns and error bars represent mean ± SE