Administration of Exogenous E2

Fish in three parallel treatment and control groups were kept in 1-ton tanks with dark blue and smooth material walls, at a density of 150 fish per tank/group. The treatment groups were directly exposed to E2 for 2 h each day. E2 (17β-estradiol, CAS: 50-28-2, ≥98%) purchased from Sigma (#E8875, Sigma-Aldrich, Shanghai, China) was dissolved in 95% ethanol as a 4 mg/ml stock solution, which was stored at 4°C. The final concentration of E2 in the treatment water was 10 μg/L. The treatment was applied from 80 to 120 dph, and the fish gonads were randomly sampled (N = 30) at 120 dph for statistical and histological analyses.