| Abstract: | This study used tiger puffer Takifugu rubripes to explore new, simple methods for lowtemperature-induced masculinization in a cultured fish without the use of ultraviolet irradiation or sex hormones. An orthogonal test L9（34） design was used to consider three factors at three levels: treatment starting times（days post-hatch, dph: factor A） of 20, 50 and 80 dph; treatment temperatures（factor B）of 13℃, 15℃ and 17℃; and treatment durations（factor C） of 30, 45 and 60 days. A control group was reared at 21±1℃. The experiments were repeated twice. At 230 dph, the gonads were removed from thirty randomly sampled fish in each group. Histological observations and analysis of single nucleotide polymorphisms（SNP） were used to identified pseudo males, which biological sex was male and genetic sex were female（XX）. Treatment group 4（A2B1C2） resulted in the highest proportion of males（75%）.According to the intuitive analysis of the orthogonal-array experiments, the optimal combination of lowtemperature-induced masculinization of T. rubripes was A2B1C2. The population sex ratio depended on the three factors in the sequence B→A→C. A comparison of the daily increases in length and weight during and after the low-temperature conditions showed that the absolute daily increases in weight and length were significantly less during treatment than after treatment. Daily increases in weight and length did not significantly differ between the treatment groups and the controls（P>0.05）, demonstrating that the growth rate could return to normal after the low-temperature conditions. This study establishes a low-temperatureinduced masculinization technology for T. rubripes and demonstrates that although the growth rate（length and weight） decreased in an array of nine treatment groups during the processing time, it returned to a normal level after processing. The results should serve as a guide for achieving the masculinization of T.rubripes in production. |
