2. Materials and methods
2.1 Experimental design
First, 240 P. vachelli (14 ± 1.25 cm in length, 22 ± 1.78 g in weight) were acclimated for three weeks. They were sequentially feed restricted for two days in an aquarium (volume of 600 L; 26 ± 1°C). All the experimental methods were the same as description in our previous article (Zhang et al.2016b). In order to reduce the sequencing error caused by the sample itself, the muscle tissue was all taken from the same spot of the fish. Each control sample (No. M0a, M0b, M0c, M0d, M0e, M0f, M0g, M0h, M0i, M0j; 6.8 mg/L) was made up of five different individual muscle from a total 50 control fish. Afterwards the dissolved oxygen in water from 6.8 mg/L to 0.7 mg/L was reduced by bubbling pure nitrogen by 30-35 min. After dissolved oxygen was maintained for 4 h at 0.7 mg/L, 50 treated fish (No. M4a, M4b, M4c, M4d, M4e, M4f, M4g, M4h, M4i, M4j; 0.7 mg/L) were rapidly acquired for muscle dissection. Control and treated samplings had 10 biological replicates respectively, which were used to conduct metabolome analysis. Meanwhile, six of the same harvested samples (No. M0a, M0b, M0c, M4a, M4b, M4c) were used to conduct analyses of transcriptome, miRNAome, proteome, and qRT-PCR.