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.