16S high-throughput sequencing of hindgut flora in grouper after soybean meal induced enteritis

The red FM used in this study contained 72.53% crude protein and 8.82% total lipid was provided by Bayovar Plant, Peru. SBM used in this study contained 48.92% crude protein was provided by Zhanjiang Haibao Feed Company (Zhanjiang, China). Three iso-nitrogenous (approximately 50% crude protein) and isolipidic (10% total lipid) experimental diets were formulated to replace 0, 20% and 40% of FM protein by SBM protein to prepare the experimental diets named FM (control), SBM20 and SBM40, respectively. When the juvenile grouper adapted to the culture environment, fish of similar size was randomly assigned to a 1000 L cylindrical fiberglass barrel with 60 fish per barrel and 4 replicates in each group. Each group diet fed twice a day (8:00 and 16:00) to apparent satiety level for 10 weeks. The culture conditions meet the normal growth requirements of fish.For 16S high-throughput sequencing, three fish were randomly taken from each barrel, and removed the mesentery and adipose tissue. Then DI tissue was washed with deionized water to remove the residue. There are three replicates in each group. The DI samples were placed in the cryopreservation tube and immediately put into liquid nitrogen for intestinal flora analysis.