At the end of trialthe trial, guts of tilapia from the Air treatment group (n = 3) and NB-O2 treatment group (n = 4) were sampled, then sliced open, and rinsed with distilled water to eliminate the contents inside. Total genome DNA samples were extracted using a lysis buffer containing proteinase K as previously described by Senapin et al. (2018). DNA concentration and purity (ranging at 1.90-2.14) was measured by NanoDrop One (Thermo Fisher, UK).
The distinct regions such as 16S rRNA, 18S rRNA, and/or ITS waswere amplified using specific primers, 515F-806R (16S-V4), 528F-706R (18S-V4), and 1380F-1510R(18S-V9). The PCR mixture was performed with Phusion® High-Fidelity PCR Master Mix (Biolabs, UK). The PCR products were detected on 2% agarose gel with fragment sizes from 400 to 450 bp, followed by purifying using an extraction kit (Qiagen, Germany). The NEBNext® UltraTM DNA Library Prep Kit for Illumina was used to generate the library. Illumina platform was used to analyze Q-PCR.
Sequences analysisSequence analyses were performed using Uparse software vs7.7.1001 with all the effective tags. OTUs abundance information was normalized according to the protocol described by…ref. Based onOn the basis of the output of normalized results, subsequent analysis of both alpha and beta diversity was conducted (Ref).
The abundance plots were generated at the level of the genus, family and order using MOTHUR outputs. Statistical Analysis of Metagenomic Profiles (STAMP) was performed to verify the abundance significance of taxontaxons between groupgroups. The significance between two groups was analyzed using the two-sided Welch’s t-test (Ref).