The use of exogenous fibrolytic enzymes (EFE) to improve feed efficiency and milk production in cattle has been previously studied with positive results. However, the effect of these enzymes on the ruminal microbiome is not well understood. This study evaluates the effects of two T. reesei EFE preparations on the taxonomic profile, diversity, relative abundance, and shifts of planktonic or free-floating (LIQ), weakly (AS), and tightly feed-adhered (SOL) ruminal bacteria of lactating cows. Three lactating cows were fed three treatments (TRT): one control (CON) without enzymes and two enzyme preparations [cellulase/xylanase mix (MIX) and high-xylanase (XYL)] sprayed daily on the total mixed ration. The experiment was designed as a 3 × 3 Latin square with 23-day periods. Rumen contents were collected on day 23, and bacteria from three ruminal content fractions (FRC) were evaluated: LIQ, AS, and SOL. The fixed effects of TRT, FRC, and their interaction were evaluated as a 3 × 3 factorial design. Sequencing analysis was performed using the ‘dada2’ package in R to infer amplicon sequence variants (ASV). Alpha diversity (Observed ASV and Chao1) was higher in XYL compared to CON. However, no TRT effect was observed on beta diversity. The relative abundance (RA) of the family Prevotellaceae increased, while that of Ruminococcaceae and Rikenellaceae decreased in XYL compared to MIX and CON. Bacterial community structure (beta diversity) was affected by FRC (P = 0.03), indicating a difference between LIQ communities compared to SOL and AS, but no effects of FRC were observed on alpha diversity. Lachnospiraceae RA was greater in SOL, followed by AS, and lower in LIQ (P<0.001), while Spirochaetaceae RA was greater in SOL and AS compared to LIQ (P = 0.003. Prevotellaceae was positively correlated with alpha diversity measures and negatively correlated with Oscillospiraceae and Rikenellaceae. Although differences in bacterial RA were observed with EFE supplementation, no significant correlations were observed between the top 15 bacterial families and previously published measurements of in situ rate of digestion, ruminal pH, total VFA, acetic acid, and propionic acid concentrations. The effects of EFE supplementation on rumen bacterial RA were independent of the ruminal content fraction.

Rumen content samples were collected from 3 lactating Holstein cows fed 3 treatments in a 3 x 3 Latin Square design: one control (CON) without enzymes, a cellulase/xylanase mix (MIX), and high-xylanase (XYL). Then, bacteria were extracted from the 3 rumen content fractions: free floating (LIQ), weakly adherent (AS), and tightly feed-adhered (SOL), resulting in 27 experimental units.

Extraction of DNA from the bacterial pellets was achieved using the PowerLyzer-PowerSoil DNA isolation kit (MO BIO Labs Inc.; Carlsbad, CA) following the manufacturer-recommended procedure. Extracted DNA was analyzed using the Illumina (San Diego, CA) MiSeq platform for 2×300 base pair-end reads (600 sequencing cycles). Amplification of the V1–V3 regions of the bacterial 16S rRNA gene was achieved using the barcoded primer pair forward F27 and reverse R519 (Stackebrandt and Goodfellow, 1991). The PCR reactions were performed in 30 cycles using the HotStarTaq Plus Master Mix Kit (Qiagen; Germantown, MD) under the following conditions: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, after which a final elongation step at 72°C for 5 minutes was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples were pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated AMPure XP beads. Then, the pooled and purified PCR product was used to prepare the DNA library by following the Illumina TruSeq DNA library preparation protocol (Illumina; San Diego, CA). Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer’s guidelines. This dataset contains the raw Illumina fastq files of each experimental unit in this study.