DOI: 10.5061/dryad.612jm64h0
title: Data from: Ruminal planktonic, weakly, and tightly feed-adhered bacterial community as affected by two <em>Trichoderma reesei</em> enzyme preparations fed to lactating cattle

description: 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&lt;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.
description: 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.
description: # Data from: Ruminal planktonic, weakly, and tightly feed-adhered
 bacterial community as affected by two *Trichoderma reesei* enzyme
 preparations fed to lactating cattle Dataset DOI:
 [10.5061/dryad.612jm64h0](10.5061/dryad.612jm64h0) ## Description of the
 data and file structure #### Dataset overview The data set published
 herein contains the forward and reverse fastq files of each experimental
 unit. Filenames end in the unique identifier of each experimental unit,
 including Period-TRT-FRC (e.g., P1.C.AS = Period1, Control, Weakly
 feed-adhered). Additionally, the csv file includes the measurements of
 rumen fermentation and digestion kinetics of each experimental unit, as
 measured by Romero et al. (2016, as well as fastq barcodes. ### Files and
 variables #### File: Rumen_comp_meta3_up.csv ##### Variables * SampleID:
 Unique identifier of each experimental unit, including Period-TRT-FRC
 (e.g., P1.C.AS = Period1, Control, Weakly feed-adhered). *
 BarcodeSequence: Barcode sequence used to identify the sample sequence. *
 LinkerPrimer: Linker primer sequences used in library preparation. *
 BarcodeName: Name of barcode sequence. * Period: Latin Square Period *
 Cow: ID of cow subject. * TRT: Treatment. CON = Control treatment with no
 enzymes; MIX = cellulase/xylanase mix; XYL = high xylanase. * Fraction:
 Rumen bacteria fraction. LIQ = planktonic or free-floating bacteria; AS =
 weakly feed-adhered bacteria; SOL = tightly feed-adhered bacteria. *
 Inter: TRT x Fraction interaction abbreviation * pH: pH of rumen fluid. *
 Ammonia: Concentration of ammonia-N in rumen fluid (mg/dL) * Washout:
 Percentage of dry matter washed out during in situ incubations (Romero et
 al. 2016). * PD: Percentage of potentially digestible dry matter from in
 situ incubations (Romero et al. 2016). * Rate: Rate of digestion of the
 potentially digestible dry matter (%/h), from in situ incubations (Romero
 et al. 2016). * Lag: Lag time to fermentation start (h), from in situ
 incubations (Romero et al. 2016). * Indigestible: Percentage of
 indigestible dry matter, from in situ incubations (Romero et al. 2016). *
 Acetic: Concentration of acetic acid in rumen fluid (mM). *
 Propionic: Concentration of propionic acid in rumen fluid (mM). *
 Isobutyric: Concentration of isobutyric acid in rumen fluid (mM). *
 Butyric: Concentration of butyric acid in rumen fluid (mM). *
 Isovaleric: Concentration of isovaleric acid in rumen fluid (mM). *
 Valeric: Concentration of valeric acid in rumen fluid (mM). * Totalvfa:
 Total volatile fatty acids (acetic, propionic, isobutyric, butyric,
 isovaleric, and valeric). All measurements from rumen fluid and rumen in
 situ incubations are described by Romero et al., 2016. Reference: Romero,
 J.J.; Macias, E.G.; Ma, Z.X.; Martins, R.M.; Staples, C.R.; Beauchemin,
 K.A.; Adesogan, A.T. Improving the performance of dairy cattle with a
 xylanase-rich exogenous enzyme preparation. *J. Dairy Sci. ***2016**,
 *99*, 3486–3496. https://doi.org/10.3168/jds.2015-10082. ### File:
 Forward.zip Description: Contains forward fastq files of each experimental
 unit. Filenames end in the unique identifier of each experimental unit,
 including Period-TRT-FRC (e.g., P1.C.AS = Period1, Control, Weakly
 feed-adhered). ### File: Reverse.zip Description: Contains reverse fastq
 files of each experimental unit. Filenames end in the unique identifier of
 each experimental unit, including Period-TRT-FRC (e.g., P1.C.AS = Period1,
 Control, Weakly feed-adhered).