Animals and sampling
Rumen fluid samples were collected from four non-lactating Holstein dairy cows (body weight, 550 ± 50 kg) fitted with ruminal fistulas. The dairy cows were owned by the Animal Care and Use Committee for Livestock of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences. Diet of cows were total mixed ration, which consisted of 36% corn silage, 16% corn, 14.6% syrup vinasse, 5.9% soybean meal, 5.7% dried distillers grains with solubles, 5.4% soybean hulls, 5.3% barley, 5.0% oat grass, 4.0% alfalfa, 0.8%, CaHPO4, 0.5% NaHCO3, 0.2% NaCl, 0.2% CaCO3, 0.1%, C5H14ClNO, 0.1% calcium fatty acid, 0.1% double beneficial element, and 0.1% rhodamine. Samples of the rumen contents of each cow were obtained at 0, 2 and 6 h after morning feeding. The rumen fluid samples were stored in liquid nitrogen prior to analysis.
gDNA extraction and cDNA reverse transcription
Total gDNA was extracted using the cetyltrimethylammonium bromide method, as described previously. Total RNA was extracted using TRIzol method. Rumen fluid samples were homogenized for 5 min with steel balls (one with a diameter of 20 mm and 10 with diameters of 5 mm) into fine powder in liquid nitrogen using a CryoMill (Retsch GmbH). Then, 3 g of the rumen fluid samples were incubated in 15 mL of TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) at room temperature for 5 min. Afterward, 4 mL of chloroform were added to the samples by vortexing for 15 s and then each was incubated at room temperature for 3 min. Following centrifugation at 13,000 × g for 15 min at 4ºC, the aqueous phase was transferred to a fresh tube. Following the addition of 10 mL of isopropyl alcohol, the tubes were vortexed for 30 s and then incubated at room temperature for 10 min. Afterward, the samples were centrifuged at 13,000 × g for 10 min at 4ºC and the supernatant was discarded. The RNA pellet was washed once with 75% ethanol and resuspended in 10 mL of 75% ethanol by hand mixing and then centrifuged at 7500 × g for 10 min at 4ºC. Once the supernatant was removed, the RNA pellet was air-dried and dissolved in RNase-free water by passing the solution a few times through a pipette tip, and then incubated for 10 min at 55ºC. Trace gDNA in the RNA samples was removed by incubation with 4 µL of DNase I (Takara Bio, Inc., Kusatsu, Shiga Prefecture, Japan) per 100 µg of total RNA for 30 min at 37 °C. Then, the RNA was further purified using an RNAclean Kit (Tiangen Biotech Co., Ltd., Beijing, China). The 16S rRNA gene was amplified using the primers 27F (5´-GAG TTT GAT CCT GGC TCA G-3´) and 1492R (5´-GGT TAC CTT GTT ACG ACT T-3´). Purified RNA was used to check whether the purified RNA samples contained residual gDNA. The integrity and concentration of the total RNA were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada). RNA samples with a RNA integrity number > 8.0 were deemed suitable for reverse transcription of cDNA, which was synthesized using FastQuant RT Super Mix (Tiangen Biotech Co., Ltd.) and random primers in accordance with established procedures.
PCR amplification and sequencing of ureC genes
UreC genes were amplified with the primer set UreC-F (5´-TGG GCC TTA AAA THC AYG ARG AYT GGG-3´) and UreC-R (5´-GGT GGT GGC ACA CCA TNA NCA TRTC-3´). Reactions were performed in a MyCycler Thermal Cycler (Bio-Rad, USA) using a 50-µL mixture containing 5 µL of 10 × PCR buffer (Invitrogen Corporation), 1 µL of dNTP mixture (10 mM), 1.5 µL of each forward and reverse primer (10 µM), 0.25 µL of Platinum Taq DNA polymerase (Invitrogen Corporation), 2 µL of gDNA or cDNA (100 ng/µL), and 38.75 µL of sterile double-distilled H2O. The PCR amplification consisted of denaturation at 94ºC for 5 min, followed by 30 cycles at 94ºC for 30 s, 50ºC for 30 s, and 72ºC for 30 s, and a final extension at 72ºC for 15 min. PCR amplicons were extracted from agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Scientific Inc., Union City, CA, USA) in accordance with the manufacturer’s instructions and quantified using the Qubit™ Assay kit (Thermo Fisher Scientific). Purified amplicons were pooled in equimolar amounts and paired ends were sequenced (2 × 300 bp) on an Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA) by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai. China).
Sequence analysis used methods described previously . Using QIIME calculated alpha and beta diversity indices and significant fold-changes of OTUs. Heatmaps of the top 50 significantly different OTUs at the genus level were generated with MicrobiomeAnalyst .
The differences between gDNA and cDNA for the top 20 ureC gene OTUs and top 10 ureolytic bacteria genera were analyzed using the paired Mann–Whitney test with GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). Differences in beta diversity between gDNA and cDNA were determined by analysis of similarity. The top 50 significant different ureC gene OTUs was identified using the paired Mann–Whitney test with the MicrobiomeAnalyst web-based tool. A probability (p) value of < 0.05 was considered statistically significant.