All animals studied in this experiment were approved by the animal protection and utilization committee of Yunnan Agricultural University, China (contract 2007-0081), and there was compliance with the guidelines of the Laboratory Animal Ethics Committee in experimental animal sources and sample collection.
The test site was located in the natural pasture of Tiancheng Lun Zhu Agricultural Products Development Co., Ltd. in the north of Shangri-La County, with an average altitude of 3600 m, and a temperate monsoon climate. Maximum average daily temperature was 13 °C, and minimum was 1 °C, annual precipitation was 600 mm, and relative humidity was 65%. Grass vegetation types were mainly Compositae, Gramineae, Sedge family, Buttercup family, and Polygonaceae. Plant types were Blysmus sinocompressus, Anemone rupestris, Kobresia setchwanensis, Kobresia pygmaea, and Penetilla saundersiana, with a forage yield of 182 g/m2. Breast milk components were as follows: milk fat 6.14% ± 0.19%, milk protein 4.38% ± 0.06%, lactose 5.07% ± 0.05%, total milk solids 17.83% ± 0.15%, non-fat 10.92% ± 0.10%, urea nitrogen 14.24% ± 0.67%, and somatic cells 39.63% ± 2.68% (103).
Experimental animals were provided by the Tiancheng Lunzhu Company, Shangri-La City, China. Three Zhongdian yaks (Bos grunniens) that had been following their mothers to graze naturally in the wild from birth to 6 months of age were selected, and six 2-year-old free-grazing Zhongdian yaks were selected to collect samples at 45, 90, and 180 days after birth. Two hours after morning grazing, a catheter was inserted into the rumen, and a vacuum sampler was used to collect rumen fluid samples. For each animal, 30 mL of rumen fluid was collected, divided into three parts, placed in 10 mL polypropylene tubes, rapidly stored in liquid nitrogen, and brought back to the laboratory for storage in a refrigerator at -80 °C.
DNA extraction and sequencing
Microbial community DNA was extracted using the EZNA Stool DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer's instructions. DNA was quantified with a Qubit Fluorometer using the Qubit dsDNA BR Assay kit (Invitrogen, USA), and the quality was checked by running an aliquot on 1% agarose gel. Variable regions V1–V9 of the bacterial 16S rRNA gene were amplified with degenerate PCR primers, 27F (5′-AGRGTTYGATYMTGGCTCAG-3′) and 1492R (5′-RGYTACCTTGTTACGACTT-3′)(Takahashi et al. 2014). The ITs2 of the internal transcribed spacer (ITS) region was amplified using degenerate PCR primers, ITs3 (5′-GCATCGATGAAGAACGCAGC-3′) and its4 (5′-TCCTCCGCTTATTGATATGC-3′)(Toju et al. 2012). Both forward and reverse primers were tagged with Illumina adapter, pad, and linker sequences. PCR enrichment was performed in a 50 μL reaction containing 30 ng template, fusion PCR primer, and PCR master mix. PCR cycling conditions were as follows: 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 56 °C for 45 s, 72 °C for 45 s, and a final extension for 10 min at 72 °C for 10 min. PCR products were purified using AmpureXP beads and eluted in elution buffer. Libraries were qualified using the Agilent 2100 Bioanalyzer (Agilent, USA). Validated libraries were used for sequencing on the Illumina MiSeq platform (BGI, Shenzhen, China) following standard pipelines of Illumina, and generated 2 × 300 bp paired-end reads. Sample Numbers and links in the NCBI database:SRP260807 :PRJNA630991
Raw data was filtered to eliminate adapter pollution and low-quality readings to obtain clean reads, with paired end reads with overlaps merged into tags. Subsequently, tags were clustered into operational taxonomic units (OTUs) at a 97% sequence similarity. Taxonomic ranks were assigned to OTU representative sequences using Ribosomal Database Project (RDP) Naive Bayesian Classifier v.2.2. Alpha diversity, beta diversity, and screening of different species were then analyzed based on OTU and taxonomic ranks.
Tags were clustered into OTUs using USEARCH (v7.0.1090) software. OTU representative sequences were taxonomically classified using Ribosomal Database Project (RDP) Classifier v.2.2, trained on the Greengene_2013_5_99 database, using 0.5% confidence values as the cutoff. Filtered tags were clustered into operational taxonomic units (OTUs) at 97% similarity, and the OTU number per sample primarily represented sample diversity degree. Based on OTU abundance, the OTU of each group was listed, Venn diagrams were drawn using Venn diagram software R (v3.1.1), and common and specific OTU IDs were summarized. Based on the OTU abundance information, relative abundance of each OTU in each sample was calculated, and principal component analysis (PCA) of OTUs was performed with the relative abundance value. The software used in this step was package ade4 software R (v3.1.1). Good‘s coverage, Alpha diversities including Inverse Simpson and Shannon index, richness (observed number of OTUs) and evenness (Shannon evenness) were calculated using Mothur V.1.31.2. Beta diversity analysis was performed using QIIME (v1.80) software. There were differences in sequencing depth in different samples; thus, normalization was introduced, with sequences extracted randomly according to the minimum sequence number for all samples. Extracted sequences formed a new ‘OTU table biom’ file, and the beta diversity distance was calculated based on the ‘OTU table biom’ file. Kyoto Encyclopedia of Genes and Genomes (KEGG); Version: 2018.01.