design: Total DNA was extracted from rumen and cecum content samples using a mechanical disruption and phenol-chloroform extraction protocol based on Stevenson and Weimer (2007). DNA quality and concentration were assessed using a Qubit Fluorometer, and all samples were normalized to a concentration of 10 ng/L.The V4 hypervariable region of the bacterial 16S rRNA gene was amplified via PCR. Each 25 L reaction contained 50 ng of template DNA, 0.4 M of each universal primer, and 12.5 L of 2X Hot Start Ready Mix (KAPA Biosystems). The PCR cycling conditions were: an initial denaturation at 95C for 3 minutes, followed by 25 cycles of 95C for 30 seconds, 55C for 30 seconds, and 72C for 30 seconds, with a final extension at 72C for 5 minutes.Amplification success was confirmed by visualizing a bright band at approximately 380 bp on a low-melt agarose gel. These target bands were excised from the gel for subsequent purification and library preparation. The final libraries were sequenced on an Illumina MiSeq instrument using v4 chemistry, with a 5% PhiX spike-in as an internal control.