Rumen Sample Collection and Measurements

At the end of the trial (day 135), rumen fluid samples were collected before the first morning feeding using an oral stomach tube. Rumen samples were strained through four layers of cheesecloth to obtain rumen liquids. Disposable sterile gloves were worn throughout the sample collection process to avoid contamination. Twenty-four samples were collected and placed in 2-ml frozen tubes to avoid cross-contamination. The samples were immediately frozen in liquid nitrogen and stored at −80°C.