Dry matter content was determined according to the National Forage Testing Association method 2.1.4 [19 ]. Neutral detergent fiber (NDF) was determined as described by Van Soest et al. [20 (link)] using heat-stable α-amylase (FAA, Ankom Technology, Macedon, NY) and sodium sulfite. Acid detergent fiber (ADF) and lignin concentrations were determined according to AOAC method 973.18 [21 ]. Ash content was determined according to AOAC method 942.05 [21 ]. Hemicellulose was calculated as the difference between NDF and ADF. Cellulose was calculated by subtracting ash and lignin from ADF.
For measurement of ruminal VFA concentrations, rumen fluid samples were thawed and composited by day, animal, and period (n = 6). The samples were centrifuged for 30 min at 2500×g at room temperature to remove solid particles, and supernatant liquid was collected. An external tracer consisting of a mix of 13C-labelled acetate, propionate, and butyrate was added to each liquid sample, then rumen liquid samples were derivatized, and the derivatives were analyzed for isotopic ratio using a Thermo Electron Polaris Q mass spectrometer in tandem with a Thermo Electron Focus gas chromatography (GC-MS; Thermo Electron Corporation, Austin, TX) as described by Kristensen [22 (link)].
For measurement of ruminal VFA concentrations, rumen fluid samples were thawed and composited by day, animal, and period (n = 6). The samples were centrifuged for 30 min at 2500×g at room temperature to remove solid particles, and supernatant liquid was collected. An external tracer consisting of a mix of 13C-labelled acetate, propionate, and butyrate was added to each liquid sample, then rumen liquid samples were derivatized, and the derivatives were analyzed for isotopic ratio using a Thermo Electron Polaris Q mass spectrometer in tandem with a Thermo Electron Focus gas chromatography (GC-MS; Thermo Electron Corporation, Austin, TX) as described by Kristensen [22 (link)].