2.3. Sample Preparation

Samples of feeds, TMR, and TMR refusals were mixed per animal and period and divided in two portions. The first was placed in duplicate in an oven for 24 h at 103 °C for DM determination and the other portion was placed in an oven for 48 h at 55 °C and then ground with a cutting mill (SM 2000, Retsch GmbH, Haan, Germany) with a 1-mm screen for further analysis. Rumen and omasal samples were thawed at room temperature, homogenized for 1 min at 24,000 rpm (Diax900, Heidolph, Nurnberg, Germany), and subsamples were taken for ammonia-N and volatile fatty acid (VFA) analyses. Homogenized omasal samples were squeezed through 1 layer of cheesecloth, and the retained solids were defined as the omasal LP. The filtrate was centrifuged at 1000× g (5 °C, 5 min), and the supernatant was carefully separated from the pellet. The supernatant was defined as the omasal FP and the pellet was defined as the omasal SP. The separated phases were frozen, freeze dried, and ground through a 1-mm screen for analyses. Fecal pooled samples per cow and period were thawed at room temperature, homogenized for 2 min manually, dried in an oven for 48 h at 55 °C, and ground with a cutting mill (SM 2000, Retsch GmbH, Haan, Germany) with a 1-mm screen for further analyses.