Rumen contents were sampled at various times to determine pH and volatile fatty acid concentrations.
Rumen contents were sampled at –2, 0, 2, 4, 6, 8, 10, 12, and 20 h after feeding for rumen fermentation measures.
In order to quantify short-chain fatty acids (SCFA), methane (CH4), NH3-N (ammonia nitrogen) production, protozoa and the ruminal dynamics, rumen contents were sampled prior to and 3, 6, 9 and 12 h after morning feeding.
Rumen contents were sampled on d 18 to 19 at 0, 1, 2, 4, 8, 12, 16, 20, and 22 h after feeding to measure pH and volatile fatty acid (VFA) concentrations.
Rumen contents were sampled on d 19 at 0, 1.5, 3, 4.5, 6, 9, 13, 17, 21, and 23 h after feeding to measure pH, volatile fatty acid (VFA), ammonia-N, and free AA concentrations.
On 15, 16, and 17 d of the experimental period, ruminal contents were sampled 0, 2, 4, 6, and 8 h after dosing, and blood samples were collected at the end of the experiment (18th and 19th d).
The rumen content was sampled on day 27 and 29 of the experiment and then subjected to volatile fatty acids (VFA) and long chain fatty acids analyses.
Enteric CH4 production was determined using respiration chambers and total rumen content was sampled for the determination of fermentation characteristics and molecular biology analyses (cDNA-based length heterogeneity PCR, quantitative PCR).
Approximately 1 liter of mixed liquid and solid ruminal content was sampled from each cow pooled together to give a composite sample from all cows.
Only feces estimated to be <1-year-old based on moisture content and color were sampled.
