The experiment had four 22-day periods, each
consisting of 20 days of diet adaptation followed by
2 days of rumen fluid sampling at 0 (before feeding), 2, 4
and 8 hours after feeding to measure the concentration
of volatile fatty acids (acetic, propionic and butyric) and
ammonia. A 5-mL ruminal fluid sample was preserved
with 1-mL 25% w/v meta-phosphoric acid for volatile fatty
acid (volatile fatty acid) analysis in gas chromatography
according to Erwin et al. (1961). To measure the ammonia
concentration, 2-mL of the rumen liquid sample was diluted
in 1-mL H
2SO4 1N and kept in a freezer for further analysis
by colorimetric phenol-hypochlorite reaction methodology.
Immediately after each sampling time, the rumen pH was
measured with a digital pH meter.