This experiment was conducted using lamb rumen fluid adapted to OPF for 30 d. Rumen contents were collected at
slaughter from Barbados Black Belly
×
Malin crossbred lambs which had been fed 150 g OPF/kg diet DM. The other part of the
diet consisted of a commercial pellet of Leucaena leaf meal, rice bran, wheat pollard, oat pollard, urea, dicalcium phosphate;
ingredients in decreasing order of importance. All treatments were incubated in duplicate for each of two consecutive runs.
For each run, two fasted (12 h) lambs were slaughtered and collected rumen fluid was filtered through four layers of
cheesecloth. Fungal colonized OPF (0.560 g), 32 ml of a 50:50 anaerobic and CO2 saturated mixture (flushed for 2.5 h with
CO2) of phosphate (per litre distilled water: 28.8 g Na2HPO4·12H2O; 6.1 g NaH2PO4·H2O; 1.4 g NH4Cl; pH 6.8) and bicarbonate
buffer (per litre of distilled water: 39.2 g NaHCO3) and 8 ml of strained rumen fluid were added to each of the 100 ml gas
tight syringes. Air was expelled and the tip of the syringe was tightly closed prior to incubation at 39 ◦C for 96 h.
Total net volatile fatty acid (VFA) production was analyzed by gas chromatography on a Shimadzu GC-14A (Shimadzu
Corporation, Hertogenbosch, The Netherlands) according to Van Ranst et al. (2010).