The influence of prolonged pre-treatment vs. Instant administration ofNatuzyme@ (Bioproton Co.) on in vitro gas production (GP. ml/200 mg OM), dry matter disappearance (IVOMO) methane production (MET. ml/200 mg OM) and fermentation efficiency as mg IVOMO per ml MET (FE) of various ruminant diets containing wheat straw (01: 6.2% wheat straw, 39.4% corn silage) or alfalfa hay (02: 21.3% alfalfa. 37.2% corn silage) at half time (tII2) of gas production. Approximately 200 mg (OM) of each diet was weighted into a 125 ml serum bottle at 0,0 or 24 h prior to incubation (TI and T2, respectively). Each bottle received 2.52 glkg OM of the enzyme in an aqueous suspension to maintain same moisture content maintain same moisture content (40%), run=3 and n=3. The gas production procedure was followed by pipetting buffered rumen fluid into the bottles and incubated at 38.6 °C for desired intervals. In a pre-trail, pressure of gas was recorded at 2, 4, 6, 8. 10, 12. 24, 48, 72 and 96 h of incubation. Pressure data was converted to volume using anexperimentalcurveandwasmodeledtoestimatetl/2. Maintrailincubationwascontinueduntiltl/2 and,/ volumes of GP and ME, and residual OM was measured. Oata were analyzed as 2x2 factorial arrangement in a completely randomized design. Results revealed that 02 comparing to 01, had higher GP (43.95 vs. 43.18) and Met (12.10 vs. 8.62) but less IVOMO (30.90 vs. 33.90) and FE (5.35 vs. 8.24) (P<0.05). In addition Tl compared to T2 had higher GP (47.43 vs. 36.70) and less IVOMD (30.90 vs, 33.90) (P<0.05) and Met and FE did not differ for TI and T2. Although T2 in 01 decreased FE (6.44 vs. 10.46) and increased Met (10.38 vs. 6.85) comparing to Tl(P<0.05), situation was different for 02 and FE was higher for T2 in 02 (6.64 vs. 4.10) (P<0.05) while Met was not affected. Therefore it seems optimal Pre-treatment duration for Natuzyme@ widely depends on diet type and forage content.

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