The effects of melamine on gas production (GP) kinetics, methane (CH 4 ) production and fermentation of diets differing in forage content (low-forage (LF) diet: 200 g/kg and high-forage (HF) diet: 800 g/kg) by rumen micro-organisms in vitro were studied using batch cultures. Rumen contents were collected from three Simmental × Luxi crossbred beef cattle. Melamine was added to the incubation bottles to achieve final concentration of 0 (control), 2, 6, 18, 54, 162 and 484 mg/kg of each diet. Cumulative GP was continuously measured in an automated gas recording instrument during 72 h of incubation, while fermentation gas endproducts were collected to determine molar proportions of carbon dioxide (CO 2 ), CH 4 and hydrogen gas (H 2 ) in manually operated batch cultures. Differences in GP kinetics and fermentation gases were observed in response to the nature of the diets incubated. Although melamine addition did not affect GP kinetics and fermentation gas pattern compared to the control, the increase of melamine addition stimulated the yield of CH 4 by decreasing CO 2 , especially during the fermentation of the HF diet. The concentrations of ammonia nitrogen (N), amino acid N and microbial N in culture fluids were greater in the fermentation of LF-than HF diets, and these concentrations were increased by the increase of melamine addition after 72-h fermentation. The concentrations of total volatile fatty acids (VFA) were greater in HF than LF diets. The addition of melamine decreased total VFA concentrations and this response was greater in HF than LF diet fermentations. Melamine addition did not affect molar proportions of acetate, butyrate, propionate and valerate compared with the control; however, branched-chain VFA production, which was lower in the HF than the LF diet, was increased by the melamine addition, especially in the HF diet fermentation. The ratio of non-glucogenic to glucogenic acids was lower in the HF than the LF diet, but it was not affected by melamine addition. In brief, the greater reduction in the rate and extent of rumen fermentation found for the HF diet in comparison with the LF diet suggested that rumen fermentation rate and extent in vitro depended mainly on the nature of the incubated substrate, and that they could be further inhibited by the increase of melamine addition.

Summary Rumen fungus Neocallimastix sp. YAK11 was isolated from yak (Bos grunniens), and three consecutive 10‐day pure cultures were anaerobically performed at 39 °C in 20‐ml Hungate’s tubes to explore ferulic acid esterase (FAE) and acetyl esterase (AE) activity profiles of the fungus grown on whole hay fraction of Chinese wildrye grass (Leymus chinensis) (WHOcw, n = 4) and its neutral detergent fibre fraction (NDFcw, n = 4), respectively. An aliquot of 0.7‐ml culture was sampled daily using a sterile syringe, and 0.7‐ml fresh medium was immediately added to the tubes to compensate for the withdrawn samples. Peak esterase activity occurred for FAE on day 5 (p < 0.001) and for AE on day 6 (p < 0.001). The mean activities of FAE and AE in WHOcw were 2.07 and 1.29 times of those in NDFcw (p < 0.001). Both FAE and AE activities were positively correlated with xylanase (r > 0.65, p < 0.001) and carboxymethyl cellulase (r > 0.57, p < 0.001) activities. Total volatile fatty acid concentration was positively correlated with enzyme activities of AE (r > 0.87, p < 0.001), FAE (r > 0.82, p < 0.001) and xylanase (r > 0.56, p < 0.001). Crude enzyme solution was harvested for the fungus grown on WHOcw, and the pH optimum of FAE activity was 8.0 while the optimum for AE was 9.0. Both FAE and AE had a broad pH stability range. The optimal temperatures for FAE and AE activity were 40 and 50 °C. The Michaelis constant (Km) and maximum velocity (Vmax) for FAE against methyl ferulate at pH 6.0 and 39 °C were 0.078 mm and 2.93 mU, respectively. The Km and Vmax for AE against p‐nitrophenyl acetate at pH 7.0 and 39 °C were 2.73 mm and 666.67 mU, respectively. Both FAE and AE may have prospective advantages for the enzymatic degradation of roughages in ruminant animals.