Leucaena was harvested and chopped into 2 to 3 cm lengths, then ensiled with urea and molasses at 1.0% and 2.0% DM, respectively, packed in plastic boxes and pressed well to remove air. The duration of ensilage time was at least two weeks before offering to experimental dairy steers.

Four, rumen fistulated dairy steers with initial weight of 167± 12 kg were randomly assigned to receive four diets according to a 4×4 Latin square design. All animals were kept in individual pens; water and mineral block were provided at all times. Animals were supplemented with concentrate at 0.2% of body weight/d. The treatments were as follows: T1 = untreated RS (Control), T2 = 70% RS+30% LS, T3 = 40% RS+60% LS, and T4 = 100% LS. Animals were fed twice per day in the morning (07:00) and afternoon (16:00). The experiment was carried out for four periods. Each period lasted for 21 days; during the first 14 days feed intake was measured, while the last 7 days dairy steers were moved to the metabolism crates for total collection of feces and urine. Feed ingredients and composition of concentrate, RS and LS are presented in Table 1.

Feed offered and refusals were recorded daily throughout the experimental period. Feed samples were randomly collected twice a week for DM analysis using hot air oven at 100°C for 24 hours [5]. Samples of feed and feces were collected daily during the last 7 days of each period by total collection method. The feces were sampled about 50 g/kg of total fresh weight and divided into two parts; the first part was for analysis of DM [5] daily during the collection days and the second part was kept in refrigerator and pooled by each steer at the end of each period. Samples of dietary feed were composited by period. All samples were stored in the freezer at −20°C for chemical analysis in the laboratory. Feeds, feed refusals and fecal samples were dried at 60°C for 48 hours and ground (1 mm screen using the Cyclotech Mill, Tecator, Höganäs, Sweden) for chemical analysis using standard methods of AOAC [5] for DM, ash, and CP. Acid detergent fiber was determined according to an AOAC method [5] and was expressed by inclusive of residual ash. Neutral detergent fiber was estimated according to Goering and Van Soest [6] with addition of α-amylase without sodium sulphite and the result was expressed with residual ash. Sample LS was analyzed for CT using the Vanillin-HCL method [7,8].

Total urine was collected on the same days as feces by using plastic container fixed with sulfuric acid (10%) to protect nitrogen (N) loss. The urinary samples were collected about 100 mL/L of the total volume urine, kept in refrigerator, and then pooled at the end of each period for total N analysis according to [5] for determining N utilization. The allantoin in the urine was determined by High Pressure Liquid Chromatography according to Chen et al [9] (HPLC, Instrument by controller water model 1525; water model 2707 auto sampler; water model 2489 UV detector and BREEZE software, Water Corp., Milford, Massachusetts, USA; A 3.9 mm×300 mm stainless-steel column, packed with ReproGel H and a pre-column, packed with the same material were used). The mobile phase consisted of 10 mM H₂PO₄ (pH 2.5) and the flow rate was 0.8 mL/min. The UV detector (at 210 nm) was used for quantification. The UV- Visible spectra were recorded at the peak maxima and were corrected for the solvent background. The results were determined, using the standard volatile acids (Merck, India) as control. The amount of microbial purines derivative (PD) absorption (X mmol/d) corresponding to the PD excretion (Y mmol/d) was calculated basing on the relationship derived by Chen and Gomes [10]: Y = 0.85X+(0.385W^0.75). The supply of microbial N (MN) was estimated by urinary excretion of PD according to Chen and Gomes [10]: MN (g/d) = 70X/(0.116×0.83×1,000) = 0.727X; where X is PD absorption in mmol/d, digestibility of microbial purine is 0.83, the N content of purines is 70 mg N/mmol, and the ratio of purine-N: total N in mixed rumen microbes is 11.6:100. The efficiency of microbial N synthesis (EMNS) to denote the MN supplied to the animal per unit of digestible organic matter apparently fermented in the rumen (DOMR) was calculated using the following formula: EMNS = MN (g/d)/DOMR (assuming that rumen digestion was 650 g/kg organic matter (OM) of digestion in total tract, DOMR = DOMI×0.65; DOMI = digestible organic matter intake).

Rumen fluid samples were collected at 0, 2, 4, 6 h post-feeding. Approximately 200 mL of rumen fluid was taken from the middle part of the rumen each time at the end of each period. Rumen fluid samples were filtered through four layers of cheesecloth. The samples were divided into two portions. The first portion of 1 mL rumen fluid was collected and kept in a plastic bottle to which 9 mL of 10 mL/L formalin solution (1:9 v/v, rumen fluid:10 mL/L formalin) was added and stored at 4°C for determining bacteria, protozoal and fungi population by using total direction counts according to Galyean [11] by haemacytometer (Boeco, Hamburg, Germany). The second portion was quantified ruminal microorganism (cellulolytic, proteolytic, amylolytic, and total viable count bacteria) using the viable count of bacteria, based on the use of a roll-tube technique [12].