This study was approved by the Institutional Animal Care and Use Committee of Khon Kaen University, Thailand. Record No. IACUC-KKU-94/61 and Reference No. 0201.2.11/73.
Preparation of Flemingia macrophylla Silage
Flemingia macrophylla was planted by stems on the experimental plots of Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand with close supervision of the advisory Professor. All plant parts were kept and stored at TROFREC. Flemingia macrophylla (FM) whole top plant was harvested from the shrub after three months of regrowth. Young stem, leaf and branch were chopped with a machine to about 3 cm in length. Molasses: urea: water at 2:1:10 were mixed well with 100 kg chopped FM and stored in the plastic barrel after pressing well (approximately 60 kg/barrel), and tightly covered for 21 days. Flemingia macrophylla silage (FMS) was sampled from various barrels, oven-dried at 60 °C for 48 hours and then ground, stored in bottles for chemical analysis. Standard methods were employed to analyze for chemical compositions including dry matter (DM), ash, crude protein (CP), neutral-detergent fiber (NDF), acid-detergent fiber (ADF) [34, 35]. While condensed tannins (CT) was by the vanillin-HCl [36], method modified by [37] FMS sample was collected weighing 40 g soaked in 200 ml of cool distilled water for 12 hours. The mixture was then filtered and supernatant divided into 4 aliquots for determination of pH using a portable pH meter (HANNA Instruments HI 8424 microcomputer, Singapore). Apart from that, a FMS sample was washed with deionizing water for analyzed lactic acid and acetic acid analysis [38].
Animals and Design
These experimental beef crossbreds belonged to TROFREC, Khon Kaen University and were provided for use to support Ph.D. students for research. After the termination the experiment, they have been maintained in good health and were used for another experiment by a student. Four, beef cattle about two year old with 172 ± 43 kg liveweight, were randomly assigned to in a 4 × 4 Latin square design. Concentrate was offered at 0.5 kg of body weight (BW)/day and rice straw offered ad libitum with supplementation of FMS at 0, 0.2, 0.4 and 0.6 kg DM/head/day. The trial was conducted for four periods each was consisted of 21 days, during the first 14 days was for matter feed intake measurement, while during the last 7 days was for sample collection using total collection method. All animals were in individual pens, clean water and mineral-salt blocks were available at all times. The diet was offered to the animals twice in the morning (07:00a.m.) and afternoon (04.00p.m.). The liveweight of each cattle was weighed at the beginning and the end of each period to calculate feed intake. Feed provided and refusals were measured daily throughout the experimental period. Feed samples were collected twice a week for DM analysis [34]. Samples of feeds including concentrate, FMS, rice straw, feces were collected randomly daily during the last 7 days of each period. A daily sample of feces of each animal (about 100 g) was collected to be analyses [34]. Urine samples were acidified immediately after collection by diluting one volume of urine with four volumes of 0.1 N H2SO4 and stored at − 20 ºC urine samples were analyzed for total Nitrogen content [34] total purine derivatives (PD) excretion per day was calculated as the sum of allantoin and uric acid the daily absorbed exogenous purines estimated, and microbial N supply (MNS) predicted [39, 40], respectively. In the last day of each period, rumen fluid samples were collected at 0 and 4 hour post-feeding. Details of sampling procedures of rumen fluid for animal of volatile fatty acids (VFA) [38], protozoal population count [41], and estimated methane (CH4) production [42] are presented in details in Wanapat et al. [43]. Blood samples (about 10 ml) were collected from the jugular vein at each rumen sampling time and kept into the tubes to which 0.1 g EDTA was added for analysis of blood urea-nitrogen (BUN) [44].
Data Management and Statistical Analysis
All data were subjected to ANOVA according to a 4 × 4 Latin square design using the General Linear Models (GLM) procedures [45] (24). The results were presented as mean values with the standard error of the means. Difference among means with P < 0.05 was accepted as statistical differences while 0.05 < P < 0.10 was accepted as a tendency. Treatment means were statistically compared by Duncan’s New Multiple Range Test [46] (25).