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R E S E A R C H A R T I C L E Zapata-Campos, et al. 724 Emir. J. Food Agric ● Vol 32 ● Issue 10 ● 2020 of these arboreal legumes present in many countries for silvopastoral use with livestock. It was considered important to deepen the chemical composition and nutritional value of three leguminous trees heavily selected by livestock. This experiment was thus conducted with three objectives: (a) to assess the nutrient composition and secondary compounds of foliage and pods from three leguminous trees heavily used by livestock, (b) to investigate the in vitro digestibility characteristics of these forage plants, and (c) to characterize the mineral content of three leguminous trees highly consumed by livestock. MATERIAL AND METHODS Study area sampling of leaves and pods This study was conducted in a subtropical zone of northeastern Mexico (23° 44’ 06 “ N; 99° 07’ 51” W) at an average altitude of 321 meters. The average annual temperature is 23.5° C and the average annual rainfall is 780 mm with a summer rainfall season. The vegetation corresponds to the Tamaulipan thorn scrub (xerophilous shrubland). The foliage and pods sampling was carried out from April to June 2017 (rainy season). The species sampled were leucaena, mesquite, and huisache (Fig. 1). The leaves and mature pods of 10 full-grown trees of each of these species were collected. Nutritional analysis Leaves and pods samples were dried in a forced-air oven at 50-60° C for 48 h, to determine DM. Subsequently, they were ground to pass a 1-mm screen using a Wiley mill (Model 4; Arthur H. Thomas Co. Philadelphia, Pa., USA). Ash content was determined in duplicate by incineration in a muffle at 600° C for 2 h; ether extract (EE) and PC, by the macro-Kjeldahl procedure (AOAC, 2000). Neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, hemicellulose, and cellulose was performed by the procedure described by Van Soest et al. (1991). The in vitro disappearance of the DM (IVDMD) was determined by the Tilley and Terry method modified by Barnes (1970). Concentration of calcium (Ca), magnesium (Mg), potassium (K), sodium (Na), copper (Cu), zinc (Zn), manganese (Mn), and iron (Fe), was determined by atomic absorption spectrophotometry. Phosphorus (P) was measured by colorimetry using a spectrophotometer (model UV-2101 PC, Shimadzu Scientific Instruments, Columbia, MD) at 650 nm (AOAC, 2000). Metabolites extraction Aqueous extraction of three sub-samples (10 g c/u) from ground leaves and pods was carried out. For this procedure, the sample was weighed in 125 mL Erlenmeyer flasks, 100 mL of distilled water was poured at 60° C and was then homogenized. Afterward, it was placed inside a stove at 60° C, stirring it every 15 min for 60 min. It was then filtered through a Whatman No. 41 membrane. The material was centrifuged at 3000 rpm for 10 min. Finally, the aqueous extract was deposited in amber bottles and stored at 4° C until further processing. Fig 1. Foliage and pods of leguminous trees collected in northeastern Mexico and used in the study, (a) Leucaena leucocephala (Lam.) de Wit, (b) Acacia farnesiana (L.) Willd, (c) Prosopis laevigata (Humb. et Bonpl. ex Willd). Zapata-Campos, et al. Emir. J. Food Agric ● Vol 32 ● Issue 10 ● 2020 725 Methanolic, acetonic, and ethanolic extraction were also carried out. One g of each sample was weighed and placed in a test tube to which 10 mL of each of these solutions (70:30 v/v) were added. They were stirred in a vortex to homogenize them and were allowed to rest for 24 h, avoiding exposure to light and were refrigerated at 4° C. Subsequently, they were centrifuged at 3000 rpm for 20 min and the supernatants were obtained for further analysis. Determination of condensed and hydrolyzable tannins The HCL-Butanol technique (Swain and Hillis, 1959) was used to obtain condensed tannins (CT). The reading of tubes with the extracts was carried out with a spectrophotometer with absorbance at 460 nm. The concentration was calculated using the catechin as standard and the results were expressed as mg/g in catechin equivalents (mg/CE/g DM). Hydrolyzable tannins (HT) were determined using the Folin Ciocalteu technique (Taga et al., 1984). The concentration was calculated using the gallic acid standard and the results were expressed as mg of gallic acid equivalent per g of DM of the plant extract (mg/GAE/g DM). Partial purification of metabolites An aqueous extraction was carried out as previously described for leaves and pods of the trees for column chromatography (Still et al., 1978). The components of plants were detected with the ProStar Varian HPLC system (Spectra Lab Scientific Inc., Markham, Ontario Canada), with a three-phase pump, a model 410 autosampler, and a diode array UV-vis detector. The column used for the analysis was a Varian Pursuit XRs c18, 4.6 mm x 250 mm, with a flow of 1 mL/min and a volume injection of 10 μL per sample. Details of this procedure were described by Ascacio-Valdés et al. (2013). Statistical analyses The effects of tree leaves, pods, and the leaves by pods interaction on nutrient content of forage, IVDMD, and condensed and hydrolyzable tannins were analyzed as a completely randomized design using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA). Individual tree samples were considered the experimental unit. The effects of tree species and the leave × pod interaction were considered to be fixed effects and individual foliage and pod samples were considered to be a random effect. Significant differences detected by ANOVA were further investigated using the PDIFF option of SAS comparing tree ́s leaves and pods.