Accurate determination of dry matter digestibility (DMD) and total faecal output (FO) is necessary for estimating ruminant dry matter intake (DMI) under grazing conditions. This study evaluated the efficacy of inductively coupled plasma optical emission spectrometry (ICP-OES) for quantifying external markers [chromium oxide (Cr₂O₃) and titanium dioxide (TiO₂)] and near-infrared reflectance spectroscopy (NIRS) for quantifying external markers and indigestible NDF (iNDF) in samples from grazing ruminant systems under diverse grazing conditions in Colombia. Cr₂O₃ and TiO₂ were processed via wet acid digestion and analysed using ICP-OES, while iNDF was determined through in vitro fermentation (288 h). NIRS equations were developed using modified partial least squares method with cross-validation to prevent model overfitting. ICP-OES methodology yielded detection limits for Ti (2.97–5.95 µg·g⁻¹) and Cr (23.08–46.16 µg·g⁻¹). Ti concentration was analysed in 285 forages samples and Cr concentration in 213 samples, with mean values of 34.52 and 7.06 µg·g⁻¹, respectively. For iNDF, 1285 total samples were processed, including 421 forages, 3 commercial feed supplements, 4 partial mixed rations and 855 faecal samples, with mean values of 18.2%, 4.37%, 2.07% and 23.66% iNDF, respectively. NIRS validation for forages showed satisfactory predictive capacity for iNDF and Ti (r² = 0.88 and 0.89, respectively) and RPD (2.85 and 2.89, respectively) compared to Cr (r² = 0.42 and RPD = 1.23). In faecal samples without marker added, Ti showed a satisfactory fit (r² = 0.86 and RPD = 2.65), while for iNDF demonstrated lower accuracy (r² = 0.64 and RPD = 1.67). The ICP-OES and NIRS techniques proved efficient methods for Ti, Cr and iNDF marker determination, offering potential for accurate estimation of ruminant DMI under grazing conditions.

Plants used as forage in livestock production are a source of secondary metabolites (SM) that are involved in important interaction in life, health, nutrition and animal performance. This study aimed to adapt and validate six spectrophotometric micro-methods for quantifying SM such as total phenols (TP), total tannins (TT), condensed tannins (CT), total saponins (TS), total alkaloids (TA), and total sterols (TE), in more than 700 Colombian tropical forage samples. The sample and reagent quantities used in the assays were reduced, preparation conditions were modified in some steps, and the colorimetric reactions were performed in 96-well microplates. The micro methods were validated for linearity, limits of detection and quantification, accuracy, precision, and percent recovery in materials such as grasses, legumes, and tree forages from six Colombian ecoregions. The coefficients of variation (CV), repeatability and reproducibility for all methods were less than 0.4 and 5.6%, respectively. The recoveries ranged from 84.8 to 88.4% for TP, 65.7 to 92.3 for TT, 59.8 to 80.2 for CT, 49.9 to 69.5 for TA, and 61.9 to 78.8% for TS. Micro assays promote minimal waste generation in the environment and allow the processing of a larger number of samples, thereby reducing reagent consumption. This is particularly useful in the analysis of forage materials when making decisions about the effects and functionality of these components in animal diets and their performance.