75 Development of a rapid screening assay for monitoring the relative nutritional value of pasture samples

Abstract

Pasture with a high biodiversity has the potential to provide a balanced diet and year-round forage availability for grazing equids. Monitoring the influence of biotic and abiotic factors on the relative proportions of key nutrients in pasture could help to inform grazing strategies. Traditional wet chemistry forage analytical methods are labor-intensive and require large sample amounts, thereby limiting their usefulness for high sample throughput. To develop a high throughput screening assay, 3 different hydrolysis methods were compared, each allowing the simultaneous extraction of total hydrolyzable carbohydrates (THC), protein, and phenolics for subsequent spectrophotometric quantification using established microassays. Three pasture samples were collected from mixed species grassland plots (3m x 2m, SRUC Aberdeen, UK). These samples represented a diverse range of THC, protein, and phenolic contents and included high- and low-sugar perennial ryegrass and forage herbs plots (collected in June 2021) and a herbal ley plot (collected in August 2021). Freeze-dried and milled samples were extracted in triplicate and subjected to a range of hydrolysis conditions (Method 1: 2.4% H2SO4, 2 h at 100°C; Method 2: 3M HCl, 14 h at 100°C; Method 3: 2M HCl, 0.5 h at 95°C) before spectrophotometric quantification (Lever assay for reducing sugars,Ninhydrin assay for proteins, Fast Blue assay for phenolics). Solvent-matched calibration curves of Bovine serum albumin (BSA), gallic acid, and glucose/fructose standards were used to quantify analytes and evaluate optimal hydrolysis conditions for each analyte to maximize yield. Extractions were repeated across 3 consecutive days to assess reproducibility of each hydrolysis method. All 3 hydrolysis methods demonstrated a reproducibility and reliability > 99% for THC and phenolics across all samples. ANOVA (using SPSS: significance level set at P < 0.05) showed significant differences between the means of phenolics and THC concentrations for method 2 vs. method 1 (P = < 0.001) and method 2 vs. method 3 (P = 0.015). Method 3 was identified as most optimal for the quantification of THC and phenolics, resulting in analyte concentrations reflective of the expected seasonal and grass species differences in the samples. However, the short hydrolysis time of this method was not sufficient for optimal protein quantification as achieved by Method 2. Therefore, a combination of Method 2 and 3 was subsequently established and is undergoing further optimization to evaluate performance in a greater range of pasture samples.