A three-step in vitro procedure for evaluating rumen-protected Lysine products

Abstract Text: The three-step in vitro procedure is used for estimating intestinal digestibility of the RUP fraction of feedstuffs. However, rumen protected amino acid products have been evaluated in various ways for estimates of bioavailability. The objective of this study was to propose a three-step in vitro procedure for rumen-protected Lysine products (RPL), which is composed of buffer solutions with enzymes and can be developed as a standardized method to evaluate RPLs. Three grams of six different RPLs were weighed into nylon bag (5 x 7 cm, pore size 53 ± 10 µm). Bags were incubated using a dissolution apparatus for drug evaluation with rotating paddles at 100 rpm at 39 °C. Three individual vessels were allocated to each RPL as ruminal, abomasal and duodenal phases, respectively. Modified McDougal’s buffer containing lipase (900 mL) was used to simulate ruminal conditions (pH 6.8). A hydrochloride buffer containing pepsin (900mL, pH 2.0) and a phosphate buffer containing pancreatin and gall powder (900 mL, pH ≈ 7.9) were used to simulate abomasal and intestinal conditions, respectively. After a 20-h incubation in ruminal vessels, aliquot samples of solution were taken, and bags containing each RPL were transferred from ruminal to abomasal vessels. These procedures were repeated after a 2-h incubation followed by incubation in duodenal vessels for 8 h; aliquot buffer samples were taken again. Each buffer sample was analyzed for Lys, and amount of lysine escaping dissolution was calculated by subtracting buffer lysine from original feed lysine provided to the assay. Residual Lys under ruminal conditions was compared with extent of in situ ruminal protection. Statistical differences (P < 0.05) were tested by a one-way ANOVA. In vitro ruminal protection measured by this procedure correlated (P < 0.05) with in situ ruminal protection with a correlation coefficient of > 0.9. The RPLs showed various characteristics; high/medium/low ruminal protection and high/low post-ruminal Lys release. However, when pH of the ruminal buffer was reduced, one of the RPLs showed an increase (P < 0.05) in ruminal protection using in vitro procedures (46.2 ± 9.3 % at pH 6.8, 90.0 ± 4.0 % at pH 6.2, n=3). Results from this study indicate that a buffer-based three-step in vitro procedure can be a useful tool to evaluate RPLs, but further research is needed to optimize pH of ruminal conditions.

Keywords: rumen-protected lysine, in vitro procedure, rumen pH