In Vitro Procedures for Estimating Rates of Ruminal Protein Degradation and Proportions of Protein Escaping the Rumen Undegraded

Abstract

An in vitro procedure, in which rates of ruminal protein degradation and proportions of protein escaping ruminal degradation were estimated from release of amino acid plus ammonia in the presence of hydrazine sulfate (HS), was developed and applied to casein. An equation was derived to estimate proportion of protein escaping ruminal degradation: estimated % escape = [k_r/(k_r + k_d)] × 100, where k_r and k_d are fractional rate constants for ruminal protein turnover (washout) and degradation, respectively. HS, when added at 1.0 mM to an incubation medium consisting of strained ruminal liquor (SRL) and McDougall's buffer, was found to effectively inhibit removal of added amino acids and ammonia. Thus, protein degradation may be estimated from accumulation of these end-products. Casein was incubated in the medium in small quantities (2 mg/ml SRL) and its degradation estimated by this procedure. Casein degradation was found to be a first-order process, with plots of log fraction undegraded versus time being linear up to 3 hours, and with mean k_d = 0.242/hour. Assuming k_r = 0.04/hour, a value of 14.2% ([0.04/(0.04 + 0.242)] × 100) ruminal escape was calculated. Alkali-labile phosphorus (ALP) naturally bound to casein was used in an alternative approach to quantitate casein disappearance. In vitro studies indicated 1.0 mM HS did not inhibit casein degradation (measured by ALP disappearance) up to 3 hours, and both the amino acid plus ammonia release and ALP procedures gave comparable results. Casein k_d value determined simultaneously in vivo and in vitro were, respectively, 0.462/hour (by ALP) and 0.300/hour (by amino acid plus ammonia release). Use of cetyltrimethyl-ammonium bromide when stopping incubations increased apparent amino acid plus ammonia release rates, presumably by rupturing microbial cell walls. Alternative k_d measurement was made from the ratio of Michaelis-Menten kinetic constants, k′_d = V_max/K_m. Results suggest that this approach may give more accurate k_d estimates. Implications of the described theory and techniques are also discussed.