Effect of diet, digesta processing, freezing and extraction procedure on some polysaccharide-degrading activities of ruminal contents

Three procedures for extracting polysaccharide-degrading enzymes (PDE) from ruminal contents were compared, and the effects of diet, inoculum preparation and freezing of samples on recovery of enzyme activities were evaluated. Four nonlactating cows and four heifers were fed either high-forage (83%, dry matter [DM] basis) or high-grain (91%, DM basis) diets. Three sample types, (1) enriched ruminal fluid; (2) blended enriched ruminai fluid; and (3) whole ruminal content, were analyzed fresh or after having been frozen (-40°C) for 48 h. Four cell-free preparations of each sample type were assayed: (1) control (no enzyme extraction); and enzymes extracted by (2) sonication; (3) lysozyme; and (4) lysozyme + EDTA. Animals fed a high-grain diet had lower carboxymethylcellulase and xylanase but higher amylase activities in the rumen. Blending ruminai solids with buffer and combining the filtrate with cheese-cloth-filtered ruminal fluid produced higher polysaccharide-degrading activities per unit of DM compared with whole ruminal contents. In lysozyme treatments, freezing inactivated from 22 (amylase) to 52% (xylanase) of the enzyme activities measured in fresh ruminal fluid. Lysozyme treatment resulted in the highest enzyme recovery (from fresh ruminal fluid) of the extraction methods tested. With frozen samples, sonication and lysozyme gave similar results. If a large number of ruminal content samples are to be processed for PDE activities, freezing and subsequent sonication appear to be a practical alternative to extraction by lysozyme.