Use of an in Vitro Fermentation Bioassay to Evaluate Improvements in Degradation of Alfalfa Hay Due to Exogenous Feed Enzymes

Document Type


Journal/Book Title/Conference

Animal Feed Science and Technology







Publication Date


First Page


Last Page



An in vitro batch culture assay was used to determine the efficacy of five developmental feed enzyme products in terms of improving ruminal degradation of alfalfa hay. A second objective was to establish whether the length of time that alfalfa hay was incubated with ruminal fluid and buffer in a batch culture in vitro system influenced enzyme efficacy. The experiment was conducted as a completely randomized design in four replications with treatments arranged as a 3 (incubation times) × 5 (enzyme products) factorial. Two of the enzyme products (P1 and P2) were proteases, while the other three products (F1, F2, and F3) contained differing proportions of endoglucanases and xylanases. Milled alfalfa hay was incubated with buffer, ruminal fluid and enzyme product (1.5 mg/g of dry matter; DM). Gas production (GP) and degradability of DM and fibre were measured after terminating the incubation at 12, 18 and 24 h. At all incubation times, P1 increased GP by 5.6–7.9%, but GP was not affected by P2. Of the polysaccharidases evaluated, F1 and F2 increased GP by 3.7–10.6% at all incubation times, while F3 had no effect. Improvements in fibre degradability depended upon enzyme product and incubation time. Degradation of neutral detergent fibre (aNDF) was increased at 12 h of incubation using F1 and F2 (12–13% increase), at 18 h of incubation using P1 (11%) and F1 (7%), and at 24 h by F1 (10.5%), F2 (16.5%), and F3 (11.3%). Improvements in acid detergent fibre degradation ranged from 17.5 to 44.4% for these enzymes, depending upon incubation time. Three of the enzyme products (F1, F2 and F3) decreased acetate to propionate ratio, suggesting that improvements in fibre degradation would increase availability of glucose precursors to the animal. Larger improvements in degradability of alfalfa hay occurred for enzyme products containing mainly fibrolytic, rather than proteolytic, activity. An in vitro bioassay that consists of incubating forage samples in the presence of ruminal fluid, buffer and enzyme can be a useful means of screening efficacy of exogenous feed enzymes for ruminants. Cost and labour of the assay can be reduced by using a single incubation time of 24 h to assess enzyme effects on GP and degradability of DM and fibre.