1a. Objectives (from AD-416):
To identify enzymes that augment the digestibility of forages by ruminal microorganisms.
1b. Approach (from AD-416):
Two approaches will be taken to identify novel enzymes for improving ruminal degradation of plant biomass: 1) The first will involve screening of enzymes or enzyme mixtures from non-ruminal sources for their ability to improve the in vitro fermentation of biomass materials by ruminal inocula. 2) The second will involve the identification of combinations of ruminal inocula, obtained from different cows, that display synergistic improvement of fermentability. The individual donor inocula that, upon combination, display the greatest synergy can subsequently serve as subjects for proteomic investigations to identify unique enzymes present in each inoculum that may be responsible for this synergy. For both approaches, primary screening of biomass fermentation will be conducted by measuring gas production during in vitro fermentation of representative biomass substrates, including stover from corn (a C4 plant), a C3 grass (e.g., timothy), and a legume (e.g., alfalfa). For Approach 1 above, gas production will be compared in the absence or presence of individual candidate enzymes. For Approach 2, gas production from combinations of ruminal inocula will be compared to that obtained from individual inocula; synergy will be defined as the ratio of gas production by the combined ruminal inocula to the average gas production from the individual donor inocula). For both approaches, the improvement in fermentation by the best candidate enzymes (Approach 1) or ruminal inoculum combinations (Approach 2) will be confirmed by measurement of residual plant cell wall material (as neutral detergent fiber, NDF) and subsequent calculation of NDF digestion (fraction of added NDF removed). For a six-month extension of this project, we will employ an in vitro gas production assay to: 1) screen candidate enzymes for their ability to enhance the fermentation of representative forages by mixed ruminal microorganisms; and 2) screen combinations of ruminal fluids from different donor cows for synergistic improvement of the fermentation of representative forages. In vitro fermentations wth enzymes or ruminal fluids that display enhanced gas production will be further tested for improved conversion of neutral detergent fiber, the primary component of plant cell walls.
3. Progress Report:
This project relates to Objective 4 of the parent project: Develop an integrated system for evaluating forage genotypes and validate the usefulness of in vitro, in situ, and small ruminant digestibility in assessing the utilization of nutrients by lactating dairy cows representing current levels of production. Research was conducted by an ARS scientist in Madison, Wisconsin in collaboration with a non-ARS cooperator who supplied enzyme materials for testing. Measurement of gas production during in vitro ruminal fermentations using gas pressure transducers was shown to be useful for screening of proprietary polysaccharide-hydrolyzing enzymes for enhanced digestibility of forages when co-incubated with mixed rumen microbes, compared to controls without added enzyme. Improvements were made in the assay to account for variations introduced by: slight differences in evaporative loss of buffer during vial preparation, slight differences in inoculum volume, and gas pressure loss during measurement. Enhancement of fermentative gas production resulting from enzyme activity was detected more readily at shorter incubation times (2, 4, or 6 hours) than at longer times (12, 24, 48 or 96 hours). Over 100 proprietary enzyme preparations were screened, several of which significantly enhanced the in vitro ruminal fermentation of corn stover or alfalfa. The most active of these preparations has been selected for scale-up of enzyme production and subsequent feeding trials in lambs.