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 NDF, the primary component of plant cell walls.
3. Progress Report:
This project relates to Objective 4 of the parent project (3655-31000-021-00D): 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. Measurements of fermentation gas in the in vitro ruminal fermentations of dairy cows were shown to be useful to screen proprietary polysaccharide-hydrolyzing enzymes for enhanced forage digestibility when co-incubated with mixed rumen microbes. These measurements were compared to those of controls without added enzyme. Enhancement of fermentative gas production was detected more readily at shorter incubation times (4 to 6 hours). Screening of proprietary enzyme preparations identified several preparations of potential commercial utility as feed additives. The most active of these preparations has been selected for scale-up of enzyme production and subsequent feeding trials in lambs.