Reducing the Environmental Impact of Beef Production Through the Use of Naturally Occurring Secondary Plant Metabolites in Southeastern Forage Systems

Specific Objective 1. Determine effects of species and location on yield potential, nutritive value, and plant secondary metabolite expression of forage forb species. To address this objective, activities will be divided into two subsections, each under the management of a different co-PD. The first subsection will consist of small-plot field experiments conducted in both the warm and cool seasons over two yeasrs. This subsection will be managed by co-PD Dr. Jim Muir (Texas A&M AgriLife Research) Native and introduced species to be included in this screening experiment are presented in Table 2. In each season of Years 1 and 2, plots will be seeded at the Chilton Research and Extension Center (Clanton, AL), the Coastal Plain Experiment Station (Tifton, GA), the Texas A&M AgriLife Research and Extension Center at Stephenville (Stephenville, TX), the Swine Research and Education Center (Auburn, AL), and the Wiregrass Research and Extension Center (Headland, AL). Each location will consist of three blocks (spatial replication). Plots will be 1.5 × 1.5 m (2.25 m²) and will have 1.5 m tilled or defoliated (herbicide) alleys between all plots and 1.5 m borders on all sides. Seeding and fertilizer rates and methods will follow Extension recommendations for each species. Irrigation and pesticide use will be avoided to limit input costs. The first forage harvest will occur when individual plant species have reached 25% flowering (warm-season; hand-plucked) or an average canopy height of 30 cm (cool-season; whole-plant ⁽³⁷⁾). Forage will be harvested on 28-d intervals (cool-season) or 25% flowering (warm-season) thereafter until vegetative growth ceases (approximately four harvests in each season). Total harvested biomass will be recorded, and a subsample will be taken for dry matter and nutritive value determination. Biomass will be assayed for dry matter, organic matter, fiber fractions, and crude protein, as well as all relevant secondary plant metabolites (condensed tannins, glucosinolates, isoflavones, and saponins).

The second subsection will address methane yield potential of forbs grown in small plots and will be under the management of co-PD Dr. Todd Callaway (University of Georgia). Concurrent in vitro gas production experiments will be conducted at the University of Georgia using harvested biomass from the small-plot experiments. Samples (0.5 g) of each forb species, with or without the addition of the accompanying seasonal grass species (cool-season: tall fescue; warm-season: bermudagrass), or sodium formate will be weighed into duplicate serum bottles. Samples will be allowed to incubate for 24 h, then transferred to a refrigerator at 4°C to stop fermentation. A gas sample (0.5 ml) will be removed from each bottle analyzed for methane and hydrogen via gas chromatography. Fermentation end production (volatile fatty acids and ammonia) will be measured following methane sampling.

Table 2. Forb species to be used in the evaluation of methane reduction potential in beef cattle production.

Specific Objective 2. Determine digestive and metabolic parameters in beef cattle exposed to grass/forb systems including naturally occurring plant secondary metabolites. This objective will be under the management of PD Dr. Brandon Smith (Auburn University). We will select the three forbs from the warm season with the highest methane reduction potential from Specific Objective 1 to address Specific Objective 2. These forbs will be planted at a single location in Alabama in Year 2 to produce the material necessary to conduct an in vivo metabolism experiment. Each of the selected forbs will be fed in combination with bahiagrass hay (30% forb/70% bahiagrass hay). This experiment will be conducted as a 4 × 4 Latin square. Columns will correspond to periods, and rows will correspond to ruminally-fistulated steers. Treatments will be randomly assigned to steers in each period such that each steer will receive each diet once throughout the experiment. Each in vivo period will last 30 days, consisting of a 21-d adaptation phase, a 5-d total fecal and urinary collection phase, a 1-d blood collection phase, and a 3-day ruminal sampling phase. For the first 14 d, treatment diets (in hay form) will be offered to steers at 110% ad libitum intake (defined as 10% daily refusal). On d 14 of each period, TiO₂ boluses will be placed through the rumen cannula for marking passage of solid material. Boluses will continue to be inserted intraruminally throughout the remainder of each in vivo period for comparison of marker technologies relative to total fecal collections. From d 22 through 26, steers will be subjected to total fecal and urine collections. Feces will be collected in bulk and subsampled for digestibility calculations while urine will be collected via Foley catheter. During this time, steers will also be fitted with collars for methane assessment using the SF₆ technique. On d 27, blood will be collected via jugular venipuncture for assessment of energy and protein status as a result of forb supplementation. On d 28, the rumen sampling period will begin. Rumen fluid will be sampled at 0, 0.25, 0.50, 0.75, 1, 2, 3, 4, 8, 12, 16, 20, and 24 h relative to feeding for determination of volatile fatty acids and ammonia. A subset of these samples will be used for ruminal microbial population analysis. On d 29, Cr(III)-EDTA (500 mL solution containing 5 g Cr) will be infused intraruminally via the cannula immediately prior to feeding. Rumen fluid will be sampled at 0, 4, 8, 12, 16, 20, and 24 h relative to infusion for calculation of liquid volume and dilution rate of the ruminal liquid fraction. On d 30, rumen evacuations will be performed at 0, 6, and 12 h relative to feeding. Ruminal contents will be weighed, and every 10^th handful will be separated, mixed, and subsampled for determination of ruminal contents and solid passage rate. Following each period, steers will be allowed a 12-d rest period. The total length of the experiment will be 156 d.

Specific Objective 3. Determine beef cattle performance using grass/forb systems including naturally occurring plant secondary metabolites. This objective will be under the joint management of PD Dr. Brandon Smith (Auburn University) and co-PDs Dr. Leanne Dillard and Dr. Kim Mullenix (Auburn University). We will select the three forbs from the cool season with the highest methane reduction potential from Specific Objective 1 to address Specific Objective 3. Forbs will be sown into existing tall fescue pastures in Year 2 at a rate necessary to achieve 30% dry matter inclusion. Each forb will be sown into three replicate pastures, and three pastures will be reserved from seeding as a control. Weaned beef calves will begin grazing these pastures when the average plant height reaches 20 cm. A put-and-take grazing experiment (used to maintain similar forage allowance) will be conducted to assess animal performance. Calves will be weighed and blood collected every 28-d throughout the grazing season. Grazing will be terminated when forage allowance falls below 1 kg DM/kg BW.

Specific Objective 4. Create and promulgate educational products on forage management practices to reduce greenhouse gas emissions in forage-based livestock systems. This objective will be jointly managed by co-PD’s Dr. Leanne Dillard and Dr. Kim Mullenix. Members of the project team will form an advisory committee with farmer cooperators in the proposal, the project leaders, and selected Extension agents from each institution to develop educational products on management practices to reduce GHG emissions in forage-based beef cattle systems. Outside of new product creation, this will also include updating existing educational products on this topic which previously focused primarily on traditional, agricultural management uses of these forage species. Resources will be updated to include timely information on sustainability perspectives of GHG emissions and the role of forage systems in emissions management. Product outputs include PowerPoint-based curriculum for incorporation into existing forage-educational programs for use by Extension agents, addition of a module on GHG to the Alabama Extension Forage Basics online course, a breakout session at regional forage field days on “Methane Mythbusters”, and web-based content pieces on this topic. Activities will begin in Year 1 to create awareness among end users of how improved forage management practices can positively benefit the environment. Web-based and in-person educational products will be generated in subsequent years to create a referenceable database on practices to enhance environmental sustainability of forage-based systems using research findings from Objectives 1 through 3. Assessment of product reach will be measured through 1) distribution and use of PowerPoint materials by Extension agents using a Qualtrics survey and 2) through web-based access activity records. The advisory committee will work with selected Extension agents to identify farmers (at least three) for one-on-one interviews regarding their perceptions of the environmental footprint of forage-based livestock systems in the region prior to and after participation in one or more of the Extension activities listed above.

Data Analysis. Data from the proposed experiments will be analyzed using SAS v. 9.4 (SAS Institute, Cary, NC). Data obtained from the forb screening will be analyzed as a completely randomized design using the generalized linear mixed models procedure (PROC GLIMMIX). The data will be analyzed as a fixed effects model in which species is the sole fixed effect and random effects are location and block within location. Data obtained from the methane yield experiment will be analyzed in the same fashion as the forb-screening study, but the additional random effect of incubation vial will be added. Data obtained from the digestion and metabolism experiment will be analyzed as a Latin square design using PROC GLIMMIX. Responses will be analyzed with the fixed effect of forb species and the random effects of period (column) and steer (row). Data obtained from the put-and-take grazing experiment will be analyzed as a generalized complete block design using PROC GLIMMIX. Pasture will be defined as the experimental unit, and tester animal within pasture will be defined as the observational unit. Response variables will be analyzed with the fixed effect of forb species and the random effects of block, replication by block, and animal within pasture (nesting the observational unit). In all experiments, strong differences among responses will be declared when P < 0.01, differences among responses will be declared when 0.01 ≤ P < 0.05, tendencies will be declared when 0.05 ≤ P < 0.10, and trends will be declared when 0.10 ≤ P < 0.15.

Pitfalls and Limitations. Although current literature would suggest that PSM-producing forbs will reduce methane emissions in beef cattle production, our working hypothesis could be rejected. In that case, our efforts will, nonetheless, present forage mixtures that may be used for beef cattle management decisions. Specific Objectives 2 and 3 rely on selection of forbs in the screening trial conducted in Specific Objective 1. Should the hypothesis of Specific Objective 1 be rejected, Specific Objectives 2 and 3 would still continue. In that case, forbs would be selected based on yield potential and current extent of use in the Southeast. To address the grazing experiment, if forage supply is limited (drops below 1 kg DM/kg BW with no grazers present), the study could be terminated prior to the projected period completion. If forage supply is in excess and cannot be controlled with put-and-take animals, pasture size (and, therefore, forage allowance) can be restricted with temporary fencing. In the case of a tester animal being removed, the experiment can be analyzed as an unbalanced design.