Beef production is at a crossroads in terms of environmental and economic sustainability.
Recently, grass-fed beef has gained popularity with consumers who are concerned about the
environmental impact of beef production and animal welfare. However, feedlot-finished beef
has been shown to have lower rates of greenhouse gas emissions than pasture-finished beef.
Here, we propose using tannin- and saponin-containing legume forages to lower greenhouse gas (GHG)
emissions and improve nitrogen (N) retention in pasture-finished beef systems. These forages
are known for reducing methane production in cows, reducing leachable N-inputs, reducing the
need for further N-additions, improving animal rate of gain, and preserving natural grassland
ecosystem services. However, it is still unclear how tannins and saponins function in the soil. Previous work
has shown that tannins reduce mineralization rates, although it is unclear whether this is a
function of tannin structure or concentration. Saponins are another secondary plant compound which have been
observed to have a similar function to tannins in the soil. To discriminate the effect of tannin or saponin source and
concentration on soil N-cycling, we propose an in vitro incubation study using varied doses of
condensed tannins from Lotus corniculatus (birdsfoot trefoil) and Onobrychis viciifolia
(sainfoin), and saponins from Medicago sativa (alfalfa) as opposed to commonly studied commercially-available
varieties, to monitor rates of N-mineralization, volatilization, and GHG production in pasture soil. Incorporating the
influence of N-cycling on soil GHG emissions will require a whole-farm environmental
sustainability assessment. Holos is a comprehensive and user friendly GHG accounting software
which uses a whole-farm approach to assess beef production environmental and economic
sustainability. Holos is currently designed for use in Canada, restricting its adoption in the U.S.
We propose to extend Holos’ geographic range and characteristics to include Utah for easier
adaptation throughout the Intermountain West. By training local producers and extension staff to
use Holos, we will facilitate its use while giving producers the ability to understand how
management changes affect environmental and economic sustainability.
1. Extract tannins from fecal samples produced by cows that have grazed on birdsfoot trefoil (BFT) and sainfoin (SFN)
tannin-containing legumes to determine a baseline tannin concentration that we would expect to see in the field in April 2018. We will also extract tannins from the leaves of BFT and SFN plants, and saponins from the leaves of ALF plants in April 2018. The tannins and saponins extracted from these leaves will be added to the soils during the incubation study and used as assay standards.
2. Perform a 84-day soil incubation study with varying concentrations of tannins extracted
from BFT and SFN leaves, and a single concentration of saponins extracted from ALF leaves (May-June 2018).
3. Determine concentrations of NH4+, NO3-, and NH3 at the start and end of the incubation,
and throughout the study on the same days as headspace sampling (May-June 2018).
These data will be used to calculate rates of N mineralization.
4. Assay the secondary plant compounds in soil samples at the start and end of the incubation study (May-June
2018) to monitor their availability in soils. Soils will be assayed for autoclaved citrate extractable protein (ACE protein) prior to KCl extraction on each sampling date to determine the
amount of protein substrate available to be bound by tannins or saponins (May-June 2018). Soil
secondary plant compound extractions and ACE protein assay trials will be performed prior to the incubation study
to determine the amounts of each substrate needed for successful assay.
5. Determine concentrations of CO2 and N2O gases using gas chromatography throughout
the incubation to determine production rates of each gas as well as cumulative production
(May-June 2018). Headspace samples will be collected on days 0, 2, 7, 14, 28, 42, 56, 70, and 84.
6. Create Holos farm scenarios for feedlot-finished, and various pasture-finished (MBG,
BFT, SFN, ALF) beef production systems for Utah using climate and soil data from Utah sites
where pasture-based beef production is or could be carried out, and quantify GHG
emissions for each scenario in units of CO2 equivalents (CO2-eq) (April-June 2018).
7. Create print and electronic resources explaining the effect of tannin-containing legume
forages on livestock health and soil nutrient cycling in Utah (March 2019).
8. Host two half-day training sessions for regional producers and outreach personnel in
partnership with USU Extension to demonstrate the use of Holos software (April 2019).
9. Create an online video tutorial with partner researchers at Agriculture and Agri-Food
Canada where Holos was developed to demonstrate how to use Holos software and adjust
it for Utah soil and climate conditions (January 2019).
10. Evaluate how producers’ skills have changed with regard to Holos software abilities as
well as their understanding of how management changes influence farm sustainability
before and after the Holos training sessions (April 2019).
In Vitro Incubation Experiment:
We are conducting an 84-day in vitro soil incubation experiment. This experiment uses various sources and doses of condensed tannins to distinguish between the effect of tannin source and concentration on N cycling dynamics. A saponin treatment with one source and concentration was added to the experiment so that the affect of tannins could be compared against the affect of other secondary plant compounds found in legume forages.
Each treatment will be added to 5 g of oven dry equivalent of uniform 0-15 cm soil under grass located adjacent to the pastures at the Utah State Intermountain Irrigated Pasture Project (USU IIPP) in Lewiston, Utah. Soil samples were collected on October 29, 2018 using a step-in soil corer. Soils were homogenized, sieved to 2 mm, and stored at 5 degrees celsius.
The experiment consists of 6 treatments:
- Soil control (Control)
- Birdsfoot trefoil tannins @ 3 mg tannins/g dry soil (BFT Low)
- Birdsfoot trefoil tannins @ 15 mg tannins/g dry soil (BFT High)
- Sainfoin tannins @ 3 mg tannins/g dry soil (SFN Low)
- Sainfoin tannins @ 15 mg tannins/g dry soil (SFN High)
- Alfalfa saponins @ 3 mg saponins/g dry soil (SAP Low)
Treatments will be added to 5 g of the uniform soil by dissolving the dry tannins or saponins in double distilled de-ionized water. All treatments will be created to deliver the correct concentration of secondary plant compounds and bring the samples to 22% moisture (field capacity). Each sample will be placed in a 40 mL borosilicate glass incubation vial and sealed with caps fitted with septa. Each treatment will contain 39 replicates: 3 replicates for ammonium (NH4+) and nitrate (NO3-) extractions on days 0, 2, 7, 14, 28, 42, 56, and 70; 3 replicates for NH4+, NO3- on day 84 and headspace (ammonia (NH3), carbon dioxide (CO2), and nitrous oxide (N2O)) sampling throughout the experiment; 6 replicates for autoclaved citrate extractable protein (ACE protein) – 3 replicates for day 0, and 3 replicates for day 84; and 6 replicates for secondary plant compound analysis – 3 replicates for day 0, and 3 replicates for day 84. In addition to the 39 replicates of each treatment, 3 empty jars will be used as blanks and be preserved throughout the experiment (237 samples total).
Tannins will be extracted from plant leaves grown at the USU IIPP according to Hagerman (2011) and purified according to Grabber et al. (2013). Tannins will also assayed be from freeze-dried fecal samples collected in June 2017 from cows grazing exclusively on each forage. This will be done to provide a reference value for tannin concentrations being deposited in the field. Saponins will be extracted and purified according to Lee et al. (2001).
On each sampling day (0, 2, 7, 14, 28, 42, 56, 70, 84), NH4+ and NO3- concentrations will be determined by performing a 2M KCl extraction on the soil. Extracts will be analyzed using a Lachat Quikchem 8500 Flow Injection analyzer (Lachat Instruments, Loveland, CO, U.S.).
On each sampling day, 7 mL headspace samples will be taken using a syringe and analyzed for concentrations of NH3, CO2, and N2O. Concentrations of NH3 will be collected on 2.5 M KHSO4 acidified filter paper traps (Stark & Hart, 1996) and then analyzed for N. Concentrations of CO2 will be analyzed on a HP 6890 Series Gas Chromatograph System (Hewlett-Packard, Palo Alto, CA, U.S.). Concentrations of N2O will be analyzed on an Agilent Technologies 6850 Series II Network GC System (Agilent Technologies, Santa Clara, CA, U.S.). Jars will be flushed to ambient atmospheric conditions after each sampling event. Headspace samples will be collected from the same 3 experimental replicates of each treatment throughout the experiment.
Samples will be analyzed for ACE protein on days 0 and 84 according to Hurisso et al. (2018). Protein analysis will be conducted to assess the amount of soil protein available in the soil at the start and end of the incubation. This will act as a proxy for the amount of protein which has been bound by secondary plant compounds throughout the experiment.
Secondary plant compounds will be analyzed according to the hot water method described in Halvorson & Gonzalez (2008). Hot water extracts will be analyzed for concentrations of dissolved organic carbon and total dissolved nitrogen. Extracts will be analyzed on a Shimadzu TOC-L Analyzer (Shimadzu Corporation, Kyoto Japan).
Concentrations of soil NH4+ and NO3- will be used to calculate rates of N mineralization. Concentrations of NH3 will be used to calculate rates of NH3 volatilization, N2O will be used to calculate rates of denitrification, and CO2 will be used to monitor microbial activity.
Data will be analyzed for significant differences using SAS statistical analysis software (SAS Institute Inc., Cary, NC, U.S.).
Grabber, J.H., W.E. Zeller, and I. Mueller-Harvey. 2013. Acetone enhances the direct analysis of procyanidin- and prodelphinidin- based condensed tannins in Lotus species by the butanol-HCl-assay. J. Agric. Food Chem. 61: 2669–2678.
Hagerman, A.E. 2002. Tannin Purification.
Halvorson, J.J., and J.M. Gonzalez. 2008. Tannic acid reduces recovery of water-soluble carbon and nitrogen from soil and affects the composition of Bradford-reactive soil protein. Soil Biol. Biochem. 40(1): 186–197.
Hurisso, T. T., Moebius-Clune, D. J., Culman, S. W., Moebius-Clune, B. N., Thies, J. E., & van Es, H. M. 2018. Soil Protein as a Rapid Soil Health Indicator of Potentially Available Organic Nitrogen. Agricultural & Environmental Letters, 3(1).
Lee, Stephen T., Bryan L. Stegelmeier, Dale R. Gardner, and Kenneth P. Vogel. 2001. The isolation and identification of steroidal sapogenins in switchgrass. Journal of Natural Toxins. 4: 273-281.
Stark, J.M., and S.C. Hart. 1996. Diffusion Salt Solutions, Kjeldahl Digests, and Persulfate Digests for Nitrogen-15 Analysis. Soil Sci. Soc. Am. 60: 1846–1855.
Holos Greenhous Gas Emission Modeling:
We will create Holos software scenarios for feedlot and various pasture-finished beef production systems for the Intermountain West. Holos is a whole-farm life cycle greenhouse gas modeling software developed by Agriculture and Agri-Food Canada for animal agriculture operations. The feedlot-finished scenario will represent typical feedlot diets and conditions in northern Utah. The 4 pasture-finished scenarios will represent beef finished on an alfalfa, birdsfoot trefoil, sainfoin, or meadow bromegrass diet. The scenarios will be adjusted to reflect soil, climate, and yield conditions in Utah based on data taken from the USU IIPP, USU Caine Dairy Farm, and the Utah Climate Center. Greenhouse gas emissions will be quantified in units of CO2 equivalents. Using a modeling approach will allow us to simulate how changes in soil N cycling dynamics as a result of the presence or absence of tannin- and saponin-containing forage legumes affect total greenhouse gas emissions at the farm scale.
Holos Software: http://www.agr.gc.ca/eng/science-and-innovation/agricultural-research-results/holos-software-program/?id=1349181297838
In Vitro Incubation Experiment:
This experiment is currently in progress. As of day 28, tannin and saponin treatments have showed some alteration of N cycling dynamics. Soil nitrate concentrations have decreased over time for all treatments (Figure 1). By day 28, the SFN Low, BFT Low, and SFN High treatments are not significantly different from the soil control.
Carbon dioxide production rates for the soil control were significantly lower than all tannin and saponin treatments by day 28 (Figure 2). The BFT Low and SFN Low treatments had the next lowest CO2 emission rates. The BFT High and SFN High had significantly higher emission rates than the BFT Low, SFN Low, and soil control treatments. However, cumulative CO2 production for BFT Low, SFN Low, and SAP Low treatments were not significantly different from the soil control by day 28 (Figure 3). The SFN High and BFT High treatments had significantly higher cumulative CO2 production as compared to all other treatments.
By day 28, nitrous oxide production rates for the SFN High treatment were significantly higher than all other treatments (Figure 4). All other treatments were not significantly different from the control. Cumulative N2O production was also significantly higher for the SFN High treatment (Figure 5). The relationships between N2O production and soil NO3- concentration, and between N2O production and treatment N content are being investigated further.
Results for NH3 volatilization, NH4+ concentration, ACE protein, and secondary plant compound analysis are currently being processed.
In the first 28 days of this experiment, all treatments experienced a decrease in NO3- concentrations. This is likely a due to microbial assimilation of N as the communities grow. As the incubation continues and microbial communities begin to turnover, we will be able to distinguish whether or not tannins and saponins inhibit net N mineralization. Cumulative production of CO2 and N2O indicate the low doses of tannins and saponins do not significantly increase greenhouse gas emissions at this time. We will continue to monitor these results as the experiment continues to determine if tannins and saponins can inhibit net N mineralization without significant greenhouse gas emissions.
Holos Greenhouse Gas Emission Modeling:
Holos software scenarios for all five beef-finishing scenarios are currently in progress. Soil, climate, yield, and forage quality data has been obtained and is currently being assimilated into the software. Once the scenarios are created, they will be run and results will be compared among treatments.
Educational & Outreach Activities
Preliminary data from the in vitro incubation experiment was presented as a poster at the Soil Science Society of America 2018-2019 International Soils Conference in San Diego, CA on January 7th, 2019 by graduate student Kathryn Slebodnik.
A Holos informational presentation and training workshop is in preparation and will be presented at the 2019 Urban and Small Farms Conference in Salt Lake City, UT on February 21st, 2019. Presenters will include graduate student Kathryn Slebodnik, Jennifer Reeve, and collaborators from Agriculture and Agri-Food Canada. A Holos video demonstration will be prepared in conjunction with this event. A survey has been prepared for presentation and workshop participants. This survey will assess how participants’ thoughts and attitudes towards Holos software and on-farm environmental and economic planning has changed over the course of the presentations and workshops.
Factsheets regarding the effectiveness of tannin- and saponin-containing legume forages will be prepared at the conclusion of the experiment in February 2019. Factsheets will be published in cooperation with Utah State University Extension.
This project will contribute to future economic, environmental, and social sustainability of beef production in the Intermountain West. By finishing beef on tannin- and saponin-containing legume forages, producers will likely be able to increase the productivity of pasture-finished beef by increasing forage quality and increasing the average daily gains of cattle. This will allow them to produce higher quantities of high-quality beef over the course of the growing season. The use of nitrogen (N)-fixing legumes will further increase the profitability of pasture-finished beef by reducing the amount of N fertilizer and additional feed required, as well as their transportation costs. The use of these tannin- and saponin-containing legumes further increase profits by reducing seeding costs. Legumes require less frequent seeding than grasses in the Intermountain West, therefore reducing equipment and labor costs.
The adaptation of these tannin- and saponin-containing legumes will also increase environmental sustainability. Based on preliminary results, low doses of tannins and saponins may be able to inhibit N mineralization. This may decrease the amount of N leached in local surface runoff as well as N lost as greenhouse gases such as nitrous oxide. After 28 days of incubation, low doses of BFT and SFN tannins and ALF saponins did not produce significantly higher cumulative carbon dioxde or nitrous oxide emissions. These preliminary results suggest that low concentrations of tannins and saponins may inhibit soil N mineralization without increasing greenhouse gas production. This will improve local water quality and potentially reduce greenhouse gas emissions on a whole-farm, life cycle basis.
This project will produce social benefits for producers and the greater community. The outreach portion of this project will give producers the skills they need to use Holos software for their own operations. This will allow producers to evaluate and share the impact that management changes have on the environmental and economic sustainability of their farm. The adaptation of tannin- and saponin-containing legumes will increase the availability of more sustainably-finished beef products to the local community, as well as improve local environmental quality.
Although the project is still in-progress, my attitude and awareness of sustainable agriculture has increased because of this project. The development and preparation of this WSARE project has allowed me to improve my field and laboratory skills, improve my ability to draw conclusions based on several data sets, facilitate new connections with researchers and outreach coordinators, and gain experience preparing outreach activities. This WSARE project experience has highlighted the importance of being able to perform specific, mechanism-driven scientific experiment, and transform the results into concise conclusions and management recommendations that will benefit scientists, producers, and the general public. The outreach portion of this project has reinforced the importance of sharing the results of scientific activities not only with other scientists, but with producers and land managers who can put these sustainable practices into action. Planning outreach workshops has supported my enthusiasm for sustainable agriculture-based extension activities which I hope to pursue in my future career. This project has also allowed me to attend conferences where I made connections with other scientists conducting sustainability research and learned about other novel sustainable agriculture research.