Jul 17, 2025
USDA LTAR Common Experiment measurement: Concentration of nutrients and in vitro organic matter digestibility in plant tissue
- Elizabeth Boughton1,
- Grégory onnier1
- 1Archbold Biological Station, Venus, Florida
External link: https://ltar.ars.usda.gov/
Protocol Citation: Elizabeth Boughton, Grégory onnier 2025. USDA LTAR Common Experiment measurement: Concentration of nutrients and in vitro organic matter digestibility in plant tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwq3r9vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 11, 2025
Last Modified: July 17, 2025
Protocol Integer ID: 219938
Keywords: plant tissue sampling, in vitro organic matter digestibility, forage, LTAR, USDA LTAR, Common Experiment, aboveground biomass, plant tissues, Long-Term Agroecosystem Research Network, digestibility, agroecosystems, grazing, organic matter, rangelands, plant growth, nutritive value, leaves, plant nutrient content, plant tissue composition data, plant tissue nutrient, temporal variation in plant nutrient content, soil nutrient analysis, understanding ecosystem nutrient, ecosystem nutrient, plant tissue sampling, organic matter digestibility in plant tissue, overarching objective of plant tissue sampling, usda ltar common experiment measurement, edaphic factors on crop, chemical composition of plant species, concentration of nutrient, plant tissue, nutrient, organic matter digestibility, rangeland plant growth, ltar common experiment, grazing animal, ecosystem service, agroecosystem function, plant species, nutritional value, whole plant, digestibility, more mineral element, crop, rangeland productivity
Funders Acknowledgements:
United States Department of Agriculture
Grant ID: -
Abstract
The overarching objective of plant tissue sampling in LTAR common experiments is to quantify the chemical composition of plant species, with particular regard to elements (N and P) or digestibility by grazing animals (e.g. In vitro organic matter digestibility (IVOMD) that may limit agroecosystem function and goods/services provision. Plant tissue composition data have broad applicability. They can be used to understand spatial and temporal variation in plant nutrient content and supply, identify the extent to which one or more mineral elements may limit crop and rangeland plant growth, and determine the influence of management, disturbance regimes, climate, or edaphic factors on crop and rangeland productivity, ecosystem services, commodity quality and nutritional value. Plant tissue nutrients are also important for understanding ecosystem nutrient use efficiency and are a complement to soil nutrient analyses.
Measurements can be taken at the leaf, whole plant, or community level depending on the research objective at the site.
Troubleshooting
Sample collection
The spatial scales of sampling will vary depending on the research objective, and to an extent vary between crop and rangeland systems. The spatial scale of interest is field scale (i.e. fetch of the eddy-covariance towers) to allow data to be used in combination with most LTAR measurements. The number and array of sub-samples necessary to accomplish this will depend on the degree of spatial heterogeneity, and variation.
This protocol assumes a once-per-year sampling at a time corresponding to peak growth for most species. Some research questions may warrant more frequent sampling, as determined by local investigators.
Sampling leaves
Sample mature leaves in full sun just below the growing tip on main branches or stems. Sample just prior to or at the beginning of reproductive growth.
Sample enough leaves to yield 10 g of dried tissue (see post-harvest handling). A suggested minimum is to sample leaves from a minimum of 5 plants, but more may be required for small-leaved plants or in location with high heterogeneity. Local investigator judgement prevails in defining adequate sampling intensity.
Avoid senesced leaves and those with signs of herbivory, disease, mechanical damage, or contamination by soil, dust or chemical residues
Sampling whole plants
To sample whole plants, select plants representative of the population, and cut as close as practical to the soil level. Clip above the lowest portions of the plant stem that may be contaminated by soil if required.
Collect enough plants to adequately represent each experimental unit. A suggested minimum is 5 plants, however for small plants or in heterogeneous locations a great number may be needed, according to local investigator judgement.
As with leaf sampling, avoid plants with substantial herbivory, disease, mechanical damage, or contamination.
Sampling communities
Collect leaves or whole plants, as appropriate, from each species in the community, or from the dominant or representative species from the functional groups of interest.
Alternatively, collect aboveground biomass from a quadrat. The ideal size of the quadrat will depend on the characteristics of the plant community and ideally capture a representative sample of aboveground biomass.
Record the quadrat dimensions for subsequent area-based calculations (see equations in the Calculations section below).
Determine locations of the sampling quadrats based on random or pre-selected stations in each treatment replicate. The number of quadrats will depend on the experiment and how best to account for spatial
variability. After placing the quadrat, collect percent cover of all species to the nearest 1 % (if applicable). All plants rooted within the boundary of the sampling quadrat are hand clipped at ground level and placed in a brown paper bag.
If desired, Live and Dead can be analyzed for nutrients separately. After placing the quadrat, remove litter and standing dead vegetation and place in a bag labeled “Dead”. The remaining live vegetation is clipped to ground level and placed in a bag labeled “Live”.
Sample processing
The purpose of post-harvest handling is to preserve samples with minimal loss of dry weight or changes in chemical composition from the living state. Tissue composition can change post-harvest when tissues become hot, desiccated, or begin to decompose.
To avoid exposing collected samples to these conditions, proceed to the following:
Immediately place collected samples on ice until return to the laboratory. Keep samples in zip-lock bags if needed to minimize desiccation; however this increases the importance of keeping the samples cool and stabilizing the samples promptly once back at the lab.
If sampling of contaminated plants cannot be avoided, follow decontamination procedures in Campbell and Plank 1988.
In the lab, immediately dry the samples at moderate temperatures not to exceed 60 °C to constant weight. The time required will vary with the size and nature of the sample, but generally 24 to 48 hours.
Record the dry weight of samples (g). Ensure the bag weight is not recorded in the final biomass weight.
Calculate the dry weight per area (kg/ha) using the equations below (in the Calculations section).
Once dried, grind the sample to pass through a 1 mm screen using a Wiley Mill. Uniform particle size is important. Alternatively, grind the sample to a size needed based on the instruments utilized.
For some instruments, subsampling of the biomass may be required. Subsampling for nutrient concentrations is conducted at the discretion of the site based on the instrumentation and methods used for analysis.
Laboratory analysis
Send samples to a commercial laboratory, a university laboratory, or a USDA ARS laboratory.
Depending on research objectives, analyze the plant tissue for % Nitrogen, % Phosphorus, % Carbon, and/or % In vitro organic matter digestibility.
In Vitro Organic Matter Digestion. This procedure is "two-stage":
Incubation of a sample with rumen microorganisms for 48 h followed by
A 44 h incubation with acid-pepsin. Results are expressed in percentage units, meaning the percentage of Organic Matter (OM) which was “digested” or disappeared.
Concurrently sampled covariate metrics
- Biomass dry weight from biomass sampling.
- Species composition of the sample.
- Date of sampling.
Calculations
For community sampling:
Dry weight per area (kg/ha) = Dry weight (g) x Sampling area (m2) x 10
Calculate the content of nutrients on an area basis using the aboveground biomass dry weight and the
concentration (percent) of C, N, and P.
See Cavigelli and Strickland 2024 for equations (https://dx.doi.org/10.17504/protocols.io.bp2l62km5gqe/v1)
DOM yield: Yield per unit land area of digestible organic matter (DOM).
DOM yield (kg/ha) = Dry Matter (DM) yield, kg/ha x (%OM/100) x (%IVOMD/100)
Crude Protein:
Crude protein is calculated as %N x 6.25.
Quality assurance and quality control
Meta-data should include the method and instruments used for analysis.
Archiving
Follow the archival methods as described in the aboveground biomass protocol (Wilke et al. 2024). dx.doi.org/10.17504/protocols.io.bp2l62zmkgqe/v1
Recommendations for data collection
| A | B | C | D | |
| Attribute | Preferred | Minimum | Comments | |
| Spatial scale | Field | Plot, plant, or leaf | ||
| Frequency | Once per year at peak growing season | Once per year at peak growing season | ||
| Covariate metrics | For community scale measurements, collect aboveground biomass per unit area and species composition |
Protocol references
Campbell and Plank. 1988. Chapter 3 in Kalra (ed) Handbook of Reference Methods for Plant Analysis. CRC Press, Boca Raton, FL.
Cavigelli, M. A., and Strickland, T.C. 2024. USDA LTAR Common Experiment measurement: Concentration of carbon and nitrogen in aboveground biomass. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62km5gqe/v1
Gallaher, R. N., C. O. Weldon and J. G. Futral. 1975. An aluminum block digester for plant and soil analysis. Soil Sci. Soc. Amer. Proc. 39:803-806.
Hambleton, L. G. 1977. Semiautomated method for simultaneous determination of phosphorus, calcium and crude protein in animal feeds. J.A.O.A.C. 60:845-852.
Moore, J. E., and G. O. Mott. 1974. Recovery of residual organic matter from in vitro digestion of forages. J. Dairy Sci. 57:1258-1259.
Wilke, B., Abendroth, L. J., and VanderWulp, S. 2024. USDA LTAR Common Experiment measurement: Aboveground biomass. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62zmkgqe/v1
Acknowledgements
This research is a contribution from the Long-Term Agroecosystem Research (LTAR) network. LTAR is supported by the United States Department of Agriculture. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.