Protocol for determining the amount of Nitrogen (N) in a substance that is in a biologically available form. The assay also measures the effects of tannins and the digestibility of dry matter.
Even though many ecologists realize that not all of the N in plants is available to animals they insist on measuring total N in studies of herbivory. Tannins are the main cause of reduced N availability because, by definition, they bind proteins at the physiological pH found in vertebrate herbivores. The effect can be dramatic; for example, when analysed using the assay described here many eucalypts give negative available N. In other words, there is more N in the digestion bag at the end of the assay than at the start because the tannin binds the protein in the plant material and also some of the protein we add in the form of digestion enzymes. While this may seem impossible it is perfectly reasonable and mimics what could occur in nature if animals ate plant material containing high concentrations of tannins that bind protein. Two recent studies demonstrate the utility of this approach. DeGabriel et al. (2009) showed that the effects of tannins on N availability in Eucalyptus leaves, within the home range of female brushtail possums, correlated with reproductive success and the growth rates of young. Similarly, McArt et al. (2009) showed that the availability of N in the summer foods of moose, at several sites in Alaska, correlated with fecundity and twinning. In both studies, tannins were the main dietary constituent that limited the availability of N and best explained reproductive output. Clearly, N is important and perhaps ecologists and plant physiologists should consider carefully what they measure – total N, available N or even individual amino acids. This protocol describes an in vitro method for measuring available N.
- drying oven
- electronic balance
- Acid, alkali and water resistant pen for labeling digestion bags
- Bag sealer (e.g., TEW impulse sealer; 310-330 W impulse; TEW Electric Heating Equipment Ltd)
- dry, ground leaf samples (or other tissue samples, about 5g of dry matter)
- dry, labeled, preweighed ANKOM F57 filter bags (4 per sample)
- 0.05 M Tris-BASE buffer, pH 7.1
- PEG 4000 (Sigma, analytical grade)
- 5l conical flask for mixing solutions
- 2.5 l beakers (2) for digestion
- magnetic stirrers (2), with grid
- evaporation dishes
- distilled water
- large plastic measuring cylinder
- 1:10,000 Pepsin (DifcoTM)
- 0.1N HCl
- sodium acetate (CH3COONa.3H2O)
- glacial acetic acid (99%)
- 100 mmol acetate buffer
- cellulase (Onazuka 3S Japan)
Units, terms, definitions
Following are our preferred abbreviations for the various traits associated with or measured by the in vitro assay. All traits should be reported on a dry matter basis.
N: Total N expressed on a dry matter basis (g per 100 g DM)
AvailN (Available N): The concentration of N that is available to a model vertebrate herbivore (the proportion digested in the in vitro assay multiplied by the concentration of total N in the starting material).
AvailNPEG: Available N after treatment with the tannin binding agent, polyethylene glycol. The difference between the available N content of a sample, with and without PEG, is a measure of the effect of tannins.
NDig(N digestibility). The proportion of the N in the starting material that disappears during the in vitro assay
NDigPEG N digestibility after treatment with polyethylene glycol. The difference between N digestibility measured with and without PEG, is a measure of the effect of tannins.
Solubles The loss of mass from the sample after incubation with buffer. This is the highly soluble material in the plant sample. We have not analysed the contents of the solubles but their concentration is often closely correlated with other traits, such as dry matter digestibility.
SolublesPEG The loss of mass from the sample after incubation with buffer containing polyethylene glycol
DMD (dry matter digestibility): The proportion of the original dry matter that disappears during the in vitro digestion.
DMDPEG: The proportion of the original dry matter that disappears during the in vitro digestion from the samples treated with polyethylene glycol
In vitro analysis
- Collect spectra of all dry, ground leaf samples and select samples for analysis a.
- Analyze the selected samples for N to a precision of less than 2%. We advise using a combustion analyzer because the large number of N analyses (at least 5 per sample) makes the technique extremely slow with most Kjeldahl digestion methods.
- Weigh 0.8050 ± 0.0050 g of air-dry, ground sample into each of four dry, labeled, preweighed ANKOM F57 filter bags b (Ankom Technology, Macedon, New York).
- Dry to constant mass and reweigh. Calculate dry matter content of sample.
- Place two bags of each sample in a beaker containing 25 ml per bag of exactly 33.33 g L-1 of PEG 4000 (Sigma, analytical grade) in 0.05 M Tris-BASE buffer, pH 7.1 (Sigma). Place the second sets of bags in a beaker containing 25 ml per bag of the 0.05 M buffer. (MW TRIS = 121.1).
NOTE: Use two 2.5 l beakers for the incubation. The beakers sit on magnetic stirrers, with a grid in each (that we cut from a kitchen strainer) to prevent the bags disturbing the stirrer. Also, an evaporation dish, which fits inside the beaker, prevents the bags floating.
- Place the flasks on magnetic stirrers in an oven at body temp (ca 34-39°C) for 24 h.
- Remove the flasks from the oven, wash about eight times in tap water and twice in distilled water.
NOTE: The washing steps are the key to the assay. Place the bags in a large plastic measuring cylinder, fill it with water, shake it hard and repeat the process until you are convinced they are free of buffer or PEG.
- Dry bags to constant mass. Weigh the two sets of bags. The loss of mass from the bags treated with buffer is what we call “solubles”.
- Add exactly 25 ml per sample of solution containing 2.00 g 1:10,000 pepsin (DifcoTM) in 1 L 0.1N HCl to each flask before incubating them on magnetic stirrers in an oven at body temp for 24 h. The pH should be 1.0 but meters are notoriously poor when calibrated outside their pH range. Thus, either make it from standard acid or make it the same way each time from AR HCL, which is 36-39% w/w) or titrate it. AR HCL ≈ 12N. Therefore, add 7.5 ml acid per litre of deionised water to make 0.1 N HCl.
- After 24 h, wash the samples in each flask as described in step 7.
- Add exactly 25 ml per sample of 100 mmol acetic acid buffer containing 6.25 g cellulase (Onazuka 3S, Japan), 6.8 g sodium acetate (CH3COONa.3H2O) and 2.9 ml glacial acetic acid (99%) per litre (pH 4.75) to each beaker. Mix thoroughly and incubate on magnetic stirrers at 37°C. (NB pH optima of cellulase is low). (NB Adjust if using CH3COONa.1H2O (5 g per litre) or anhydrous (4.1 g per litre)).
- After 48 h, wash the samples eight times in tap water and twice in distilled water and then dry the bags to constant mass in an oven at 50°C. Weigh them to determine the dry residue.
- Analyze the residue for N to a precision of less than 2%.
- Calculate digestible N, dry matter digestibility (DMD), etc. (equations below)
- Build NIR Spectrometry models and predict these traits in the remaining samples. (Related protocol: Measuring chemical composition with NIR spectrophotometry )
a This assumes that the analysis is to calibrate NIRS. If not, proceed to step 2 and ignore step 14.
b This assumes that the researcher wants to measure the effect of PEG. If not, weigh out two samples in step 4 and continue from step 8 to obtain digestibility values for N and DMD.
N digestibility (%) = 100*((N content of sample*Dry mass of sample)-(N content of residue*Dry mass of residue))/ (N content of sample*Dry mass of sample)
Dry matter digestibility (%) = 100*(Dry mass of sample-Dry mass of residue)/(Dry mass of sample)
Available N content of sample = (N content of sample*N digestibility)/100
Note: we calculate many of these factors with and without PEG. Thus the difference between N digestibility or available N content of a sample, with and without PEG, is a measure of the effect of tannins.
Notes and troubleshooting tips
As we point out above, washing is a key step in the process; careful washing greatly reduces the coefficient of variation (CV) between duplicate digestion bags. The mean CV between duplicate bags for N digestibility is typically less than 2% (provided the N digestibility is above 20%). The CVs will be much higher when the digestibility of N is negligible.
Links to resources and suppliers
DEGABRIEL, J. L., WALLIS, I. R., MOORE, B. D., and FOLEY, W. J. 2008. A simple, integrative assay to quantify nutritional quality of browses for herbivores. Oecologia 156:107-116.
Health, safety & hazardous waste disposal considerations
Follow MSDS guidelines, particularly when using:
- Polyethylene glycol (PEG)
- Hydrochloric acid
- sodium acetate
- glacial acetic acid (99%)
Note that enzymes may require import permits and customs documents