Reference:
Goering, H.K. and P.J. Van Soest. 1970. Forage fiber analysis
(apparatus, reagents, procedures, and some applications). USDA
Agricultural Research Service. Handbook number 379 as modified by
D.R. Mertens (1992, Personal Communication).
Van Soest, P.J, J.B. Robertson, and B.A. Lewis. 1991. Methods for
dietary fiber, neutral detergent fiber and non-starch
polysaccharides in relation to animal nutrition. J. Dairy Science
74:3583-3597.
Mertens, D.R. 1992. Critical conditions in determining detergent
fiber. Proceedings of NFTA Forage Analysis Workshop. Denver, CO.
p C1-C8.
Scope:
This method is applicable for the determination of neutral
detergent fiber in all types of forages and feeds.
Basic Principle:
A neutral detergent solution is used to dissolve the easily
digested pectins and plant cell contents (proteins, sugars and
lipids), leaving a fibrous residue (aNDF) that is primarily cell
wall components of plants (cellulose, hemicellulose and lignin).
Detergent is used to solubilize the proteins and sodium sulfite
also helps remove some nitrogenous matter; EDTA is used to
chelate calcium and remove pectins at boiling temperatures;
triethylene glycol helps to remove some nonfibrous matter from
concentrate feeds; and heat-stable amylase is used to remove
starch. Two additions of amylase (one during refluxing and one
during filtration) have been observed to aid aNDF analyses and
minimize filtering difficulties. Heat-stable amylases are used in
hot solutions to inactivate potential contaminating enzymes that
might degrade fibrous constituents.
Equipment: Refluxing apparatus
Berzelius beakers (600 mL)
Fritted glass (Gooch) crucibles (coarse porosity, 50 mL)
Analytical electronic balance, accurate to 0.1 mg
Suction filtering device with trap in line and
valve to break vacuum Forced-air drying oven set at 100oC
Reagents:
Neutral detergent solution - To make approximately 18 liters mix:
17.82 L Distilled water 540 g Sodium lauryl sulfate, USP 335 g
Ethylenediami netetraacetic acid (EDTA), disodium salt (may
substitute 72 g sodium hydroxide (NaOH) and 263 g free acid EDTA
as a less expensive alternative.)
122.6 g Sodium borate, decahydrate (Na2B4O7.10H2O), reagent
grade 82.1 g Sodium phosphate, dibasic (Na2HPO4), anhydrous,
reagent grade 180 mL Triethylene glycol, reagent grade
Preparing a NDF Solution
Pour one-half of distilled water into mixing container. Place on
stir plate in hood and begin stirring. Add remaining reagents
except for triethylene glycol. (Caution: wear dust mask when
weighing and transferring the sodium lauryl sulfate.) Slowly add
remaining distilled water to container to limit foaming of the
detergent. When approximately three-fourths of the distilled
water has been added to the container, add the triethylene
glycol. The triethylene glycol will reduce foaming of the
detergent solution. Allow to stir overnight. Use heated stirrer
if material fails to dissolve. Keep container at 20oC or higher
to avoid precipitation of the solution. Verify pH of solution to
be between 6.95 and 7.05. Adjust with HCl or NaOH as required if
not within range. Reagents, Sodium sulfite, anhydrous (Na2SO3),
reagent grade Acetone, reagent grade Heat-stable alpha-amylase
solution (standardized by method 5.1.1)
Safety Precautions:
- Acetone is highly flammable. Do not let vapors accumulate
in work area. Use effective fume removal device. Also
avoid inhaling or contact with skin. Make sure all traces
of acetone have evaporated from crucibles containing the
fiber residue before placing them into the oven.
- Sodium lauryl sulfate is irritating to mucous membranes.
Wear dust mask and gloves while handling.
Procedure:
- Samples should be microwave or oven dried at 55oC to
greater than 85% dry matter, then ground to pass a 1 mm
screen.
- Dry 50 mL fritted glass crucibles overnight at 100oC and
hot weigh (W1), recording weight to nearest 0.1 mg.
(Refer to method 2.2.2.2
for description of hot weighing techniques.)
- Thoroughly mix sample, then weigh 0.45 to 0.55 g,
recording to nearest 0.1 mg, (W2) into 600 mL Berzelius
beaker. Weigh a second subsample for laboratory dry
matter determination.
- Preheat extraction heating (reflux) unit to a temperature
that permits boiling of neutral detergent solution within
5 min.
- Add 0.5 g of sodium sulfite using previously calibrated
scoop.
- Add 50 mL of neutral detergent solution and swirl beaker
until the sample and sodium sulfite are completely
suspended.
- Place beaker on the heating unit under a cool water
condenser. Samples should come to a boil in 4 to 5 min.
Samples normally foam vigorously for 1 to 2 min. Do not
reduce temperature of heating unit during this time.
- After 5 min, remove beaker from the reflux unit and add 2
mL of the standardized amylase solution.
- Swirl beaker to thoroughly mix the amylase in the neutral
detergent solution and resuspend any particles that have
crept up the sides of the beaker. Detach any sample
attached to the sides or bottom of the beaker using a
rubber policeman. Rinse off policeman with aNDF solution.
- Return beaker to the reflux unit and allow to come to a
boil. Reflux for 60 min. Five to 10 min after adding
amylase, rinse down the sides of the beaker with neutral
detergent solution.
- Remove sample from heating unit and allow to settle for
30-60 sec before filtering.
- Preheat the fritted glass crucible for filtering by
adding 40 mL of boiling water. Remove water with vacuum.
- Carefully decant the first 30-40 mL of solution from the
Berzelius beaker into the crucible. Rinse lip of beaker
to prevent solution with particles from running down
outside of beaker. Keep beaker in "decant"
position while emptying. Remove the solution with a
minimum amount of vacuum.
- Close vacuum and rinse the remaining residue from the
beaker into the crucible using a fine stream of boiling
water. Be certain that no particles remain in the beaker
or on the lip to run down the outside as the beaker is
turned upright. Apply minimum vacuum to filter.
- Immediately fill crucible half full of hot water and add
2 mL of standardized amylase solution. Allow to react for
approximately 45 to 60 sec, while policing particles from
sides of Berzelius beaker.
- Rinse Berzelius beaker with boiling water while inverted
over the crucible until all residue is transferred.
- Filter and wash twice by adding 30 to 40 mL boiling water
to residue in fritted glass crucible and allowing to soak
for 2 min each time.
- Rinse sample twice with 30 mL of acetone, allowing at
least 2 min soaking time between rinses.
- Rinse policeman, vacuum sample dry, and remove sample
from manifold.
- Dry crucibles at 100oC for 8 hr or overnight and hot
weigh recording weight (W3) to nearest 0.1 mg.
Comments:
- Difficult filtration may result from plugging of the
fritted glass crucibles. Crucibles should be cleaned
regularly with acid or alkaline cleaning solution. The
filtration rate of crucibles should be as uniform as
possible for a given set of samples. To check the
filtration rate of crucibles, fill them with 50 mL of
distilled water and record the time required to drain
completely without vacuum. This should be about 180 sec.
If filtration takes more than 240 sec, crucibles need
cleaning. If cleaning does not improve the filtration
rate, the crucible should be discarded. If filtering
takes less than 120 sec, check crucible for cracks or
holes in the fritted disk. If filtering takes less than
100 sec, the crucible should be discarded.
- The proper vacuum is critical to good filtering. It
should be sufficient to remove the solutions rapidly but
not so great that fiber particles plug the fritted glass
disc.
- Rinse water must be in excess of 95oC. This is
particularly true for samples containing pectic
substances, mucilages or glycoproteins.
- Some sample types are consistently difficult to filter
(corn silage, citrus pulp, sunflower meal, meat
by-products and feces). Experience has shown that any
sample that takes longer than 10 min to filter will
provide erroneous results and must be repeated using
modifications described by Mertens or Van Soest.
- Many amylase extracts are crude mixtures that may contain
fiber degrading enzymes. Because heat will inactivate
these contaminating enzymes, it is recommended that a
heat-stable amylase be used in hot solution to minimize
fiber loss.
Calculation: Percent Amylase Neutral Detergent Fiber (aNDF)
aNDF (DM basis) = W3 - W1 / W2 X Lab DM / 100
X 100
- W1 = tare weight of crucible in grams
- W2 = initial sample weight in grams
- W3 = dry weight of crucible and dry fiber in grams
Quality Control:
Include one or more quality control (QC) samples in each run,
choosing QC samples by matching analyte levels and matrices of QC
samples to the samples in the run. Include at least one set of
duplicates in each run if single determinations are being made.
An acceptable average standard deviation among replicated
analyses for neutral detergent fiber ranges from about ±0.35 for
samples with 40% NDF to ±0.60 for samples with 70% NDF, which
results in warning limits (2s) ranging from ±0.70 to 1.20 and
control limits (3s) ranging from ±1.05 to 1.80. Plot the results
of the control sample(s) on an X-control chart and examine the
chart for trends. Results outside of upper or lower warning
limits, ±2s (95 percent confidence limits), are evidence of
possible problems with the analytical system. Results outside of
upper or lower control limits, ±3s (99 percent confidence
limits), indicate loss of control and results of the run should
be discarded. Two consecutive analyses falling on one side of the
mean between the warning limits and the control limits also
indicate loss of control.
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