Measuring detergent fibre and insoluble protein in corn silage using ...

Abstract
Animal Feed Science and Technology
133 (2007) 335–340
Short communication
Measuring detergent fibre and insoluble
protein in corn silage using crucibles
or filter bags
Gonzalo Ferreira a,∗ , David R. Mertens b
a Dairy Science Department, University of Wisconsin, Madison, WI 53706, United States
b USDA-ARS, US Dairy Forage Research Center, Madison, WI 53706, United States
Received 2 January 2006; received in revised form 12 April 2006; accepted 25 April 2006
Different methods exist for the determination of fibre concentration in feeds. To determine, whether
fibre recovery and the contamination of fibre by nitrogenous compounds are altered, we measured
fibre concentrations in a diverse set of corn silages using three method modifications and two extraction/filtration
systems. Thirty-three corn silages, obtained from a commercial feed analysis laboratory,
were dried (55 ◦ C for 24 h) and ground to pass through a 1-mm screen of a cutter mill before analysis.
All samples were extracted in neutral detergent with the inclusion of sodium sulphite (neutral detergent
fibre or NDF), �-amylase (neutral detergent residue or NDR) or both (amylase-treated neutral
detergent fibre or aNDF), and using either Gooch crucible (CRUC) or filter bag (FBAG) systems.
The aNDF method obtained the lowest and similar average fibre concentrations for both CRUC and
FBAG (433 and 433 g/kg, respectively). Fibre concentration of NDR was greater (456 and 449 g/kg
for CRUC and FBAG, respectively) than aNDF. Fibre concentration was greater for NDF (473 g/kg)
than for NDR and aNDF (449 and 433 g/kg, respectively) when using FBAG. Poor extraction occurred
for FBAG when �-amylase was not used. For CRUC, NDF and NDR concentrations were similar
(456 g/kg), although filtration of fibre residue after extraction without �-amylase was difficult. Neutral
detergent insoluble crude protein (ICP) was similar for NDF and aNDF, and slightly greater
Abbreviations: ADF, acid detergent fibre; aNDF, amylase-treated neutral detergent fibre; CRUC, Gooch
crucibles system; FBAG, filter bag system; ICP, insoluble crude protein; NDF, neutral detergent fibre; NDICP,
neutral detergent insoluble crude protein; NDR, neutral detergent residue
∗ Corresponding author. Present address: Casilla de Correo 115, (6070) Lincoln, Buenos Aires, Argentina.
Tel.: +54 2355 42 3382.
E-mail address: gferreira@nutrep.com (G. Ferreira).
0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2006.04.010

336 G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) 335–340
FBAG than for CRUC systems (8.4 and 9.6 g/kg, respectively). With both FBAG and CRUC, ICP
was greater for NDR determined without sulphite (12.5 and 14.2 g/kg, respectively) than for aNDF
(8.6 and 9.7 g/kg, respectively). The lower fibre concentration for aNDF method was attributed to
less starch contamination when compared to NDF and to less protein contamination and possibly
to extraction of phenolic compounds when compared to NDR. Concentration of acid detergent fibre
(ADF) was greater for CRUC than for FBAG, although this difference was minimal (266 and 261 g/kg,
respectively). Acid detergent ICP was similar for CRUC and FBAG systems and averaged 4 g/kg. We
observed that amylase and sulphite affect the measurement of NDF concentration in corn silage. It is
crucial that authors and laboratories accurately describe how they measure NDF and clearly indicate
by acronym the method they used. Although the extraction/filtration system did not affect the determination
of NDR and aNDF, the FBAG system generated higher NDF concentrations for corn silage
when amylase is not used.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Neutral detergent fibre; Insoluble crude protein; Fibre analysis
1. Introduction
The neutral detergent fibre (NDF) method (Van Soest and Wine, 1967) was developed
to separate feed’s dry matter into a soluble fraction, that is readily digested and a fibrous
fraction, that is slowly and incompletely digested. In plant materials, NDF consists primarily
of hemicellulose, cellulose and lignin, but also contains small amounts of protein
and ash. Sodium sulphite was included in the original NDF method to reduce the protein
contamination of fibre (Hintz et al., 1996). Because the original NDF method did
not adequately remove starch, the neutral detergent residue (NDR) method (Robertson
and Van Soest, 1981) was developed to solubilize starch during neutral detergent extraction
using a heat-stable �-amylase. However, sodium sulphite was removed in the NDR
method to minimize the loss of phenolic compounds in feeds. Sodium sulphite is necessary
to remove protein contamination of cooked or heated feeds (Hintz et al., 1996).
Because removal of sodium sulphite results in incomplete removal of protein from NDR,
the amylase-treated NDF (aNDF) method (Mertens, 2002) that uses both heat-stable �amylase
and sodium sulphite was developed to measure insoluble dietary fibre in all
feeds.
The NDF, NDR and aNDF methods are based on the isolation of fibrous residues using
coarse porosity Gooch crucibles (CRUC). The Ankom Technology Corp. (Ankom Technology
Corp., Fairport, NY) developed an alternative method for isolating fibrous residues
based on the extraction of samples in filter bags (FBAG) using a pressurized chamber. To
our knowledge no direct comparisons among methods have been made for corn silage. To
determine whether each of the modifications of the NDF method has potential for altering
the concentration of fibre recovered, we measured fibre concentrations in a diverse set of
corn silages using each of the three methods with each of the two extraction/filtration systems.
In addition, to determine whether protein contamination affects fibre concentration
of corn silages, we measured neutral detergent insoluble crude protein (NDICP) and acid
detergent insoluble crude protein.

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2. Materials and methods
2.1. Corn silage samples
Thirty-three diverse corn silages were obtained from a commercial feed analysis laboratory.
No information regarding genotype and growing or harvesting (e.g., chop-length and
kernel-processing) conditions were obtained.
2.2. Fibre analysis
All corn silage samples were dried in a forced-air oven (55 ◦ C for 24 h) and ground to pass
through a 1-mm screen of a cutter mill (Wiley mill, Arthur H. Thomas, Philadelphia, PA)
before analysis. For the CRUC and the FBAG systems, all corn silages were analyzed using
three neutral detergent fibre modifications (i.e., NDF, NDR and aNDF). Acid detergent fibre
(ADF) was also determined using both CRUC and FBAG systems.
Extraction of detergent fibre using crucibles followed AOAC methods (Mertens, 2002).
Briefly, 0.5 g (NDF) or 1.0 g (ADF) of test samples was heated in neutral detergent or acid
detergent to boiling within 5 min and subsequently, refluxed for 60 min. Fibre residues were
transferred into coarse porosity (50 �m) 50-mL Gooch crucibles, filtered and soaked in hot
(90–100 ◦ C) water three times for 5 min each. Residues were soaked two times in acetone for
5 min each. To measure NDF, sodium sulphite (0.5 g) was added to the test sample and ND
prior to refluxing. Sand was added to Gooch crucibles as a filtering aid to isolate NDF. For
NDR, sodium sulphite was omitted and �-amylase (Ankom Technology Corp.) previously
diluted (10%, v/v) was added in two 2-mL doses (the first after 5 min of heating and the
second during the first water soaking). For aNDF, both sodium sulphite and �-amylase were
used.
Extraction of detergent fibre using filter bags was based on an in-house procedure using
a fibre analyzer (Ankom 220 , Ankom Technology Corp.). Briefly, 2 L of neutral detergent
or acid detergent was poured into the extraction chamber. Filter bags (F57, 25 �m, Ankom
Technology Corp.) containing 0.5 g of test sample were placed in plastic trays. After inserting
the plastic trays, the chamber lid was sealed, the heat turned on and the solution was
heated to 100 ◦ C within 15 min. After 60 min of extraction (75 min total time) the detergent
was expelled. Filter bags were washed four times within the chamber with 2 L of water at
80–90 ◦ C. All washes were performed for 5 min each time with the chamber lid sealed and
heat and stirrer turned on. After the last water wash, extracted filter bags were removed
from the chamber and placed between two absorbent pads, and gently pressed to remove
water. Filter bags were placed in a 500 mL jar, and approximately 250 mL of acetone was
added. The jar was sealed with a lid and shaken three times during a 5-min extraction.
Acetone was removed and replaced with 250 mL of acetone for a second 5-min extraction.
Acetone residues were allowed to evaporate (air-drying) and bags were then dried in
forced-air oven at 105 ◦ C for at least 8 h before being weighed. For NDF, 20 g of sodium
sulphite was mixed with ND solution before it was added to the chamber for extraction. For
NDR, 5 mL of heat-stable �-amylase was mixed with ND solution before it was added to
the chamber for extraction. In addition, 5 mL of heat-stable �-amylase was added to each
of the first two water washes. For the aNDF method, 20 g of sodium sulphite and 5 mL of

338 G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) 335–340
heat-stable �-amylase were mixed with ND solution before this was added to the chamber
for extraction. Also, 5 mL of heat-stable �-amylase was added to each of the first two water
washes.
For all methods and systems single samples were analyzed (i.e., no replication).
2.3. Insoluble crude protein analysis
For the CRUC system, fibre residues in crucibles were scraped into ceramic boats and
the recovered residue was weighed. Concentration of CP was measured as N × 6.25 after
analysis with a N analyzer (Leco FP-2000; Leco Corp., St. Joseph, MI). Insoluble N was
corrected for recovery of residues from crucibles by assuming that residue remaining in
the fritted disk of the crucible had the same N content as recovered residues. For the
FBAG system, the N content of the filter bag plus residue was determined as described
above and corrected for the N content in blank bags. For both CRUC and FBAG, detergent
insoluble CP (ICP) was expressed as g of CP per kg of the original dry sample
weight.
2.4. Statistical analysis
Data were analyzed as a randomized complete block design using the MIXED procedure
of SAS (release 8.2.; SAS Institute Inc., Cary, NC). The model included the effect
of treatments (fixed; degrees of freedom, d.f. = 5), the effect of block or silage (random;
d.f. = 32) and the residual error (d.f. = 160). Protected multiple comparisons were performed
according to the method of Tukey. Statistical difference was declared at P

G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) 335–340 339
Table 1
Concentration of fibre and neutral detergent insoluble crude protein (NDICP) in corn silage (n = 33) determined
using six method modifications
FBAG CRUC
NDF NDR aNDF NDF NDR aNDF S.E.M.
Fibre (g/kg dry matter) 473a 449b 433c 455b 456b 433c 14
NDICP (g/kg dry matter) 9.5c 14.2a 9.7c 8.1d 12.5b 8.6d 0.7
CRUC, residue filtration after extraction in neutral detergent using Gooch crucibles; FBAG, extraction in neutral
detergent within filter bags; NDF, neutral detergent fibre (i.e., residue after extraction with neutral detergent with
the use of sodium sulphite); NDR, neutral detergent residue (i.e., residue after extraction with neutral detergent
with addition of �-amylase); aNDF, amylase-treated NDF (i.e., residue after extraction with neutral detergent
with addition of sodium sulphite and �-amylase). Different letters (a–d) in the same row differ (P0.09) or FBAG (P>0.95) systems (i.e., across systems).
Insoluble CP was similar for aNDF and NDF (Table 1), and slightly greater for FBAG
than for CRUC systems. Insoluble CP was greater for the NDR than for the aNDF method.
Insoluble CP was greater for the FBAG than for the CRUC system, although these differences
were small (less than 2 g/kg) and inconsequential. The lower fibre concentration for aNDF
method can be attributed to less protein contamination of the fibre residue (Hintz et al.,
1996), although the magnitudes of these differences suggest that protein contamination
explains these observations only partially. The differences in fibre concentration for NDR
and aNDF when using the CRUC and the FBAG systems were 23 and 16 g/kg, respectively.
The differences in NDICP concentration for NDR and aNDF when using the CRUC and
the FBAG systems were 3.9 and 4.5 g/kg, respectively. This means that ICP accounted
for 17–28% of the total difference between NDR and aNDF. Mertens (2003) reported that
76% of the difference between NDR and aNDF of grasses was due to differences in lignin
recoveries. Sulphite attack to lignin may account for some of difference between NDR and
aNDF methods (Van Soest, 1994; Mertens, 2003) after they are adjusted for differences in
protein contamination. It is also possible that sulphite might attack proteins and weaken the
protein matrix in starch granules; thereby allowing greater extraction of starch.
Concentration of ADF was greater for the CRUC than for the FBAG system (P0.45) for CRUC and
FBAG systems and averaged 4 g/kg.
4. Conclusions
Poor filtration of starchy materials can result in overestimates of fibre concentration. This
overestimation can be increased when using filter bags with fibre analyzer. Consequently,
the NDF method should be avoided when measuring fibre concentration of corn silage. For
the NDR and aNDF methods, fibre concentrations measured using filter bags with fibre
analyzer were similar to those measured using coarse porosity Gooch crucibles. The aNDF

340 G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) 335–340
method reduces protein contamination of the fibre residue. However, this reduction does
not account for all the reduction in fibre concentration due to the use of sodium sulphite.
We demonstrated that the use of amylase and sulphite affects the measurement of fibre
concentration in corn silage. Implications of this study are that it is crucial that authors and
laboratories accurately describe how they measure fibre concentration and clearly indicate
by acronym the method they used. We also conclude that the extraction/filtration system
did not affect the determination of NDR and aNDF, but that the FBAG system generated
higher NDF concentrations for corn silage when amylase was not used.
References
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in beakers or crucibles: collaborative study. J. AOAC 85, 1217–1240.
Mertens, D.R., 2003. Challenges in measuring insoluble dietary fiber. J. Anim. Sci. 81, 3233–3249.
Robertson, J.B., Van Soest, P.J., 1981. The detergent system of analysis and its application to human food. In:
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Van Soest, P.J., Wine, R.H., 1967. The use of detergents in analysis of fibrous feeds: IV. Determination of plant
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