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Animal Feed Science and Technology

146 (2008) 169–174

Short communication
Evaluation of autoclave procedures for fibre
analysis in forage and concentrate feedstuffs
Clóvis C.D. Senger, Gilberto V. Kozloski ∗ ,
Luis M. Bonnecarrère Sanchez, Francisco R. Mesquita,
Tiago P. Alves, Douglas S. Castagnino
Animal Science Department, Universidade Federal de Santa Maria (UFSM),
Campus Camobi, Santa Maria 97105-900, RS, Brazil

Received 12 June 2007; received in revised form 18 December 2007; accepted 21 December 2007

Abstract

Autoclave procedures for amylase-treated neutral (aNDF) and acid detergent fibre (ADF) analysis
in concentrate and forage feedstuffs were evaluated. Dried and ground samples were weighed in
polyester filter-bags, heat sealed, placed into a 500 ml Erlenmeyer flask and treated with neutral
or acid detergent solution in an autoclave. Different times and temperatures were tested: 40 min at
110 ◦ C (1); 60 min at 110 ◦ C (2); 40 min at 120 ◦ C (3) or 60 min at 120 ◦ C (4). Results from autoclave
treatments were compared to those obtained with the conventional standard method by regression.
Conventional method included the use of Berzelius beakers and Gooch-crucibles, as well as refluxing
and filtration apparatus. For aNDF analysis, all autoclave methods showed high level of precision as
indicated by low standard deviation (S.D.) from regressions (mean of 40.4 g aNDF/kg dry matter).
Slope of the regression for autoclave treatment at 110 ◦ C during 60 min, however, differed from 1
(P<0.05). Autoclaving treatment at 110 ◦ C during 40 min had the lowest (24.1 g aNDF/kg dry matter)
and at 120 ◦ C during 60 min had the highest (75.6 g aNDF/kg dry matter) bias. For ADF analysis,
although the level of precision of regressions for all treatments was relatively high (S.D. mean of
34.5 g ADF/kg dry matter), the slope of regression did not differ from 1 only for 110 ◦ C during 40 min
treatment. Moreover, bias using this treatment was near 0 while it varied from 73 to 174 g ADF/kg
dry matter using the others autoclave treatments. In conclusion, aNDF and ADF analysis in forage

Abbreviations: ADF, acid detergent fibre; DM, dry matter; NDF, neutral detergent fibre; aNDF, neutral
detergent fibre (with a heat stable amylase, ash included).
∗ Corresponding author. Tel.: +55 55 220 8355; fax: +55 55 220 8355.

E-mail address: kozloski@smail.ufsm.br (G.V. Kozloski).

0377-8401/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2007.12.008
170 C.C.D. Senger et al. / Animal Feed Science and Technology 146 (2008) 169–174

and concentrate feedstuffs may be carried out simultaneously in an autoclave and not biased from
Gooch-crucible conventional method, using filter bags and a temperature of 110 ◦ C for 40 min.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Acid detergent fibre; Autoclave; Method; Neutral detergent fibre

1. Introduction

Fibre concentration has been a useful measure to describe and estimate the energetic
value of ruminant feedstuffs. One of the most important sets of fibre assays is the deter-
gent analysis system, which was initially proposed by Peter Van Soest at the United States
Department of Agriculture (USDA) unit in Beltsville (MD, USA). A historical description of
the development of the detergent system has been previously published (Udén et al., 2005).
Initially the original method was developed to determine acid (ADF) and neutral deter-
gent fibre (NDF) in forage samples. Subsequently, its application to concentrate feedstuffs
resulted in modifications such as the inclusion of amylases and sodium sulphite, as well as
the adaptation of the new equipments, apparatus and sampling procedures. For example,
Komarek (1993) developed a filter-bag procedure that excluded the use of beakers, crucibles
and filtering apparatus, which was recently compared to original Gooch-crucible method
for fibre analysis in silage samples by Ferreira and Mertens (2007). The use of the filter-bag
technique allows a significant increase in the number of samples simultaneously analysed
and eliminates the filtration process in individual Gooch glass crucibles, which is sometimes
troublesome. However, the technique requires specialized equipment, which is expensive
and not always available in all laboratories. Autoclaves are, in turn, relatively common
laboratorial equipment. Pell and Schofield (1993) used 50 ml serum bottles for micro-NDF
analysis, where detergent treatment of samples was in autoclave at 105 ◦ C during 60 min,
and Deschamps (1999) adapted the filter-bag methodology for use in autoclave where, based
on his personal observations, samples were autoclaved at 120 ◦ C during 40 min. The use of
filter-bags and autoclaving treatment for NDF or ADF analysis results in a more practical
and rapid assay, compared with the Gooch-crucible conventional method. It may also be
less expensive for many laboratories, compared to the use of Ankom apparatus. However,
the adequate time and temperature of the autoclave treatment, as well as the accuracy and
precision of this alternative method has still not been evaluated. This experiment was carried
out to test different autoclave times and temperatures for NDF and ADF analysis to find an
alternative procedure which is not biased from the Gooch-crucible conventional method.

2. Materials and methods

2.1. Feed samples

Seventeen forages (Arachis sp., Echinochloa sp., Lolium sp. (two samples were taken at
the vegetative stage and one sample at the end of growth season), maize silage, Sorghum sp.,
Hemarthria altissima, Pennisetum sp. (dwarf elephant grass and elephant grass), Eragrostis
spp., Cynodon spp. (two samples of tifton 85 and one of the coast cross), Trifolium repens,
 C.C.D. Senger et al. / Animal Feed Science and Technology 146 (2008) 169–174 171

Desmodium sp., Paspalum notatum) and fifteen concentrate (corn grain (two samples), soy-
bean meal (three samples), soybean residue, corn grain residue, wet corn grain silage, grape
residue, rice bran, palm kernel meal, sorghum grain, wheat bran, linseed meal, rapeseed
meal) samples were used. All samples were force-air oven (55 ◦ C for 72 h) and ground to
pass through a 1-mm screen. Dry matter (DM) content of samples was determined by drying
at 105 ◦ C for at least 8 h.

2.2. Fibre analysis

Extraction of detergent fibre using crucibles was carried out in triplicate for each sample
and followed AOAC conventional methods (Mertens (2002) and Method 973.18 (AOAC,
1997) for NDF and ADF, respectively). Briefly, 1.0 g 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 Porosity 2 (50 ␮m) 50-mL Gooch-sintered glass
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, oven-dried at 105 ◦ C during at least 8 h and
weighed. To measure amylase-treated NDF (aNDF), sodium sulphite was omitted and
␣-amylase (Thermamyl 120 L, Novozymes Latin America Ltda.) was added in two 50-␮L
doses (the first after 5 min of heating and the second during the first water soaking). Sand
was added to Gooch-crucibles as a filtering aid to isolate aNDF. To measure ADF asbesto
was omitted. Both aNDF and ADF content of experimental feed samples as analysed by
conventional method are shown in Table 1.
For fibre extraction using filter bags and autoclave, approximately 350 mg of forages and
500 mg of concentrates samples were weighed in polyester filter-bags (4 cm × 5 cm, 50 ␮m
porosity), heat sealed, placed into a 500 mL Erlenmeyer flask and treated with neutral or
acid detergent solution in an vertical autoclave (capacity of 50 L, Prismatec Autoclaves,
Itu, SP, Brazil). Acid and neutral detergent solutions were the same used in conventional
methods. In each Erlenmeyer 10 or 15 filter-bags containing concentrate or forage sam-
ples, respectively, and 400 mL of detergent solution were added. The flasks were covered
with aluminium paper and inserted into a pre-heated autoclave. For aNDF analysis sodium
sulphite was omitted, but a heat stable ␣-amylase (Thermamyl 120 L, Novozymes Latin
America Ltda., Brazil) was added in two 300-␮L doses (the first at the start and the second
at the end of autoclaving treatment). Four autoclave procedures, using different times and
temperatures, were tested: 60 min at 120 ◦ C (equivalent to 1.0 kgf/cm2 ); 60 min at 110 ◦ C
(equivalent to 0.5 kgf/cm2 ); 40 min at 120 ◦ C or 40 min at 110 ◦ C. Three runs, with sam-
ples triplicates in each run, were carried out on each procedure. After autoclaving treatment,
flasks were removed from the autoclave and bags were washed at least three times with boil-
ing distilled water, soaked in acetone for 5 min, oven-dried at 105 ◦ C during at least 8 h and
weighed. Because detergent residues can be used in a sequential step to N or lignin analysis,
bags and crucibles containing the neutral and acid detergent residues were not ashed.
Analysis of variance including the effects of sample type, method, run and interaction
run × method, was initially carried out on aNDF and ADF concentrations data within
autoclave methods. Once there was not effect of run or interaction run × method, mean
values of aNDF and ADF concentrations from the three runs for a given feed within each
autoclave method were randomly paired to one observation selected from the conventional
172 C.C.D. Senger et al. / Animal Feed Science and Technology 146 (2008) 169–174

method. Two regressions between observations from the conventional method and from
each autoclave method were used to test the validity of the new methods. Precision
was estimated as the standard deviation (S.D.) from the first regression, and the second
regression with a forced 0 intercept yielded an estimate of the bias of the autoclave
methods. Deviation of the regression slope from 1 was assessed using a two-tailed t test.
For variance and regression analysis the PROC GLM and PROC REG of statistical analysis
systems (SAS, 2002) were respectively used.

3. Results

The relationship between aNDF or ADF concentrations in feed samples as analysed by

Gooch-crucible conventional and autoclave methods are shown in Tables 1 and 2, respec-

Table 1
Neutral (aNDF) and acid (ADF) detergent fibre concentrations (g/kg dry matter) in experimental feedstuffs
Feedstuff aNDFa ADF
Arachis pintoi 390 287
Coast-cross (Cynodon sp.) 677 334
Corn grain I 137 33
Corn grain II 123 30
Corn silage 437 297
Corn residue 281 74
Desmodium sp. 529 372
Dwarf elephant grass (Pennisetum sp.) 665 395
Elephant grass (Pennisetum sp.) 593 336
Eragrostis sp. 725 360
Grape residue 583 397
Hemarthria altissima 860 376
Linseed meal 371 246
Palm kernel meal 741 470
Paspalum notatum 653 375
Rapeseed meal 421 nd
Rice bran 264 105
Rice grass (Echinochloa sp.) 680 439
Ryegrass I (Lolium sp.) 456 309
Ryegrass II (Lolium sp.) 584 371
Ryegrass III (Lolium sp.) 481 303
Sorghum grain 166 48
Sorghum forage (Sorghum sp.) 748 518
Soybean meal I 130 59
Soybean meal II 202 131
Soybean meal III 180 112
Soybean residue 387 158
Tifton 85 I (Cynodon sp.) 760 421
Tifton 85 II (Cynodon sp.) 777 357
Trifolium repens 346 228
Wet corn grain silage 71 25
Wheat bran 431 126
a Alpha-amylase treated NDF.
 C.C.D. Senger et al. / Animal Feed Science and Technology 146 (2008) 169–174 173

Table 2
Relationship between amylase-treated neutral detergent fibre (aNDF) concentrations (g/kg dry matter) in feed
samples as analysed using the Gooch-crucible conventional method (X) vs. autoclave procedures (Y): 120 ◦ C
during 60 min, 120 ◦ C during 40 min, 110 ◦ C during 60 min or 110 ◦ C during 40 min
Treatment Regression Slope S.E.a R2 S.D.b Biasc
120 ◦ C, 60 Y = −29.3 + 0.97X 0.018 0.97 39.1 75.6
120 ◦ C, 40 Y = −8.8 + 0.97X 0.018 0.97 39.8 40.7
110 ◦ C, 60 Y = −2.2 + 0.95Xd 0.019 0.96 41.2 51.6
110 ◦ C, 40 Y = 6.31 + 0.97X 0.019 0.96 41.8 24.1
a Slope standard error where n = 96 per treatment.
b Standard deviation from regression.
c g aNDF/kg dry matter.
d Slope different from 1 (P<0.05).

Table 3
Relationship between acid detergent fibre (ADF) concentrations (g/kg dry matter) in feed samples as analysed
using the Gooch-crucible conventional method (X) vs. autoclave procedures (Y): 120 ◦ C during 60 min, 120 ◦ C
during 40 min, 110 ◦ C during 60 min or 110 ◦ C during 40 min
Treatment Regression Slope S.E.a R2 S.D.b Biasc
120 ◦ C, 60 Y = 5.4 + 0.81Xd 0.024 0.93 33.5 174.2
120 ◦ C, 40 Y = 13.0 + 0.85Xd 0.027 0.92 36.6 113.3
110 ◦ C, 60 Y = 10.3 + 0.90Xd 0.023 0.95 31.1 73.5
110 ◦ C, 40 Y = 15.9 + 0.97X 0.027 0.95 36.8 2.0
a Slope standard error where n = 90 per treatment.
b Standard deviation from regression.
c g ADF/kg dry matter.
d Slope different from 1 (P<0.05).

tively. For aNDF analysis, all autoclave methods showed high level of precision as indicated
by low standard deviation (S.D.) from regressions (mean of 40.4 g aNDF/kg dry matter).
Slope of the regression for autoclave treatment at 110 ◦ C during 60 min, however, differed
from 1 (P<0.05). Autoclaving treatment at 110 ◦ C during 40 min had the lowest (24.1 g
aNDF/kg dry matter) and, at 120 ◦ C during 60 min had the highest (75.6 g aNDF/kg dry
matter) bias. For ADF analysis, although the level of precision of regressions for all treat-
ments was relatively high (S.D. mean of 34.5 g ADF/kg dry matter), the slope of regression
did not differ from 1 only for 110 ◦ C during 40 min treatment. Moreover, bias using this
treatment was near 0 while it varied from 73 to 174 g ADF/kg dry matter using the others
autoclave treatments (Table 3).

4. Discussion

The use of the filter-bag technique (Komarek, 1993) has become an alternative method for
fibre analysis, which allows a significant increase in the number of samples simultaneously
analysed and eliminates the filtration process in individual Gooch glass crucibles, which is
troublesome for some feedstuffs. However, the technique requires specialized equipment,
which is expensive and not always available in all laboratories. Autoclaves are, in turn,
174 C.C.D. Senger et al. / Animal Feed Science and Technology 146 (2008) 169–174

relatively common laboratorial equipment and has been used as an alternative equipment
for fibre analysis (Pell and Schofield, 1993; Deschamps, 1999). Time and temperature
affected the results of the present experiment and ADF values were more sensitive to these
factors than aNDF. Almost all autoclave treatments tested could be used for aNDF while,
for ADF, only 110 ◦ C during 40 min was as accurate as the Gooch-crucible conventional
method. Increasing temperature from 110 to 120 ◦ C and/or time of the autoclave treatment
from 40 to 60 min underestimated ADF concentrations of the forage samples.
Autoclaving at 110 ◦ C during 40 min, samples for both aNDF and ADF analysis can be
treated simultaneously. Both polyester and nylon filter-bags can be used for aNDF. For ADF
analysis, nylon bags cannot be used, as polyamides are not resistant to hot acid detergent
solutions. Furthermore, as polyester filter-bags are used, the nitrogen (N) content of residue
can be analysed in a subsequent step by a Kjeldhal method.

5. Conclusion

Neutral and acid detergent fibre analysis in forage and concentrate feedstuffs may be
carried out simultaneously in an autoclave and not biased from Gooch-crucible conventional
method, using filter bags and a temperature of 110 ◦ C for 40 min.

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