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MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

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AGRICULTURAL MATERIALS

Gravimetric Determination of Amylase-Treated Neutral Detergent Fiber in Feeds with Refluxing in Beakers or Crucibles:

Collaborative Study

DAVID R. MERTENS

U.S. Department of Agriculture, Agricultural Research Service, U.S. Dairy Forage Research Center, 1925 Linden Dr West, Madison, WI 53706-1108

Collaborators: M. Allen; J. Carmany; J. Clegg; A. Davidowicz; M. Drouches; K. Frank; D. Gambin; M. Garkie; B. Gildemeister; D. Jeffress; C-S. Jeon; D. Jones; D. Kaplan; G-N. Kim; S. Kobata; D. Main; X. Moua; B. Paul; J. Robertson; D. Taysom; N. Thiex; J. Williams; M. Wolf

As an important constituent of animal feeds, fiber represents the portion of feeds that is bulky and difficult to digest. The neutral detergent fiber (NDF) method, developed over 30 years ago, is the method of choice for measuring total fiber in for- ages and other feeds. Several modifications that were made to improve its general applicability to all feeds and others developed in individual labora- tories often resulted in variability among laborato- ries in measuring NDF. The amylase-treated NDF (aNDF) method, therefore, was developed as an ac- curate and precise method of measuring total in- soluble fiber in feeds. A collaborative study was conducted to evaluate the repeatability and reproducibility of the aNDF method over the full range of animal feed materials. Twelve laboratories representing research, feed company, regulatory, and commercial feed testing laboratories analyzed 11 materials as blind duplicates. The materials rep- resented feed matrixes, including animal products; high-protein, high-fat, and high-pectin feeds; oil seeds; grains; heated by-product feeds; and le- gume and grass hays and silages. Materials se- lected varied in chemical composition and con- tained 0–90% aNDF, 1–16% ash, 1–20% crude fat, 1–40% crude protein, and 0–50% starch. Cor- recting results for changes in blanks and reporting results as ash-free aNDF organic matter (aNDFom) improved the repeatability and reproducibility of results when aNDF was <25%. The within-labora- tory repeatability standard deviation (s r ) for per- centage aNDFom in feeds varied from 0.21 to 1.82

Submitted for publication July 2002. The recommendation was approved by the Methods Committee on Feeds and Fertilizers and Agricultural Related Products as First Action. See “Official Methods Program Actions,” (2002) Inside Laboratory Management, September/October issue. Corresponding author’s e-mail: davem@dfrc.wisc.edu.

and among-laboratory reproducibility standard de- viation (s R ) varied from 0.37 to 2.24. The HORRAT was <2 for all materials except feed materials con- taining >10% fat. However, standard deviations of repeatability and reproducibility for feeds with >10% fat were similar to those of other materials. It is recommended that the aNDF method be ac- cepted for Official First Action status.

F iber is nutritionally important because it represents the

organic portion of feeds and foods that is the most diffi-

cult to digest; nonfiber fractions of feeds are easily and

almost completely digestible by most animals. There is a need for a rapid and simple assay to determine the total insoluble fi- ber content of feeds. Originally fiber was related to the cell wall fraction of plants; however, because fiber occurs in all feeds, fiber methods must be applicable to all feeds and foods. Although neutral detergent (ND) extraction solubilizes some material that is not digested by mammalian enzymes, neutral detergent soluble fiber is often fermented by bacteria in the gut of animals and is digested. Thus, neutral detergent fiber (NDF) is the insoluble fiber in feeds that is either indigestible or slowly digested, and occupies space in the digestive tract of animals. The NDF method was originally developed by Van Soest and Wine (1) for the analysis of total fiber in forages, and nu- merous modifications have been proposed to extend its appli- cation to grains, concentrated feeds, and human foods (2–5). Most of these modifications were based on conceptual im- provement in total fiber analysis without ruggedness testing to determine their suitability for all feed matrixes or practical ap- plication as a routine method. Unfortunately, this resulted in fiber methods that differed, yet were all called NDF. The diffi- culty in extracting and washing fibrous residues in some mate- rials and the variety in modifications of the method have led to the perception that NDF is difficult to measure precisely.

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Collaborative Study

Optimization and Ruggedness of NDF Methods

The critical steps in the NDF and amylase-treated NDF (aNDF) procedures were investigated by the original develop- ers, who proposed modifications, and the Study Director, who developed the method evaluated in this study. The following factors were evaluated:

(1) Time of refluxing.—Sixty min of refluxing at boiling temperatures achieves asymptotic extraction for most materi- als and was selected as an optimum compromise between analysis time and complete extraction (1). It should not vary more than 5 min. (2) Neutral detergent concentration.—Sodium lauryl sul- fate is used at near saturation concentrations to maximize solubilization of nonfiber with a minimal volume of liquid that must be filtered (1). (3) Neutral detergent pH.—Phosphate and borate buffers are used to maintain pH near 7.0 because alkaline or acid con- ditions can solubilize fiber. The Study Director observed that laboratories were not confirming that pH was near 7.0 and conducted a test to evaluate the effects of pH variation. Re- sults indicated that the pH should be maintained within the range of 6.95–7.05 (6). (4) Triethylene glycol.—The original NDF method used ethylene glycol monoethylether to remove nonfiber soluble material from concentrated feeds. This compound is a poten- tial mutagen and was replaced with triethylene glycol. Re- search confirmed that changing to triethylene glycol did not alter results (J.B. Robertson, Cornell University, Ithaca, NY, personal communication, 1988). (5) Sodium sulfite.—Sodium sulfite was included to re- move proteinaceous material from fiber residues. When heat-stable amylase was included as a part of the NDF method, sodium sulfite was removed because it has the potential to de- stroy phenolic compounds (2). However, research by the Study Director indicated that sodium sulfite is critical for removal of proteinaceous matter in heated or cooked feeds, and it was rein- troduced in the method that is being evaluated (7). (6) Heat-stable -amylase.—Starch was not completely removed by the original NDF method. Numerous modifica- tions of the method used various amylases to remove starch before or during ND extraction. Both the amylase source and the method of using the enzyme alter NDF values, and pre-ex- traction by incubation has the potential to solubilize fiber. Heating samples and gelatinizing the starch may improve am- ylase effectiveness in removing starch contamination. Many amylase solutions are crude extracts that contain enzymes that can degrade fiber and must be deactivated by near-boiling temperatures. Alternative methods of using heat-stable amylases have been evaluated by the Study Director and oth- ers (4, 8). The approach used in the proposed method uses 2 additions of heat-stable amylase: one in ND after initiation of boiling, and one in the first residue washing step. Unfortunately enzyme sources changed or were discontin- ued and new specifications for the type and amount of amy- lase to use had to be rediscovered. The Study Director, there-

fore, developed a visual assessment method for determining the amount of any heat-stable amylase source that is active un- der the conditions used in aNDF analysis, and this is included as part of the method. (7) Washing method.—Rapid rinsing of fibers removes only surface contamination of detergent and soluble matter and results in high fiber values. Research by the Study Direc- tor’s laboratory indicated that multiple soakings of fibrous residues are required to obtain consistent results and uncon- taminated fibrous residues. (8) Reporting fiber as organic matter.—Ashing fiber resi- dues and reporting results as aNDF organic matter (aNDFom) eliminates some differences in results associated with inade- quate washing of fibrous residues. In addition, it allows nutri- tionists to estimate nonfibrous carbohydrates more accurately by difference (NFC = 100 – crude protein – crude fat – ash – aNDFom), because a portion of ash is not double-subtracted as it would be if aNDF were used in the calculation. Calcula- tions in the proposed method allow results to be reported to meet the needs of the user. (9) Sample amount and ratio to ND solution.—The origi- nal NDF method used 100 mL ND with 1 g sample (9). Halving the amount of sample reduced aNDF by about 1 per- centage unit. To reduce filtering problems, the same ratio of sample to ND was maintained, but sample and ND solution amounts were reduced to 0.5 g in 50 mL. (10) Sample grinding.—In most near infrared reflectance spectroscopy, samples are ground through a cyclone mill with 1 mm screen. Discrepancies in fiber analysis were observed when these samples were analyzed by chemical methods. Re- search by the Study Director confirmed that particle size of the sample and aNDF results were affected by both screen size and grinder type. Although samples that are ground finer are ex- tracted more completely by ND, they are often more difficult to filter. Thus, the proposed method specifies that materials must be ground through a 1 mm screen with a cutter mill or equivalent. (11) Sample drying.—Artifact fiber can be created during sample preparation by drying materials at temperatures >60°C (10). Therefore, the method to be evaluated specifies that wet samples be dried at temperatures <60°C. (12) Modifications for problem samples.—Samples that are high in starch, pectin, fat, or animal proteins are difficult to filter. Modifications to the basic method have overcome the difficulties inherent in these types of samples (11). As indicated by Horwitz et al. (12), empirical methods of analysis, such as fiber methods, must be followed exactly. This suggests that the various modifications of the original NDF method should have different names because they are different measures of fiber. The proposed fiber method is called aNDF to distinguish it from the original method and its modifications. The objective of this research was to evaluate the repeatability and reproducibility of the aNDF method in a collaborative study involving laboratories with different missions and using materials representing the full scope of feed ingredients.

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Table 1.

Characteristics of collaborating laboratories

 

Amylase

Lab No.

Type

Method apparatus

source

0

Research (Study Director)

Reflux beaker–crucible

A

1

Research

Reflux beaker–crucible

B

2

Research (dropped out)

3

Research

Reflux beaker–crucible

A

4

Regulatory

Reflux in crucible

A

5

Regulatory

Reflux beaker–crucible

B

6

Testing

Reflux in crucible

B

7

Feed company

Reflux beaker–crucible

B

8

Testing

Reflux beaker–crucible

A

9

Testing

Reflux beaker–crucible

A

10

Testing (dropped out)

11

Feed company

Reflux beaker–crucible

B

12

Feed company

Reflux beaker–crucible

B

13

Feed company

Reflux in crucible

A

14

Regulatory

Reflux beaker–crucible

B

Collaborating Laboratories

The 14 laboratories which agreed to participate in the study represented 3 feed company laboratories, 5 commercial feed testing laboratories, 3 feed regulatory laboratories, and 4 re- search laboratories. Two feed testing and one research labora- tory failed to complete the study; the Study Director’s labora- tory was substituted for the missing research laboratory and a volunteer feed company laboratory was substituted for one of the feed testing laboratories (Table 1). Participants received no compensation. Each laboratory provided reflux apparatus,

filtration vessels, balances, and drying ovens. Collaborators were provided with test samples, heat-stable α-amylase, and chemicals, but were required to mix and standardize the neces- sary reagents. They were asked to respond to several question- naires about equipment and routine procedures in their labora- tories, provide results of standardizing the amylase working solution, analyze 5 familiarization samples, and provide aNDF results for 11 collaborative study materials and one blank ana- lyzed as blind duplicates. Collaborators were asked to perform singles analyses of each sample, to report all weights to 4 signif- icant digits, and to submit data on an as-is basis.

Materials

There are no officially recognized reference materials for fiber, and it is not possible to prepare spiked samples. To de- termine the general applicability of the aNDF method, study

materials were selected to represent the full range of fiber con- centrations and types of animal feeds (Table 2). In addition, materials were included that required the use of each of the modifications described in the method. Materials representing feed matrixes from animal products; high-protein, high-fat, and high-pectin feeds; oil seeds; grains; heated by-product feeds; legume forages; and grass forages (hays and silages) were included in the study. A mixed dairy feed that contained

a mixture of animal and plant supplements with added fat was

used to represent a variety of feed ingredients. Materials selected for the collaborative study encompassed

a wide range of aNDF concentrations in feeds, varying from 0

to 90%. The materials also varied in concentration of ash (1–16%), fat as measured by ether extract (1–20%), crude pro- tein (1–40%), and starch (0–50%). The diverse chemical com- position of the materials used as blind duplicates permitted evaluation of most, if not all, possible interactions of feed composition on aNDF analysis. Blanks were included to eval-

Table 2.

Materials used in the study, representing full range of fiber concentrations and types of animal feeds a

Material

Description

DM, %

Ash, %

EE, %

CP, %

Starch, % NFC, %

ADF, %

aNDFom, %

Alfalfa silage

Forage, fermented legume

91.0

9.7

3.0

20.1

0.2

20.1

37.4

38.1

Brewer’s grains

Concentrate, heated by-product

92.6

5.1

6.3

24.7

2.8

9.2

20.0

47.2

Citrus and beet pulp

Concentrate, pectic by-product

91.4

7.8

3.9

7.3

1.3

45.7

25.4

26.7

Corn grain with cob

Concentrate, containing starch

92.4

1.3

4.5

6.4

50.4

59.6

10.6

20.6

Corn silage

Forage, grass with starch

92.3

3.8

5.0

6.5

33.4

41.5

22.3

35.4

Corn stalks

Forage, high-fiber grass

92.0

7.4

1.0

4.1

0.2

11.2

46.5

68.2

Dairy mixed feed

Concentrate, mixture with fat

92.6

16.1

13.5

39.8

3.9

11.3

7.4

11.9

Grass hay

Forage, temperate grass

92.0

8.6

5.4

8.5

0.5

14.1

38.1

55.5

Milk replacer

Concentrate, animal fat

93.5

7.0

17.6

18.6

0.6

50.2

0.3

0.1

Roasted soybeans

Concentrate, plant fat

93.9

5.2

18.0

38.2

2.6

19.5

6.2

13.0

Sawdust

Reference, high cellulose

92.5

1.6

0.8

0.6

0.6

0.4

77.6

89.1

Blank

No material

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Table 3.

Homogeneity of aNDFom (blank-corrected) for 4 sample sets for each material analyzed a

Material

n

Mean, %

s r

s R

RSD r , %

RSD R , %

2.8 × s r

2.8 × s R

HORRAT

Alfalfa silage

4

38.1

0.50

0.52

1.31

1.37

1.40

1.46

0.59

Brewer’s grains

4

47.2

0.46

0.59

0.98

1.24

1.30

1.64

0.55

Citrus and beet pulp

4

26.7

0.36

0.50

1.33

1.87

1.00

1.40

0.77

Corn grain with cob

4

20.6

0.65

0.65

3.17

3.17

1.83

1.83

1.25

Corn silage

4

35.4

0.18

0.34

0.51

0.97

0.51

0.96

0.42

Corn stalks

4

68.2

0.82

0.84

1.20

1.23

2.29

2.35

0.58

Dairy mixed feed

4

11.9

0.30

0.31

2.52

2.57

0.84

0.86

0.93

Grass hay

4

55.5

0.22

0.42

0.39

0.76

0.61

1.18

0.35

Milk replacer

4

0.1

0.29

0.32

373.29

409.78

0.81

0.89

69.70

Roasted soybeans

4

13.0

0.36

0.53

2.76

4.07

1.01

1.49

1.50

Sawdust

4

89.1

0.56

0.60

0.63

0.68

1.58

1.69

0.33

Blank, g

4

0.0009

0.0012

0.0015

0.0035

0.0042

a s r = Standard deviation of replicated analyses within sample set; s R = standard deviation within and among sample sets; RSD r = repeatability relative standard deviation; RSD R = reproducibility relative standard deviation.

uate weighing technique and determine if blank-correction improved precision. Single batches of homogenous materials were obtained from commercial feed suppliers or the U.S. Dairy Forage Research Center (Madison, WI). Materials were dried as needed at 55°C and were ground through a 1 mm screen with a Wiley cutter mill, and thoroughly mixed. Forty sets of samples containing about 2 g of each of the 11 materials were prepared. Samples were measured into glass vials that were sealed in mois- ture-proof pouches for storage until shipment. All samples were numbered in the order in which they were to be analyzed. Homogeneity of sample sets was verified by selecting 4 sets of samples at random for each material and analyzing them in duplicate in the Study Director’s laboratory to provide an esti- mate of random variation within and among sets of samples. The results of the homogeneity study were analyzed by using the AOAC spreadsheet statistical software recommended for collaborative studies to determine among-sample set instead of among-laboratory variation (Table 3). For most materials, the variation between duplicate analyses within a sample set (s r ) was similar to the variation between samples sets (s R ). This analysis allows variability in homogeneity of sample sets to be compared directly with the variation among collaborating labo- ratories. Except for milk replacer (MR), which had an aNDF concentration near zero, the Horwitz ratio (HORRAT; 12) of observed versus expected relative standard deviations (RSDs) among results was below the acceptable threshold of 2.0. For most samples, the HORRAT was between 0.33 and 0.58. Within the Study Director’s laboratory, the aNDF variation was similar to that of other AOAC Official Methods. The HORRAT values for roasted soybeans (RS), corn grain (CG), dairy mixed feed (DM), and citrus and beet pulp (CB) were between 0.80 and 1.50, suggesting that these materials were less homoge- neous or more difficult to analyze for aNDF.

Familiarization samples were sent to each collaborator in a preliminary study to acquaint them with the method and eval- uate their ability to handle difficult samples. Familiarization materials included alfalfa silage (AS), barley hay (BH), CG, meat and bone meal (MB), and RS. The latter 3 materials were selected to represent materials that were extremely difficult to analyze for aNDF. Heat-stable amylase is a critical reagent in the aNDF method. To verify that alternative sources of amylase can be standardized and used, 3 amylase stock solutions from 2 sources were standardized by each collaborator during the familiarization study, and half of the laboratories were as- signed to use one of the 2 sources during the collaborative study (Table 4).

Statistical Analyses

The experiment was designed as a randomized complete block design using blind duplicates of 11 unidentified materi- als. Although feeds were not identified, milk replacer, dairy feed, and roasted soybeans were labeled to indicate >10% fat in the first blind duplicate, but not the second. Only one ana- lyst from each laboratory was asked to submit results, and blind duplicates were analyzed on the same day within the

Table 4.

evaluated and used in the study

Sources of stock heat-stable -amylases

Amylase type

Source

Dilution

Taka-Therm L-340

ANKOM Technology Corp. (Fairport, NY)

Full strength

Termamyl 120 L

Novo Nordisk Biochem (Raleigh, NC)

Full strength;

one-half strength

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same run. Block was defined as first replicate, second repli- cate, and rerun analyses using modifications. In the first repli- cate, all laboratories were asked to analyze one duplicate of each of the samples in a fixed order of decreasing filtration ease. In the second replicate, one of 12 random orders of anal- ysis for the samples was assigned to each laboratory. Ordering samples in the first replicate was used to determine the effect of a difficult-to-filter sample on the subsequently analyzed samples. The statistical model for analysis of variance (ANOVA) was:

Y = µ + Material + Lab + Block + Material*Lab + error

(1)

where µ = the mean, Material = variation due to materials, Lab = variation among laboratories, Block = variation due to replicate or rerun, Material*Lab = variation due to Material by Laboratory interaction, and error = random variation within laboratory. Block was included in the model to test the effect of order of analysis and was removed from the model when not significant. There are several procedures in the aNDF method that may not be followed exactly among laboratories. For example, identi- fying the proper level of amylase to use when determining aNDF is an integral part of the method because the source and level of enzyme is not specified in the method. A portion of the collabo- rative study was designed to evaluate the effects of amylase source (A or B) and dose (independently determined by each col- laborator). The effect of source and dose of amylase was evalu- ated on all materials and only on materials containing significant starch [corn silage (CS) and CG] using the ANOVA model:

Y = µ + Material + Amylase + Dose + Amylase*Dose + error (2)

where Amylase = variation due to source of amylase, Dose and Amylase*Dose = variation explained by linear regression of amylase dose for each source, and all other variables as de- scribed previously. Because aNDF is an empirical method it is important to evaluate the effects of factors that are left to the discretion of laboratories and may differ among them. Each collaborator submitted answers to a questionnaire about factors that might affect results. In addition to amylase source and dose, collabo- rators were asked to describe for each result the apparatus, fil- tering aid, and pre-extraction technique used, and to describe the temperature of washing water and the methods for residue washing and weighing. This information was evaluated using appropriate ANOVA models. Initially, outlying results were detected with spreadsheet statistical software provided by AOAC for a blind duplicates design using a 2.5% significance level. The Cochran test iden- tifies replicate results within a laboratory that are suspect, and the Grubb test determines if the average result of laboratories deviates from those of all laboratories. Cycles of Cochran, sin- gle Grubb, and pair Grubb tests were used to identify outliers until no additional removal was necessary or no more than 2/9 of the laboratories were flagged. The ANOVA identified which of the 2 blind replicates was the outlier by using a main effects ANOVA with no interaction terms:

Y = Material + Lab + Block + error

(3)

Predicted results were compared with observed results, and the replicate with the largest residual deviation was identified as the outlier. Laboratory ranking scores (after removal of in- dividual replicate outliers) were used to identify laboratories that were outliers across all materials according to Wernimont (13). All outlying results were checked for calcu- lation and data entry errors, and answers to the questionnaire were scrutinized to determine if the collaborator’s procedure differed from the aNDF method that was being evaluated. Within laboratory repeatability (s r ), among laboratory sys- tematic variation (s L ), and total reproducibility variation (s R ; where s R 2 = s r 2 + s L 2 ) associated with a single analysis were determined by approaches described by Youden and Steiner (14) and Wernimont (13) with spreadsheet statistical software recommended by AOAC. Statistical results were de- termined for individual materials. In addition, ANOVA using the interaction model (Model 1) was used to determine if re- sults from all materials or classes of materials (forage, concen- trates with <10% fat and concentrates with >10% fat) could be pooled. If the interaction term in Model 1 was not significant, it was assumed that the method obtained consistent results among materials, and pooled precision statistics were calcu- lated according to Wernimont (13). True values cannot be determined for fiber because the em- pirical method defines the fraction. Therefore, there is no direct measure of systematic bias (accuracy) for fiber, and consensus values derived from the collaborative study were used as the point of reference. As suggested by Youden (15), variability in systematic bias was equated with among-laboratory variability (s L ) that does not include within-laboratory variability. Repeat- ability (s r ) is the within-laboratory precision that was calculated from variation between blind duplicates within laboratories. Reproducibility (s R ) is the total variation associated with a single analysis, which is the sum of systematic bias and re- peatability, i.e., s R 2 = s r 2 + s L 2 . Repeatability within a labora- tory is expected to be 1/2–2/3 of the total reproducibility varia- tion among laboratories. Although specific criteria for the acceptability of a fiber method cannot be imposed, it is ex- pected that the variation among laboratories will be a function of aNDF concentration. The ratio (HORRAT) of the observed reproducibility relative standard deviation (RSD R ) divided by the expected RSD from the Horwitz equation (16) was used to evaluate the acceptability of the method. A HORRAT <2 was used as an indicator of acceptability (12).

AOAC Official Method 2002.04 Amylase-Treated Neutral Detergent Fiber in Feeds

Using Refluxing in Beakers or Crucibles First Action 2002

[This method is applicable for the determination of amy- lase-treated neutral detergent fiber (aNDF) in forages, grains, grain by-product feeds, animal by-product feeds, oil seeds, oilseed meals and mixed feeds that range in concentration from 1.5 to 100%.]

1222 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

Caution: Enzyme (amylase) preparations can cause allergic reactions in hypersensitive individuals; avoid in- haling aerosols or dusts. Cover eyes, nose, mouth, and exposed skin as needed to prevent irritation. Sodium lauryl sulfate is mildly irritating to mucous membranes and may cause allergic reactions in hy- persensitive individuals. Do not inhale or allow material to contact skin or eyes. Wear a dust mask and gloves while handling. Triethylene glycol is harmful if inhaled, ingested, or absorbed through the skin. Vapors or mists are irritating to the eyes, mucous membranes, and upper respiratory tract. Contact is irritating to the skin. Wear appropriate clothing and mix in a hood or well-ventilated area. Acetone is a flammable solvent; do not use near open flames. Use in an effective fume hood and do not let vapors accumulate in work area. Avoid in- haling or contact with skin. Evaporate all traces of acetone before placing fiber residues into an oven. Avoid skin contact with hot neutral detergent solu- tion and reagents. Maintain all electrical equipment in suitable working order and ensure that it is prop- erly grounded.

See Table 2002.04 for the results of the interlaboratory study supporting acceptance of the method.

A. Principle

Fiber in feeds is a nutritionally defined entity that repre- sents the indigestible and slowly digesting fraction of feeds. Neutral detergent (ND) solution and heat-stable α-amylase are used to dissolve easily digested proteins, lipids, sugars, starches, and pectins in feeds, leaving a fibrous residue that is primarily cell wall components in plant materials (cellulose, hemicellulose, and lignin) and indigestible nitrogenous matter in animal products. aNDF is a gravimetric method that best es- timates the total insoluble fiber, and is inversely related to di- gestibility and intake potential of a feed. ND soluble matter is almost completely digested by most animals; however, the di- gestibility of aNDF is variable among feeds and is related to lignin and other constituents in fiber. Sodium lauryl sulfate, an anionic detergent, and sodium sul- fite are used to solubilize nitrogenous matter; EDTA is used to chelate calcium and enhance removal of pectins at boiling tem- peratures; triethylene glycol helps remove some nonfibrous matter from concentrated feeds; disodium phosphate and so- dium borate are used as buffers to maintain a neutral pH. Hot ND solution has limited ability to solubilize starch; therefore, a heat-stable amylase is used to hydrolyze starch to saccharides that can be easily removed from fiber by filtration. Heat-stable amylases are used in hot solutions to inactivate potential contaminating enzymes in crude amylase extracts that might degrade fibrous constituents. To ensure that the am- ylase activity is sufficient to remove most starch and to reduce filtering difficulties, the amount of any specific amylase source needed to measure aNDF is determined under the con- ditions of the aNDF method.

Although boiling ND solution dissolves most proteins, lipids, and nonfibrous carbohydrates, these constituents are nonviscous only in water that is near boiling temperature. Therefore, boiling water is needed for washing fibrous residues and removing nonfibrous matter. Because fiber is particulate matter, mass-ac- tion equilibration during soaking is needed to migrate ND and contaminating soluble matter from the interior of fiber particles. Fibrous residues must be soaked, instead of being simply rinsed, in near boiling water to remove nonfibrous matter. Boiling ND solution solubilizes lipids, and acetone soak- ing of the residue completes the extraction of lipids and pig- ments in most materials. However, excessive amounts of lipids in materials can complex with the detergent and reduce extraction efficiency. Because extraction of lipids by ND and acetone may be incomplete when feeds contain >10% lipid, these materials are pre-extracted to ensure complete removal of lipid contamination from fiber. Pre-extract materials with 5–10% lipid to minimize filtration difficulties and avoid variable aNDF results.

B. Apparatus

(a) Refluxing apparatus.—Any conventional apparatus

suitable for crude fiber or acid-detergent fiber determinations. Test samples should be extracted in 500 or 600 mL beakers without spouts (Pyrex No. 1040, or equivalent) that are covered with a round cold-water condenser to minimize evaporation. Calibrate heating unit setting so that 50 mL water boils within 4–5 min when cold water condensers are used. Fibertec appara- tus 2010 or M6 (Foss North America, Eden Prairie, MN 55344) can be used and should boil 50 mL water within 10 min.

(b) Fritted-disk Gooch crucibles.—Coarse porosity (pore

size 40–60 µm) crucibles, high-form, 40–50 mL capacity or Fibertec USP2 (pore size 40–90 µm, 26–28 mL capacity). Clean new crucibles and ash at 500°C for 1 h. Clean crucibles after each use by ashing at 500°C for 3 h, removing ash, in- verting in detergent solution, and sonicating for 7–10 min. Rinse crucibles in hot water, and soak in room temperature water for at least 30 min. Back-flush crucibles by connecting top of each crucible to a No. 9 ½ stopper or a No. 9 stopper fit- ted with a No. 4 filter adapter (Cat. No. 24035-087; VWR, West Chester, PA 19380, www.vwr.com) that has a port that is connected to a trap and vacuum line. Back-flush each cruci- ble with water by rapidly plunging and removing the bottom of the crucible into water to create a vigorous rinsing action.

Occasionally test filtration rate as follows: Fill each cruci- ble with 50 mL distilled water (25 mL for Fibertec USP2 cru- cibles) and record time required to drain completely without vacuum (should be 180 ± 60 s for Gooch or 75 ± 30 s for USP2). If <100 s (or <35 s for USP2), discard crucible. If <120 s (or <45 s for USP2), check for cracks in fritted disk. If filtration >240 s (or >105 s for USP2), clean crucibles with slow filtration rates using acid or alkaline cleaning solutions.

(c) Vacuum filter manifold.—Any apparatus similar to that

described by Mertens et al. (17) or suitable alternative that al- lows adequate soaking of fibrous residues (Figure 2002.04A). Manifold should provide vacuum-tight seal with crucible to re- duce foam formation in vacuum lines. Use thick-walled vac-

Table 2002.04.

MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

1223

Interlaboratory study results for amylase-treated neutral detergent fiber in feeds

Feed

Fiber

Mean, %

s r

s R

RSD r , %

RSD R , %

r a

R b

Forages

aNDF c

52.2

0.84

1.10

1.61

2.10

2.36

3.08

Forages

aNDFbc d

52.2

0.84

1.08

1.61

2.08

2.36

3.03

Forages

aNDFom e

50.4

0.93

1.18

1.85

2.34

2.61

3.31

Forages

aNDFombc f

50.1

0.93

1.16

1.85

2.32

2.60

3.26

Concentrates <10% fat

aNDF

33.5

1.18

1.42

3.51

4.25

3.29

3.98

Concentrates <10% fat

aNDFbc

33.2

1.25

1.57

3.76

4.72

3.50

4.38

Concentrates <10% fat

aNDFom

32.5

1.16

1.46

3.56

4.48

3.24

4.08

Concentrates <10% fat

aNDFombc

32.2

1.14

1.47

3.55

4.56

3.20

4.11

Concentrates >10% fat

aNDF

8.7

1.01

1.61

11.66

18.57

2.83

4.50

Concentrates >10% fat

aNDFbc

8.7

0.79

1.24

9.06

14.26

2.21

3.47

Concentrates >10% fat

aNDFom

8.8

0.58

1.20

6.54

13.68

1.61

3.37

Concentrates >10% fat

aNDFombc

8.5

0.53

1.10

6.25

12.94

1.49

3.09

All materials

aNDF

38.7

1.05

1.33

2.72

3.44

2.94

3.72

All materials

aNDFbc

38.6

1.02

1.28

2.64

3.32

2.86

3.59

All materials

aNDFom

37.7

1.02

1.28

2.70

3.39

2.85

3.58

All materials

aNDFombc

37.4

1.00

1.24

2.67

3.32

2.80

3.48

a 2.8 × s r .

b 2.8 × s R .

c Amylase-treated neutral detergent fiber (as-received basis).

d Amylase-treated neutral detergent fiber, blank-corrected (as-received basis).

e Amylase-treated neutral detergent fiber organic matter (as-received basis).

f Amylase-treated neutral detergent fiber organic matter, blank-corrected (as-received basis).

uum tubing to connect manifold to trap (4–18 L) and vacuum source. Place a vacuum reservoir (18 L) between trap and vac- uum source to ensure adequate vacuum capacity to remove foam. Fibertec apparatus may be used for filtration.

(d) Boiling water supply.—Use continuous boiling water

generator as described by Goering and Van Soest (9) or suit- able alternative (Figure 2002.04B). Apparatus must be capa- ble of supplying boiling water (>95°C) in quantity sufficient for all residues to be washed at one time through a nozzle pro- ducing a fine stream (flow rate, 35–40 mL/10 s; a 2.5 mL dis- posable plastic pipet tip makes an acceptable nozzle). A fine nozzle minimizes water needed to transfer particles to cruci- ble, but provides water pressure needed to remove residues at- tached to side of beaker. It is critical that water is boiling when added to crucibles, especially for products containing starches, pectic substances, mucilages, or glycoproteins. (Fibertec users:

Use syringe with cone-spray nozzle to rinse condensers and 60 mL disposable syringe with 12 gauge needle 10 cm long to dislodge any residues adhering to condensers.)

(e) Wash bottle for ND solution.—Rinse down particles

that are carried up the side of beakers by foaming during refluxing. Use fine nozzle to minimize addition of ND solu- tion during rinsing (flow rate, 10–15 mL in 3 s with moderate pressure; unclipped nozzle on 500 mL Nalgene Unitary wash bottle or equivalent is acceptable). (Fibertec users: Use ex-

tended nozzle with larger opening to adequately rinse particles into ND solution during extraction.)

C. Reagents

(a)

Sodium sulfite.—Na 2 SO 3 anhydrous.

(b)

Dried hominy corn (corn grits, raw).—Obtained from

food or grocery store. Must be raw or uncooked grits; pre- cooked or instant grits are not acceptable. Grind grits to pass 1 mm screen in a Wiley cutter mill.

(c) Burke’s iodine solution.—2 g KI and 1 g I in 100 mL

water. Store in amber or opaque bottle.

(d) Stock heat-stable -amylase solution.—Termamyl

120 by Novozyme Corp. (Cat. No. A3403, Sigma-Aldrich, St. Louis, MO 63178), Spezyme FRED by Genecor International (Cat. No. FAA, Ankom Technology Corp., Fairport, NY 14450) or equivalent liquid heat-stable α-amylases or water extracts of heat-stable α-amylase powders.

(e) Working heat-stable -amylase solution.—Standard-

ize heat-stable α-amylase stock solution or enzyme powder extract so that 2 additions of 2 mL will remove raw corn starch from 0.5 g of corn hominy grits. Assay as follows:

(1) Weigh 0.5 g (±0.005 g) ground, dried hominy corn into

each of 6 beakers similar to those used to extract fiber residues described in B(a).

1224 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

: J OURNAL OF AOAC I NTERNATIONAL V OL . 85, N O . 6, 2002

MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

1225

OF AOAC I NTERNATIONAL V OL . 85, N O . 6, 2002 1225 Figure 2002.04B.

Figure 2002.04B.

right consists ofa3L round flask modified to contain 2 standard taper female joints for cold water condensers at the top and a glass column (6 cm diameter, 17 cm length) at the bottom to accommodate a 1000 watt quartz immersion heater, 455 mm long and 19 mm diameter (Cat. No. 16790-1L, Vycor sheathed heating element, Corning Glass, Corning, NY, vycor@corning.com, or equivalent), inserted through the original joint at top. Cold water enters through a port at the bottom of the column and leaves through a port in the upper 2/3 of the round flask. The tank at the left contains a float valve adjusted to maintain a water level in the heating unit about 3/4 full.

Water heating unit. Heating unit on

(2) Preheat calibrated reflux units, prepare ice bath for cooling beakers (must contain enough ice to maintain temper- ature below 1°C), and prepare tempering bath [shallow pan containing enough water at exactly 20°C to exceed depth of solutions in beakers and maintain a final temperature between 19.5 and 20.5°C at the end of step (8)]. (3) Add 50 mL ND solution, (f), (do not add sodium sul- fite), swirl beaker, and place on preheated refluxing apparatus at 1 min intervals. (4) After ND begins to boil (ca 5 min), add one of 6 doses stock or powder extract solution (geometric progression, e.g., 0, 0.025, 0.05, 0.10, 0.20, and 0.40 mL; exact doses will de- pend on source of amylase) to beakers in ascending order. (5) Reflux for 10 min, remove at 1 min intervals; add sec- ond dose of amylase (matching the first), swirl, and rinse sides of beaker using minimum of room temperature ND solution. (6) React 60 s and filter through glass wool or 2 layers of cheesecloth into 100 mL glass beaker. Prepare blank by add- ing 2 intermediate doses, e.g., 0.10 and 0.20 mL stock solu- tion, to 40 mL room temperature ND in 100 mL glass beaker. (7) Place beakers, except blank, in ice bath. Remove from ice bath after 5 min (temperature of solutions should be ca 21°C) and place all beakers in tempering bath (20°C).

(8) After solutions are 20 ± 0.5°C (may take 5 min or more), remove beakers from tempering bath and arrange in or- der of increasing enzyme doses on white background. (9) Quickly add 0.5 mL Burke’s iodine solution to beak- ers, and mix. (10) Do not look at beakers. After 90 s, view through solu- tions from above and make a quick decision (within 30 s) about color of each solution, using the following scale: Purple = not adequate enzyme; pink-amber or amber = not adequate enzyme; pale yellow = adequate enzyme. Compare to blank. Brown tint of enzyme solution should not be confused with pink-amber or amber. If color differ- ences are unclear, place beakers in tempering bath for 5 min and repeat steps (8)–(10). (11) After lowest dose that is pale yellow (V 2 ) and next lowest dose (V 1 ) that is pink-amber or amber are identified us- ing a geometric progression of doses, do final standardization using the dose below the pink-amber one (V 1 ) and a linear pro- gression of doses (0.25*V 1 ) between V 1 and V 2 (e.g., if 0.05 mL treatment is amber [V 1 ] and 0.10 mL treatment is pale yellow [V 2 ], use doses of 0.025, 0.05, 0.0625, 0.075, 0.0875, and 0.10 mL) stock solution in the final standardization. (12) The lowest dose that is pale yellow (and exceeds next highest inadequate dose with pink-amber or amber solution) represents the volume of amylase stock solution or extract (Vs) used to make amylase working solution. (13) Record date, batch or lot of amylase, doses tested, amount of iodine solution used, and color and final tempera- ture (before iodine addition) of each dose in reagent log book. (14) Determine number (n) of test samples to be analyzed in the following 5 days or less. To add 2 mL amylase working solution twice for each test requires a total volume of amylase working solution of (n) × 4 mL. Mix (n) × 2 × (Vs) mL amy- lase stock solution with (n) × [4 – 2 × (Vs)] mL water. Store working amylase solution in refrigerator using stoppered con- tainer no longer than 5 days. (15) Confirm adequacy of amylase working solution by re- peating standardization procedure using 0.5 g corn grits with 0, 2, and 4 mL working solution (each added at boiling and after removal from refluxing apparatus). If there is no appreciable difference in color between 2 and 4 mL working solution, then 2 additions of 2 mL are adequate for the aNDF method. Notes: Time and temperature are critical for proper assess- ment of adequate amylase dose. The solutions must be 20°C and the decision about color must be made within 90–120 s af- ter addition of iodine solution. Pink-amber or amber colors fade quickly, and waiting longer than 120 s before making a decision will result in a dosage that is too low. Initial doses of stock solution or extract may have to be ad- justed, and the standardization rerun. If maximum dose (0.4 mL) results in purple or pink-amber color, extend the geo- metric progression of doses starting at 0.4 mL, and rerun ini- tial standardization. If minimum dose (0.025 mL) is yellow, decrease the geometric progression to between 0 and 0.025 mL, and rerun initial standardization. Each new source or lot of enzyme should be standardized; a single lot that is being used over a period of time should be

1226 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

checked every 6 months for activity. Excess enzyme is not ben- eficial and can be detrimental. Concentrated enzyme solutions are not recommended as working solutions because a 1 drop er- ror when dispensing enzyme contains significant activity that can affect results. Many amylase extracts are crude mixtures that may contain fibrolytic and proteolytic activities. Heat-sta- ble amylase solution must be used in hot liquids (>80°C) to in- activate contaminating enzymes and minimize fiber loss.

(f) Neutral-detergent (ND) solution.—Measure 990 mL

water. Pour ca half of water into a flask, add 4.0 g NaOH,

14.6

g EDTA, 4.56 g dibasic sodium phosphate (Na 2 HPO 4 ),

6.81

g sodium borate decahydrate (Na 2 B 4 O 7 10H 2 O), and mix

until dissolved (heat if necessary). Under a hood, add 30 g so- dium lauryl sulfate and ½ of remaining measured water. Mix until detergent is dissolved and add 10 mL triethylene glycol to suppress foam. Add remaining water and thoroughly mix. Verify that pH is between 6.95 and 7.05, and adjust with con- centrated HCl or NaOH, as required. If pH is off by >0.5, dis- card. Store ND solution at room temperature or, if cool storage causes precipitation, warm to 25°C, and mix before use. Re- cord date ND solution was prepared, pH measurements, and adjustments in reagent log book.

Note: NaOH and EDTA can be replaced with 18.6 g disod- ium EDTA.

(g) Filter aid.—(1) Silica sand.—Sand, cristobalite, acid

purified, 40–200 mesh (Fluka, Buchs, Switzerland, Cat. No. 84880 or equivalent). (2) Glass microfiber mats.—4.25 cm Whatman GF/D or equivalent. (h) Crucible cleaning solutions.—(1) Acid.—Chromic acid [see Definition of Terms (13)] or prepare from 2.5 L H 2 SO 4 and one package Nochromix (Godax Laboratories, Inc., Takoma Park, MD 20912, www.godax.com), or equiva- lent inorganic oxidizer. (2) Alkaline.—Dissolve 5 g Na 2 EDTA2H 2 O 50 g Na 3 PO 4 , and 200 g KOH in 1 L water. Cleaning procedure.—After testing filtration rate of cruci- bles, place crucibles with slow filtration rates in a shallow glass or enamel pan and add ca 40 mL acid cleaning solution to each crucible. Let acid cleaning solution filter through cru- cible and soak for 1 h. Rinse with water by back flushing and retest filtration rate. Clean crucibles with slow rates with the alkaline cleaning solution. Place crucibles in a shallow pan and add 50 mL alkaline cleaning solution and heat to 70–80°C. Let alkaline cleaning solution filter through the cru- cible and then back flush each crucible until it is one-half full of solution. Let solution filter through crucible and back flush crucible until it is one-half full. Let solution filter through cru- cible. Remove from alkaline solution and back flush each cru- cible with water. After it has cooled, alkaline cleaning solution can be saved and reused. Use alkaline cleaning solution spar- ingly because it dissolves glass and weakens the fritted disks in crucibles. Retest crucible filtration rate; do not use those with slow rates for aNDF analysis.

D. Test Sample Preparation

Dry wet laboratory samples at <60°C to prevent creation of artifact fiber. Fiber extraction is affected by particle size of test sample. Grind representative samples to obtain geometric

mean particle size of 220–260 mm. A 1 mm screen in a cutter mill (Wiley) or 2 mm screen in cyclone or centrifugal mill is generally acceptable. Cyclone or centrifugal mills throw parti- cles through the screen at an angle, which effectively reduces the size of the aperture that particles pass through. On material ground to finer particle size, fiber must be determined by microfiber filter mats to minimize particle loss; however, re- sults may have negative bias. Grinding segregates the material, with highest fiber material passing out of grinder last. Do not discard material in grinder; combine it with mate- rial in grinder receptacle. Mix ground material by placing on creased sheet of paper (ca 40 × 40 cm). Lift corners of paper to roll material to diagonal corner, turn paper 90°, and roll; repeat 10 times. Transfer material to suitable container. Ground test samples can be pre-extracted with acetone or ether to remove fat, but do not dry at >60°C. Products contain- ing >5% fat should be pre-extracted; those with >10% fat must be pre-extracted to remove fat. To pre-extract with acetone, weigh test portion into crucible, place on filtering manifold, extract with 40–50 mL acetone 4 times (allow material to soak at least 5 min, and stir 3 times during each soaking), apply vac- uum to remove traces of acetone, air-dry for 10–15 min, en- sure that all traces of acetone are removed, and transfer to re- flux beaker. Use same crucible to collect fiber residue for test portion after ND extraction. If filtering aid is used, dry and weigh it with crucible, and then transfer it to a small beaker before test sample is weighed into crucible. After pre-ex- tracted residues have been transferred from the crucible into the refluxing beaker, replace filtering aid in crucible before fil- tering ND-extracted residues. [Fibertec users: Add silica sand to USP2 crucible, dry and weigh it before adding test sample; then pre-extract with 20–30 mL acetone 4 times (allow mate- rial to soak at least 5 min and stir with back pressure 3 times) using cold filtration device. After removing all traces of ace- tone, transfer crucible containing silica sand and pre-extracted test portion to the hot extraction device for ND extraction.]

E. Determination

Dry at 105°C for >4 h and weigh empty crucibles (hot di- rectly from oven or room temperature after desiccation). Re- cord empty crucible weight for test portions (We) or blanks (Be) to nearest 0.0001 g. Mix material thoroughly and weigh 0.5 (±0.0500) g air-dry feed, or equivalent amount of wet ma- terial into refluxing beaker. (Fibertec users: Weigh test portion into dried and preweighed USP2 crucible.) Record fi- nal weight of test portion to nearest 0.0001 g (S). If results are to be reported on dry matter basis, weigh a second test portion at the same time for dry matter determination. Include in-house reference sample and 2 blanks for first 20–30 test samples in a run and add one reference and one blank for each additional 20–30 test samples. Preheat calibrated reflux units. Add 0.5 (±0.1) g sodium sulfite using calibrated scoop (EKCO Housewares “Pinch” measuring spoon, World Kitchen, Inc., Elmira, NY, www.worldkitchen.com or equivalent) and 50 (±5) mL ND to each refluxing beaker and swirl (critical for starchy feeds that stick to bottom during refluxing). (Fibertec users: Use back

MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

1227

pressure to mix reagents.) Do not add ND and sodium sulfite to test portions more than 60 min before refluxing. Heat to boiling within 4–5 min, add 2 mL working amylase solution, resuspend particles stuck to bottom or sides, and swirl. (Fibertec users: Use back pressure to mix amylase with ND solution and test portion.) Reflux for 60 min at boiling temperature that creates vigorous particle movement, but not excessive foaming that carries parti- cles up the side of the beaker. Mixtures may foam vigorously for 1–2 min (do not reduce temperature of heating unit). Rinse sides of beaker with minimum amount of ND using bottle with fine nozzle 5–10 min after amylase is added, and rinse as needed to resuspend particles on side of beaker (twice maximum). Remove extracted mixture from heating unit and let parti- cles settle for 30–60 s. Before transfer (Fibertec users: Before initial filtration), observe mixture to determine if lipid globules are present on surface or if solution is milky, which indicates that test sample should be rerun after acetone pre-extraction. Place Teflon stirring rod in crucible and preheat by adding 40 mL boiling water for 30–60 s. (Fibertec users: Ignore.) Remove water with vacuum, and immediately decant top 30–40 mL of solution, keeping beaker inverted over crucible. (Fibertec users: Ignore.) Use minimum vacuum to evacuate ex- cess liquid, but close vacuum before residue becomes dry. Note: Excessive vacuum and evacuating to dryness cause some residues to clog crucible and not wash properly. Rinse all unat- tached particles into crucible using fine stream of boiling water. (Fibertec users: Ignore.) Fill crucible half full with hot water. Add 2 mL working amylase solution and stir. (Fibertec users:

Use back pressure to mix amylase in initial water soak.) React with amylase for minimum of 45–60 s while scrap- ing remaining particles from bottom and sides of reflux beaker with rubber policeman. Evacuate amylase solution and trans- fer any remaining residue from reflux beaker into crucible with 20–30 mL boiling water. Two rinses are usually suffi- cient. After transferring residues from beaker, fill crucible 3/4 full with boiling water and soak for 1–3 min. (Fibertec users:

Remove amylase-water soak after minimum reaction of 60 s. Crucibles can be removed from hot to cold filtration unit for remaining hot water soaks for residues that are easy to filter. This allows next set of test samples to begin ND extraction on hot filtration unit. Residues that are difficult to filter can be washed on Fibertec heating unit with heat reduced to mini- mize particle agitation.) Evacuate water, add 40–50 mL boiling water, soak 3–5 min, and repeat. If residues are difficult to filter after first soak, add additional 2 mL working amylase solution. If resi- dues appear translucent and become more difficult to filter with each additional soaking, eliminate third water soak. If plugged, crucible can be back-flushed by removing it from fil- ter manifold and reinserting it. (Fibertec users: Use minimal back pressure to open crucibles and improve filtration.) Evacuate water, refill crucible with 40–50 mL acetone, stir to disperse particles, soak 3–5 min, and repeat, rinsing stir rod to remove attached fiber particles. (Fibertec users: Move cru- cibles to cold extraction unit for acetone soaks.) Note: After the last water soak, do not evacuate fiber residues to dryness,

but remove water to leave a damp or moist residue, before adding acetone. Excessive evacuation clumps the residues and makes acetone extraction difficult. Vacuum residue dry, remove crucible from manifold, and air dry for 10–60 min to remove acetone. Dry crucibles at 105°C for minimum of 8 h and weigh (Wf and Bf). Ignite cruci- ble and fiber in 500°C furnace for 5 h or until C-free. Temper in 105°C oven for at least 1 h and weigh (Wa and Ba). Weigh cru- cibles containing fiber or ash residues (hot or desiccated to room temperature) in same order as empty crucibles. Modifications for specific types of test samples.—(a) If extracted ND solution appears milky and opaque and filtration is slow during transfer of residues or after first water soaking, high starch is suspected. Add additional treatment with 2 mL amylase during second water soaking. Shorten soaking times to minimum to keep soaking solutions as hot as possible (>85°C).

(b) If residue clogs crucible during transfer and additional

amylase does not improve filtration, feed material may contain proteinaceous, gum, or mucilage residues (meat products and some oil seed meals). Preheating crucible with boiling water is crucial for filtering these materials. The best filter aid for these materials is 12–15 g (6–8 g for Fibertec USP2) of silica sand,

C(g)(1). The gummy substances in these feed materials will stick to sand particles which prevents them from clogging the fritted disk and allows residues to be washed. All filter aids must be added to crucibles (including blanks) before initial weights are recorded.

(c) If fiber residue has glossy, translucent sheen and filtra-

tion becomes more difficult with each water soaking, pectic substances are suspected. Preheat crucible with boiling water and transfer residues as quickly as possible without settling when removed from reflux unit. Reduce all soaking times to minimum to maintain temperature >85°C to prevent cooling and jelling in crucible. Filtering aids may improve filtration (in order of preference): 12–15 g (6–8 g for Fibertec USP2) silica sand, 0.25 g (0.15 g for Fibertec USP2) glass wool, and glass microfiber mats, C(g)(2).

(d) If fat globules are observed floating on surface of ND or

wash water and residue is difficult to filter, or if material is known to contain >10% fat, pre-extract it with acetone or ether (see D).

(e) If material contains fine particles, flocculant precipitates,

dirt (fine clay), or fecal matter, but not pectic substances or starch, increase settling time to maximum of 2 min after removal from refluxing unit and use filter aid in crucible. Filter aids (in or- der of preference) include glass microfiber mats, ceramic fiber, 12–15 g silica sand, and 0.25 g glass wool. Microfiber mats can be gently scraped to renew surface during filtration.

(f) If all other modifications fail, reduce test portion amount

to 0.3 g and repeat analysis with filter aid in crucible. Reducing test portion will magnify effects of weighing errors and increase variation in results. Sometimes reducing test portion amount and increasing ND to 70–100 mL is beneficial. If fiber is <1.5%, do not reduce test portion amount, and if filtration is not possible, report results as “difficult to analyze, fiber <1.5%.”

(g) Do not add acetone before all rinse water has been re-

moved. Although this occasionally improves filtering, it does not remove detergent or detergent solubles from residues.

1228 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

Adding acetone before water washing is complete will give inflated fiber values.

F. Calculations

Without blank correction:

% aNDF (as-is basis) = 100 (Wf – We)/S

where aNDF is amylase-treated neutral detergent fiber and S is g as-is test portion weight.

% aNDF (DM basis) = 100 (Wf – We)/(S*DM)

where DM is (g oven-dried matter weight/g air-dried or wet test portion weight).

% aNDFom (as-is basis) = 100 (Wf – Wa)/S

where aNDFom is aNDF organic matter.

% aNDFom (DM basis) = 100 (Wf – Wa)/(S*DM)

With blank correction (average all blanks before calculat- ing results):

% aNDF (as-is basis) = 100 (Wf – We – Bf + Be)/S

where aNDF is amylase-treated neutral detergent fiber, S is g as-is test portion weight, and Bf and Be are averages of all blanks in the run.

% aNDF (DM basis) = 100 (Wf – We – Bf + Be)/(S*DM)

where DM is (g oven-dried matter weight/g air-dried or wet test portion weight).

% aNDFom (as-is basis) = 100 (Wf – Wa – Bf + Ba)/S

where aNDFom is aNDF organic matter.

% aNDFom (DM basis) = 100 (Wf – Wa – Bf + Ba)/(S*DM)

Results should be reported to nearest 0.1%, and results <1.5% aNDF or aNDFom should be reported as “aNDF <1.5%” or “aNDFom <1.5%.” When fiber is <25%, blank correction is required. Crucible weights after ashing (Wa) that are less than empty crucible weights (We) indicate that crucibles lost weight dur- ing the aNDF procedure. The crucible weight after ashing (Wa) is the most accurate estimate of the correct crucible weight for calculating fiber and must be used to calculate aNDFom. In this circumstance, aNDFom is a more accurate estimate of fiber than is aNDF. If results for in-house reference or quality control samples are outside of control limits, do not report results; re-analyze test samples.

G. Quality Assurance

Maintain log of reagent preparation and amylase standard- ization. Check pH of each batch or lot of ND solution and ad- just as needed. Determine activity of each amylase source and

lot in hot ND solution and adjust amylase working solution ac- cordingly. Check activity of stock amylase every 6 months during storage, and adjust working solutions accordingly. Include at least one in-house reference or quality control (QC) sample and 2 blanks for the first 20–30 test samples in a run, and add one QC and one blank for each additional 20–30 test samples analyzed. Suitable QC materials include brewer’s grains, grass hay, or corn silage that has been dried at <60°C. Each of these materials is sensitive to changes in reagents and technique, but corn silage is preferred because it contains starch and grass cell walls. Acceptable standard deviation (SD) among repeated analyses of the reference material should be ±1.00% aNDF. Plot QC sample results on X-control chart and examine chart for trends. Results outside of upper or lower warning limits (±2.00 standard deviations) are evidence of possible problems with the analytical system. Results out- side of upper or lower control limits of ±3.00 SDs indicate loss of QC, and results of the run should be verified. Two consecu- tive analyses falling on one side of the mean between warning limits and control limits also indicate loss of QC. Include at least one set of duplicates in each run if single determinations are being made. Duplicates should not be run consecutively, but one replicate should be spaced at the begining and end of the run. Acceptable differences among duplicate analyses ranges from 1.50%-units for materials with <20% NDF to 3.00%-units for materials with >70% NDF. Change in weights of blank crucibles should be <±0.0100 g after either ND extraction or ashing. If weights blank crucibles change by >10 mg, or crucible weights after ashing are less than empty crucible weights, suspect inadequate cleaning of crucibles or problems with weighing technique. Ref.: J. AOAC Int. 85, 1221–1228(2002)

Results and Discussion

Familiarization Study

In the familiarization study, collaborators were provided 6 materials with a mean value and acceptable range for each. The mean value and acceptable range were determined in the Study Director’s laboratory based on duplicate or triplicate analyses of 4 randomly selected sets of materials (Table 5). Except for MB and RS, variation among sample sets was less than replication variation within sets, as indicated by the ob- servation that s R was equal to s r . For RS there was a small, but insignificant variation among sample sets. The variation among samples sets of MB was about 40% of the reproducibility standard deviation (RSD R ), indicating a lack of homogeneity among sample sets. The large s r and HORRAT for MB and RS indicate that samples of these mate- rials were less homogeneous and more difficult to analyze for aNDF than other familiarization materials. Collaborators were asked to analyze the materials using the aNDF method and to repeat analyses using modifications given in the method until they achieved acceptable results. The collaborators’ results in Table 5 are the best 2 or 3 results selected from all analyses they performed. However, several participants did not follow instructions and only provided one set of duplicate results.

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Table 5.

best 2 or 3 results for collaborator laboratories on materials in the familiarization study

Statistical data for 4 random sample sets of materials analyzed in the Study Director’s laboratory and the

Sample ID a

n

Mean, %

s r

s R

RSD r , %

RSD R , %

r b

R c

HORRAT d

SD–BH

4

54.3

0.78

0.78

1.44

1.44

2.20

2.20

0.66

SD–AS

4

43.4

0.36

0.36

0.82

0.82

0.99

0.99

0.36

SD–CS

4

38.9

0.20

0.20

0.52

0.52

0.57

0.57

0.23

SD–CG

4

8.0

0.18

0.18

2.29

2.29

0.51

0.51

0.78

SD–MB

4

19.5

1.50

2.42

7.70

12.39

4.20

6.76

4.84

SD–RS

4

19.5

0.81

0.88

4.13

4.49

2.26

2.46

1.76

Col–BH

13

54.6

0.69

0.98

1.26

1.79

1.93

2.73

0.82

Col–AS

13

43.4

0.79

1.06

1.82

2.45

2.21

2.98

1.08

Col–CS

13

39.2

0.73

0.85

1.85

2.17

2.03

2.38

0.94

Col–CG

12

8.1

0.41

0.62

5.08

7.60

1.16

1.73

2.60

Col–MB

11

22.4

2.77

6.68

12.40

29.85

7.77

18.70

11.91

Col–RS

10

20.3

1.15

3.29

5.67

16.19

3.23

9.21

6.37

a SD = Study Director’s laboratory; BH = barley hay; AS = alfalfa silage; CS = corn silage; CG = corn grain; MB = meat and bone meal; RS = roasted soybeans; Col = collaborators’ laboratories.

b 2.8 × s r .

c 2.8 × s R .

d Ratio of RSD R divided by expected RSD from the Horwitz equation (16).

Two forage samples, BH and AS were selected to represent feeds that were easy to analyze. Deviations from the results of the Study Director’s laboratory were used to determine if labo- ratories were following the aNDF procedure exactly. The NDF method is commonly used for feed analysis, and most of the participating laboratories were determining NDF routinely. There was concern that these laboratories would use their method rather than the aNDF method that was being evaluated. For BH, only one participant exceeded the Study Director’s mean +2.8 × s R , and both the mean and s R of all collaborators agreed closely with those of the Study Director (Table 5). For AS, the results of the collaborators were more variable than that of the Study Director. Two laboratories exceeded the Study Di- rector’s mean +2.8 × s R , and 2 were less than the Study Direc- tor’s mean –2.8 × s R . The s R of all collaborators for AS was nearly triple that of the Study Director, and their HORRAT ap- proached 2 (Table 5), suggesting that they were not following the aNDF method exactly. Comments on a questionnaire ask- ing about specifics of their technique confirmed that collabora- tors were deviating from the proposed method, and they were requested to follow the aNDF method exactly. Two samples, CS and CG, were included in the familiar- ization set to determine the ability of collaborating laborato- ries to analyze feeds containing starch. The mean values for the collaborators were similar to the Study Director’s values, but the collaborators’ s R was 3–4 times higher than the Study Director’s (Table 5). Several laboratories had to analyze the samples numerous times to achieve acceptable results. Some participants only analyzed the samples in duplicate one time whether or not they achieved acceptable results. Thus, 4 of the

14 collaborators had unacceptably high values for CS and 3 were unacceptably high for CG. One participant obtained un- acceptably low values for CS or CG. The high aNDF results suggest that starch was not adequately removed from fiber residues. This raised concern that amylase standardization by the participants was not adequate. However, from question- naire responses there did not seem to be a relationship be- tween high results and the dose of amylase used by collabora- tors. If high results were not related to amylase, this suggests that filtering difficulties caused problems or that participants were not following the aNDF method exactly. Two samples, MB and RS, were known to be extremely difficult to analyze and were included in the familiarization study to test the limits of the collaborator’s ability to handle difficult samples. The mean results of the collaborators were higher than those determined by the Study Director’s labora- tory (Table 5). For MB and RS, 3 participants reported unac- ceptably high values. For RS, 2 collaborators reported unac- ceptably low values. The s R of the collaborators was 3–4 times greater than that observed by the Study Director. Some partici- pants analyzed these materials 5 or more times without obtaining acceptable results. Their inability to match the Study Director’s results for CS, CG, MB, and RS convinced most laboratories that their method of measuring NDF was inadequate. All collabora- tors were requested to follow the aNDF method exactly when an- alyzing the collaborative study samples. It was evident from both the results of the familiarization study and comments made by the collaborators that the aNDF method needed to be modified so that laboratories could ana- lyze extremely difficult samples. After the familiarization

1230 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

study, the aNDF method was modified to improve the analysis of materials that are difficult to filter. Although glass microfiber GF/D mats were used successfully in the Study Director’s labo- ratory, they filtered slowly and were not an acceptable filter aid for some collaborators. Sand had been tried previously by the Study Director as a filter aid without success when it was mixed with the sample. There also were concerns that fine sand parti- cles or celite might plug the pores in the fritted disks of Gooch crucibles. However, a source of acid-washed silica sand with particle size distribution between 40 and 200 mesh was found, which cannot penetrate and plug the pores of coarse porosity Gooch crucibles. The sand was stable to ND extraction and ashing, and had a negligible effect on blank values. This sand was evaluated as a filter aid for the difficult-to-filter samples in the familiarization and collaborative studies (MB, RS, CB, and DM) and greatly improved filtration and duplicate standard de- viations (SDs). The sand was also evaluated with 6 other mate- rials that are extremely difficult to filter. In all cases, filtration was easy and SDs of duplicate analyses were much smaller (pooled SD = 0.44 versus 0.77) than was possible without the sand filter aid. The aNDF protocol was modified to include sil- ica sand as a filtering aid. Collaborating laboratories were asked to reanalyze MB and RS using sand as a filtering aid. With sand, their s r decreased from 1.15 to 0.77 for RS and their s R de- creased from 6.68 to 2.68 for MB.

Amylase Standardization

Development of the aNDF method was hampered by changes in the sources of heat-stable α-amylase. Sources would become unavailable or simply be changed by the pro- vider, making it difficult to specify a source and level of en- zyme needed for the aNDF method. To eliminate dependence of the aNDF method on a specific source of amylase and to comply with AOAC policy for Determination of Equivalency of Products Used in Official Methods, we developed a proce- dure for standardizing the heat-stable amylase. Because many feed testing laboratories do not have colorimeters, we devised a simple visual test for standardizing the amylase. In addition, we found that ND interferes with measurement of starch in most colorimetric methods, and concluded that a visual test would encourage routine evaluation of the amylase. The procedure for standardizing amylase for the aNDF method is based on the reaction of iodine with starch. A proce- dure was developed that would ensure that raw corn starch (one of the most difficult starches to remove from fiber) would be solubilized and extracted in hot ND solution. It became evi- dent that the starch-iodine complex that creates a purple color was unstable in ND solution, making visual detection of resid- ual starch difficult and time dependent. The original amylase standardization procedure was evaluated in the Study Direc- tor’s laboratory and gave variable results among technicians. Additional research indicated that the procedure was sensitive to both time and temperature; therefore, it was extensively re- written before the collaborative study began. Collaborating laboratories were asked to evaluate 3 amylase stock solutions during the familiarization study to evaluate the adequacy of the amylase standardization procedure (Table 6).

Doses of 3 amylase stock solutions

determined by collaborating laboratories to provide adequate enzymatic activity for the aNDF method during the familiarization study and actual doses used in the collaborative study

Table 6.

Familiarization study

doses, µL

Collaborative study

doses, µL

Lab

Amylase A Amylase B Amylase C

Amylase A Amylase B

0

100

60

120

100

1

75

500

400

200

3

100

200

400

200

4

300

500

500

300

5

75

125

150

250

6

200

150

300

150

7

50

125

200

175

8

30

150

100

31

9

200

200

200

100

10

400

88

200

11

88

88

200

88

12

300

200

400

200

13

300

200

200

250

14

200

100

150

125

Mean

178

202

262

176

170

SD

119

139

124

110

54

The mean and median dose of amylase selected by the par- ticipants were higher than those selected in the Study Director’s laboratory. Most collaborators overestimated the dose of amy- lase needed for the aNDF method. This is acceptable because too much amylase is better than too little, although there is a concern that doses >5 times the minimum dose needed may cause retrogradation of starch hydrolysis. Except for Labora- tories 4, 5, and 8, standardization of amylase for the collabora- tive study was much more consistent. Laboratories 4 and 8 may not have restandardized the amylase for the collaborative study samples and simply used the information they collected during the familiarization study. Laboratories 3, 6, 7, and 10–12 ob- served that amylase C was half the activity of amylase B. Several collaborators commented that amylase standard- ization was difficult. The procedure was rewritten to clarify the steps. There was some indication that participants could not discriminate between the pink-amber of unhydrolyzed starch and the brown of the stock enzyme solutions. Based on results of the familiarization and collaborative studies, we modified the procedure for standardizing amylase for the aNDF method to indicate that a control solution of amylase in blank ND (no grits) should be run with the test doses to improve discrimination between pink-amber (inadequate amylase) and brown (amylase solution). Although the range in doses of amy- lase used was greater than desired, there was no significant rela-

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tionship in the familiarization study between dose of amylase and aNDF results across samples and laboratories.

Evaluation of the aNDF Method

Results of aNDF analyses were calculated 4 different ways to evaluate the effects of blank correction and adjusting fiber to an organic matter (ash-free) basis (Tables 7–10). Outliers were detected by the Cochran and Grubb’s tests in AOAC spreadsheet software and are shown in Tables 7–10. Suspect results were identified as having large residuals (>3*root mean squared error) in an ANOVA using Model 3 (see Statis- tical Analyses section) with main effects of Material, Lab, and Rep. These latter results were not removed when AOAC sta- tistics were calculated. To reduce the effect of single suspect results on laboratory ranking and means, the results in bold font were removed. An ANOVA using the main-effects model (Model 3) was used to calculate least-squared means for laboratories with all suspect data removed. All outlier data were double checked to verify data entry and to determine if collaborators’ comments could explain the aberrant results. The differences between blind duplicates for grass hay (GH) and sawdust (SD) reported by Laboratory 11 were not de- tected as outliers by ANOVA or AOAC statistical methods. Laboratory 12 had significant or nearly significant Grubb tests for several materials and reported the highest results for most materials. The laboratory ranking test (14) indicated that Laboratory 12 was a statistical outlier. Although it reported consistent results for blind duplicates (except for RS), it had an average bias of +1.99 (±1.21) %-units for all materials, and questionnaire results indicated that it used medium porosity Gooch crucibles with ceramic fiber as a filter aid. Neither of these modifications was indicated in the aNDF method for collaborative study. Based on both statistical and methodolog- ical reasons, the results of Laboratory 12 were not used in the statistical evaluation of the aNDF method. Most outliers occurred in 2 laboratories (8 and 9), and in each case only one of the blind duplicates was suspect. This suggests that the aNDF method and its description may not be at fault, because most remaining collaborators produced ac- ceptable results; even the 2 laboratories with problems gener- ated one acceptable result for all materials. Horwitz et al. (12) reported that the reproducibility of empirical methods such as fiber is generally much poorer than for analytes such as nitro- gen, which are well defined and have suitable reference stan- dards. Because the analyte is defined by the method, it is cru- cial that fiber methods be followed exactly. As Horwitz et al. (12) surmized, laboratories often modify methods to fit lab- oratory conditions, attempt to improve the method by elimi- nating or changing steps or apparatus, or simply ignore details of the method that are thought to be superfluous. Although the familiarization study helped to establish that the aNDF method should be followed as described, we suspected that some laboratories used modifications to accommodate their unique laboratory situation. A questionnaire was used to dis- cover exactly how each participant measured aNDF and the responses were used to explain atypical results.

For aNDF, the HORRAT was <2 for all materials except DM, RS, and MR (Table 11). Horwitz suggested that a HORRAT of 2 is a reasonable, if not liberal, criterion for ac- cepting a method. The feeds with HORRAT >2 had mean aNDF <15%. The smaller means for aNDF of these materials greatly inflated the observed RSD R . The Horwitz (16) equa- tion for calculating the expected RSD was developed by re- gressing s R versus the mean concentration across analytes and methods. Implicit in this equation is the assumption that limits of detection (LODs) decrease and precision increases as the mean of the analyte decreases. In general, instrumental meth- ods for measuring analytes in parts-per-billion and smaller concentrations (near zero analyte) have very small LODs and very good precision. However, within a method, the relation- ship between precision and mean concentration may differ from the Horwitz equation, especially if the method is gravimetric and attempts to measure analytes from 0 to 100% concentrations. As discussed by Horwitz et al. (12), gravimetric methods have LODs and precision that are related to weighing error. Thus, within the aNDF method, it may be more valid to evalu- ate acceptability across materials by comparing s R rather than RSD R . For aNDF, the s R for DM, RS, and MR are similar to those for other materials, indicating that the method can be used to analyze feeds with <15% aNDF with precision similar to those with >15% aNDF. Blanks may account for systematic weighing variation among runs. The potential magnitude of blank variation within a single laboratory is indicated in Tables 3 and 4. Blank varia- tion among runs in the Study Director’s laboratory (2.8 × s R ) is about 4 mg. The blank variation among collaborating laborato- ries was greater with a 2.8 × s R of 10 mg (Tables 9 and 10). Be- cause weights of fiber residues are small, the effect of adjusting for systematic weighing variation using blanks should be greater for materials with low aNDF. Assuming a 0.5 g sample and 25% aNDF in the material yields a fiber residue of 125 mg, this could result in a range in blank-correction of about 8%. If the material contains 10% aNDF, the fiber residue would weigh only 50 mg and the range in blank-correction could be 20%. When aNDF was blank-corrected (aNDFbc), the average s R for materials containing <25% aNDF decreased from 1.30 to 1.01 (Table 11). Thus, blank-correction increases analytical precision for materials with <25% aNDF. For unexplained reasons the s R for materials containing >25% aNDF increased from 1.29 to 1.36 due primarily to an increase in s R for brewer’s grains (BG). It is difficult to envision how correcting for blanks could harm the precision of materials with >25% aNDF, given that the magnitude of blank-correction diminishes as the fiber residue increases. The HORRAT of aNDFbc was <2 for all materials except BG, DM, MR, and RS (Table 11). The HORRAT increased slightly for BG, but decreased for DM, MR, and RS when aNDF was blank-corrected. The nutritional definition of fiber, which is the guide for developing appropriate methods, indicates that fiber is the in- digestible or slowly digesting fraction of a feed that occupies space in the digestive tract of animals; therefore, indigestible ash should be included in fiber. However, fiber traditionally

1232

Table 7.

Results of collaborating laboratories for amylase-treated neutral detergent fiber (aNDF) expressed on as-received basis

MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002

aNDF, % Collaborating laboratory

Feed

Duplicate

0

1

3

4

5

6

7

8

9

11

12 a

13

14

Alfalfa silage

1

40.51

41.13

41.11

40.08

42.36

38.43

41.49

41.29

39.19

40.92

44.57

39.60

39.84

2

40.26

40.20

40.82

40.88

41.26

40.66

41.43

42.57

40.71

42.20

45.85

41.40

41.20

Brewer’s grains

1

49.67

46.55

50.19

49.00

52.76

49.25

49.46

48.60

b

46.28

51.41

52.25

52.00

48.87

2

47.11

49.30

50.37

48.25

50.64

49.10

44.92 c

16.18

b

46.78

47.05

54.11

50.40

47.98

Citrus and beet pulp

1

29.30

30.13

29.69

28.73

31.42

29.06

27.57

29.33

28.45

29.96

31.91

31.20

30.46

2

28.64

30.77

30.63

31.68

30.05

30.87

29.58

29.17

29.67

28.58

32.73

32.00

28.84

Corn grain with cob

1

21.13

21.91

22.79

21.89

21.79

19.60

21.57

22.20

25.50

b

22.08

23.72

19.80

21.92

2

22.03

21.60

23.06

21.69

21.81

19.93

21.62

21.47

20.61

b

21.12

25.80

21.40

22.10

Corn silage

1

36.43

36.82

38.38

37.08

37.89

36.81

35.94

37.24

35.98

38.88

39.28

37.20

37.10

2

36.05

35.92

38.03

36.42

37.36

35.52

38.66

38.22

35.54

37.23

39.64

37.60

37.42

Corn stalks

1

71.42

72.68

74.30

70.85

74.00

72.05

73.92

74.20

b

72.48

74.56

75.03

74.40

73.04

2

70.83

73.40

74.15

71.93

72.83

72.21

73.38

98.79

b

73.22

73.05

76.49

74.00

72.26

Dairy mixed feed

1

12.30

12.40

12.90

11.79

14.22

10.87

11.79

13.27

11.14

13.45

16.26

14.60

12.23

2

11.74

12.78

11.62

9.06

12.17

13.18

13.90

14.44

11.76

12.92

15.86

14.80

12.50

Grass hay

1

55.66

57.57

58.06

58.21

57.57

57.75

58.79

57.40

48.46

b

59.65

59.96

59.60

57.56

2

55.41

57.56

58.90

59.48

58.44

58.21

57.62

59.52

56.87

b

55.92

62.16

60.40

58.46

Milk replacer

1

0.14

0.52

0.62

–0.45

0.14

–0.38

–1.36

0.66

d

0.00

0.18

1.37

–2.40

0.36

2

0.02

0.12

0.28

0.38

0.12

–1.83

0.78

3.72

d

0.40

0.12

1.65

–0.80

0.30

Roasted soybeans

1

12.45

12.75

13.95

8.59

e

19.13

b

11.31

14.59

11.91

15.73

15.09

19.11

12.40

13.54

2

11.96

13.63

15.92

11.93

e

12.05

b

10.82

16.78

11.74

17.83

c

14.69

26.00 c

12.20

13.24

Sawdust

1

92.01

88.23

90.54

90.49

90.59

91.01

87.93

e

87.93

90.61

b

88.01

90.19

90.20

90.60

2

89.87

89.33

90.44

90.99

90.48

90.98

83.70

e

89.79

75.10

b

93.46

92.15

92.60

91.32

Least-squared means f

37.95

38.42

39.40

38.33

39.27

37.97

38.88

38.80

37.99

39.12

41.66

39.30

38.69

a Outlier detected by laboratory ranking.

b Outlier detected by Cochran test.

c Outlier detected by analysis of variance to determine least-squared means, but not removed for AOAC statistics.

d Outlier in Tables 8–10.

e Outlier detected by single Grubb test.

f Least-squared means calculated without data in bold font.

Table 8.

Results of collaborating laboratories for amylase-treated neutral detergent fiber organic matter (aNDFom) expressed on as-received basis

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aNDF, % Collaborating laboratory

Feed

Duplicate

0

1

3

4

5

6

7

8

9

11

12 a

13

14

Alfalfa silage

1

38.70

39.37

39.70

39.96

40.48

37.24

39.73

39.55

38.15

39.66

42.02

40.80

37.88

2

38.25

38.45

39.40

39.46

39.48

40.35

39.23

40.62

39.75

40.41

43.04

39.60

39.28

Brewer’s grains

1

48.20

45.30

49.27

48.74

51.64

48.36

48.10

48.04

b

45.94

50.26

50.72

53.00

49.20

2

45.51

48.05

49.20

49.60

49.36

48.79

43.52 c

16.02

b

45.30

46.29

52.34

49.40

47.20

Citrus and beet pulp

1

27.20

27.80

27.54

27.68

28.80

27.92

25.49

26.95

27.11

28.40

28.22

29.40

27.42

2

25.95

28.50

28.90

28.53

27.15

29.25

26.74

27.66

26.93

26.98

29.03

28.60

27.12

Corn grain with cob

1

20.88

21.55

22.49

22.71

21.57

20.89

21.23

21.45

25.76

b

21.71

22.76

21.20

21.40

2

21.32

21.25

23.02

22.16

21.63

21.55

20.96

21.25

20.87

b

21.04

24.57

21.60

21.78

Corn silage

1

35.76