ANIMAL FEED

SCIENCE AND
TECHNOLOGY

ELSEVIER Animal Feed Science Technology 57 (1996) 347-358

Standardization of procedures for nitrogen

fractionation of ruminant feeds
G. Licitra a, T.M. Hernandez b, P.J. Van Soest bp*
a Uniuersitci di Catania, I.S.T.PA. - Progetto IBLEO, Via Val di Savoia 5, 95123 Catania, Italy
b Cornell University, 324 Morrison Hall, Ithaca, NY 14853 USA

Received 1 November 1994;accepted 26 April 1995

Abstract

The Cornell Net Carbohydrate Protein Model (Chalupa et al., 1991; Sniffen et al., 1992) has
developed the need for uniform procedures to partition feed nitrogen into A, B, and C fractions
(Pichard and Van Soest, 1977). While carbohydrate fractions are relatively standardized (based on
NDF, ADF with corrections for ash, protein, and lignin), the fractionation of plant nitrogen has
been open to considerable variation in procedures. This has led to non-uniformity among reported
values for nitrogen fractions. This paper recommends reliable procedures for nonprotein nitrogen
(NPN) and buffer-soluble protein. These procedures have been examined for reproducibility and
relevance to biological expectations. Procedures for acid-detergent insoluble nitrogen (ADIN), and
neutral-detergent insoluble nitrogen (NDIN) am also included as they are required for the model.
Some alternatives in certain procedures are offered.

1. Introduction

Specific information on the contents of nonprotein nitrogen (NPN), true protein,

protein degradability, cell wall bound protein, etc. are dependent on preliminary
preparation and separation of nitrogenous components in the sample. The separation of
protein and nitrogen fractions as used by the Cornell Net Carbohydrate Protein Model is
shown in Table 1. Nonprotein nitrogen is denoted as the A fraction while true protein is
broken down into B,, B, and B, fractions based on decreasing solubility. The respective
fractions are dependent upon the estimation of insoluble nitrogen, true protein, and the
nitrogen residual in ADF and NDF. The nitrogen that is insoluble in acid detergent is

* Corresponding author.

0377-8401/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved

SSDI 0377-8401(95)00837-3
348 G. Licitra et al./Animal Feed Science Technology 57 (1996) 347-358

Table 1
Partition of nitrogen and protein fractions in feedstuffs
Fraction Abbr. Estimation or definition Enzymatic Classification a
Degradation
Nonprotein N NPN Not precipitable Not applicable A
True protein TP Precipitate with tungstic
acid
Tme soluble protein BSP Buffer soluble but precip- Fast B,
itable (TP-IP)
Insoluble protein IP Insoluble in buffer
Neutral detergent soluble IP-NDIP Difference between IP Variable B2
protein and protein insoluble in
neutral detergent (ND)
ND insoluble protein, but NDIP- Protein insoluble in ND Variable to Slow B,
soluble in AD ADIP but soluble in acid deter-
gent (AD)
Insoluble in acid deter- ADIP or Includes heat-damaged Indigestible C
gent ADIN protein and nitrogen asso-
ciated with lignin

a According to Pichard and Van Soest, 1977, and Van Soest, 1994.

denoted as the C fraction, and is assumed to be indigestible. A series of preparatory

procedures to obtain respective NPN and protein components follow with some altema-
tives.

2. Nonprotein nitrogen OWN)

2.1. Materials and methods

2.1 .I. Definition

Nonprotein nitrogen is traditionally the nitrogen passing into the filtrate after
precipitation with a protein specific reagent. Feedstuffs may contain a wide variety of
low molecular weight nitrogenous substances (Hughes, 1970; Hegarty and Peterson,
1973). Another issue is the inclusion of peptides in NPN. Krishnamoorthy et al. (1982)
used trichloroacetic acid (TCA) which includes them in NPN since they are not
precipitated. The view here is that they are metabolically closer to soluble protein. Many
rumen bacteria (particularly cellulolytics) do not take up single amino acids, but require
peptides, an observation that favors the tungstic acid method which recovers peptides in
the protein precipitate (Greenberg and Shipe, 1979). Certain protein sources particularly
fish meal and blood-feather-meat-bone mix exhibit important differences between the
two methods indicating presence of peptides. The TCA method is included here as it
may be relevant to a future partition of the B, class into peptides and soluble protein.

2.1.2. Review of procedures for determination

The determination of NPN depends upon precipitation of true protein by a suitable
precipitant, filtration and determination of the insoluble nitrogen in the residue. The
 G. Licitra et al. /Animal Feed Science Technology 57 (1996) 347-358 349

NPN is calculated as the difference between the total crude protein nitrogen and the
value of the precipitated true protein nitrogen. A variety of precipitating agents for
protein have been employed (Hawk et al., 1947; Greenberg and Shipe, 1979). These
include tungstic acid, trichloroacetic acid, copper hydroxide, zinc-barium hydroxide and
others. The choice of method depends on the kind of procedure and objectives being
followed. Tungstic and trichloroacetic acids (TCA) are the most common precipitants
applied to feeds. As mentioned, these reagents differ in respect to the cut-off in
molecular size. Tungstic acid cuts off at about a peptide size of 3 amino acids, while the
TCA cuts off around 10 amino acids, depending on the amino acid profile of the peptide
(Greenberg and Shipe, 1979; Marais and Evenwell, 1983).

2.1.3. Recommended procedure for determination of NPN using tungstic acid

2.1.3.1. Apparatus. Erlenmeyer flask (125 ml>, Whatman #54 or 541 filter paper,
analytical balance, pH meter, filter funnels, Kjeldahl apparatus.

2.1.3.2. Reagents.
1. Sodium tungstate (Na,W0,2H,O) (100 g 1-l) solution in water, 0.30 M.
2. 0.5 M sulfuric acid (H,SO,).

2.1.3.3. Procedure.
1. Weigh 0.5 g dry ground sample into a 125 ml Erlenmeyer flask.
2. Add 50 ml of cold distilled water.
3. Add 8 ml of 10% sodium tungstate solution ‘.
4. Let flask stand at 20-25°C for 30 min.
5. Bring pH to 2 by adding 10 ml of 0.5 M sulfuric acid (check pH with pH meter).
6. Let flask stand overnight at room temperature.
7. Fold Whatman #54 or 541 filter paper and place in a conical funnel. Thoroughly
wet paper with distilled water before adding any sample. Filter by gravity; or with
mild vacuum. If first filtrate is cloudy return to the filter funnel and refilter. If
vacuum is used, separate filter flasks must be used, so that any cloudy filtrate can be
recycled through the funnel.
8. Wash residue twice with cold distilled water.
9. Transfer paper to Kjeldahl flask and determine residual nitrogen.
10. Calculate NPN by subtracting residual nitrogen from total nitrogen. Value of NPN
may be expressed as crude protein (N X 6.25) or as percent of total feed nitrogen.

2.1.4. Alternative determination of NPN using trichloroacetic acid (TCA)

2.1.4.1. Apparatus.
1. Erlenmeyer flask (125 ml), Whatman #54 or 541 filter paper, analytical balance,
filter funnels, Kjeldabl apparatus.

’Samples of low protein content ( < 20% CP) can be treated with 5 ml of sodium hmgstate solution and
6-7 ml of 0.5 M sulfuric acid.
350 G. Licitra et al./Animal Feed Science Technology 57 (1996) 347-358

Table 2
Comoarison of NPN values. All values as oercent (N X 6.25) of drv matter
Feeds Crude NPN by hmgstic acid NPN by
protein l/2 h b ppt 16 h 3 h ’ ppt 16 h trichloro-
old ’ method
acetic acid

Alfalfa silage 20.8 9.2 9.4 8.9 9.8

Blood feather meat bone mix 80.6 3.2 0.2 1.3 5.6
Cottonseed meal 45.1 2.8 3.2 3.3 4.4
Distiller’s grains 26.4 2.9 2.4 2.2 2.2
Fish meal 64.3 9.8 4.4 6.5 14.1
Grass silage 10.9 4.2 3.9 3.1 4.3
Linseed meal 33.8 0.2 0.9 0 2.3
Soybean meal 48.4 1.4 1.1 0 1.9
Trypticase 84.4 62.5 58.4 52.4 83.9

a 10 ml of 0.3 M hmgstic acid are used (30 min) pH adjusted to pH 2 with 0.5 M H,SO,. Let stand 30 min
before filtering.
b Soak in water with 8 ml 0.3 M Na,WO, 30 min, add acid to pH 2, set overnight before filtering.
’ Soak in water with 8 ml 0.3 M Na,WO, for 3 h, add acid to pH 2; set overnight before filtering.

2.1.4.2. Reagents.
1. Trichloroacetic acid 10% w/v in water. Keep refrigerated!

2.1.4.3. Procedure.
1. Weigh 0.5 g ground dry sample into a 125 Erlenmeyer flask.
2. Add 50 ml of distilled water. Allow to stand 30 min.
3. Add 10 ml 10% trichloroacetic acid. Let stand 20-30 min.
4. Filter on Whatman #54 or 541 paper by gravity.
5. Wash twice with trichloroacetic acid solution.
6. Transfer paper to Kjeldahl flask and determine residual nitrogen.
7. Calculate NPN as in the tungstic acid procedure.

2.2. Results and discussion

A comparison of trichloroacetic acid and tungstic acid precipitations is shown in

Table 2. A hydrolyzed protein source like trypticase (a tryptic digest of casein) produces
no precipitate with TCA, but gives an insoluble fraction of about 52% with tungstic
acid. The tungstic acid value is variable depending upon conditions. Several physico-
chemical factors can be discerned in this variability. One is the length of time needed in
the mildly alkaline tungstate extraction to solubilize protein. This could be a problem in
dried feeds, as polymers like proteins need time to swell and dissolve. If this is
inadequate, underestimation of soluble protein results. A second factor is the final pH
and time of the acidic precipitation. Recommendation is to check pH in case of highly
buffered feeds. Overnight precipitation decreases the NPN estimate and signifies more
complete precipitation.
Precipitates of soluble protein by tungstic acid are finely divided and sometimes
difficult to filter. There is a danger that fine material may pass the filter and time of
 G. Licitra et al./Animal Feed Science Technology 57 (1996) 347-358 351

Table 3
Comparison of NPN values obtained by filtration with and without vacuum (value in percent of crude protein)
Feeds With Without Without Mean” P value
vacuum vacuum vacuum
and cover

Trypticase 56.06 55.81 56.17 56.01 0.8754

Concentrates
Soybean 2.43 2.05 2.21 2.24
Raw soybean 2.84 3.13 3.05 3.00 0.4288
Fish meal 11.67 10.38 10.02 10.69 0.0166
Corn meal 0.69 0.69 0.93 0.77 0.323 1
Corn glut. F 11.00 10.62 10.60 10.74 0.0001
Brewers 0.07 0.27 0.22 0.19 0.3400
Hays
Alfalfa 6.21 6.58 6.13 6.29 0.0069
Vetch 6.49 6.40 6.43 6.44 0.9508
Silages
Hay silage 7.45 7.40 7.47 7.44 0.5500
Corn silage 4.32 4.39 4.53 4.42 0.8300
Triticale 5.13 5.10 5.21 5.14 0.6690
Green Forages
Alfalfa 6.67 6.16 6.62 6.48 0.2600
Vetch 5.53 5.67 5.28 5.50 0.1529
Barley 5.48 5.17 5.29 5.32 0.4378

a Statistical analysis by SAS using tbe general linear model.

filtration can be lengthy. A comparison of filtering by gravity and with mild vacuum is
shown in Table 3. Filtration using vacuum can lose up to 10 percent of the soluble
protein, which is recoverable if the first filtrate containing any cloudy matter is returned
to the funnel. Thus individual filtration flasks must be used. If samples are filtered by
gravity and the time is very long, funnels need to be covered to avoid evaporation which
can lead to variable results (see Table 3). Centrifugation as an alternative procedure
requires more steps in the preliminary preparation, especially in the case of forages that
do not form definite pellets. To get a good pellet with forages require pretreatment with
ultrasonication under mild vacuum, to take out most of the air trapped in forage structure
(P. Schofield personal communication). A swinging bracket centrifuge at 5000 rpm for
15 min. at 4°C is required. After decanting the supematant, the pellet is resuspended in
distilled water (15 ml) and recentrifuged.

3. Soluble nitrogen and protein

3.1. Materials and methods

3.1 .I. Dejnition

Soluble protein is defined here as true protein that is soluble in buffer at rumen pH.
This definition differs from others in that NPN components are excluded from the
352 G. Licitra et al./Animal Feed Science Technology 57 (1996) 347-358

fraction. The procedure offered here is for total insoluble nitrogen which in combination
with a measurement of NPN allows estimation of soluble true protein by difference.

3.1.2. Review of procedures for determination.

Soluble crude protein (nitrogen) is a simplistic concept that evolved out of the
observation that most soluble nitrogen components were rapidly degraded in the rumen,
and therefore, reduced protein that could be passed to the lower tract. The early
procedures of Wohlt et al. (1973); Waldo and Goering (1979); Crooker et al. (1978)
claim to measure soluble protein, the soluble fraction in fact includes NPN, which
supports the need for a determination of NPN as well as soluble protein and peptides
(Section 2) (Krishnamoorthy et al., 1982; Roe et al., 1990). These procedures were
applied from a biological point of view, which attempted to mimic rumen environment,
i.e., use of rumen type buffers (McDougall’s solution) high in bicarbonates, in addition
to incubation at physiological temperature (37°C). Bicarbonate buffers are unstable
evolving CO, with pH rise. Krishnamoorthy et al. (1982) introduced a borate-phosphate
buffer to insure pH stabilization. This buffer is used in this study. Incubation at
physiological pH of nonsterile feeds provides the opportunity of microbial growth and
utilization of nitrogenous feed components, as well as activation of indigenous enzymes
present in the sample. None of these factors were examined in the original work for their
effectiveness or their necessity. In these papers it was generally assumed that adherence
to physiological conditions of the rumen would ensure a correct biological measurement.

3.1.3. Recommended procedure for soluble protein (buffer-soluble nitrogen)

3.1.3.1. Apparatus.
1. Erlenmeyer flask (125 ml>, Whatman #54 or 541 filter paper, analytical balance,
waterbath, vacuum source, filter manifold fitted with conical funnels (50 ml),
Kjeldahl apparatus.

3.1.3.2. Reagents.
1. Borate-phosphate buffer, pH 6.7-6.8 including
1.1. monosodium phosphate (NaI-I,PO,.H,O) 12.20 g 1-i
1.2. sodium tetraborate (Na,B,O,.lOH,O) 8.91 g 1-l
1.3. tertiary butyl alcohol 100 ml 1-l
2. Sodium azide 10% solution freshly prepared.

3.1.3.3. Procedure.
1. Weigh 0.5 g ground dry sample into a 125 ml Erlenmeyer flask.
2. Add 50 ml borate-phosphate buffer.
3. Add 1 ml of sodium azide solution.
4. Let stand at room temperature for 3 h.
5. Filter through Whatman #54 or #541 filter paper using mild vacuum.
6. Wash the residue with 250 ml cold distilled water.
7. Estimate N in residue by Kjeldahl. This gives the insoluble protein fraction. Soluble
protein is calculated by difference from total crude protein. The soluble true protein
 G. Licitra et al./Animal Feed Science Technology 57 (1996) 347-358 353

Table 4
Comparison of protein solubility obtained by varying the temperature and the hours of incubation (value in
percent of crude protein) ’
Soak time 3h 3h 3h lh Mean 2 P value
Incub. temp. room temp temp 4°C temp 37°C temp 37°C

FEEDS
Corn silage 702 29.53 28.06 30.67 31.03 29.80 0.023
Corn silage 7 14 48.91 50.69 49.54 51.60 50.18 0.012
Corn silage 570 50.15 49.39 49.28 50.15 49.74 0.45 I
Corn silage 627 62.27 62.27 62.45 62.73 62.43 0.975
Corn silage 70 1 61.66 61.72 62.12 60.56 61.51 0.479
Brewers 30.24 29.55 31.92 30.67 30.59 0.010
Soybean meal 44% 6.43 5.49 10.65 7.97 7.64 0.001
Raw soyean 60.35 61.32 59.50 63.17 61.08 0.094
Linseed whole 24.45 21.91 26.48 26.44 24.83 0.001
Corn gluten feed 67.40 67.90 67.06 66.71 67.26 0.009
Barley green forage 31.29 28.83 29.40 30.52 30.01 0.001
Alfalfa green forage 31.23 31.58 31.92 30.54 31.32 0.058
Vetch green forage 35.98 34.57 36.80 36.76 36.03 0.001
Overall experiment 41.39 a 41.07 a 42.26 b 42.37 b 41.81 0.0001

’ The fmal wash has been done with water for all the samples; Tertiary butanol and sodium azide have been
included in the buffer.
2 Statistical analysis by SAS using the general linear model.
ab Mean values with different superscripts are significantly different.

can be obtained by subtracting the NPN by tungstic acid procedure, Section lc. Note
that when tungstic acid is used the soluble protein will include the shorter peptides.

3.2. Results and discussion

A comparison of the effect of temperature and time upon protein solubility is shown
in Table 4. Borate-phosphate buffer was used to minimize drift in pH during incubation.
Protein solubility was significantly different among treatments (P < 0.001). Somewhat
higher values were obtained at 37°C. There was no statistical difference between the
protein solubilities obtained at room temperature compared to 4°C.
In these comparisons it is assumed that the analyses of feed introduced into a
fermentation system will represent the feed as presented to the rumen. Therefore, any
biological activity whether microbial or enzymatic that will influence the laboratory
value is irrelevant to the biological interpretation. The analytical requirement is to
provide an estimate of what was actually introduced into the rumen.
Roe et al. (1990) chose incubation at 37°C because it was assumed that conditions of
the assay need to conform to that of the rumen. This leads to a conflict between
analytical criteria and biological relevance. Incubation at 37°C in a non sterile system
will lead to confounded results from (a) fermentation during the incubation and (b)
indigenous enzymatic activity provided by the samples. Sodium azide was used to
control microbial growth. However, there is no easy way to inhibit indigenous enzymes
other than incubation at lower temperatures. Incubation at physiological temperature
354 G. Licitra et al. / Animal Feed Science Technology 57 (1996) 347-358

Table 5
Comparison of protein solubility obtained by washing the residue with cold distilled water and with buffer
solution (all values ate in percent of total nitrogen)
Feeds Dist. water a SD Buffer solution a SD
Corn silage 62.21 1.33 63.41 1.06
Soyabean meal 60.35 2.22 59.26 0.95
Corn gluten meal 67.40 0.16 68.56 0.06
Alfalfa green forage 31.23 0.25 31.03 0.29
Vetch green forage 35.98 0.21 36.21 1.11

’ Each combination represents the average results of three determinations. Samples were incubated for 3 h at
room temperature.

may not be stable relative to indigenous biological activity. As noted feeds may contain
enzymes as well as microorganisms. Attempts to sterilize by heat will induce further
problems by altering the sample through denaturation reactions involving proteins that
can lead to unrealistic results. Proteolytic activity will increase protein solubility, while
microbial activity in most plant products would use soluble nitrogen by using it for
microbial growth. As with the NPN determination there is a need to maximize
extraction, leading to a longer (3 h) extraction time that risks the biological hazards of
hydrolysis and microbiological growth.
There is no advantage in using buffer in the final wash (Table 5). Variation between
the wash treatments is not statistically significant. The procedure of choice is the
incubation at room temperature (20-25°C) which involves the least work and equip-
ment. Tertiary butanol has been included to facilitate wetting of the feed although it may
also serve as an inhibitor in some feeds.

4. Determination of acid-detergent insoluble nitrogen (ADIN)

4.1. Materials and metho&

4.1.1. Definition
It is not possible to completely extract all nitrogen from plant cell wall. A residual
core appears to be resistant, indigestible and associated with lignin even in fresh forages
that do not contain tannins. Tannins, if present, are one possibility for increased
insoluble protein associated with plant cell wall. Another is the Maillard or nonenzy-
matic browning reaction caused by heating and drying. These fractions have low
biological availability and tend to be recovered in acid-detergent fiber (Van Soest, 1965;
Van Soest and Mason, 1991).
Heat-drying of forages at temperatures above 6&C shows analytically significant
increases in yield of lignin and fiber. The increased yield of acid-detergent fiber (ADF)
can be accounted for largely by the production of artifact lignin via the nonenzymic
browning reaction (Van Soest, 1965). The nitrogen content of the ADF is suggested as a
sensitive assay for nonenzymic browning due to overheating of feeds (Van Soest and
Mason, 1991).
 G. Licitra et al./Animal Feed Science Technology 57 (1996) 347-358 355

4.12. Review of current procedures

The original procedures for ADF and ADIN (Van Soest, 1963; Van Soest, 1965)
were included in the USDA handbook 379 (Goering and Van Soest, 1970) which is no
longer in print. An AOAC collaborative study on ADF and lignin (Van Soest, 1973)
improved the procedure for ADF by deleting the use of decalin. The procedure
recommended here is based on ADF prepared according to this AOAC method. An
additional change is the filtration on paper to facilitate transfer to the Kjeldahl digestion
(Robertson et al., 1972). An alternative procedure is offered for a procedure compatible
with the Fibertec apparatus.

4.1.3. Recommended procedure for acid-detergent insoluble nitrogen (ADIN)

4.1.3.1. Reagents.
1. Acid-detergent solution
2. Acetone

4.1.3.2. Procedure.
1. Follow the procedure for acid-detergent fiber using a l-2 g sample (Van Soest,
1973).
2. Filter with suction on 12.5 cm Whatman #54 paper. The paper may be weighed if an
ADF value is desired. Fold paper into a cone and use 60” angle funnel and a filter
cone (Fisher Cat. No. 9-760) to protect tip.
3. Wash paper with hot water until acid-free and then acetone. Place folded paper in a
tared crucible. Dry at 105°C for 8 h or overnight and hot weigh if determining ADF.
4. Transfer paper residue into a Kjeldahl flask. Determine nitrogen on residue according
to standard Kjeldahl procedure. Titrate distillate with 0.01 N standard acid.
5. Express ADIN as percent of total nitrogen or as N X 6.25.

4.1.4. Alternate procedure for acid-detergent insoluble nitrogen using Fibertec appara-
h4S

4.1.4.1. Apparatus.
1. Fibertec system (PBI), crucibles, analytical balance, forced-air oven, Whatman #54
or 541 filter paper, filter manifold fitted with conical funnels, Kjeldahl apparatus.

4.1.4.2. Reagents.
1. Acid-detergent solution
2. Acetone

4.1.4.3. Procedure.
1. Weigh the crucible hot. Record the weight.
2. A 0.5 g sample is heated to boiling in 100 ml of AD solution, in Fibertec System for
1 h.
3. Wash with hot distilled water to remove all the AD solution.
4. Wash with acetone.
356 G. Licitra et al./Animal Feed Science Technology 57 (1996) 347-358

5. Leave in the forced draft oven for 4 h, or better overnight.

6. Weigh the hot crucible with AD residue, record the weight.
7. Wash residue from crucible to Whatman #54 or #541 filter paper with distilled
water. Suck dry and transfer to a Kjeldahl flask.
8. Return empty crucible to oven, dry and reweigh. Calculate sample weight as the
difference between weight in step 6-step 8.
9. Estimate nitrogen in residue by standard Kjeldahl.
10. Titrate distillate with 0.01 N standard acid.

4.2. Discussion

The procedure for ADIN has not been studied for analytical variation in this paper;
however, it is included here because original publication USDA Handbook 379 (Goering
and Van Soest, 1970) is no longer in print. In addition these procedures combine the
recommendations of the AOAC study (Van Soest, 1973) with that of the nitrogen
determination.
Manual filtration on paper facilitates transfer to Kjeldahl digestion. However, an
alternative method is provided using the Fibertec apparatus where transfer from a
sintered glass crucible has to be accomplished. If this is done weighing the crucible after
transfer is necessary because a complete transfer is hardly possible.

4.3. Determination of neutral-detergent insoluble nitrogen (NDIN)

4.3.1. Dejinition
The nitrogen associated with NDF is normally cell wall-bound protein which also
includes the indigestible nitrogen found in the acid-detergent residue. The protein
insoluble in the neutral-detergent solution, but soluble in acid-detergent is digestible, but
slowly degradable and has been termed the B, fraction in the Cornell Net Carbohydrate
Protein Model. Generally the cell wall-associated protein is extensin covalently linked to
hemicellulosic carbohydrate (glycoproteins) that are involved in cross linking carbohy-
drate chains in plant cell walls (Fry, 1988). Heating denatures B, proteins and may
render them insoluble thus increasing the B, fraction as well as the C fraction obtained
as the ADIN.

4.3.2. Review of procedures

Krishnamoorthy et al. (1982) determined NDIN by preparing NDF filtration on paper
followed by Kjeldahl determination of nitrogen. The variations in this procedure depend
on which procedure for NDF was followed. There are no less than 14 published
variations on NDF procedures (Mascarenhas-Ferreira et al., 1983; Van Soest et al.,
1991). The procedures published here follow that of Van Soest et al. (1991).
The determination of NDF has the optional use of sodium sulfite to reduce protein
content of NDF. Sulfite cleaves disulfide bridges in cystine. Because this is not a
biological possibility, use of sodium sulfite is precluded. For example, sulfite will
dissolve keratins (skin, hair etc. from animal products that are completely indigestible)
(Van Soest and Wine, 1967). Similarly the use of urea-amylase to remove resistant
 G. Licitra et al./Animal Feed Science Technology 57 (1996) 347-358 357

starch cannot be used, because the chaotropic character of 8 molar urea will dissolve
proteins belonging in the B, fraction (Van Soest et al., 1991).

4.3.3. Recommended procedure using manualjltration on paper

The manual procedure follows exactly as that for acid-detergent insoluble nitrogen
except that neutral-detergent solution is substituted for acid-detergent reagent (Van Soest
et al., 1991). Sodium sulfite is omitted. Pretreatment with urea-amylase should not be
done, although amylase may be used in the boiling step to aid filtration. Filtration is
preferable on Whatman 54 paper, although the alternative procedure using sintered glass
crucibles may be followed as described in Section 4.

4.3.4. Alternate procedure for neutral-detergent insoluble nitrogen using Fibertec

apparatus
The procedure using the Fibertec apparatus for insoluble nitrogen in neutral-detergent
solution is the same as described for ADIN except that neutral-detergent reagent is
substituted for acid detergent. Amylase is added at the beginning in the case of starchy
foods. This procedure has the disadvantage that removal of sample from crucible may be
incomplete and it is necessary to reweigh the crucible after transfer to paper as in
procedure 4.3.2.

4.3.5. Discussion
This procedure unlike that for ADIN was not included in the Agriculture handbook
379. However, it was conducted by Krishnamoorthy et al. (1982). The procedure
described here utilizes the latest recommendations for NDF (Van Soest et al., 19911,
filtration on paper followed by nitrogen determination by Kjeldahl.

Acknowledgements

The help of Stefania Carpino, Francesca Lauria, Elisa Tumino, Patrizia Campo and
other Staff of the Progetto IBLEO, I.S.T.P.A. Facolta di Agraria, University of Catania
at Ragusa, Sicily who performed some of the comparative analyses and helped on the
statistical evaluation of the results, is gratefully acknowledged. This work was supported
in part by the Sicilian Government Agriculture Department who funded the Progetto
IBLEO.

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