FORAGE FIBER ANALYSES

(Apparatus, Reagents, Procedures,

and Some Applications)

<>j

Agriculture Handbook No. 379

Agricultural Research Service

UNITED STATES DEPARTMENT OF AGRICULTURE
Trade names are used in this publication solely

for the purpose of providing specific information.

Mention of a trade name does not constitute a

guarantee or warranty of the product by the U.S.

Department of Agriculture or an endorsement

by the Department over other products not

mentioned.

II

Jacket No. 387-598
ACKNOWLEDGMENTS

The authors wish to acknowledge the following for their contributions in developing the detergent apparatus:

W. R. Delauder, formerly of the Animal Husbandry Research Division* and presently research technician, Jello Division, General

Foods, Dover, Del.

W. P. Flatt, formerly of the Animal Husbandry Research Division

and presently Director of the Experiment Station, University of

Georgia, Athens, Ga.

B. Deinum, who formerly worked in the Animal Husbandry Research Division on a fellowship of The Netherlands Organization

for the Advancement of Pure Science, and now at the Department

of Field Crops and Grassland Husbandry, Agricultural University, Wageningen, The Netherlands.

*The name has been changed to Animal Science Research Division.

Ill

Jacket No. 387-598
CONTENTS

Page

Introduction 1

Chemical-fiber determinations 1

Apparatus 1

Materials 2

Description 4

Reagents 5

Analytical procedures 8

Neutral-detergent (cell-wall) 8

Acid-detergent fiber 8

Acid-detergent lignin 9

Permanganate lignin, cellulose, insoluble ash, and silica 9

Acid-detergent cutin 11

Crucible cleaning 11

Acid-detergent nitrogen 11

Pepsin-insoluble nitrogen 12

Hot-water-insoluble matter and its nitrogen content 12

In vitro rumen digestibility determination 12

Apparatus 12

Materials 12

Description 13

Reagents 13

Procedure 14

Sampling techniques 15

Sampling of dry feeds 15

Sampling of wet materials 15

Comparison of hot and cold sample weighing 17

Estimation of nutritive value from chemical data 18

Literature cited 20

Washington, D.C. Issued December 1970

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402 • Price 20 cents

IV

Jacket No. 387-598
FORAGE FIBER ANALYSES

(Apparatus, Reagents, Procedures, and Some Applications)

By H. K. GOERING, research dairy husbandman, and P. J. VAN SOEST, formerly ^ chemist,

Animal Science Research Division, Agricultural Research Service

INTRODUCTION

This handbook has been written as a guide

for those who wish to (1) set up the newer

detergent system of fiber fractionation (^, 5, P,

13)j^ (2) adapt their apparatus and equipment

to the new analyses, and (3) estimate the digestibility directly by a modified {15) Tilley and

Terry {3) two-stage in vitro rumen fermentation procedure.

Laboratory evaluation of forage is essentially

aimed at obtaining analytical data that predict

the extent of biological degradation under specified conditions, animals, organisms, and time.

In general, an in vitro rumen fermentation reflects the factors known and unknown limiting

availability of forage to the digesting organism.

The analyst can assay only known constituents.

The assays are for things that are expedient to

handle, are known, or are considered important,

and its evaluation is valid only if the principals

concerned are dominant in the sample. For example, it makes little sense to expect lignin to

be a good predictor of digestibility if silica or

some other factor is a more important variable.

In vitro fermentations will be influenced by all

factors and inhibitors known and unknown. The

in vitro, however, does not disclose anything regarding the nature of the limiting factor. This

latter task of identification remains the duty of

chemical studies.

An attempt has been made to cover the principal problems in technique encountered in these

procedures. These include sample preparation,

filtration and washing, and efficiency in handling large numbers of samples. Ultimate laboratory efficiency is tied to the precision of duplicates and the repeatability of values. Handling

of samples in drying and weighing and in ashing

and reweighing is also discussed.

CHEMICAL-FIBER DETERMINATIONS

Apparatus

In general, equipment that is used for crude

fiber may be adapted to the detergent fiber procedures. The reflux apparatus described is more

convenient and cheaper than many other types

available. However, if a laboratory contains

a reflux apparatus for crude fiber, it may be

used without modification. Other types of reflux

apparatus probably are suitable if they conform

to the following criteria: (1) a minimum of six

^ Presently associate professor, Animal Science Department, Cornell University, Ithaca, N.Y.

2 Italic numbers in parentheses refer to Literature

Cited, p. 20.

plates for reflux, (2) each plate or burner is

individually regulatable and has suflicient power

so that boiling solutions keep fibers in continuous suspension, (3) reflux containers are

straight-sided and not conical, and (4) the condenser system is sufliicient to keep volume of

boiling solutions constant. In general, oil baths

and large hotplates are not satisfactory for

quantitative work.

The filtration apparatus described is highly

recommended. In general, filtration manifolds

suitable for crude fiber do not adapt well unless

they conform to the following specifications.

Samples that contain appreciable amounts of

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2 AGRICULTUEE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

starch, protein, or other mucilaginous substance

are often hard to filter when detergent-fiber

analyses are being performed. Much difficulty

is avoided by having an adequate filtering apparatus and by using proper technique. The

main requirement is a filter manifold system

with receivers for at least six crucibles. Filtration must be individually controlled for each

crucible. Experience has shown that filtration

of a difficult sample niust begin with little to no

vacuum and then be gradually increased. Often

a very significant volume can be filtered without

any vacuum. The addition of asbestos to the

sample may help filtration, but this involves the

laborious operation of preparing the asbestos

before use and also makes subsequent analyses

on the residue more difficult or impossible.

For convenience and most efficient routine

operations, equipment must be situated so that

the operator can perform on a regularly timed

basis; also some extra time for incidental operations that arise should be allowed. It is very important to have solutions near areas of use and

quantities large enough to last for several days

of continuous analyses. An ashing oven equipped with a temperature regulator to prevent

the glass crucibles from melting is required for

cleaning the crucibles. With temperatures from

500° to 550° C. complete ashing of organic

matter is accomplished in 2 to 3 hours.

Standardized methods in the laboratory are

mandatory for obtaining precise analytical results. Enough equipment is required to adequately run a basic analysis, such as cell walls

(neutral-detergent fiber), continuously for an

8-hour period. A 12-unit refluxing apparatus

and a drying oven large enough to hold 80 crucibles make this possible. Technicians tend to

make fewer mistakes when a standard laboratory procedure is used for all activities; and,

if erratic results arise, the cause can be detected

more easily. The possibility of overlooking a

step in the procedure'is also reduced.

Materials

1. Refluxing apparatus (figs. 1 and 2):

a. Beakers, Berzelius without spout, 600-ml.

capacity

b. Crucibles, Gooch-type, high form Pyrex

fritted glass, 50-ml. capacity, coarse porosity. Type sold by Fisher Scientific, Silver

Spring, Md., Cat. No. 8-237

c. Scimatco rubber tubing, black, thick wall,

Yi6-m. (8.0-mm.) bore, %2-in. (8.5-mm.)

wall

d. Hotplate, Thermolyne (HP-A8805B),blueporcelain, steel flattop, 120 v., 3.3 a., 400

w., 3 %-m. (9.5-cm.) diameter

e. Multielectric 6-receptacle box, 20 a., 115 v.

(needed if more than one hotplate is going

to be used)

f. Condensers, reflux, crude-fiber, Pyrex. E.

H. Sargent and Company, Philadelphia,

Pa., Cat. No. S-22742 or equivalent

BN-36467

FIGURE 1.—Arrangement of hotplates, rings, beakers,

condensers, and hose.

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FORAGE FIBER ANALYSES

J

BN-36455

FIGURE 2.—Arrangement of six units of refluxing apparatus, with flexaframe support.

g. Flexaframe hook connectors (need approximately 36 for a set of six hotplates)

h. Rings, cast iron, 4-in. (10-cm.) OD

i. Flexaframe rods:

12 in. (30 cm.) (need 5)

24 in. (60 cm.) (need 3)

36 in. (90 cm.) (need 2)

2. Filter manifold (figs. 3-5) :

a. Polyethylene pipe, 1-in. (2.5-cm.) ID, %-

in. (3-mm.) wall, 2 ft. (60 cm.)

b. Funnels, Büchner, porcelain, Coors 490,

size-0

c. Flasks, filtering. Pyrex, 4-1. capacity

d. Clamps, pinchcocks, Mohr

e. Rubber adapter, size-B. Fisher Scientific

Co., Silver Spring, Md., Cat. No. 8-239

f. Rubber tubing, 34-in. (6-mm.) ID, %-in.

(3-mm.) wall

g. Glass tubing, 5-mm. OD, 3-mm. ID

h. Polyethylene tubing, %-in. (3-mm.) ID,

ViQ-m. (1.5-mm.) wall

b.

c.

i. Screw clamp

Water heater (fig. 6) :

a. Ring support, cast iron with clamp, 6-in.

(15-cm.) OD

Flask, distilling, 2 1., 3 neck

Quartz immersion heater, 1,000 or 500 w.,

120 V. Ace Glass, Inc., Vineland, N. J., Cat.

No. 2145-12 or 2145-10

Crucible holder, filter tubes. Pyrex, 10-

mm. diameter (for automatic filling of

large flask)

Bottle, Pyrex, 5-gal. (18-1.) capacity

Transformer, variable, 10 a.

Condenser

Flexaframe rods, 18 in. and 48 in. (45 cm.

and 120 cm.)

Clamp, versatile, vinylized jaws, medium

size (need 2)

Clamps, pinchcock, Day

Y connection, %6-in. (48-mm.) OD, metal

Burette tip

Jk.

1.

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AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

^^^V^^^^^^^H

X1 km^^^

^^^^^^^^B^^^^^MKBBBfer-''"^' ^J • " ' ■■■- ■■• "--^^'^^^^^^^^^^^^^^^^^^^^^^^^^^ flH

BN-36409

FIGURE 3.—Filter manifold.

Description

Figure 1 shows a section of the refluxing apparatus. Ceramic hotplates, 3%-inch, 120 v.,

are used to produce sufficient heat to boil 200

ml. of liquid in the beaker. Ceramic plates protect coils from liquids spilled on the plate, which

avoids electrical problems. Special Pyrex glass

reflux condensers rest on top of the beaker,

which are otherwise supported by the rubber

tubing connected to the condensers. Thick-wall

%Q-m. (8.0-mm.) bore rubber tubing is used to

connect the condensers in a series of six. Parts

of refluxing apparatus are supported with flexaframe unit. Rings covered with rubber tubing

are placed above the hotplate to prevent knocking off the beaker. Figure 2 shows the complete

six-unit refluxing apparatus, which requires a

multielectric six-receptable box with a minimum

of 20 a.

The filter manifold is shown in figures 3, 4,

and 5. The filter manifold is constructed to hold

six crucibles, so that six samples can be filtered

at one time. The manifold is made from large

polyethylene pipe, 2 feet (60 cm.) in length

with a 1-inch (2.5-cm.) inside diameter (ID).

Six polyethylene tubes are attached into the

larger tube. A glass lining (5-mm. outside diameter (OD) tubing) is inserted inside each small

polyethylene tube. The joints are welded with a

polyethylene welding rod to prevent leakage and

maintain vacuum. Thick-wall rubber tubing

connects each of these small tubes to size 0

Büchner funnels, to prevent collapsing from the

vacuum. The funnels are supported on a 1- x

4-in. wooden frame, constructed as shown in

figures 3 and 4. Holes are drilled to hold the funnels to give adequate working room for filtering

the sample. The rubber adapters (Fisher 8-239)

are inserted into the Büchner funnels to give a

vacuum seal. Heavy-wall rubber tubing connects from the manifold to two 4-1. side-arm

flasks, which serve as traps. Vacuum level is

controlled by attaching a rubber tube with a

screw clamp to the opposite end of the polyethylene manifold. A pinch type clamp closes

each tube coming from the Büchner funnel,

which makes it possible to control each crucible

individually.

The third component of the detergent apparatus, the hot-water system, is constructed directly above the filtering manifold (fig. 6).

Water is stored in an elevated carboy reservoir

above and to the side of the heater. A closed

siphon is connected with a leveling device (seen

on the left of the large flask in figure 6), a 2-1.,

3-neck distilling fiask with a well at the bottom

to allow space for the immersion heater (about

50 X 120 mm.), and a water inlet to the bottom of the well. Water enters from the reservoir

into the leveling device and then goes directly to

the bottom of the well. A tube connects from the

leveling device to the reservoir to serve as an

-^'"r vent. Also, an air vent is placed on the levelJacket No. 387-598
FORAGE FIBER ANALYSES

RUBBER RING

630 mm.

2x

- 20 mm.

'^ ^

POLYETHYLENE

TUBE

TAPERED END

OF GLASS TUBE

BRACE

RUBBER STOPPERS

152 mm.

i¥:

RUBBER

TUBE

FIGURE 4.—Side diagram of filter manifold.

ing device. The 2-1. flask then fills automatically; a constant supply of hot water is thus

maintained. A condenser at the top of the flask

prevents loss of steam. The water-heating apparatus is supported on one flexaframe rod. Vinylized clamps are used to allow for expansion of

glassware due to temperature change. A thickwall hose feeds water to the filtering manifold.

t

47 mm.

\

29 mm.

t

58 mm.

\

18 mm.

The hose terminates with a Y connector, fitted

on one end with a burette tip end and on the

other with a tube, which allows each to be

clamped off. The Y is insulated and wrapped

with asbestos tape so that the hot water can be

manipulated without protection for the hands.

A workable water system is essential for the

fast washing of the detergent from the residues

in the crucibles. A variable transformer with a

capacity of 10 a. and a 1,000-w. water heater

(Ace glass quartz heater) should be used to

control water temperature.

A convenient setup is shown in figure 7. The

water system is directly above the filter manifold with two sets of six refluxing units on each

side. This setup allows for refluxing detergents

continuously and also allows the technician

sufficient time for filtering and washing of the

residues.

FIGURE 5.—End diagram of filter-manifold frame.

Reagents

Neutral-detergent solution—

Distilled water 1.... 1

Sodium lauryl

sulfate, USP .... g .... 30

Disodium ethylenediaminetetraace18 40

540 1,200

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AGEICULTURE HANDBOOK 379, U.S. DEPARTMENT OP AGRICULTURE

18.61 335.0 744.4

täte (EDTA),

dihydrate crystal,

reagent grade.... g

Sodium borate decahydrate, reagent grade .... g.... 6.81 122.6 272.4

Disodium hydrogen

phosphate, anhydrous, reagent

grade g.... 4.56 82.1 182.4

2-ethoxyethanol

(ethylene glycol

monoethyl ether),

purified grade .. ml.... 10 180 400

Put EDTA and NaaBiOr • IOH2O together

in a large beaker, add some of the distilled

water, and heat until dissolved; then add to

solution containing sodium lauryl sulfate and

2-ethoxyethanol (ethylene glycol monoethyl

ether). Put Na2HP04 in beaker, add some of

the distilled water, and heat until dissolved;

then add to solution containing other ingredients.

Check pH to range 6.9 to 7.1. If solution is

properly made, pH adjustment will rarely be

required.

Decahydronaphthalene—Reagent grade.

Acetone—Use grade that is free from color

and leaves no residue upon evaporation.

Sodium sulfite—Anhydrous, reagent grade.

Acid-detergent solution— 11. 18 1.

Sulfuric acid, reagent

grade standardized to

1 N. (100 = percent

assay) g .... 49.04 882.72

Cetyl trimethylammonium bromide

(CTAB), technical

grade g .... 20 360

Weigh sulfuric acid and make up to volume

with distilled water at 20° C. Check normality

by titration before addition of detergent. Then

add CTAB and stir.

6. n-Hexane—Technical grade.

7. Asbestos—Place 100 g. (long fiber) in a 3-1.

flask with 850 ml. water. Add 1,400 ml. concentrated H2SO4 (technical grade), mix, and

let cool at room temperature for 2 hours. Filter on a large Büchner funnel, and wash with

water. Resuspend mat in water and pour into

a square bag sewn from a rectangle of fiberglass window screening of 14 x 18 mesh

(about 1 mm.) (the bag should be at least 18

inches (46 cm.) wide by 12 inches (30 cm.)

deep). Wash by immersion and agitation in

water to remove fine particles. Ash the recovered asbestos in a furnace at 800° C. for

16 hours. Store in dry form until needed.

Used asbestos can be rewashed, ashed, and

reused.

8. Sulfuric acid, 72 percent by weight—Calculate grams acid and water needed in 1 1. of

solution by:

BN-36456

FIGURE 6—Hot-water heating system.

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FORAGE FIBER ANALYSES

BN-36458

FIGURE 7.—Assembly of 12 units of refluxing apparatus with filter manifold and hot-water system.

100 X 98.08 X 12 moles

H2SO4 assay (percent) = grams acid needed

(1,000 X 1.634)3 -grams acid =

grams water needed

Weigh amount of water into a 1-1. MCA volumetric flask (with a bulb in the neck) and add

the calculated amount of H2SO4 slowly with

occasional swirling. Caution! Flask must be

cooled in a water bath (sink) in order to add

the required weight of sulfuric acid. Cool to

20° C. and check if volume is correct. If volume is too small, take out about 1.5 ml. and

add 2.5 ml. water. Repeat, if necessary. If

volume is too large, take out 5 ml. and add

4.45 ml. H2SO4. Meniscus should be within a

0.5 cm. of calibration mark at 20°.

^ Weight of 1 1. of 72 percent H2SO4.

9. Saturated potassium permanganate—

Distilled water 1.... 1 18

KMn04, reagent grade .. g — 50 900

Ag2S04, reagent grade .. g .— 0.05 0.9

Dissolve KMn04 and Ag2S04 in distilled

water. Keep out of direct sunlight. Add silver

sulfate to dehalogenate the reagent.

10. Lignin buffer solution—

1 I. 12 I.

Ferric nitrate

nonahydrate g.... 6 72

Silver nitrate g 15 1.8

Acetic acid, glacial .. ml.... 500 6,000

Potassium acetate .... g.... 5 60

Tertiary butyl

alcohol ml.... 400 4,800

Distilled water ml.... 100 1,200

Dissolve ferric nitrate nonahydrate [Fe

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8 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

(N03)3 • 9H2O] and silver nitrate in distilled water. Combine with acetic acid and

potassium acetate. Add tertiary butyl alcohol and mix. Use grades of acid and solvent

passing dicromate test (ACS).

11. Combined permanganate solution—Combine

and mix saturated potassium permanganate

and lignin buffer solution in the ratio of 2:1,

by volume, before use. Unused mixed solution may be kept about a week in a refrigerator in the absence of light. Solution is

usable if purple and containing no precipitate. Old solutions assume a reddish color

and should be discarded.

12. Demineralizing solution—

1 L 18 L

Oxalic acid dihydrate g —. 50 900

95 percent ethanol ml.... 700 12,600

Concentrated (about

12 N) hydrochloric

acid ml.... 50 900

Distilled water ml.... 250 4,500

Dissolve oxalic acid dihydrate in 95 percent ethanol. Add concentrated hydrochloric

acid and distilled water and mix.

13. Ethanol 80 percent—

1 L 18 L

95 percent ethanol .... ml .... 845 15,200

Distilled water ml.... 155 2,800

14. Hydrobromic acid, reagent grade.

15. Cleaning solution—

Distilled water 1 -.. 1

Disodium ethylenediaminetetraacetate (EDTA), dihydrate

crystal, reagent grade g.... 5

Trisodium phosphate,

laboratory grade g .... 50

Potassium hydroxide g.... 200

16. 0.1 N HCl—Add 17 ml. 6 N HCl to 1 1. of

distilled water. Need not be standardized.

17. Pepsin—NF (Fisher Scientific, Cat. No.

P-53).

Analytical Procedures^

NeutraUdetergent (cell-wall)

The neutral-detergent procedure for cell-wall

constituents is a rapid method for analyzing the

* Numbers given after "Reagents required" refer to

numbers in the reagent section, p. 5.

total fiber in vegetable feedstuffs (IS), It appears to divide the dry matter of feeds very near

the point that separates the nutritively available (98 percent) and soluble constituents from

those that are incompletely available and dependent on a microbial fermentation.

Reagents required: 1 through 4.

1. Weigh 0.5- to 1.0-g. air-dry sample ground to

pass 20 to 30 mesh (1-mm.) or equivalent

into a beaker of the reñuxing apparatus.

2. Add in order, 100 ml. cold (room temperature) neutral-detergent solution, 2 ml. decahydronaphthalene, and 0.5 g. sodium sulfite

with a calibrated scoop. Heat to boiling in 5

to 10 minutes. Reduce heat as boiling begins,

to avoid foaming. Adjust boiling to an even

level and reflux for 60 minutes, timed from

onset of boiling.

3. Place previously tared Gooch crucibles on filter manifold. Swirl beaker to suspend solids,

and fill crucible. Do not admit vacuum until

after crucible has been filled. Use low vacuum

at-first and increase it only as more force is

needed. Rinse sample into crucible with minimum of hot (90°-100° C.) water. Remove

vacuum, break up mat, and fill crucible with

hot water. Filter liquid and repeat washing

procedure.

4. Wash twice with acetone in same manner and

suck dry. Dry crucibles at 100° C. for 8 hours

or overnight and weigh.

5. Report yield of recovered neutral-detergent

fiber as percent of cell-wall constituents. Estimate cell soluble material by subtracting this

value from 100.

6. Ash residue in the crucible for 3 hours at

500° to 550° C. and weigh. Report ash content

as ash insoluble in neutral-detergent.

Acid-detergent fiber

The acid-detergent fiber procedure provides

a rapid method for lignocellulose determination

in feedstuffs {i, 5). The residue also includes

silica. The difference between the cell walls and

acid-detergent fiber is an estimate of hemicellulose; however, this difference does include

some protein attached to cell walls. The aciddetergent fiber is used as a preparatory step for

lignin determination.

Jacket No. 387-598
FORAGE FIBER ANALYSES 9

Reagents required: 2, 3, and 5.

1. Weigh 1-g. air-dry sample ground to pass

20- to 30-mesh (1-mm.) screen or the approximate equivalent of wet material into a

beaker suitable for refluxing.

2. Add 100 ml. cold (room temperature) aciddetergent solution and 2 ml. decahydronaphthalene. Heat to boiling in 5 to 10 minutes.

Reduce heat as boiling begins, to avoid foaming. Reflux 60 minutes from onset of boiling ;

adjust boiling to a slow, even level.

3. Filter on a previously tared Gooch crucible,

which is set on the filter manifold; use light

suction. Break up the filtered mat with a rod

and wash twice with hot water (90°-100°

C). Rinse sides of the crucible in the same

manner.

4. Repeat wash with acetone until it removes no

more color; break up all lumps so that the

solvent comes into contact with all particles

of fiber.

5. Optional wash with hexane. Hexane should be

added while crucible still contains some acetone. (Hexane can be omitted if lumping is

not a problem in lignin analysis.) Suck the

acid-detergent fiber free of hexane and dry at

100° C. for 8 hours or overnight and weigh.

6. Calculate acid-detergent fiber:

(Wo-Wt) (100)/S = ADF

where: Wo=weight of oven-dry crucible including fiber ;

Wt=tared weight of oven-dry crucible;

iS = oven-dry sample weight

Acid-detergent lignin

In the acid-detergent lignin procedure, the

acid-detergent fiber (ADF) procedure is used as

a preparatory step (5). The detergent removes

the protein and other acid-soluble material that

would interfere with the lignin determination.

The ADF residue consists of cellulose, lignin,

cutin, and acid-insoluble ash (mainly silica).

Treatment with 72 percent sulfuric acid dissolves cellulose. Ashing of the residue will determine the crude lignin fraction including

cutin. For silica determination and separation

of cutin and lignin, see the Permanganate and

Acid-Detergent Cutin Procedures.

Reagents required: 2, 3, 5, 7, and 8.

1. Prepare the acid-detergent fiber (p. 8).

2. Add to the crucible containing the acid-detergent fiber an amount of asbestos about equal

to the volume of fiber. Cover the contents of

the crucible with cooled (15° C.) 72 percent

H2SO4 and stir with a glass rod to a smooth

paste, breaking all lumps. Fill crucible about

half full with acid and stir. Let glass rod remain in crucible; refill with 72 percent H2SO4

and stir at hourly intervals as acid drains

away. Crucibles do not need to be kept full at

all times. Three additions suffice. Keep crucible at 20° to 23° C. After 3 hours, filter off

as much acid as possible with vacuum; then

wash contents with hot water until free from

acid. Rinse and remove stirring rod.

3. Dry crucible at 100° C. and weigh.

4. Ignite crucible in a muffle furnace at 500° to

550° C. for 3 hours, and then cool to 100°

and weigh.

5. Calculate acid-detergent lignin:

(LxlOO)/S = ADL

where: L = loss upon ignition after 72 percent H2SO4 treatment ;

S = oven-dry sample weight

Permanganate lignin^ cellulose^ insoluble

ash, and silica

An indirect method to determine lignin was

developed that makes possible the preparation

of cellulose and insoluble ash in the same sample

(li). The insoluble ash is an estimate of total

silica content, which in many grasses is a primary factor in reducing digestibility. The permanganate lignin method is an alternative

procedure to the 72 percent sulfuric acid

method; each has its own advantages. Choice of

methods depends on materials analyzed and on

the purpose for which the values are to be used.

Advantages of the permanganate method over

the 72 percent acid method include a shorter

procedure for lignin per se while the residue is

reserved for further analysis of cellulose and

silica. The permanganate reagents are much

less corrosive and require no standardization.

The residue requires no filter aids, and lignin

values are not subject to some interferences that

affect 72 percent sulfuric acid lignin. Values are

less affected by heat-damage artifacts and are

closer to a true lignin figure.

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10 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

However, cutin, which is important in many

seed hulls, is not measured. A variation for the

analysis of seed hulls is to prepare the permanganate cellulose and treat with 72 percent H2SO4

and asbestos for 3 hours as described in the

acid-detergent lignin procedure. This results in

the partitioning of crude lignin into two fractions as described in the acid-detergent cutin

procedure.

One disadvantage to permanganate lignin is

that large particles are poorly penetrated by the

reagents and yield low values. Consequently, all

materials must be dried and ground to pass

through a 20- to 30-mesh (less than 1 mm.)

screen, and the method is not applicable to fresh

feces and forages that have been ground in a

meat grinder. Because of high sensitivity to heat

damage, 72 percent acid is preferred for assaying artifact lignin.

Theory of the method—Interfering matter is

removed by preparing acid-detergent fiber,

which is chieñy composed of lignin, cellulose,

and insoluble minerals. Lignin is oxidized with

an excess of acetic acid-buffered potassium permanganate solution, containing trivalent iron

and monovalent silver as catalysts. Deposited

manganese and iron oxides are dissolved with

an alcoholic solution of oxalic and hydrochloric

acids, which leaves cellulose and insoluble minerals. Lignin is measured as the weight lost by

these treatments; whereas, cellulose is determined as the weight loss upon ashing. The ash

residue is mainly silica and much of the nonsilica matter can be removed by leaching with

concentrated hydrobromic acid.

Reagents required: 2, 3, 5, 6, 9 through 14.

1. Dry samples at less than 65° C. and grind

through 20- to 30-mesh (1-mm.) screen.

Prepare and determine acid-detergent fiber

according to standard procedure. Use a 1-g.

sample, except on samples containing a high

amount of lignin (15 percent or more) use

0.5-g. sample. Place previously weighed crucibles in a shallow enamel pan containing

cold water to a depth of about 1 cm. Fiber in

crucibles should not be wet.

2. Add about 25 ml. of combined saturated

potassium permanganate and lignin buffer

solution (2:1 by volume) to the crucibles in

the enamel pan containing cold water. Adjust

level (2-3 cm.) of water in pan to reduce flow

of solution out of crucibles. Place a short

glass rod in each crucible to stir contents, to

break lumps, and to draw permanganate

solution up on sides of crucibles to wet all

particles.

3. Allow crucibles to stand at 20° to 25° C. for

90 ±1 10 minutes; add more mixed permanganate solution if necessary. Purple color

must be present at all times.

4. Remove crucibles to filtering apparatus. Suck

dry. Do not wash. Place in a clean enamel pan,

and fill crucibles no more than half full with

demineralizing solution. Demineralizing solution may be added directly to crucibles in

case filtering is difficult. Care must be taken

to avoid spillage by foaming. After about 5

minutes, suck dry on filter and refill half full

with demineralizing solution. Repeat after

second interval if solution is very brown.

Rinse sides of crucibles with solution from a

wash bottle with a fine stream. Treat until

fiber is white. Total time required is 20 to

30 minutes.

5. Fill and thoroughly wash crucible and contents with 80 percent ethanol. Suck dry and

repeat two times. Wash twice in similar

manner with acetone. Suck dry.

6. Dry at 100° C. overnight and weigh. Calculate lignin content as loss in weight from

acid-detergent fiber.

7. Ash at 500° C. for 3 hours, cool, and weigh.

Calculate residual ash as the difference between this weight and original tare of crucible. Calculate cellulose by weight loss upon

ashing.

8. A presumptive analysis for silica may be

obtained by hydrobromic acid treatment of

the ashed permanganate lignin or ADF residue. This determination has its greatest value

when the residual ash is greater than 2 percent. Ash and weigh, then add enough drops

of 48 percent HBr to moisten all particles.

Use no more than 4 ml. acid. Allow to stand

1 to 2 hours. Add more drops of HBr if much

red color forms. Suck off excess acid on

vacuum and wash once with acetone. Use no

water. Dry and ash briefly at 500° C. Cool

and weigh. Report silica as the difference

between this weight and the original tare.

Jacket No. 387-598
FORAGE FIBER ANALYSES 11

Precautions—Crucibles containing fiber of

a high lignin content require more permanganate solution ; however, avoid additions of more

solution than is necessary. Appearance of a

yellow or brown color indicates exhaustion of

permanganate. If crucible is full, filter solution

OÍF on a vacuum and add more reagent. A yellow

color persisting after treatment of fiber with

demineralizing solution indicates incomplete removal of lignin. This occurs only in materials

of a very high lignin content. Cutin material

present in seedcoats and other plant parts is not

oxidized by permanganate; thus, it is neither

determined as lignin nor bleached with the

treatments. Seedcoats appear as colored flecks

among white cellulose particles, and thus

they should not be confused with incomplete

oxidation.

Excessive flow of permanganate solution

through the crucibles should be avoided with

samples of low lignin content, particularly in

samples of immature grasses. With these, a

single addition of permanganate solution suffices. Fiber from immature grasses is very

rapidly delignified, and, therefore, there is

danger of loss of cellulosic carbohydrates if the

flow is too great. Reduction in flow is accomplished by adjusting the water level in the pan

to near that in crucibles. These precautions are

not needed with the demineralizing solution.

Acid-detergent cutin (2)

The fraction of plant material referred to as

cutin is the fraction that is not oxidized by

KMnOé and resists hydrolysis by 72 percent

H2SO4 acid. This fraction can be very large, as

in some seed hulls, or not important, as in the

common forages. The relation of cutin to the

nutritive value of the other plant constituents

is not understood. However, the cutin factor is

resistant to microbial degradation.

Reagents required: 2, 3, 5 through 13.

1. Follow procedure for KMn04 lignin and cellulose preparation up to the step where lignin

can be calculated but the residue has not been

ashed (steps 1-6, p. 10).

2. Treat the unashed residue with 72 percent

H2SO4, as in the acid-detergent lignin procedure.

3. Calculate cutin as loss in weight upon ashing.

Crucible cleaning

Reagents required: 15.

1. Empty contents and ash crucibles briefly, 1

to 2 hours, at 500° to 550° C. (not necessary

if crucible already ashed in lignin or silica

determination).

2. Wash crucible with tapwater.

3. Force distilled water upward through the

crucible; use a No. 7 rubber stopper with tube

through middle connected to the distilled

water outlet. Rinse outside with distilled

water and place in oven. Proceed to next step

if the crucible does not give normal filtering

properties.

4. Place crucible that has been ashed in 500°

to 550° C. in a shallow enamel pan. Add about

50 ml. crucible cleaning solution to each crucible. Heat (such as steam bath) should be

placed under the pan. Let cleaning solution

filter through crucible. Bore hole in a No. 9%

rubber stopper and insert one end of a 50-ml.

pipette and attach a tube to the upper end of

the pipette. Place the stopper assembly in the

top of the crucible and apply vacuum until the

crucible is approximately one-half full of

cleaning solution. Wait for solution to filter

through crucible again. Refill the crucible

with the solution again. The cleaning solution

may be saved and reused.

5. Repeat steps 2 and 3.

Acid-detergent nitrogen

Heat-drying of forages at temperatures above

50° 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. Value

for ADF and lignin in dried forages can be corrected on the basis of the nitrogen content of

the ADF (1, 9), The nitrogen content of the

ADF is suggested as a sensitive assay for nonenzymic browning due to overheating of feeds.

Reagents required: 2, 3, and 5.

1. Follow step 1 and 2 of acid-detergent fiber

procedure using a 2-g. sample, page 8.

2. Filter with suction on previously tared 12.5-

cm. Whatman No. 54 paper. Fold paper into

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12 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

a cone and use 60° angle funnel and a filter

cone (Fisher No. 9-760) to protect tip.

3. Wash paper with hot water and then acetone

until acid free. Dry at 100° C. for 8 hours or

overnight and weigh.

4. Transfer paper residue into Kjeldahl ñask.

Run nitrogen on residue in flask according to

standard Kjeldahl procedure.

5. Calculate ADF:

(Wo-Wt){im/S^ADF

where : Wo = weight of oven-dry filter paper

including fiber ;

Tft = tared weight of oven-dry filter

paper ;

S = oven-dry sample weight

6. Express ADF-N as percent of total DM :

AT (100)/S = ADF-N

where: N — grams of nitrogen;

S = oven-dry sample weight

Another way of expressing the ADF-N is

ADF-N/total nitrogen (100).

Pepsin-insoluble nitrogen (1)

Reagents required: 16 and 17.

1. Weight 0.5-g. of air dry sample ground to pass

20- to 30-mesh (less than 1-mm.) screen, into

125-ml. Erlenmeyer flask.

2. Add 0.5 g. of pepsin and 50 ml. of 0.1 N HCl

to flask, stopper, and swirl. Place in oven or

water bath at about 39° C. for 20 hours.

3. Filter on 12.5-cm. Whatman No. 4, 41, or 54

filter paper. Leach thoroughly with distilled

water. One way to ascertain if all soluble

nitrogen is leached out is to determine if all

traces of HCl has been removed from the

filter paper. The taste test can be used for this.

4. Take filter paper containing pepsin insoluble

residue and place in a Kjeldahl flask. Run

nitrogen according to standard Kjeldahl

procedure.

5. Calcualte pepsin-insoluble nitrogen:

(g. N/oven-dry sample) (100)

which can be expressed as:

(pepsin-insoluble N/total sample N) (100)

Hot'tvater-insoluble matter and

its nitrogen content

This procedure gives an estimate of true protein nitrogen in forages. The forage sample is

boiled in water, which coagulates the protein

causing it to become water insoluble and remain

in the cell of the forage.

1. Weigh 2.0-g. sample into 600-ml. Berzelius

beaker.

2. Add 200 ml. distilled water to beaker and boil

for 1 hour on refluxing apparatus.

3. Filter with vacuum on a previously tared

12.5-cm. Whatman No. 54 filter paper on a

60° funnel and a filter cone tp protect tip.

Wash four times with hot water. Place in

100° C. oven for 8 hours and weigh.

4. Take weighed filter paper residue and transfer to a Kjeldahl flask. Run nitrogen on resi

due and filter paper according to standard

Kjeldahl procedure. Correct for filter by running Kjeldahl on filter paper itself.

5. Calculate hot-water-insoluble msitier (HwIM)

including nitrogen (HwIN) :

(Wr- Wt/oven-dry sample weight) (100) =

HwIM

(g.N/oven-dry sample weight) (100) =

HwIN;

where : Wr = weight of filter paper plus insoluble residue;

Wt = weight of filter paper

This insoluble nitrogen can be used as an

estimate of true protein in forages only (hot

water insoluble N x 6.25).

IN VITRO RUMEN DIGESTIBILITY DETERMINATION

Apparatus

Materials

1. Rumen-content source from an animal on a

high cell-wall roughage (preferably a fistulated animal)

2. Erlenmeyer flasks, 125 ml. (Pyrex, requiring

No. 6 stoppers)

3. Shaking water bath at 40° C. with holder for

18 flasks

4. Manifold for 18 flasks constructed over water

bath

Jacket No. 387-598
FORAGE FIBER ANALYSES 13

5. Waring Blendor

6. CO2 source

7. Cheesecloth

8. Glass wool and enclosed funnel assembly

9. Automatic syringe, 10 ml.

10. Glassware refluxing apparatus as used for

detergent preparations

Description

Fermentations are conducted in 125-ml.

Pyrex Erlenmeyer flasks (wide-mouth); 0.5-g.

substrate, 40-ml. medium, and 10-ml. inoculum

are used. Fermentation flasks are placed in a

shaking water bath (capacity 18) and closed

with No. 6 rubber stoppers. Stoppers are fitted

with three openings: an inlet tube, a bunsen

valve—both flush with the bottom of the stopper—and a gassing tube connected to a common

manifold. The inlet tube is closed on the outside

with a rubber sleeve and glass rod. The gassing

tube should stop about 1 cm. above the surface

of the liquid. The manifold is connected to a

supply of carbon dioxide and in parallel with a

water manometer with a capacity of 60-cm.

water pressure. Figure 8 shows the shaking

water bath and CO2 manifold apparatus.

Reagents

Trypticase—A pancreatic digest of casein, USP.

Sodium sulfide nonahydrate—Reagent grade.

1 N sodium hydroxide—Dissolve 4 g. in water

and dilute to a liter.

Cysteine-HCl—J. T. Baker Go. grade or equivalent.

Resazurin—0.1 percent w/v solution.

6 N HCl—Dilute concentrated HCl (about 12

N) with an equal amount of water. Need not

be standardized.

Pepsin—NF (Fisher Scientific, Cat. No. P-53).

Toluene—Commercial grade.

In vitro rumen bufl:er solution —

Distilled water 1.- 18

Ammonium bicarbonate .. g —. 72

Sodium bicarbonate g .— 630

BN-36454

FIGURE 8.—Arrangement of water bath for in vitro fermentations.

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14 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

In vitro rumen macromineral solution —

Distilled water 1.- 1 18

Na2HP04, anhydrous g .... 5.7 102.6

KH2PO4, anhydrous ...... g.... 6.2 111.6

MgS04-7H20 g 6 10.5

In vitro micromineral solution —

CaCl2-2H20 g.-.. 13.2

MnCl2-4H20 g... 10.0

CoCl2-6H20 g .... 1.0

FeCl3-6H20 g .... 8.0

Add to volumetric and bring volume to 100 ml.

with distilled water.

Procedure

The in vitro rumen procedure is designed so

that a true or apparent digestibility value can be

obtained. The predicted true digestibility value

is based on undigested cell-wall constituents.

The predicted apparent digestibility value or

value that is equal in magnitude to in vivo

apparent values is based on the Tilley and Terry

in vitro digestion technique that may contain

bacterial residues and other pepsin-insoluble

material. The in vitro procedure yielding true

digestibility values is a faster method and requires little extra equipment in a laboratory

containing a detergent apparatus.

The first five steps are common to both procedures.

1. Weigh sample,—Weigh 0.5-g. sample (20

mesh or 1 mm.) into 125-ml. Erlenmeyer flask.

2. Prepare medium,—Add in order 2 g. trypticase, 400 ml. water, and 0.1 ml. micromineral

solution, and agitate to dissolve. Then add

200 ml. buffer solution, 200 ml. macromineral

solution, and 1 ml. resazurin. Mix and add 40

ml. per 125-ml. flask.

3. Equilibration,—Assemble and put stoppers

and flasks in bath, admit carbon dioxide pressure (about 30 to 40 cm. water), and check

bunsen valves. Open the inlet tubes and swirl

flask while open and then close. Next prepare the reducing solution. Add 625 mg. cysteine hydrochloric acid, 95 ml. water, 4 ml.

1 N sodium hydroxide and dissolve ; then add

625 mg. sodium sulfide nonahydrate and dissolve. Reduce carbon dioxide pressure to 3 or

4 cm., and inject 2 ml. reducing solution

through the inlet tube with an automatic

syringe; open and close each tube in turn.

Swirl all flasks. Watch for reduction of

medium, which is a change from a red color

(oxidized) to colorless (reduced).

4. Prepare inoculum,—Collect ingesta from a

fistulated animal in a liter beaker, fill, cover

with a watchglass to eliminate airspace. Discard the top layer of the ingesta, and blend

400 ml. of the remainder in a Waring Blendor

for 2 minutes under carbon dioxide. Squeeze

the blended mass through cheesecloth and

filter through glass wool into a warm flask;

thereafter keep the filtrate under carbon dioxide. Inoculate 10 ml. of the filtrate with an

automatic syringe through inlet tubes of each

fermentation flask.

5. Fermentation,—Seal tubes and incubate 48

hours with shaking at a rate not to produce

splashing. Adjust carbon dioxide pressure to

2 cm. water.

At the end of fermentation one of two procedures can be followed : The Tillery and Terry

(3) filtration procedure (step 6) or treatment with neutral detergent (step 7). Flasks

may be stored before proceeding with step 6

or 7. Add 1 ml. toluene as a preservative and

refrigerate. Stopper with cork.

6. Tilley and Terry procedure (3, 15),—Add 2

ml. 6 N HCl to each flask carefully to avoid

excessive foaming. (This is sufficient to lower

pH below 2.) Add 0.5 g. pepsin National

Formulary grade (may be measured with a

scoop). Swirl to dissolve. Add 1 ml. toluene,

replace flasks in water bath, and incubate 48

hours. Remove flasks from water bath and

filter on previously tared Whatman No. 4, 41,

or 54 filter paper without suction. Rinse filter

twice by filling (almost to overflowing) and

allowing to drain to a low level. Fill filter with

acetone and allow to drain and air-dry. Fold

papers, dry at 100° C, and weigh. Dry matter

on paper is done on separate circles. Use drymatter factor to calculate dry weight for

tared paper circles used in filtering. Separate

blanks containing inoculum and medium, but

not substrate, must be run simultaneously.

Whatman No. 4, 41, or 54 filter paper and a

Jacket No. 387-598
FORAGE FIBER ANALYSES 15

common forage such as orchardgrass are

used as standards.

Neutral-detergent procedure for estimation

of true digestibility.—Remove flasks from

water bath after digestion or from refrigerator if stored. Wash with 100 ml. neutraldetergent solution into 600-ml. Berzelius beaker to make a total volume of 150 ml. Add 2 ml.

decahydronaphthalene. Reflux for 1 hour, and

filter on previously tared 50-ml., 40-mm. plate,

coarse-porosity fritted-glass crucibles. Wash

twice with hot water and twice with acetone.

and suck dry. Dry in oven at 100° C. and

weigh. Blanks are not necessary.

8. Calculations,—Calculate true dry-matter digestibility:

100 - percent ND residue = true digestibility

Calculate by Tilley and Terry method:

[1.00 - [(R-F) - blank/oven-dry sample

weight]] 100=percentage digestibility

where: R = weight of residue and filter paper;

F = weight of filter paper;

Blank value is R-F when substrate is

not added to medium.

SAMPLING TECHNIQUES

Preparatory and drying procedures cannot be

standardized to one method that would be satisfactory for all conditions; the experimenter must

choose intelligently those that suit his purpose.

A variety of preparatory and drying methods

are presented. Incompatibilities are generally

noted.

Sampling of Dry Feeds

Digestion trial

Chop hay to 1% inches (about 3.5 cm.)

through 1-inch (2.5-cm.) screen. Weigh and bag

hay for individual feedings before digestion

trial but after intake level has been established.

Composite samplings from the bags to comprise

not less than 5 kg. Dry sampled forage if necessary, but at less than 65° C. Grind in a large

Wiley mill through 2-mm. (10-mesh) screen.

Return all contents of mill to the sample. Collect

in a large plastic bag and mix, by rolling partially filled bag on floor. Subsample from all parts

of the bag to an amount of at least 2 kg. and

grind through 1-mm. (about 20-mesh) screen.

Allow ground material to equilibrate with air

overnight before placing in enclosed containers.

Suhsampling

Spread material on a smooth surface, preferably metal, and divide the pile into quarters.

Select at least eight increments from all parts

equally to obtain subsample. Samples for collaborative work should be obtained from existing

stocks in this manner.

Suhsampling for chemical analyses

Roll bottle thoroughly to obtain mixing. Insert

spatula into different places to obtain sample for

analytical work.

Sampling of Wet Materials

Gross sampling

Cattle feces.—Mix (by hand or with a mechanical mixer) daily collection thoroughly on a

clean surface, quarter, and subsample 1 kg. or

10 percent of wet weight. Store in a plastic container in a freezer. Daily samples may be

thawed, composited, mixed, and subsampled at

a later date.

Sheep feces.—Collect cumulative feces from

at least 5 days' collection, and pass through a

meat grinder with a 6-mm. plate. Clean grinder,

add all contents to the ground mass, mix thoroughly, and subsample 1 kg. A salad chopper

can be used in place of meat grinder. Store in a

plastic container in a freezer.

Silages and fresh forages.—Pass not less than

2 kg. frozen silage through meat grinder with

6-mm. plate. Tie large plastic bag over the end

of the grinder to collect ground material. Clean

grinder and add all contents to ground mass, and

mix thoroughly. A salad chopper can be used in

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16 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

place of a meat grinder. Store bag and contents

in a freezer.

Subsampling and handling of wet samples

for laboratory analyses ^

Thaw and empty contents of forage subsamples from the gross sampling onto a clean

surface and cover with a large sheet of plastic.

Mix material with hands under the plastic.

Quarter and subsample forage and fill an 8-oz.

(250-ml.) wide-mouth plastic polyethylene bottle not more than two-thirds full. For fecal

material, mix in a plastic beaker with a food

mixer (electric eggbeater) and subsample material to fill bottle two-thirds full. Close bottle

with a plastic cap (puncture the cap with a

spatula allowing the spatula to remain in place

primarily within the bottle). Weigh closed container to 1 mg. and remove an amount of material with spatula equivalent to 0.5 to 2 gm. of

dry matter to a requisite sample container. Replace cap-spatula assembly and reweigh bottle.

Take difference between first and second weights

as weight of sample taken. Weigh samples for

dry-matter determination in conjunction with

matter weighed for other determinations. Mix

sample contents of bottle between weighing by

stirring with spatula.

Technique is devised so that the loss of weight

in wet sampled material and material adhering

to the spatula does not effect the determination

of net sample weight. Weighing the cap-spatula

assembly as well as the sample bottle and contents before and after removing the sample

eliminates this source of error. The cap-spatula

assembly is removed after weighing is completed

and then replaced with an ordinary cap. Bottles

are refrozen; they may be rethawed, mixed, and

subsampled for further analyses at a later time.

Alternate techniques for handling

wet samples

Wet grinding tvith a Wiley mill.—Grind frozen material through an intermediate Wiley mill

with a 2-mm. (10-mesh) screen. Add charges of

dry ice to keep sample frozen. This procedure

is equivalent to the grinding in a meat grinder,

but it is not amenable to the handling of large

samples.

Oven drying.—Weigh 500-g. sample into a 18-

X 30-cm. tared pan and dry at 65° C. in a forceddraft oven. Remove pan and allow to equilibrate

with air at room temperature for 24 hours.

Weigh and calculate yield. Grind dried material

through 1-mm. (about 25-mesh) screen in a

Wiley mill. Grinding through finer screens (30-

to 40-mesh) may tend to induce filtering

problems.

Caution.—In feces and silages, loss of nitrogen

as ammonia results from oven drying. In silages

there is also a serious loss of volatile organic

acids and caloric value. Damage to lignin, protein, and carbohydrates can occur in all ovendried materials, so that true values of individual

components may not be obtained.

Freeze drying.—Wet samples can be freeze

dried and ground through a Wiley mill. Heatdamage is avoided, but loss of nitrogen in the

form of ammonia occurs in feces and silages.

Volatile fatty acids are lost in freeze drying.

Acetone drying (Suitahle for lignin and other

components of fiber and cell wall.).—Weigh 100

g. of wet ingesta into a 500-ml. wide-mouth

Erlenmeyer flask and add 400 ml. of reagentgrade acetone. Shake thoroughly and allow to

stand with occasional shaking for 1 hour. Shake

and pour mixed contents into a 10-cm. frittedglass, coarse-porosity, Büchner funnel previously tared to 0.1 g. Allow to settle before applying

suction. Suck off excess acetone with vacuum.

Remove vacuum and add 400 ml. fresh acetone,

while stirring to wash any remaining fiber from

Erlenmeyer flask. One washing is sufficient.

Preparation need not be washed free of pigment.

Preparation is sucked dry on the filter. Allow

to air-dry for 24 hours at room temperature.

If humidity is high, funnels containing fiber

may be dried at 40° C. for 4 hours in a forceddraft oven. Weigh funnel plus contents and calculate yield of acetone-dried powder. Dry-matter determinations should be made on original

wet ingesta. Dried acetone powders are ground

in an intermediate Wiley mill. Store in a tightly

stoppered container. Calculate oven-dry sample

weight by multiplying acetone dry sample

weight with the following correction factor:

' See below for alternate techniques. OD sample factor dry matter of wet ingesta

acetone powder yield

Jacket No. 387-598
FORAGE FIBER ANALYSES 17

COMPARISON OF HOT AND COLD SAMPLE WEIGHING

Problem.—Fiber and lignin determinations

are made in open crucibles that cannot be covered or otherwise protected from the pickup of

moisture. Since fiber and lignin are hydroscopic

materials, great care must be taken to avoid

errors due to moisture pickup. A ''hot'' weighing

procedure was compared with a ''cold" one when

two types of desiccators were used.

Apparatus.—A forced-draft oven set at 100°

C. in proximity to a single-pan automatic balance

sensitive to 0.1 mg. and two desiccators, one

containing silica gel and the other phosphorous

pentoxide, were used.

Procedure.—For hot weighing, dry crucibles

were placed in a forced-draft oven for a minimum of 2 hours before weighing. Crucibles were

removed one at a time for weighing, placed on

balance pan, and weighed rapidly. This weight

was obtained 20 to 30 seconds after placing

crucible on balance pan. The minimum weight

attained on the vernier scale was recorded. After

removal of the crucible, any deflection of the

balance setting from zero was recorded. The

zero deflection due to temperature change of the

balance was subtracted or added (positive or

negative) for each weight. The balance was

readjusted to zero before weighing the next

crucible.

For cold weighing, dry crucibles were placed

in a forced-draft oven a minimum of 2 hours

before placing in desiccators. Crucibles were

placed in dessicators for a minimum of 20

minutes and then weighed.

Conclusions.—Since water absorption will be

evidenced as an apparently higher fiber yield,

the technique resulting in the lowest fiber yield

is assumed to be most accurate. The data show

that the hot-weighing technique is reproducible

(table 1) and this technique gives lighter crucibles than the cold technique (table 2). Crucibles

weighed from desiccators picked up moisture;

hence, they weighed 2.8 (P2O5 desiccant) and

3.8 percent (silica gel desiccant) higher than

those weighed hot.

TABLE 1.—Reproducibility with the hot

weighing procedure

Crucible Hot weight after drying for — Average

3 hr. 6 hr. 24 hr. deviation

G. G. G. G.

1 34.6911 34.6924 34.6928 0.0007

2 34.7861 34.7856 34.7850 .0006

3 34.0462 34.0451 34.0470 .0015

4 34.1868 34.1877 34.1867 .0009

5 35.4202 35.4203 35.4205 .0001

From this comparison it is evident that the

hot-weighing procedure is superior. The precision of the hot-weighing technique is dependent on reading the minimum weight within 20

to 30 seconds. The shifting from zero can be

nonsignificant if a series of crucibles are

weighed with a minimum but equal time between crucibles. Precision can be attained with

practice. In the Beltsville laboratory, the procedure was found to be more rapid and precise

than cold weighing. This technique is also used

on filter paper.

TABLE 2.—Difference in weight and yield of fiber from the hot technique

when weighed by the cold technique with two desiccants

Tare weight ^ Tare plus sample ^ Relative fiber yield ^

Crucible Silica gel P2O5 Silica gel P2O5 Silica gel P2O5

Mg. Mg. Mg. Mg. Percent Percent

1 22.2 19.8 36.0 26.1 103,8 102.7

2 20.5 17.6 35.0 26.2 103.7 102.7

3 20.4 17.3 35.9 26.3 103.7 102.7

4 23.2 22.1 36.7 26.4 103.8 102.8

5 23.1 21.5 35.2 26.4 103.7 102.8

6 22.5 22.7 35.8 26.6 103.8 102.8

7 20.9 2L0 36^6 26J 103.8 102.7

^ Difference from hot weight.

2 Apparent fiber as percentage of yield obtained by hot technique.

Jacket No. 387-598
18 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

ESTIMATION OF NUTRITIVE VALUE FROM CHEMICAL DATA

A summative system of calculation of nutritive value is based on the assumption that

individual chemical factors additively limit

nutritive value. The most basic division in plant

dry matter is between cellular contents and

plant cell wall (7, 10, 13). As a result the summative digestibility equation can be formulated:

Percent digestible dry matter =

0.98S+T^Dc-M

where: S = cellular contents of an average digestibility of 98 percent ;

W^ = the percent cell-wall contents;

i)c = the estimated digestion coefficient

of the cell walls ;

Misestimated metabolic fecal losses.

The estimated metabolic losses for sheep average

12.9 units of digestibility, which value is always

used. In estimating metabolic losses for cattle

the following regression should be used:

M = 36.57 - 0.275Z

where: M — estimated metabolic fecal losses;

X = estimated true digestibility;

if: X = 50, 60, 70, 80, 90

then: M = 22.8, 20.1, 17.3, 14.5, 11.8,

respectively.

Digestibility of cell walls is variable and depends on lignification, silicification, and other

factors. Lignin is most important and is best

expressed as a percentage of acid-detergent fiber

(L/ADF), Cell-wall digestibility (Dc) can be

estimated by the use of the appropriate equation

for either permanganate or 72 percent acid

lignin or, alternatively, values may be interpolated from table 3. Values above 80 percent

digestibility deviate from the equations; the

table allows for this.

There is one precaution to the use of lignin

to estimate cell-wall digestibility; that is, artifact lignins produced by heating or drying forage can cause errors (6, 9). Corrections should

be made if the forage in question is known to

have undergone a history of heating. In order

to correct lignin and acid-detergent fiber for

heat damage, it is necessary to know the nitrogen content of acid-detergent fiber. This requires an extra Kjeldahl determination. Correction of lignin is made through a regression

equation :

TABLE 3.—Conversion of KMn04 and 72 percent

acid L/ADF ^ ratios to estimated cell-wall

digestibility

Estimated Estimated

72 percent cell-wall KMnO, cell-wall

acid digestibility - digestibility ^

{Dc) (Dc)

4 90 5 92

6 85 8 88

7 81 10 84

8 76 11 80

9 72 12 76

10 68 13 73

11 65 14 70

12 62 15 67

13 59 16 65

14 57 17 62

15 55 18 60

16 52 19 57

17 50 20 55

18 48 21 53

19 46 22 51

21 43 24 48

23 40 26 44

25 37 28 41

27 34 30 38

30 30 35 32

35 25 40 28

40 21 45 24

45 17 50 21

50 13 60 14

^ L/ADF, percentage of lignin in acid-detergent fiber.

^ 147.3-78.9 logio [{L/ADF) 100].

n80.8-96.6 logio [{L/ADF) 100].

Lc-1.208 Lo - 10.75 No + 0.42

where : Lc = corrected lignin ;

Loathe observed lignin;

No = the amount of nitrogen in aciddetergent fiber expressed on a

whole-feed basis.

Artifact lignin — Lo - Lc =" La. Acid-detergent

fiber and cell-wall values are corrected by subtracting La. If heat damage has occurred, corrected lignin and acid-detergent fiber values

must be used in the summative scheme.

In grasses, silica (SÍO2) is an important factor

affecting digestibility, and a special term is introduced into the summative equation (11, 12),-

Plant metabolic silica causes a decline of 3.0

units per 1 percent of silica. This factor must

not be applied when there is sand or soil contamination ; the factor 1.4 should be used in such

cases.

A detailed scheme of calculations is shown in

table 4, where the successive effects of lignificaJacket No. 387-598
FORAGE FIBER ANALYSES 19

TABLE 4.—Scheme for using the summative equation

Component Analytical value ^ Factor Digestible amount '

Cellular contents 100—cell-wall constituents

Lignification of cell wall ^ Cell-wall constituent analysis

Silica correction SÍO2 analysis

Heat-damage effect _. Artifact lignin ^

Estimated true DDM ^

Metabolic fecal matter^

Estimated apparent DDM ^

0.98 Add

From table 3 Add

*3.0 Subtract

1.0 Subtract

Sum

Subtract

Difference

^ All values must be expressed as percentage of whole dry matter.

2 Analytical value is multiplied by factor to obtain the digestible amount and then the process indicated in this

column is performed.

^ Acid-detergent fiber and lignin must be corrected if heat damage exists. Follow directions as given with

equation.

* The factor 3.0 is not appropriate for silicates of sand or soil contamination in forage. The silica correction

can be ignored if insoluble ash is less than 2.0 percent.

^ DDM, digestible dry matter.

^ See discussion on variation of metabolic fecal matter, p. 18.

tion, silicification, heat damage, and metabolic

losses from digestion are outlined.

An example of a poor-quality orchardgrass

hay is given as a sample calculation. Forage was

brown, showing visible evidence of having heated; it yielded the following analysis: cell walls

of 72.0 percent, acid-detergent fiber 43.1 percent, and apparent acid-detergent lignin 5.51

percent, silica 5.40 percent (from appearance,

obviously metabolic),^ and nitrogen content of

acid-detergent fiber 0.58 percent.

First step is to calculate the correct lignin.

This is done by substitution in the lignin-correction equation:

Lc = 1.208 X 5.51 - (0.58 x 43.1/100 x 10.75)

+ 0.42 = 4.4

Then

Artifact lignin = 5.5-4.4 = 1.1.

and

Corrected acid-detergent fiber—43.1-1.1 = 42.0.

Next step is to calculate the estimated cellwall digestibility. This is done by dividing the

corrected lignin by the corrected ADF to yield :

(L/ADF) 100 = (4.4/42.0) 100 = 10.5

This value is substituted in the regression:

^ Metabolic silica appears fibrous and white like filter

paper cellulose. Soil contamination is usually colored

and shows grains of sand.

Z)c = 147.3 - 78.9 logio[ (L/ADF) 100] =67 percent.

Values may also be interpolated from table 3.

The summative relation is then set up as shown

in table 5.

Estimation of voluntary intake (8),—Voluntary intake based on sheep data and cell-wall

contents (CWC) has given the following regression:

1,716 Intake as g/kg'^^ = 110.4 -

(100^ CWC)

Intake estimates based on this regression are

subject to large errors, and hence, should be

used with caution. The relations between chemical compositions and intake are fairly consistent

in some forage species but unpredictable in

others.

TABLE 5.—Example of calculation of digestible

dry matter (DDM) for orchardgrass hay

Component

Analytical

value Factor

Digestible

amount ^

Cellular contents 28 0.98 -f27.4

Lignification of

cell wall _. 72 .67 +48.2

Silica correction .. 5.4 3.0 -16.2

Heat-damage effect .. 1.1 1.0 - 1.1

Estimated true DDM . 58.3

Metabolic fecal

matter -12.9

Estimated DDM 45.4

^ Analytical value is multiplied by factor to obtain the

digestible amount and then the process indicated in this

column is performed.

Jacket No. 387-598
20 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

LITERATURE CITED

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

GoERiNG, H. K., and VAN SOEST, P. J.

1967. EFFECT OF MOISTURE, TEMPERATURE, AND

pH ON THE RELATIVE SUSCEPTIBILITY OF

FORAGES TO NON-ENZYMIC BROWNING.

Jour. Dairy Sei. 50:989 (Paper No. 103).

MEARA, M.L.

1955. THE WAXES, CUTIN AND SUBERIN. Peach, K.,

and Tracy, M. V., Eds., Moderne Methoden der Pflanzenanalyse 2:380-402.

TiLLEY, J. M. A., and TERRY, R. A.

1963. A TWO-STAGE TECHNIQUE FOR THE IN VITRO

DIGESTION OF FORAGE CROPS. Jour. Brit.

Grassland Soc. 18:104-111,

VAN SOEST, P. J.

1963. USE OF DETERGENTS IN THE ANALYSIS OF

FIBROUS FEEDS. I. PREPARATION OF FIBER

RESIDUES OF LOW NITROGEN CONTENT.

Assoc. Off. Agr. Chem. Jour. 46:825-829.

1963. USE OF DETERGENTS IN THE ANALYSIS OF

FIBROUS FEEDS. II. A RAPID METHOD FOR THE

DETERMINATION OF FIBER AND LIGNIN.

Assoc. Off. Agr. Chem. Jour. 46:829-835.

1964. SYMPOSIUM ON NUTRITION AND FORAGE AND

PASTURES: NEW CHEMICAL PROCEDURES

FOR EVALUATING FORAGES. Jour. Anim. Sci.

23:838-845.

1965. NON-NUTRITIVE RESIDUES: A SYSTEM OF

ANALYSIS FOR THE REPLACEMENT OF CRUDE

FIBER. Assoc. Off. Agr. Chem. Jour. 49:

546-551.

1965. SYMPOSIUM ON FACTORS INFLUENCING THE

VOLUNTARY INTAKE IN RELATION TO CHEMICAL COMPOSITION AND DIGESTIBILITY. Jour.

Anim. Sei. 24:834-843.

(9)

(10)

(11)

(12)

(13)

(14)

(15)

1965. USE OF DETERGENTS IN ANALYSIS OF FIBROUS FEEDS. III. STUDY OF EFFECTS OF

HEATING AND DRYING ON YIELD OF FIBER

AND LIGNIN IN FORAGES. AssOC. Off. Agr.

Chem. Jour. 48:785-790.

1967. DEVELOPMENT OF COMPREHENSIVE SYSTEM

OF FEED ANALYSES AND ITS APPLICATION TO

FORAGES. Jour. Anim. Sei. 26:119-128.

1968. STRUCTURAL AND CHEMICAL CHARACTERISTICS WHICH LIMIT THE NUTRITIVE VALUE OF

FORAGES. Forage: Economics/Quality,'

ASA Spec. Pub. No. 13:63-76.

and JONES, L. H. P.

1968. EFFECT OF SILICA IN FORAGES UPON DIGESTÍ- ,

BILITY. Jour. Dairy Sei. 51:1644-1648.

and WINE, R. H.

1967. USE OF DETERGENTS IN THE ANALYSIS OF

FIBROUS FEEDS. IV. DETERMINATION OF

PLANT CELL-WALL CONSTITUENTS. AssOC.

Off. Analytical Chem. Jour. 50:50-55.

and WINE, R. H.

1968. DETERMINATION OF LIGNIN AND CELLULOSE

IN ACID-DETERGENT FIBER WITH PERMANGANATE. Assoc. Off. Analytical Chem.

Jour. 51:780-785.

WINE, R. H., and MOORE, L. A.

1966. ESTIMATION OF THE TRUE DIGESTIBILITY OF

FORAGES BY THE IN VITRO DIGESTION OF

CELL WALLS. Proc. 10th Internatl. Grassland Cong., pp. 438-441.

lîrU.S. GOVERNMENT PRINTING OFFICE: 1970 O 387-598

Jacket No. 387-598