FORAGE FIBER ANALYSES (Apparatus, Reagents, Procedures, and Some Applications)

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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.

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Jacket No. 387-598 ACKNOWLEDGMENTS

The authors wish to acknowledge the following for their contribu- tions in developing the detergent apparatus: W. R. Delauder, formerly of the Animal Husbandry Research Divi- sion* 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 Re- search 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 Univer- sity, Wageningen, The Netherlands.

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

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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 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

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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 concerned are dominant in the sample. For ex- for those who wish to (1) set up the newer ample, it makes little sense to expect lignin to detergent system of fiber fractionation (^, 5, P, be a good predictor of digestibility if silica or 13)j^ (2) adapt their apparatus and equipment some other factor is a more important variable. to the new analyses, and (3) estimate the di- In vitro fermentations will be influenced by all gestibility directly by a modified {15) Tilley and factors and inhibitors known and unknown. The Terry {3) two-stage in vitro rumen fermenta- in vitro, however, does not disclose anything re- tion procedure. garding the nature of the limiting factor. This evaluation of forage is essentially latter task of identification remains the duty of aimed at obtaining analytical data that predict chemical studies. the extent of biological degradation under speci- An attempt has been made to cover the prin- fied conditions, animals, organisms, and time. cipal problems in technique encountered in these In general, an in vitro rumen fermentation re- procedures. These include sample preparation, flects the factors known and unknown limiting filtration and washing, and efficiency in han- availability of forage to the digesting organism. dling large numbers of samples. Ultimate lab- The analyst can assay only known constituents. oratory efficiency is tied to the precision of dupli- The assays are for things that are expedient to cates and the repeatability of values. Handling handle, are known, or are considered important, of samples in drying and weighing and in ashing and its evaluation is valid only if the principals and reweighing is also discussed.

CHEMICAL-FIBER DETERMINATIONS Apparatus plates for reflux, (2) each plate or burner is individually regulatable and has suflicient power In general, equipment that is used for crude so that boiling solutions keep fibers in con- fiber may be adapted to the detergent fiber pro- tinuous suspension, (3) reflux containers are cedures. The reflux apparatus described is more straight-sided and not conical, and (4) the con- convenient and cheaper than many other types denser system is sufliicient to keep volume of available. However, if a laboratory contains boiling solutions constant. In general, oil baths a reflux apparatus for crude fiber, it may be and large hotplates are not satisfactory for used without modification. Other types of reflux quantitative work. apparatus probably are suitable if they conform The filtration apparatus described is highly to the following criteria: (1) a minimum of six recommended. In general, filtration manifolds ^ Presently associate professor, Animal Science De- suitable for crude fiber do not adapt well unless partment, Cornell University, Ithaca, N.Y. 2 Italic numbers in parentheses refer to Literature they conform to the following specifications. Cited, p. 20. Samples that contain appreciable amounts of

Jacket No. 387-598 2 AGRICULTUEE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE starch, protein, or other mucilaginous substance fritted glass, 50-ml. capacity, coarse poros- are often hard to filter when detergent-fiber ity. Type sold by Fisher Scientific, Silver analyses are being performed. Much difficulty Spring, Md., Cat. No. 8-237 is avoided by having an adequate filtering ap- c. Scimatco rubber tubing, black, thick wall, paratus and by using proper technique. The Yi6-m. (8.0-mm.) bore, %2-in. (8.5-mm.) main requirement is a filter manifold system wall with receivers for at least six . Filtra- d. Hotplate, Thermolyne (HP-A8805B),blue- tion must be individually controlled for each porcelain, steel flattop, 120 v., 3.3 a., 400 crucible. Experience has shown that filtration w., 3 %-m. (9.5-cm.) diameter of a difficult sample niust begin with little to no e. Multielectric 6-receptacle box, 20 a., 115 v. vacuum and then be gradually increased. Often (needed if more than one hotplate is going a very significant volume can be filtered without to be used) any vacuum. The addition of asbestos to the f. Condensers, reflux, crude-fiber, Pyrex. E. sample may help filtration, but this involves the H. Sargent and Company, Philadelphia, laborious operation of preparing the asbestos Pa., Cat. No. S-22742 or equivalent 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 opera- tions that arise should be allowed. It is very im- portant to have solutions near areas of use and quantities large enough to last for several days of continuous analyses. An ashing oven equip- ped 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 re- sults. Enough equipment is required to ade- quately 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 cru- cibles make this possible. Technicians tend to make fewer mistakes when a standard labora- tory 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. BN-36467 capacity FIGURE 1.—Arrangement of hotplates, rings, beakers, b. Crucibles, Gooch-type, high form Pyrex condensers, and hose.

Jacket No. 387-598 FORAGE FIBER ANALYSES

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BN-36455 FIGURE 2.—Arrangement of six units of refluxing apparatus, with flexaframe support.

g. Flexaframe hook connectors (need approx- i. Screw clamp imately 36 for a set of six hotplates) Water heater (fig. 6) : h. Rings, cast iron, 4-in. (10-cm.) OD a. Ring support, cast iron with clamp, 6-in. i. Flexaframe rods: (15-cm.) OD 12 in. (30 cm.) (need 5) b. Flask, distilling, 2 1., 3 neck 24 in. (60 cm.) (need 3) c. Quartz immersion heater, 1,000 or 500 w., 36 in. (90 cm.) (need 2) 120 V. Ace Glass, Inc., Vineland, N. J., Cat. 2. Filter manifold (figs. 3-5) : No. 2145-12 or 2145-10 a. Polyethylene pipe, 1-in. (2.5-cm.) ID, %- Crucible holder, filter tubes. Pyrex, 10- in. (3-mm.) wall, 2 ft. (60 cm.) mm. diameter (for automatic filling of b. Funnels, Büchner, porcelain, Coors 490, large flask) size-0 Bottle, Pyrex, 5-gal. (18-1.) capacity c. Flasks, filtering. Pyrex, 4-1. capacity Transformer, variable, 10 a. d. Clamps, pinchcocks, Mohr e. Rubber adapter, size-B. Fisher Scientific Flexaframe rods, 18 in. and 48 in. (45 cm. Co., Silver Spring, Md., Cat. No. 8-239 and 120 cm.) f. Rubber tubing, 34-in. (6-mm.) ID, %-in. Clamp, versatile, vinylized jaws, medium (3-mm.) wall size (need 2) g. Glass tubing, 5-mm. OD, 3-mm. ID J- Clamps, pinchcock, Day h. Polyethylene tubing, %-in. (3-mm.) ID, k. Y connection, %6-in. (48-mm.) OD, metal ViQ-m. (1.5-mm.) wall 1. tip

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

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1 km^^^ ^^^^^^^^B^^^^^MKBBBfer-''"^' ^J • " ' ■■■-■■■• "--^^'^^^^^^^^^^^^^^^^^^^^^^^^^^■X flH BN-36409 FIGURE 3.—Filter manifold.

Description connects each of these small tubes to size 0 Büchner funnels, to prevent collapsing from the Figure 1 shows a section of the refluxing ap- vacuum. The funnels are supported on a 1- x paratus. Ceramic hotplates, 3%-inch, 120 v., 4-in. wooden frame, constructed as shown in are used to produce sufficient heat to boil 200 figures 3 and 4. Holes are drilled to hold the fun- ml. of liquid in the beaker. Ceramic plates pro- nels to give adequate working room for filtering tect coils from liquids spilled on the plate, which the sample. The rubber adapters (Fisher 8-239) avoids electrical problems. Special Pyrex glass are inserted into the Büchner funnels to give a reflux condensers rest on top of the beaker, vacuum seal. Heavy-wall rubber tubing con- which are otherwise supported by the rubber nects from the manifold to two 4-1. side-arm tubing connected to the condensers. Thick-wall flasks, which serve as traps. Vacuum level is %Q-m. (8.0-mm.) bore rubber tubing is used to controlled by attaching a rubber tube with a connect the condensers in a series of six. Parts screw clamp to the opposite end of the poly- of refluxing apparatus are supported with flex- ethylene manifold. A pinch type clamp closes aframe unit. Rings covered with rubber tubing each tube coming from the Büchner funnel, are placed above the hotplate to prevent knock- which makes it possible to control each crucible ing off the beaker. Figure 2 shows the complete individually. six-unit refluxing apparatus, which requires a The third component of the detergent appa- multielectric six-receptable box with a minimum ratus, the hot-water system, is constructed di- of 20 a. rectly above the filtering manifold (fig. 6). The filter manifold is shown in figures 3, 4, Water is stored in an elevated carboy reservoir and 5. The filter manifold is constructed to hold above and to the side of the heater. A closed six crucibles, so that six samples can be filtered siphon is connected with a leveling device (seen at one time. The manifold is made from large on the left of the large flask in figure 6), a 2-1., polyethylene pipe, 2 feet (60 cm.) in length 3-neck distilling fiask with a well at the bottom with a 1-inch (2.5-cm.) inside diameter (ID). to allow space for the immersion heater (about Six polyethylene tubes are attached into the 50 X 120 mm.), and a water inlet to the bot- larger tube. A glass lining (5-mm. outside diam- tom of the well. Water enters from the reservoir eter (OD) tubing) is inserted inside each small into the leveling device and then goes directly to polyethylene tube. The joints are welded with a the bottom of the well. A tube connects from the polyethylene welding rod to prevent leakage and leveling device to the reservoir to serve as an maintain vacuum. Thick-wall rubber tubing -^'"r vent. Also, an air vent is placed on the level-

Jacket No. 387-598 FORAGE FIBER ANALYSES

RUBBER RING

630 mm. -♦20 mm.

2x '^ ^

152 mm. i¥:

TAPERED END POLYETHYLENE RUBBER OF TUBE TUBE BRACE RUBBER STOPPERS

FIGURE 4.—Side diagram of filter manifold. ing device. The 2-1. flask then fills automati- The hose terminates with a Y connector, fitted cally; a constant supply of hot water is thus on one end with a burette tip end and on the maintained. A condenser at the top of the flask other with a tube, which allows each to be prevents loss of steam. The water-heating appa- clamped off. The Y is insulated and wrapped ratus is supported on one flexaf rame rod. Vinyl- with asbestos tape so that the hot water can be ized clamps are used to allow for expansion of manipulated without protection for the hands. glassware due to temperature change. A thick- A workable water system is essential for the wall hose feeds water to the filtering manifold. 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 t control water temperature. 47 mm. A convenient setup is shown in figure 7. The \ water system is directly above the filter mani- fold with two sets of six refluxing units on each 29 mm. side. This setup allows for refluxing detergents continuously and also allows the technician sufficient time for filtering and washing of the t residues. 58 mm.

\ Reagents 18 mm. Neutral-detergent solution— Distilled water 1.... 1 18 40 Sodium lauryl sulfate, USP .... g .... 30 540 1,200 Disodium ethylene- FIGURE 5.—End diagram of filter-manifold frame. diaminetetraace-

Jacket No. 387-598 AGEICULTURE HANDBOOK 379, U.S. DEPARTMENT OP AGRICULTURE

täte (EDTA), a square bag sewn from a rectangle of fiber- dihydrate crystal, glass window screening of 14 x 18 mesh reagent grade.... g 18.61 335.0 744.4 (about 1 mm.) (the bag should be at least 18 Sodium borate de- inches (46 cm.) wide by 12 inches (30 cm.) cahydrate, re- deep). Wash by immersion and agitation in agent grade .... g.... 6.81 122.6 272.4 water to remove fine particles. Ash the re- Disodium hydrogen covered asbestos in a furnace at 800° C. for phosphate, anhy- 16 hours. Store in dry form until needed. drous, reagent Used asbestos can be rewashed, ashed, and grade g.... 4.56 82.1 182.4 reused. 2-ethoxyethanol 8. Sulfuric acid, 72 percent by weight—Calcu- (ethylene glycol late grams acid and water needed in 1 1. of monoethyl ether), solution by: 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 in- gredients. 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 trimethylam- monium 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. con- centrated H2SO4 (technical grade), mix, and let cool at room temperature for 2 hours. Fil- ter on a large Büchner funnel, and wash with BN-36456 water. Resuspend mat in water and pour into FIGURE 6—Hot-water heating system.

Jacket No. 387-598 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 9. Saturated potassium permanganate— = grams acid needed H2SO4 assay (percent) Distilled water 1.... 1 18 KMn04, reagent grade .. g — 50 900 (1,000 X 1.634)3 -grams acid = Ag2S04, reagent grade .. g .— 0.05 0.9 grams water needed Dissolve KMn04 and Ag2S04 in distilled Weigh amount of water into a 1-1. MCA volu- water. Keep out of direct sunlight. Add silver metric flask (with a bulb in the neck) and add sulfate to dehalogenate the reagent. the calculated amount of H2SO4 slowly with 10. Lignin buffer solution— occasional swirling. Caution! Flask must be 1 I. 12 I. cooled in a water bath (sink) in order to add Ferric nitrate the required weight of sulfuric acid. Cool to nonahydrate g.... 6 72 20° C. and check if volume is correct. If vol- Silver nitrate g 15 1.8 ume is too small, take out about 1.5 ml. and Acetic acid, glacial .. ml.... 500 6,000 add 2.5 ml. water. Repeat, if necessary. If Potassium acetate .... g.... 5 60 volume is too large, take out 5 ml. and add Tertiary butyl 4.45 ml. H2SO4. Meniscus should be within a alcohol ml.... 400 4,800 0.5 cm. of calibration mark at 20°. Distilled water ml.... 100 1,200 ^ Weight of 1 1. of 72 percent H2SO4. Dissolve ferric nitrate nonahydrate [Fe

Jacket No. 387-598 8 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

(N03)3 • 9H2O] and silver nitrate in dis- total fiber in vegetable feedstuff s (IS), It ap- tilled water. Combine with acetic acid and pears to divide the dry matter of feeds very near potassium acetate. Add tertiary butyl alco- the point that separates the nutritively avail- hol and mix. Use grades of acid and solvent able (98 percent) and soluble constituents from passing dicromate test (ACS). those that are incompletely available and de- 11. Combined permanganate solution—Combine pendent on a microbial fermentation. and mix saturated potassium permanganate Reagents required: 1 through 4. and lignin buffer solution in the ratio of 2:1, 1. Weigh 0.5- to 1.0-g. air-dry sample ground to by volume, before use. Unused mixed solu- pass 20 to 30 mesh (1-mm.) or equivalent tion may be kept about a week in a refrig- into a beaker of the reñuxing apparatus. erator in the absence of light. Solution is 2. Add in order, 100 ml. cold (room tempera- usable if purple and containing no precipi- ture) neutral-detergent solution, 2 ml. deca- tate. Old solutions assume a reddish color hydronaphthalene, and 0.5 g. sodium sulfite and should be discarded. with a calibrated scoop. Heat to boiling in 5 12. Demineralizing solution— to 10 minutes. Reduce heat as boiling begins, 1 L 18 L Oxalic acid dihydrate g —. 50 900 to avoid foaming. Adjust boiling to an even 95 percent ethanol ml.... 700 12,600 level and reflux for 60 minutes, timed from Concentrated (about onset of boiling. 12 N) hydrochloric 3. Place previously tared Gooch crucibles on fil- acid ml.... 50 900 ter manifold. Swirl beaker to suspend solids, Distilled water ml.... 250 4,500 and fill crucible. Do not admit vacuum until Dissolve oxalic acid dihydrate in 95 per- after crucible has been filled. Use low vacuum cent ethanol. Add concentrated hydrochloric at-first and increase it only as more force is acid and distilled water and mix. needed. Rinse sample into crucible with mini- 13. Ethanol 80 percent— mum of hot (90°-100° C.) water. Remove 1 L 18 L vacuum, break up mat, and fill crucible with 95 percent ethanol .... ml .... 845 15,200 hot water. Filter liquid and repeat washing Distilled water ml.... 155 2,800 procedure. 14. Hydrobromic acid, reagent grade. 4. Wash twice with acetone in same manner and 15. Cleaning solution— suck dry. Dry crucibles at 100° C. for 8 hours Distilled water 1 -.. 1 or overnight and weigh. Disodium ethylenediaminetetra- 5. Report yield of recovered neutral-detergent acetate (EDTA), dihydrate fiber as percent of cell-wall constituents. Esti- crystal, reagent grade g.... 5 mate cell soluble material by subtracting this Trisodium phosphate, value from 100. laboratory grade g .... 50 6. Ash residue in the crucible for 3 hours at Potassium hydroxide g.... 200 500° to 550° C. and weigh. Report ash content 16. 0.1 N HCl—Add 17 ml. 6 N HCl to 1 1. of as ash insoluble in neutral-detergent. distilled water. Need not be standardized. 17. Pepsin—NF (Fisher Scientific, Cat. No. Acid-detergent fiber P-53). The acid-detergent fiber procedure provides a rapid method for lignocellulose determination Analytical Procedures^ in feedstuffs {i, 5). The residue also includes NeutraUdetergent (cell-wall) silica. The difference between the cell walls and acid-detergent fiber is an estimate of hemicel- The neutral-detergent procedure for cell-wall lulose; however, this difference does include constituents is a rapid method for analyzing the some protein attached to cell walls. The acid- * Numbers given after "Reagents required" refer to detergent fiber is used as a preparatory step for numbers in the reagent section, p. 5. lignin determination.

Jacket No. 387-598 FORAGE FIBER ANALYSES 9

Reagents required: 2, 3, and 5. 1. Prepare the acid-detergent fiber (p. 8). 1. Weigh 1-g. air-dry sample ground to pass 2. Add to the crucible containing the acid-deter- 20- to 30-mesh (1-mm.) screen or the ap- gent fiber an amount of asbestos about equal proximate equivalent of wet material into a to the volume of fiber. Cover the contents of beaker suitable for refluxing. the crucible with cooled (15° C.) 72 percent 2. Add 100 ml. cold (room temperature) acid- H2SO4 and stir with a to a smooth detergent solution and 2 ml. decahydronaph- paste, breaking all lumps. Fill crucible about thalene. Heat to boiling in 5 to 10 minutes. half full with acid and stir. Let glass rod re- Reduce heat as boiling begins, to avoid foam- main in crucible; refill with 72 percent H2SO4 ing. Reflux 60 minutes from onset of boiling ; and stir at hourly intervals as acid drains adjust boiling to a slow, even level. away. Crucibles do not need to be kept full at 3. Filter on a previously tared Gooch crucible, all times. Three additions suffice. Keep cru- which is set on the filter manifold; use light cible at 20° to 23° C. After 3 hours, filter off suction. Break up the filtered mat with a rod as much acid as possible with vacuum; then and wash twice with hot water (90°-100° wash contents with hot water until free from C). Rinse sides of the crucible in the same acid. Rinse and remove stirring rod. manner. 3. Dry crucible at 100° C. and weigh. 4. Repeat wash with acetone until it removes no 4. Ignite crucible in a muffle furnace at 500° to more color; break up all lumps so that the 550° C. for 3 hours, and then cool to 100° solvent comes into contact with all particles and weigh. of fiber. 5. Calculate acid-detergent lignin: 5. Optional wash with hexane. Hexane should be (LxlOO)/S = ADL added while crucible still contains some ace- where: L = loss upon ignition after 72 per- tone. (Hexane can be omitted if lumping is cent H2SO4 treatment ; not a problem in lignin analysis.) Suck the S = oven-dry sample weight acid-detergent fiber free of hexane and dry at 100° C. for 8 hours or overnight and weigh. Permanganate lignin^ cellulose^ insoluble 6. Calculate acid-detergent fiber: ash, and silica (Wo-Wt) (100)/S = ADF An indirect method to determine lignin was where: Wo=weight of oven-dry crucible in- developed that makes possible the preparation cluding fiber ; of cellulose and insoluble ash in the same sample Wt=tared weight of oven-dry cruci- (li). The insoluble ash is an estimate of total ble; silica content, which in many grasses is a pri- iS = oven-dry sample weight mary factor in reducing digestibility. The per- manganate lignin method is an alternative Acid-detergent lignin procedure to the 72 percent sulfuric acid In the acid-detergent lignin procedure, the method; each has its own advantages. Choice of acid-detergent fiber (ADF) procedure is used as methods depends on materials analyzed and on a preparatory step (5). The detergent removes the purpose for which the values are to be used. the protein and other acid-soluble material that Advantages of the permanganate method over would interfere with the lignin determination. the 72 percent acid method include a shorter The ADF residue consists of cellulose, lignin, procedure for lignin per se while the residue is cutin, and acid-insoluble ash (mainly silica). reserved for further analysis of cellulose and Treatment with 72 percent sulfuric acid dis- silica. The permanganate reagents are much solves cellulose. Ashing of the residue will de- less corrosive and require no standardization. termine the crude lignin fraction including The residue requires no filter aids, and lignin cutin. For silica determination and separation values are not subject to some interferences that of cutin and lignin, see the Permanganate and affect 72 percent sulfuric acid lignin. Values are Acid-Detergent Cutin Procedures. less affected by heat-damage artifacts and are Reagents required: 2, 3, 5, 7, and 8. closer to a true lignin figure.

Jacket No. 387-598 10 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

However, cutin, which is important in many level (2-3 cm.) of water in pan to reduce flow seed hulls, is not measured. A variation for the of solution out of crucibles. Place a short analysis of seed hulls is to prepare the perman- glass rod in each crucible to stir contents, to ganate cellulose and treat with 72 percent H2SO4 break lumps, and to draw permanganate and asbestos for 3 hours as described in the solution up on sides of crucibles to wet all acid-detergent lignin procedure. This results in particles. the partitioning of crude lignin into two frac- 3. Allow crucibles to stand at 20° to 25° C. for tions as described in the acid-detergent cutin 90 ±1 10 minutes; add more mixed perman- procedure. ganate solution if necessary. Purple color One disadvantage to permanganate lignin is must be present at all times. that large particles are poorly penetrated by the 4. Remove crucibles to filtering apparatus. Suck reagents and yield low values. Consequently, all dry. Do not wash. Place in a clean enamel pan, materials must be dried and ground to pass and fill crucibles no more than half full with through a 20- to 30-mesh (less than 1 mm.) demineralizing solution. Demineralizing so- screen, and the method is not applicable to fresh lution may be added directly to crucibles in feces and forages that have been ground in a case filtering is difficult. Care must be taken meat grinder. Because of high sensitivity to heat to avoid spillage by foaming. After about 5 damage, 72 percent acid is preferred for assay- minutes, suck dry on filter and refill half full ing artifact lignin. with demineralizing solution. Repeat after Theory of the method—Interfering matter is second interval if solution is very brown. removed by preparing acid-detergent fiber, Rinse sides of crucibles with solution from a which is chieñy composed of lignin, cellulose, with a fine stream. Treat until and insoluble minerals. Lignin is oxidized with fiber is white. Total time required is 20 to an excess of acetic acid-buffered potassium per- 30 minutes. manganate solution, containing trivalent iron 5. Fill and thoroughly wash crucible and con- and monovalent silver as catalysts. Deposited tents with 80 percent ethanol. Suck dry and manganese and iron oxides are dissolved with repeat two times. Wash twice in similar an alcoholic solution of oxalic and hydrochloric manner with acetone. Suck dry. acids, which leaves cellulose and insoluble min- 6. Dry at 100° C. overnight and weigh. Calcu- erals. Lignin is measured as the weight lost by late lignin content as loss in weight from these treatments; whereas, cellulose is deter- acid-detergent fiber. mined as the weight loss upon ashing. The ash 7. Ash at 500° C. for 3 hours, cool, and weigh. residue is mainly silica and much of the non- Calculate residual ash as the difference be- silica matter can be removed by leaching with tween this weight and original tare of cru- concentrated hydrobromic acid. cible. Calculate cellulose by weight loss upon Reagents required: 2, 3, 5, 6, 9 through 14. ashing. 1. Dry samples at less than 65° C. and grind 8. A presumptive analysis for silica may be through 20- to 30-mesh (1-mm.) screen. obtained by hydrobromic acid treatment of Prepare and determine acid-detergent fiber the ashed permanganate lignin or ADF resi- according to standard procedure. Use a 1-g. due. This determination has its greatest value sample, except on samples containing a high when the residual ash is greater than 2 per- amount of lignin (15 percent or more) use cent. Ash and weigh, then add enough drops 0.5-g. sample. Place previously weighed cru- of 48 percent HBr to moisten all particles. cibles in a shallow enamel pan containing Use no more than 4 ml. acid. Allow to stand cold water to a depth of about 1 cm. Fiber in 1 to 2 hours. Add more drops of HBr if much crucibles should not be wet. red color forms. Suck off excess acid on 2. Add about 25 ml. of combined saturated vacuum and wash once with acetone. Use no potassium permanganate and lignin buffer water. Dry and ash briefly at 500° C. Cool solution (2:1 by volume) to the crucibles in and weigh. Report silica as the difference the enamel pan containing cold water. Adjust between this weight and the original tare.

Jacket No. 387-598 FORAGE FIBER ANALYSES 11

Precautions—Crucibles containing fiber of Crucible cleaning a high lignin content require more permanga- nate solution ; however, avoid additions of more Reagents required: 15. solution than is necessary. Appearance of a 1. Empty contents and ash crucibles briefly, 1 yellow or brown color indicates exhaustion of to 2 hours, at 500° to 550° C. (not necessary permanganate. If crucible is full, filter solution if crucible already ashed in lignin or silica OÍF on a vacuum and add more reagent. A yellow determination). color persisting after treatment of fiber with 2. Wash crucible with tapwater. demineralizing solution indicates incomplete re- 3. Force distilled water upward through the moval of lignin. This occurs only in materials crucible; use a No. 7 rubber stopper with tube of a very high lignin content. Cutin material through middle connected to the distilled present in seedcoats and other plant parts is not water outlet. Rinse outside with distilled oxidized by permanganate; thus, it is neither water and place in oven. Proceed to next step determined as lignin nor bleached with the if the crucible does not give normal filtering treatments. Seedcoats appear as colored flecks properties. among white cellulose particles, and thus 4. Place crucible that has been ashed in 500° they should not be confused with incomplete to 550° C. in a shallow enamel pan. Add about oxidation. 50 ml. crucible cleaning solution to each cru- Excessive flow of permanganate solution cible. Heat (such as steam bath) should be through the crucibles should be avoided with placed under the pan. Let cleaning solution samples of low lignin content, particularly in filter through crucible. Bore hole in a No. 9% samples of immature grasses. With these, a rubber stopper and insert one end of a 50-ml. single addition of permanganate solution suf- and attach a tube to the upper end of fices. Fiber from immature grasses is very the pipette. Place the stopper assembly in the rapidly delignified, and, therefore, there is top of the crucible and apply vacuum until the danger of loss of cellulosic carbohydrates if the crucible is approximately one-half full of flow is too great. Reduction in flow is accom- cleaning solution. Wait for solution to filter plished by adjusting the water level in the pan through crucible again. Refill the crucible to near that in crucibles. These precautions are with the solution again. The cleaning solution not needed with the demineralizing solution. may be saved and reused. 5. Repeat steps 2 and 3. Acid-detergent cutin (2) The fraction of plant material referred to as Acid-detergent nitrogen cutin is the fraction that is not oxidized by Heat-drying of forages at temperatures above KMnOé and resists hydrolysis by 72 percent 50° C. shows analytically significant increases H2SO4 acid. This fraction can be very large, as in yield of lignin and fiber. The increased yield in some seed hulls, or not important, as in the of acid-detergent fiber (ADF) can be accounted common forages. The relation of cutin to the for largely by the production of artifact lignin nutritive value of the other plant constituents via the nonenzymic browning reaction. Value is not understood. However, the cutin factor is for ADF and lignin in dried forages can be cor- resistant to microbial degradation. rected on the basis of the nitrogen content of Reagents required: 2, 3, 5 through 13. the ADF (1, 9), The nitrogen content of the 1. Follow procedure for KMn04 lignin and cel- ADF is suggested as a sensitive assay for non- lulose preparation up to the step where lignin enzymic browning due to overheating of feeds. can be calculated but the residue has not been ashed (steps 1-6, p. 10). Reagents required: 2, 3, and 5. 2. Treat the unashed residue with 72 percent 1. Follow step 1 and 2 of acid-detergent fiber H2SO4, as in the acid-detergent lignin pro- procedure using a 2-g. sample, page 8. cedure. 2. Filter with suction on previously tared 12.5- 3. Calculate cutin as loss in weight upon ashing. cm. Whatman No. 54 paper. Fold paper into

Jacket No. 387-598 12 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE a cone and use 60° angle funnel and a filter nitrogen according to standard Kjeldahl cone (Fisher No. 9-760) to protect tip. procedure. 3. Wash paper with hot water and then acetone 5. Calcualte pepsin-insoluble nitrogen: until acid free. Dry at 100° C. for 8 hours or (g. N/oven-dry sample) (100) overnight and weigh. which can be expressed as: (pepsin-insoluble N/total sample N) (100) 4. Transfer paper residue into Kjeldahl ñask. Run nitrogen on residue in flask according to Hot'tvater-insoluble matter and standard Kjeldahl procedure. its nitrogen content 5. Calculate ADF: This procedure gives an estimate of true pro- (Wo-Wt){im/S^ADF tein nitrogen in forages. The forage sample is where : Wo = weight of oven-dry boiled in water, which coagulates the protein including fiber ; causing it to become water insoluble and remain Tft = tared weight of oven-dry filter in the cell of the forage. paper ; 1. Weigh 2.0-g. sample into 600-ml. Berzelius S = oven-dry sample weight beaker. 6. Express ADF-N as percent of total DM : 2. Add 200 ml. distilled water to beaker and boil AT (100)/S = ADF-N for 1 hour on refluxing apparatus. where: N — grams of nitrogen; 3. Filter with vacuum on a previously tared S = oven-dry sample weight 12.5-cm. Whatman No. 54 filter paper on a Another way of expressing the ADF-N is 60° funnel and a filter cone tp protect tip. ADF-N/total nitrogen (100). Wash four times with hot water. Place in 100° C. oven for 8 hours and weigh. Pepsin-insoluble nitrogen (1) 4. Take weighed filter paper residue and trans- fer to a Kjeldahl flask. Run nitrogen on resi Reagents required: 16 and 17. due and filter paper according to standard 1. Weight 0.5-g. of air dry sample ground to pass Kjeldahl procedure. Correct for filter by run- 20- to 30-mesh (less than 1-mm.) screen, into ning Kjeldahl on filter paper itself. 125-ml. . 5. Calculate hot-water-insoluble msitier (HwIM) 2. Add 0.5 g. of pepsin and 50 ml. of 0.1 N HCl including nitrogen (HwIN) : to flask, stopper, and swirl. Place in oven or (Wr- Wt/oven-dry sample weight) (100) = water bath at about 39° C. for 20 hours. HwIM 3. Filter on 12.5-cm. Whatman No. 4, 41, or 54 (g.N/oven-dry sample weight) (100) = filter paper. Leach thoroughly with distilled HwIN; water. One way to ascertain if all soluble where : Wr = weight of filter paper plus in- nitrogen is leached out is to determine if all soluble residue; traces of HCl has been removed from the Wt = weight of filter paper filter paper. The taste test can be used for this. This insoluble nitrogen can be used as an 4. Take filter paper containing pepsin insoluble estimate of true protein in forages only (hot residue and place in a Kjeldahl flask. Run water insoluble N x 6.25).

IN VITRO RUMEN DIGESTIBILITY DETERMINATION

Apparatus 2. Erlenmeyer flasks, 125 ml. (Pyrex, requiring No. 6 stoppers) Materials 3. Shaking water bath at 40° C. with holder for 1. Rumen-content source from an animal on a 18 flasks high cell-wall roughage (preferably a fistu- 4. Manifold for 18 flasks constructed over water lated animal) bath

Jacket No. 387-598 FORAGE FIBER ANALYSES 13

5. Waring Blendor supply of carbon dioxide and in parallel with a 6. CO2 source water manometer with a capacity of 60-cm. 7. Cheesecloth water pressure. Figure 8 shows the shaking water bath and CO2 manifold apparatus. 8. Glass wool and enclosed funnel assembly 9. Automatic syringe, 10 ml. 10. Glassware refluxing apparatus as used for Reagents detergent preparations Trypticase—A pancreatic digest of casein, USP. Sodium sulfide nonahydrate—Reagent grade. Description 1 N sodium hydroxide—Dissolve 4 g. in water and dilute to a liter. Fermentations are conducted in 125-ml. Cysteine-HCl—J. T. Baker Go. grade or equiva- Pyrex Erlenmeyer flasks (wide-mouth); 0.5-g. lent. substrate, 40-ml. medium, and 10-ml. inoculum Resazurin—0.1 percent w/v solution. are used. Fermentation flasks are placed in a 6 N HCl—Dilute concentrated HCl (about 12 shaking water bath (capacity 18) and closed N) with an equal amount of water. Need not with No. 6 rubber stoppers. Stoppers are fitted be standardized. with three openings: an inlet tube, a bunsen Pepsin—NF (Fisher Scientific, Cat. No. P-53). valve—both flush with the bottom of the stop- per—and a gassing tube connected to a common Toluene—Commercial grade. manifold. The inlet tube is closed on the outside In vitro rumen bufl:er solution — with a rubber sleeve and glass rod. The gassing Distilled water 1.- 18 tube should stop about 1 cm. above the surface Ammonium bicarbonate .. g —. 72 of the liquid. The manifold is connected to a Sodium bicarbonate g .— 630

BN-36454

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

Jacket No. 387-598 14 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE

In vitro rumen macromineral solution — 4 cm., and inject 2 ml. reducing solution Distilled water 1.- 1 18 through the inlet tube with an automatic Na2HP04, anhydrous g .... 5.7 102.6 syringe; open and close each tube in turn. KH2PO4, anhydrous ...... g.... 6.2 111.6 Swirl all flasks. Watch for reduction of MgS04-7H20 g 6 10.5 medium, which is a change from a red color In vitro micromineral solution — (oxidized) to colorless (reduced). CaCl2-2H20 g.-.. 13.2 4. Prepare inoculum,—Collect ingesta from a MnCl2-4H20 g... 10.0 fistulated animal in a liter beaker, fill, cover CoCl2-6H20 g .... 1.0 with a watchglass to eliminate airspace. Dis- FeCl3-6H20 g .... 8.0 card the top layer of the ingesta, and blend Add to volumetric and bring volume to 100 ml. 400 ml. of the remainder in a Waring Blendor with distilled water. for 2 minutes under carbon dioxide. Squeeze the blended mass through cheesecloth and filter through glass wool into a warm flask; Procedure thereafter keep the filtrate under carbon di- oxide. Inoculate 10 ml. of the filtrate with an The in vitro rumen procedure is designed so automatic syringe through inlet tubes of each that a true or apparent digestibility value can be fermentation flask. obtained. The predicted true digestibility value 5. Fermentation,—Seal tubes and incubate 48 is based on undigested cell-wall constituents. hours with shaking at a rate not to produce The predicted apparent digestibility value or splashing. Adjust carbon dioxide pressure to value that is equal in magnitude to in vivo 2 cm. water. apparent values is based on the Tilley and Terry At the end of fermentation one of two proce- in vitro digestion technique that may contain dures can be followed : The Tillery and Terry bacterial residues and other pepsin-insoluble (3) filtration procedure (step 6) or treat- material. The in vitro procedure yielding true ment with neutral detergent (step 7). Flasks digestibility values is a faster method and re- may be stored before proceeding with step 6 quires little extra equipment in a laboratory or 7. Add 1 ml. toluene as a preservative and containing a detergent apparatus. refrigerate. Stopper with cork. The first five steps are common to both pro- 6. Tilley and Terry procedure (3, 15),—Add 2 cedures. ml. 6 N HCl to each flask carefully to avoid 1. Weigh sample,—Weigh 0.5-g. sample (20 excessive foaming. (This is sufficient to lower mesh or 1 mm.) into 125-ml. Erlenmeyer flask. pH below 2.) Add 0.5 g. pepsin National 2. Prepare medium,—Add in order 2 g. trypti- Formulary grade (may be measured with a case, 400 ml. water, and 0.1 ml. micromineral scoop). Swirl to dissolve. Add 1 ml. toluene, solution, and agitate to dissolve. Then add replace flasks in water bath, and incubate 48 200 ml. buffer solution, 200 ml. macromineral hours. Remove flasks from water bath and solution, and 1 ml. resazurin. Mix and add 40 filter on previously tared Whatman No. 4, 41, ml. per 125-ml. flask. or 54 filter paper without suction. Rinse filter 3. Equilibration,—Assemble and put stoppers twice by filling (almost to overflowing) and and flasks in bath, admit carbon dioxide pres- allowing to drain to a low level. Fill filter with sure (about 30 to 40 cm. water), and check acetone and allow to drain and air-dry. Fold bunsen valves. Open the inlet tubes and swirl papers, dry at 100° C, and weigh. Dry matter flask while open and then close. Next pre- on paper is done on separate circles. Use dry- pare the reducing solution. Add 625 mg. cys- matter factor to calculate dry weight for teine hydrochloric acid, 95 ml. water, 4 ml. tared paper circles used in filtering. Separate 1 N sodium hydroxide and dissolve ; then add blanks containing inoculum and medium, but 625 mg. sodium sulfide nonahydrate and dis- not substrate, must be run simultaneously. solve. Reduce carbon dioxide pressure to 3 or Whatman No. 4, 41, or 54 filter paper and a

Jacket No. 387-598 FORAGE FIBER ANALYSES 15 common forage such as orchardgrass are and suck dry. Dry in oven at 100° C. and used as standards. weigh. Blanks are not necessary. Neutral-detergent procedure for estimation 8. Calculations,—Calculate true dry-matter di- of true digestibility.—Remove flasks from gestibility: water bath after digestion or from refrigera- 100 - percent ND residue = true digestibility tor if stored. Wash with 100 ml. neutral- Calculate by Tilley and Terry method: detergent solution into 600-ml. Berzelius beak- [1.00 - [(R-F) - blank/oven-dry sample er to make a total volume of 150 ml. Add 2 ml. weight]] 100=percentage digestibility decahydronaphthalene. Reflux for 1 hour, and where: R = weight of residue and filter paper; filter on previously tared 50-ml., 40-mm. plate, F = weight of filter paper; coarse-porosity fritted-glass crucibles. Wash Blank value is R-F when substrate is twice with hot water and twice with acetone. not added to medium.

SAMPLING TECHNIQUES Preparatory and drying procedures cannot be laborative work should be obtained from existing standardized to one method that would be satis- stocks in this manner. factory for all conditions; the experimenter must choose intelligently those that suit his purpose. Suhsampling for chemical analyses A variety of preparatory and drying methods are presented. Incompatibilities are generally Roll bottle thoroughly to obtain mixing. Insert noted. spatula into different places to obtain sample for analytical work. Sampling of Dry Feeds Sampling of Wet Materials Digestion trial Chop hay to 1% inches (about 3.5 cm.) Gross sampling through 1-inch (2.5-cm.) screen. Weigh and bag Cattle feces.—Mix (by hand or with a mech- hay for individual feedings before digestion anical mixer) daily collection thoroughly on a trial but after intake level has been established. clean surface, quarter, and subsample 1 kg. or Composite samplings from the bags to comprise 10 percent of wet weight. Store in a plastic con- not less than 5 kg. Dry sampled forage if neces- tainer in a freezer. Daily samples may be sary, but at less than 65° C. Grind in a large thawed, composited, mixed, and subsampled at Wiley mill through 2-mm. (10-mesh) screen. a later date. Return all contents of mill to the sample. Collect Sheep feces.—Collect cumulative feces from in a large plastic bag and mix, by rolling partial- at least 5 days' collection, and pass through a ly filled bag on floor. Subsample from all parts meat grinder with a 6-mm. plate. Clean grinder, of the bag to an amount of at least 2 kg. and add all contents to the ground mass, mix thor- grind through 1-mm. (about 20-mesh) screen. oughly, and subsample 1 kg. A salad chopper Allow ground material to equilibrate with air can be used in place of meat grinder. Store in a overnight before placing in enclosed containers. plastic container in a freezer. Silages and fresh forages.—Pass not less than Suhsampling 2 kg. frozen silage through meat grinder with Spread material on a smooth surface, prefer- 6-mm. plate. Tie large plastic bag over the end ably metal, and divide the pile into quarters. of the grinder to collect ground material. Clean Select at least eight increments from all parts grinder and add all contents to ground mass, and equally to obtain subsample. Samples for col- mix thoroughly. A salad chopper can be used in

Jacket No. 387-598 16 AGRICULTURE HANDBOOK 379, U.S. DEPARTMENT OF AGRICULTURE place of a meat grinder. Store bag and contents Oven drying.—Weigh 500-g. sample into a 18- in a freezer. X 30-cm. tared pan and dry at 65° C. in a forced- draft oven. Remove pan and allow to equilibrate Subsampling and handling of wet samples with air at room temperature for 24 hours. for laboratory analyses ^ Weigh and calculate yield. Grind dried material Thaw and empty contents of forage sub- through 1-mm. (about 25-mesh) screen in a samples from the gross sampling onto a clean Wiley mill. Grinding through finer screens (30- surface and cover with a large sheet of plastic. to 40-mesh) may tend to induce filtering Mix material with hands under the plastic. problems. Quarter and subsample forage and fill an 8-oz. Caution.—In feces and silages, loss of nitrogen (250-ml.) wide-mouth plastic polyethylene bot- as ammonia results from oven drying. In silages tle not more than two-thirds full. For fecal there is also a serious loss of volatile organic material, mix in a plastic beaker with a food acids and caloric value. Damage to lignin, pro- mixer (electric eggbeater) and subsample ma- tein, and carbohydrates can occur in all oven- terial to fill bottle two-thirds full. Close bottle dried materials, so that true values of individual with a plastic cap (puncture the cap with a components may not be obtained. spatula allowing the spatula to remain in place Freeze drying.—Wet samples can be freeze primarily within the bottle). Weigh closed con- dried and ground through a Wiley mill. Heat- tainer to 1 mg. and remove an amount of ma- damage is avoided, but loss of nitrogen in the terial with spatula equivalent to 0.5 to 2 gm. of form of ammonia occurs in feces and silages. dry matter to a requisite sample container. Re- Volatile fatty acids are lost in freeze drying. place cap-spatula assembly and reweigh bottle. Take difference between first and second weights Acetone drying (Suitahle for lignin and other components of fiber and cell wall.).—Weigh 100 as weight of sample taken. Weigh samples for g. of wet ingesta into a 500-ml. wide-mouth dry-matter determination in conjunction with Erlenmeyer flask and add 400 ml. of reagent- matter weighed for other determinations. Mix grade acetone. Shake thoroughly and allow to sample contents of bottle between weighing by stirring with spatula. stand with occasional shaking for 1 hour. Shake Technique is devised so that the loss of weight and pour mixed contents into a 10-cm. fritted- in wet sampled material and material adhering glass, coarse-porosity, Büchner funnel previous- to the spatula does not effect the determination ly tared to 0.1 g. Allow to settle before applying of net sample weight. Weighing the cap-spatula suction. Suck off excess acetone with vacuum. assembly as well as the sample bottle and con- Remove vacuum and add 400 ml. fresh acetone, tents before and after removing the sample while stirring to wash any remaining fiber from eliminates this source of error. The cap-spatula Erlenmeyer flask. One washing is sufficient. assembly is removed after weighing is completed Preparation need not be washed free of pigment. and then replaced with an ordinary cap. Bottles Preparation is sucked dry on the filter. Allow are refrozen; they may be rethawed, mixed, and to air-dry for 24 hours at room temperature. subsampled for further analyses at a later time. If humidity is high, funnels containing fiber may be dried at 40° C. for 4 hours in a forced- Alternate techniques for handling draft oven. Weigh funnel plus contents and cal- wet samples culate yield of acetone-dried powder. Dry-mat- Wet grinding tvith a Wiley mill.—Grind fro- ter determinations should be made on original zen material through an intermediate Wiley mill wet ingesta. Dried acetone powders are ground with a 2-mm. (10-mesh) screen. Add charges of in an intermediate Wiley mill. Store in a tightly dry ice to keep sample frozen. This procedure stoppered container. Calculate oven-dry sample is equivalent to the grinding in a meat grinder, weight by multiplying acetone dry sample but it is not amenable to the handling of large weight with the following correction factor: samples. dry matter of wet ingesta OD sample factor ' See below for alternate techniques. acetone powder yield

Jacket No. 387-598 FORAGE FIBER ANALYSES 17 COMPARISON OF HOT AND COLD SAMPLE WEIGHING Problem.—Fiber and lignin determinations Conclusions.—Since water absorption will be are made in open crucibles that cannot be cover- evidenced as an apparently higher fiber yield, ed or otherwise protected from the pickup of the technique resulting in the lowest fiber yield moisture. Since fiber and lignin are hydroscopic is assumed to be most accurate. The data show materials, great care must be taken to avoid that the hot-weighing technique is reproducible errors due to moisture pickup. A ''hot'' weighing (table 1) and this technique gives lighter cruci- procedure was compared with a ''cold" one when bles than the cold technique (table 2). Crucibles two types of desiccators were used. weighed from desiccators picked up moisture; Apparatus.—A forced-draft oven set at 100° hence, they weighed 2.8 (P2O5 desiccant) and C. in proximity to a single-pan automatic balance 3.8 percent (silica gel desiccant) higher than sensitive to 0.1 mg. and two desiccators, one those weighed hot. containing silica gel and the other phosphorous pentoxide, were used. TABLE 1.—Reproducibility with the hot Procedure.—For hot weighing, dry crucibles weighing procedure were placed in a forced-draft oven for a mini- Crucible Hot weight after drying for — Average mum of 2 hours before weighing. Crucibles were 3 hr. 6 hr. 24 hr. deviation removed one at a time for weighing, placed on G. G. G. G. 1 34.6911 34.6924 34.6928 0.0007 balance pan, and weighed rapidly. This weight 2 34.7861 34.7856 34.7850 .0006 was obtained 20 to 30 seconds after placing 3 34.0462 34.0451 34.0470 .0015 4 34.1868 34.1877 34.1867 .0009 crucible on balance pan. The minimum weight 5 35.4202 35.4203 35.4205 .0001 attained on the vernier scale was recorded. After removal of the crucible, any deflection of the balance setting from zero was recorded. The From this comparison it is evident that the hot-weighing procedure is superior. The pre- zero deflection due to temperature change of the cision of the hot-weighing technique is depend- balance was subtracted or added (positive or ent on reading the minimum weight within 20 negative) for each weight. The balance was to 30 seconds. The shifting from zero can be readjusted to zero before weighing the next nonsignificant if a series of crucibles are crucible. weighed with a minimum but equal time be- For cold weighing, dry crucibles were placed tween crucibles. Precision can be attained with in a forced-draft oven a minimum of 2 hours practice. In the Beltsville laboratory, the pro- before placing in desiccators. Crucibles were cedure was found to be more rapid and precise placed in dessicators for a minimum of 20 than cold weighing. This technique is also used minutes and then weighed. 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 nutri- TABLE 3.—Conversion of KMn04 and 72 percent tive value is based on the assumption that acid L/ADF ^ ratios to estimated cell-wall individual chemical factors additively limit digestibility nutritive value. The most basic division in plant Estimated Estimated dry matter is between cellular contents and 72 percent cell-wall KMnO, cell-wall plant cell wall (7, 10, 13). As a result the sum- acid digestibility - digestibility ^ {Dc) (Dc) mative digestibility equation can be formulated: 4 90 5 92 Percent digestible dry matter = 6 85 8 88 0.98S+T^Dc-M 7 81 10 84 8 76 11 80 where: S = cellular contents of an average di- 9 72 12 76 gestibility of 98 percent ; 10 68 13 73 11 65 14 70 W^ = the percent cell-wall contents; 12 62 15 67 i)c = the estimated digestion coefficient 13 59 16 65 14 57 17 62 of the cell walls ; 15 55 18 60 Misestimated metabolic fecal losses. 16 52 19 57 17 50 20 55 The estimated metabolic losses for sheep average 18 48 21 53 12.9 units of digestibility, which value is always 19 46 22 51 21 43 24 48 used. In estimating metabolic losses for cattle 23 40 26 44 the following regression should be used: 25 37 28 41 27 34 30 38 M = 36.57 - 0.275Z 30 30 35 32 where: M — estimated metabolic fecal losses; 35 25 40 28 40 21 45 24 X = estimated true digestibility; 45 17 50 21 if: X = 50, 60, 70, 80, 90 50 13 60 14 then: M = 22.8, 20.1, 17.3, 14.5, 11.8, ^ L/ADF, percentage of lignin in acid-detergent fiber. respectively. ^ 147.3-78.9 logio [{L/ADF) 100]. n80.8-96.6 logio [{L/ADF) 100]. Digestibility of cell walls is variable and de- pends on lignification, silicification, and other Lc-1.208 Lo - 10.75 No + 0.42 factors. Lignin is most important and is best where : Lc = corrected lignin ; expressed as a percentage of acid-detergent fiber Loathe observed lignin; (L/ADF), Cell-wall digestibility (Dc) can be No = the amount of nitrogen in acid- estimated by the use of the appropriate equation detergent fiber expressed on a for either permanganate or 72 percent acid whole-feed basis. lignin or, alternatively, values may be interpo- lated from table 3. Values above 80 percent Artifact lignin — Lo - Lc =" La. Acid-detergent digestibility deviate from the equations; the fiber and cell-wall values are corrected by sub- table allows for this. tracting La. If heat damage has occurred, cor- There is one precaution to the use of lignin rected lignin and acid-detergent fiber values to estimate cell-wall digestibility; that is, arti- must be used in the summative scheme. fact lignins produced by heating or drying for- In grasses, silica (SÍO2) is an important factor age can cause errors (6, 9). Corrections should affecting digestibility, and a special term is in- be made if the forage in question is known to troduced into the summative equation (11, 12),- have undergone a history of heating. In order Plant metabolic silica causes a decline of 3.0 to correct lignin and acid-detergent fiber for units per 1 percent of silica. This factor must heat damage, it is necessary to know the nitro- not be applied when there is sand or soil con- gen content of acid-detergent fiber. This re- tamination ; the factor 1.4 should be used in such quires an extra Kjeldahl determination. Correc- cases. tion of lignin is made through a regression A detailed scheme of calculations is shown in equation : table 4, where the successive effects of lignifica-

Jacket 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 0.98 Add Lignification of cell wall ^ Cell-wall constituent analysis From table 3 Add Silica correction SÍO2 analysis *3.0 Subtract Heat-damage effect _. Artifact lignin ^ 1.0 Subtract Estimated true DDM ^ Sum Metabolic fecal matter^ Subtract Estimated apparent DDM ^ 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 Z)c = 147.3 - 78.9 logio[ (L/ADF) 100] =67 per- losses from digestion are outlined. cent. An example of a poor-quality orchardgrass Values may also be interpolated from table 3. hay is given as a sample calculation. Forage was The summative relation is then set up as shown brown, showing visible evidence of having heat- in table 5. ed; it yielded the following analysis: cell walls Estimation of voluntary intake (8),—Volun- of 72.0 percent, acid-detergent fiber 43.1 per- tary intake based on sheep data and cell-wall cent, and apparent acid-detergent lignin 5.51 contents (CWC) has given the following regres- percent, silica 5.40 percent (from appearance, sion: 1,716 obviously metabolic),^ and nitrogen content of Intake as g/kg'^^ = 110.4 - acid-detergent fiber 0.58 percent. (100^ CWC) First step is to calculate the correct lignin. Intake estimates based on this regression are This is done by substitution in the lignin-cor- subject to large errors, and hence, should be rection equation: used with caution. The relations between chemi- cal compositions and intake are fairly consistent Lc = 1.208 X 5.51 - (0.58 x 43.1/100 x 10.75) in some forage species but unpredictable in + 0.42 = 4.4 others. Then Artifact lignin = 5.5-4.4 = 1.1. TABLE 5.—Example of calculation of digestible dry matter (DDM) for orchardgrass hay and Analytical Digestible Corrected acid-detergent fiber —43.1-1.1 = 42.0. Component value Factor amount ^ Cellular contents 28 0.98 -f27.4 Next step is to calculate the estimated cell- Lignification of wall digestibility. This is done by dividing the cell wall _. 72 .67 + 48.2 Silica correction .. 5.4 3.0 -16.2 corrected lignin by the corrected ADF to yield : Heat-damage effect .. 1.1 1.0 - 1.1 Estimated true DDM . 58.3 (L/ADF) 100 = (4.4/42.0) 100 = 10.5 Metabolic fecal matter -12.9 This value is substituted in the regression: Estimated DDM 45.4 ^ Metabolic silica appears fibrous and white like filter ^ Analytical value is multiplied by factor to obtain the paper cellulose. Soil contamination is usually colored digestible amount and then the process indicated in this and shows grains of sand. column is performed.

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

(1) GoERiNG, H. K., and VAN SOEST, P. J. (9) 1967. EFFECT OF MOISTURE, TEMPERATURE, AND 1965. USE OF DETERGENTS IN ANALYSIS OF FI- pH ON THE RELATIVE SUSCEPTIBILITY OF BROUS FEEDS. III. STUDY OF EFFECTS OF FORAGES TO NON-ENZYMIC BROWNING. HEATING AND DRYING ON YIELD OF FIBER Jour. Dairy Sei. 50:989 (Paper No. 103). AND LIGNIN IN FORAGES. AssOC. Off. Agr. (2) MEARA, M.L. Chem. Jour. 48:785-790. 1955. THE WAXES, CUTIN AND SUBERIN. Peach, K., (10) and Tracy, M. V., Eds., Moderne Meth- 1967. DEVELOPMENT OF COMPREHENSIVE SYSTEM oden der Pflanzenanalyse 2:380-402. OF FEED ANALYSES AND ITS APPLICATION TO (3) TiLLEY, J. M. A., and TERRY, R. A. FORAGES. Jour. Anim. Sei. 26:119-128. 1963. A TWO-STAGE TECHNIQUE FOR THE IN VITRO (11) DIGESTION OF FORAGE CROPS. Jour. Brit. 1968. STRUCTURAL AND CHEMICAL CHARACTERIS- Grassland Soc. 18:104-111, TICS WHICH LIMIT THE NUTRITIVE VALUE OF (4) VAN SOEST, P. J. FORAGES. Forage: Economics/Quality,' 1963. USE OF DETERGENTS IN THE ANALYSIS OF ASA Spec. Pub. No. 13:63-76. FIBROUS FEEDS. I. PREPARATION OF FIBER (12) and JONES, L. H. P. RESIDUES OF LOW NITROGEN CONTENT. 1968. EFFECT OF SILICA IN FORAGES UPON DIGESTÍ- , Assoc. Off. Agr. Chem. Jour. 46:825-829. BILITY. Jour. Dairy Sei. 51:1644-1648. (5) (13) and WINE, R. H. 1963. USE OF DETERGENTS IN THE ANALYSIS OF 1967. USE OF DETERGENTS IN THE ANALYSIS OF FIBROUS FEEDS. II. A RAPID METHOD FOR THE FIBROUS FEEDS. IV. DETERMINATION OF DETERMINATION OF FIBER AND LIGNIN. PLANT CELL-WALL CONSTITUENTS. AssOC. Assoc. Off. Agr. Chem. Jour. 46:829-835. Off. Analytical Chem. Jour. 50:50-55. (6) (14) and WINE, R. H. 1964. SYMPOSIUM ON NUTRITION AND FORAGE AND 1968. DETERMINATION OF LIGNIN AND CELLULOSE PASTURES: NEW CHEMICAL PROCEDURES IN ACID-DETERGENT FIBER WITH PERMAN- FOR EVALUATING FORAGES. Jour. Anim. Sci. GANATE. Assoc. Off. Analytical Chem. 23:838-845. Jour. 51:780-785. (7) (15) WINE, R. H., and MOORE, L. A. 1965. NON-NUTRITIVE RESIDUES: A SYSTEM OF 1966. ESTIMATION OF THE TRUE DIGESTIBILITY OF ANALYSIS FOR THE REPLACEMENT OF CRUDE FORAGES BY THE IN VITRO DIGESTION OF FIBER. Assoc. Off. Agr. Chem. Jour. 49: CELL WALLS. Proc. 10th Internatl. Grass- 546-551. land Cong., pp. 438-441. (8) 1965. SYMPOSIUM ON FACTORS INFLUENCING THE VOLUNTARY INTAKE IN RELATION TO CHEM- ICAL COMPOSITION AND DIGESTIBILITY. Jour. Anim. Sei. 24:834-843.

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Jacket No. 387-598