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A CHEMICAL ANALYSIS OF FIBER IN FEEDS
AND ITS APPLICATION TO FEEDING STUDIES AND
PURE CULTURE ANALYSIS

By

George Frederick Collings

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Husbandry

1979

 

ABSTRACT

A CHEMICAL ANALYSIS OF FIBER IN FEEDS
AND ITS APPLICATION TO FEEDING STUDIES AND
PURE CULTURE ANALYSIS

By

George Frederick Collings

Sodium chlorite treatment, a technique of plant research for
30 years, is the oxidation of the phenyl ring of lignin without re—
moval of polysaccharides. This method was applied to the analysis
of forages and feeds and compared to permanganate lignin. One gram
samples were despersed in 0.5% ammonium oxalate, boiled for 2 hr
and filtered. The extracted fiber was resuspended in 1% acetic acid
at 70 C, sodium chlorite (1.25 g) was added, and lignin was oxidized
for a maximum length of 45 min. Oxidation was stopped by adding
ascorbic acid, and the suspension was filtered and dried at 60 C
for 48 hr. The difference in weight was defined as sodium chlorite
lignin. Differences in lignin values were considerable with the
various substrates, but sodium chlorite lignin values generally
tended to be higher than permanganate lignin values. Sodium chlorite
oxidation also removes some structural protein and may require pep-
sin—hydrochloric acid digestion prior to oxidation.

Gas-liquid chromatography methodology employed in this study

involved the hydrolysis of the hemicellulose of the plant cell wall
with trifluoroacetic acid into individual monsaccharides of all 20
substrates. The monosaccharides are converted to the corresponding
alditiol acetates which can be quantitated by GLC. VSubstrates were
boiled in ammonium oxalate solution to remove cell wall cytoplasm and
some uronic acids. Lignin was removed by sodium chlorite oxidation
prior to hydrolysis. Uronic acids removed in the various extracts
were determined by spectrometry. Values for lignin, cellulose, hemi-
cellulose, hemicellulosic sugars and uronic acids were calculated.
Hemicellulose values as determined by detergent analyses was, in gen-
eral, higher than hemicellulose values determined by GLC. Cellulose
values as determined after hydrolysis with trifluoroacetic acid
yielded similar results as detergent cellulose in most substrates.
Neutral detergent fiber values for all substrates were lower than
ammonium oxalate fiber values and correlated significantly (P<.05).
Analysis of the neutral detergent fiber and acid detergent fiber
fractions of the feeds and forages showed substantial hemicellulose
losses in the cell walls treated with neutral detergent and recovery
of hemicellulose in the cell walls treated with acid detergent.
Digestion trials with beef cattle, pigs and ponies were designed
to examine the fiber component digestibility in each animal. Four
beef cattle were fed a corn silage-supplemented diet for a 7 day col-
lection period. Pigs were fed a corn-soybean meal diet during a 5 day
collection period. Three ponies were limit-fed (80% ad libitum) al-
falfa-grass hay and oats during a 7 day collection period. Feed and

feces were collected and analyzed for NDF, ADF, and GLC fiber compon—

ents. Cattle digested 95.3% glucose, 54.5% galactose, 50.0% arabinose
and 37.4% xylose. The digestibilities of individual sugars by the pig
decreased in the following order: glucose, galactose, arabinose and
xylose. Ponies digested 92.9% arabinose, 68.9% glucose, 68.6% xylose
and 57.5% galactose. NDF digestibility was significantly correlated
with the digestibility of ammonium oxalate fiber (r=0.92), ADF (r=0.96)
and dry matter (r=0.92) in all species.

Two predominant rumen cellulolytic bacteria, Ruminococcus flave-

 

faciens C94 and Bacteroides succinogenes S85 were cultivated with

 

alfalfa, bromegrass, corn silage, cattle manure fiber, wheat straw
and Whatman filter paper (no 1) as substrates. GLC analyses, before

and after fermentation, showed that R. flavefaciens fermented a mean

 

of 35.6% of the hemicellulose and 29.7% of the cellulose in the sub-
strates, while B. succinogenes fermented a mean of 31.6% and 17.4%,
respectively. Electron microscopy showed that there were some
differences in the adherence of these species to wheat straw and
filter paper and no adherence to cattle manure fiber. Arabinose

and galactose composition of the cell wall was significantly (P<.05)

correlated with utilization only with R. flavefaciens (r=0.87 and

 

r=0.76, respectively.
The examination of fiber structure, digestibility in animals
and utilization by bacteria was enhanced by GLC analyses and coma

pared favorably to detergent fiber analyses.

ACKNOWLEGMENTS

This thesis is the end result of much work by many people
that I would like to acknowledge. First of all, my special thanks
are expressed to Dr. M. T. Yokoyama for all of his patience and time
that he has had for me in my graduate program. This is something
that will not be forgotten. Thanks are also given to the members
of my committee, Drs. M. R. Bennink, D. T. A. Lamporte, W. G. Bergen
and especially E. R. Miller for all of his extra help, guidance and
friendship. Special thanks are expressed to Dr. D. T. A. Lamporte
for all of his 'help!’ sessions with me. Gratitude is also extended
to Dr. R. H. Nelson for financing and allowing me to use the facili-
ties of the Department of Animal Husbandry. Special thanks are given
to S. Wiseley for putting up with my havoc and to Dr. S. Flegler for
his help with the electron microscope. I would also like to thank my
dear friends, Dr. W. Hart, C. Sisto, L. Margarella, C. Isichei, G.
Hawes and others who helped me in many ways. I would like to especial—
ly thank my wife, Laurie, for her constant giving, support and love
during my course of study, research and manuscript preparation. Last
but not least, I would like to thank God for His ever-present hand

in my life and for the narrow gate (John 10) he promised us.

TABLE OF CONTENTS

LIST OF TABLES ...................................................
LIST OF FIGURES ............................................ . .....
INTRODUCTION .............................................. . ......
REVIEW OF LITERATURE .............................................

Plant cell wall constituents .................................
Cellulose ...................................................
Hemicellulose .. .............................................
Lignin ......................................................
Other constituents ..........................................

Historical perspective .......................................
Fiber analyses ..............................................
Gas chromatography .................. . ................... q...
Utilization of fiber by animals and bacteria ................

MATERIALS AND METHODS .u ..........................................
Preparation of samples ......................................
Delignification of plant samples ............................
Second dry matter of plant samples ..........................
Preparation of alditol acetates .............................
GLC quantification of alditol acetates ......................
Trifluoroacetic acid hydrolysis .............................
Methanol additions ..... .....................................
Cellulose composition .......................................
Neutral and acid detergent fiber analyses ...................
Other fiber analyses ........................................
Pony digestion trial ........................................
Beef cattle digestion trials ................................
Swine digestion trials ......................................
Digestion trial samples .....................................
Pure culture analyses .......................................
Electron microscopy .........................................
Soluble carbohydrates in corn silage ........................
Statistical analyses ........................................

RESULTS ..........................................................

Preparation of plant samples ................................
Second dry matter in plant samples ..........................

iii

Page

viii

14
24

28

33
33
36
36
37
38
38
39
39
39
39
4O
41
43
43
44
45
46

47
59

Page

Trifluoroacetic acid hydrolysis ............................. 59
Methanol additions .......................................... 63
Cellulose composition ....................................... 63
Neutral and acid detergent fiber analyses ................... 63
Protein and ash composition ................................. 68
Electron microscopy of fiber fractions ...................... 71
Pony digestion trial ........................................ 75
Beef cattle digestion trials ................................ 80
Swine digestion trials ...................................... 87
Pure culture studies ........................................ 100
Electron microscopy ......................................... 108
Soluble carbohydrates in corn silage ........................ 111
DISCUSSION ....................................................... 116
Fiber analyses .............................................. 116
Fiber digestibility trials .................................. 125
Pure culture ................................................ 130
Corn silage analyses ........................................ 133
CONCLUSIONS ...................................................... 134
APPENDIX ..... . ................................................... 136
LIST OF REFERENCES ............................................... 141
VITA ............................................................. 159

iv

Table

10.

11.

12.

13.

14.

15.

LIST OF TABLES

Common and scientific names and dry matter of substrates

Composition of swine diets .................................
Detergent fiber fractions of substrates ....................
MSU fiber fractions of substrates ..........................

Individual sugars of the hemicellulose fraction of substrate
as determined by gas chromatography ......................

Individual sugars of the hemicellulose fraction as expressed
as a percent of hemicellulose ........... . ................

Comparison of total fiber values of detergent fiber and MSU
fiber systems ............................................

Ratios between fiber fractions in detergent and MSU fiber
systems ..................................................

Uronic acids in ammonium oxalate and T.F.A.A. filtrates of
substrates .... ............ . ..............................

Alditol acetate analysis of neutral detergent fiber frac—
tion of substrates and percent recovery of each compon—

ent 0 OOOOOOOOOOO O OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Alditol acetate analysis of acid detergent fiber fractions
of substrates and percent recovery of each component .....

Protein and ash content in MSU fiber fractions .............

Amino acid composition in detergent and MSU fiber fractions
of wheat straw (g/16 g N ) ...............................

Amino acid composition and loss in detergent and MSU fiber
fractions of wheat straw (mg/g d.m.) .. ...................

Apparent dry matter and crude protein digestibility in the
pony .....................................................

Page
34
42

48

54

55

57

58

6O

67

69

7O

70

Table Page
16. Fiber composition of feed and feces in pony digestion trial 79
17. Apparent digestibility of fiber fractions in the pony ...... 81

18. Apparent dry matter and crude protein digestibility in beef
cattle digestion trials .................................. 82

19. Composition of feed and feces in beef cattle trials ........ 84
20. Apparent digestibility of fiber fractions in beef cattle ... 85

21. Fiber component composition of feed and feces in starter
pigs ..................................................... 90

22. Apparent dry matter and crude protein digestibility in
starter pigs .......................... . .................. 91

23. Apparent digestibility of fiber fractions in starter pigs .. 93
24. Fiber component composition of feed and feces in grower pigs 95

25. Apparent dry matter and crude protein digestibility in
grower pigs .............................................. 96

26. Apparent digestibility of fiber fractions in grower pigs ... 98

27. Fiber component composition of feed and feces in finisher
pigs ..................................................... 99

28. Apparent dry matter and crude protein digestibility in
finisher pigs ............................................ 101

29. Apparent digestibility of fiber fractions in finisher pigs . 102

30. Fermentation of substrates by pure cultures of rumen cellu-
lolytic bacteria ......................................... 104

31. Composition of substrates used in pure culture analysis .... 105

32. Compositional analysis of recovered substrates after fer-
mentation by rumen cellulolytic bacteria ................. 106

33. Percent fermented of MSU fiber fractions in pure culture
analysis ... .............................................. 107

34. Soluble sugars, pH and dry matter of corn silage over time . 115

A1. Correlation coefficients of fiber fractions of all sub—
strates .................................................. 136

vi

Table

A2.

A3.

A4.

A5.

A6.

A7.

A8.

Page
Correlation coefficients of MSU T.F.A.A. fiber fractions of
all substrates ... ........ . ..................... . ......... 136
Correlation coefficients of MSU T.F.A.A. fiber fractions in
grasses and legumes ........................ . ............. 137

Correlation coefficients of fiber digestibility coefficients 137

Correlation coefficients of fiber fractions fermented in
pure culture analysis . .......... .......... ..... .......... 138

Correlation coefficients between fiber components in sample
and digestibility in pure culture analysis ............... 138

Abbreviations and definitions .............................. 139

Cost comparison between the detergent fiber system and the
MSU T.F.A.A. fiber system ................................ 140

vii

Figure

10.

11.

12.
13.
14.
15.

16.

LIST OF FIGURES

Changes in dry matter with time at room temperature ........
Length of hydrolysis time and sugar yield ..................

Yield of hemicellulosic sugars as affected by methanol add-
itions ...................................................

Effect of neutral detergent treatment on wheat straw .......
Effect of ammonium oxalate treatment on wheat straw ........
Effect of acid detergent treatment on wheat straw ..........

Transmission electron photomicrograph of the effect of acid
detergent on wheat straw .................................

/

Effect of sodium chlorite and trifluoroacetic acid on wheat
straw ....................................................

Effect of washing and drying on fiber of beef cattle feces .

Effect of neutral and acid detergent treatment on fiber of
beef cattle feces ........................................

Effect of ammonium oxalate treatment on fiber of beef cattle
feces ....................................................

Attachment of Ruminococcus flavefaciens C94 on wheat straw .

 

Attachment of Ruminococcus flavefaciens C94 on alfalfa .....

 

Attachment of Ruminococcus flavefaciens C94 on corn silage .

 

Attachment of Bacteroides succinogenes S85 on alfalfa ......

 

Attachment of Bacteroides succinogenes 885 on Whatman filter
paper and Kentucky bluegrass .............................

 

"iii

Page
61

62

64
72
73

74

76

77

86

88

109

113

114

INTRODUCTION

In the disciplines of nutrition and waste management, there is
a need for more definitive and descriptive methods of fiber analysis
to interpret experiments with dietary fiber and waste residues. Pre-
vious methodology for fiber analysis can only be used for proximate
analysis, therefore, it is desirable to develop a new laboratory pro—
cedure to examine the fiber structure of a feed, its effect in the
gastrointestinal tract and the possibility of recycling many fibrous
waste residues.

The word "fiber" is loosely defined as an element that gives
texture or substance with a basic toughness. Many fibers have been
described such as: crude (91), normal acid (216), acid detergent (194),
neutral detergent (19D and dietary (18». Each of these methods can
be used for an approximation of some fiber components, but can not be
used to determine the effect of a particular fiber upon utilization
of nutrients or microbial fermentation in the gastrointestinal tract.

In 1965, Salo (LSD presented a review of carbohydrate and fiber
techniques used for animal feed and feces. In this review, he intro—
duced another method of fiber analysis referred to as fiber fraction-
ation. This procedure varies widely between laboratories, but may
consist of similar fractions such as: cellulose, hemicellulose, holo—

cellulose, lignin, starch, water-soluble carbohydrates and fructosan.

Once again, it is difficult with this methodology to ascertain the
effect of fiber in nutritional studies. In general, the approach to
fiber chemistry by animal and human nutritionists has been one of
availability and fractionation (64, 173, 179, whereas the botanists
and cereal chemists have been more concerned with better understand—
ing the complex structure of fiber through finite chemical analyses
(11,34,53,61,182)-

Each chemical analysis differs in design, however, the outcome
is usually similar in that the procedure separates the fiber into
individual monosaccharides, disaccharides or oligosaccharides for
further study. The measurement of each fraction can be done gravi—
metrically (19), colorimetrically (152 or by chromatography (163.

The objectives of this study were: (1) to examine fiber more
fully by developing and modifying the earlier techniques of Alber—
sheim (11), (2) to examine the detergent fiber components of 20
forages and feeds and compare them to the fiber components of the
developed technique, (3) to conduct feeding trials with beef cattle,
swine and ponies to demonstrate the feasibility of the developed
technique, (4) to conduct pure culture studies with two rumen cell—
ulolytic bacterial species to examine mode of attack and (5) to use
electron microscopy to further elucidate differences of techniques

and bacterial attachment.

REVIEW OF LITERATURE

There exists today many definitions of fiber which depend upon
the perspective of the particular field of study. The American Col-
lege Dictionary G3) has eight definitions, of which none refer to a
nutritional component. These definitions are: (1) a fine thread-
like piece, as of cotton, jute or asbestos; (2) a slender filament;
(3) filaments collectively; (4) matter composed of filaments; (5)
fibrous structure; (6) character: moral fiber; (7) filamentous
matter from the bast tissue or other parts of plants and (8) vulcan-
ized fiber. This lack of definition of the most basic of sources
typifies the understanding of fiber in nutritional sciences, i.e.,
many techniques and definitions exist with very little agreement.

In 1682, Nehemiah Grew (189) wrote in his "The Anatomy of Plantes”:
”So in... plants: not only the threads of which the bladders (sub—
sequently renamed cells): but also the single fibers... may sometimes
with the help of a good glass (renamed microscope), be distinctly
seen." Grew recognized the complexity of the fibrous structure with—
in the plants he examined. The definition of what fiber is, has re—
mained just as complex. Cereal chemists have defined plant fiber as
cellulose ( 34,61 ) whereas botanists have described it as a dispers—
ed phase of microfibrils packed round with a continuous matrix ( 10,

53, 182 ). The nutritional term of fiber has received a great deal

of attention by animal scientists. In the ruminant, it represents
the plant cell wall which is utilized as an energy source by the
rumen microflora and is extensively degraded whereas in the non—
ruminant fiber represents the insoluble matter of plant cell walls
indigestible by animal enzymes, but partially degraded by gastro-
intestinal microflora. In either case, the plant fiber is determined
with neutral (197) or acid detergent (195).

Human nutritionists have defined fiber in the diet many ways.
Trowell (187,183 first defined dietary fiber as the plant food re—
sistant to hydrolysis by human alimentary enzymes and composed of
cellulose, hemicellulose and lignin. He continued by defining a
second term called dietary fiber complex which represented the diet-
'ary fiber plus all chemical compounds associated with the structural
polymers of the plant such as pectins, waxes, gums, minerals and non-
available protein.

An alternative term, plantix, has been proposed by Spiller (176)
which would represent cellulose, hemicellulose, mucilages, pectins,
gums and lignin. Another term, coplantix, represented the plantix
plus associated cell wall factors such as waxes, cutin, cell wall
bound protein and minerals. It was suggested that the term fiber
remain as a popular term whereas the term plantix remain as a scien-
tific term. In partial agreement with this, it was proposed that the
term fiber be changed to partially digestible plant polymers (PDPP),
partially digestible biopolymers (PDB) or plantix (79 ).

Although the term dietary fiber has been very controversial,

there is a general agreement in the qualitative definition of Trowell

(51,64,93,96,119 ), however, the definition excluded the possibility
of microbial utilization (151). This controversy has been magnified
with the difficulties in established analyses and failure to develop
quantitative techniques to examine the plant cell wall more complete-
ly. Van Soest (205) suggested that there are two things which are
not always compatible in fiber studies: (1) for research purposes
one needs a detailed system of structural analysis that is definitive
in character for the individual plant fiber sources and (2) for
quality control, the methods must be rapid, convenient and must per-
mit the handling of large numbers of samples. Southgate (175) has
stated that any procedure for the measurement of fiber must repre—
sent the compromise between a complete fractionation and measurement
of all the various species and a simplified system involving group-
ing of different compounds in some arbitrary and often empirical way.
These compounds should include cellulose, hemicellulose, lignin and

pectin as the primary sources of fiber.

Plant cell wall constituents

Cellulose. The most abundant constituent found in the plant cell
wall is cellulose. Cellulose, a linear polymer of glucose, is found
in nature in close association with other polysaccharides (47,75 ).
It is the main constituent of the cell walls and serves therein as
the primary structural element. Recognition of cellulose was first
made by Payen in 1839 (219).

The ideal cellulose is a linear polymer composed of individual

anhydroglucose units linked at the C-1 and C—4 of glucose through

glucosidic bonds with the beta configuration. The number of units
range from about 100 to 10,000 or more (143,145). The glucan chains
in the cellulose fibers are held tightly together by hydrogen bonds
(10,78). These bonds form between the hydrogen atom of a hydroxyl
group in one sugar unit and an oxygen atom on another unit. Linear
molecules of cellulose are also held together by Van der Waals forces.
Highly oriented cellulose is termed crystalline whereas randomly or-
iented cellulose is termed amorphous (46). Cellulose chains undergo
a local motion in the amorphous regions of the polymer which involves
the anhydroglucose rings. Hydrogen bonds result in rigidity of the
crystalline regions which do not permit partial rotation of the
anhydroglucose rings without breaking hydrogen bonds. Nuclear mag-
netic resonance and dielectric constant data have confirmed that re-
orientation of anhydroglucose of the cellulose chains is sufficiently
rapid and vigorous at room temperature to disrupt the structure of
cellulose. This apparently involves the amorphous regions, breaking
the weak hydrogen bonds in amorphous regions which allow large scale
changes in the physical properties of cellulose (78).

Crystallinity and lignification have been shown to be the most
important determinants of the susceptibility of cellulosic materials
to enzymatic conversion processes (47). Enzymes specific for the
bonds in a glucan chain are not very effective in degrading the in—
tact cellulose fiber (10). The accessibility of cellulose to chem-
ical reagents and the extracellular enzymes or other metabolic cata—
lysts of cellulolytic organisms is determined in part by its dis—

tribution within the cell wall and the nature of the structural

relationships among various cell wall constituents (47 ).

The classical method for the determination of cellulose was de-
veloped by Cross and Bevan in 1903 (6K)) and involved alternate
chlorination and sodium sulfite extraction. Since then, many other
methods have evolved using hypochlorite (141), potassium perman-
ganate and ashing ( 21D, sodium.chlorite and trifluoroacetic acid
(44 ), sodium hydroxide and ashing (IJMD, nitric acid-acetic acid
digesthmn (48) and hydrolysis to glucose in sulfuric acid (122).
In any method, the effect upon the cellulose bonding is most import-
ant. Trifluoroacetic acid (T.F.A.A.) hydrolysis appeared not to
hydrolyze cellulose, but only the hemicellulose chains (Hi3). Po-
tassium permanganate has been used widely in nutritional studies,
however, the mode of action upon cellulose has never been fully elu-
cidated. Other chemical treatments upon cellulose that have been
shown to alter cellulose include hydrochloric acid, liquid ethyl-
amine, ammonia (120) and hydrochloric acid in benzene (138).

Hemicellulose. Hemicelluloses are widely distributed in the

 

vegetable kingdom and, next to cellulose, are undoubtedly the most
abundant of the materials of plant origin (150). Hemicellulose is

a name given by Schulze in 1891 (218) to a group of polysaccharides
in plant material which dissolves in dilute alkaline solution.

The term hemicellulose as used today has been given many definitions
by different authors. Phillips (150) defined it as carbohydrate
substances that are insoluble in boiling water but are soluble in
dilute aqueous solutions of alkali. Reid and Wilkie (154) defined

it as the polysaccharides in a plant tissue other than cellulose

which is extracted with alkali and hydrolyzed in acid. Nutritionists
have looked at hemicellulose as a unit since the fractionation pro—
cedures have produced no meaningful nutritional division QMML). The
very name is a misnomer, since it stems from an erroneous early idea
that hemicellulose is a carbohydrate in the process of being convert-
ed to a cellulose (ZMMD. Some hemicelluloses are relatively branch—
ed molecules and are therefore more soluble than a linear molecule
such as cellulose (210).

Most hemicelluloses are heteroglycans containing two to six
different types of sugar residues. Common heteroglycans include:
arabinoxylans (.53), arabinoglucuronoxylans GKH3), methylglucurono-
xylans (168), glucans (139), galactoglucomannans (218), glucomannans
(168) and arabinogalactans (218). Keegstra $2.1: (102) suggested
that the plant cell wall is held together by non—covalent interactions
between macromolecular components. He presented evidence that sug—
gested that macromolecules of the wall are covalently cross-linked
(except cellulose) and that the linkage between cellulose and other
cell wall polymers has the strength of a covalent bond. Theander (183)
divided plant structural polysaccharides into two main classes: (1)
the fiber polysaccharides and (2) the matrix polysaccharides. The
former compounds are largely crystalline and are present as micro-
fibrils. These microfibrils, mostly cellulose, are held together by
hydrogen bonds in cement of largely amorphous matrix polysaccharides,
lignin and some protein. Matrix polysaccharides are usually separated
into two groups: pectic polysaccharides and hemicelluloses.

The fractionation of hemicellulose has been difficult depending

upon the solubility of each polysaccharide and has been complicated
with the fact that these polysaccharides are not uniform. This may
be influenced by the molecular weight and distribution, shape, type
and configuration of the functional groups (137). The more highly
branched and heterogenous composed hemicelluloses are usually re-
movable with a dilute alkaline solution whereas a more homogenous
polysaccharide is removed by extraction with strong alkali (22,35 ).
The most common method for hemicellulose extraction has been with
a 10% alkaline solution and reprecipitation with ethanol. Five
techniques for measuring hemicellulose have been described. The
first involves the boiling of the cell wall in 12% hydrochloric acid
with a subsequent determination of the furfural evolved (.IED. The
second procedure is dependent upon the isolation of a holocellulose
or delignified plant cell wall and hydrolysis of the hemicellulose-
in alkali ( 22». A.third technique produces hydrolyzed sugars in
acid and quantitation with paper chromatography and colorimetry (156,
178,179,180). The fourth technique has been used in many nutritional
studies. It is based on the subtracted value between two fiber val—
ues, i.e., neutral and acid detergent fiber ( 82). The newest pro-
cedure is based upon a complete fractionation of the hemicellulose
and involves delignification of the cell wall, acid hydrolysis, con—
version to a volatile derivative and measurement on a gas chromato—
graph ( 44).

Lignin. Lignin, the third most abundant component of the plant
cell wall, is widely distributed in the plant kingdom. Two classes,

namely guaiacyl lignins and guaiacyl-syringyl lignins, for the gymno-

10

sperms and angiosperms respectively have been suggested (2).
Lignin was first recognized as an encrusting material by Payen in
1838, but was later named by Schulze in 1865 (2).

It has been difficult to define lignin. Hartley (88) defined

it as a polyphenolic polymer containing phenyl propane structure.

Van Soest and Robertson (210) defined lignin as a condensed poly-

mer of substituted phenyl propyl alcohols and acids. Definitions
have tended to differ with point of view. Botanists have regarded
lignin as a plastic, three—dimensional, substituted phenyl propane
polymer. Wood chemists have defined it as a plastic substance giving
distinctive properties to wood. Nutritionists have regarded lignin
as a structural substance protecting plant cell walls from microbial
degradation (203).

h The structure and bonding in a particular lignin will depend
upon the plant. Lignin has been shown to be intimately associated
(with the polysaccharides of the cell wall and has a covalent link
with cellulose (88). It has also been shown to be linked with a
hydroxyproline—rich protein (221) and hemicellulose (131,132). Lig—
nin is relatively easily oxidized since it contains many ether link-
ages and polyphenolic substitutions. Most phenolic compounds in
plants occur as glycosides or esters with carbohydrates (204).

There are several procedures which have been reported in the
analysis of lignin. The earliest lignin procedure used fuming hydro-
chloric acid to dissolve the cell wall carbohydrates leaving lignin
(204). A similar procedure was based upon the gravimetric removal of

all the structural polysaccharides by hydrolysis in 72% sulfuric acid

11

followed by ashing (70, 2“». A third method is that which was
proposed by Morrison (127,123. Crude cell walls are heated in
acetyl bromide which reacts with lignin to produce a series of
soluble products which are then estimated by measuring the absorp-
tion at 280 nm. Two other methods of lignin determination have
been developed involving a preliminary treatment of the plant mater-
ial with acid detergent. Lignin is then determined by measuring
the weight of material removed by heating the acid detergent fiber
with a mixture of triethylene glycol and hydrochloric acid (65 )

or oxidation with potassium permanganate (221D. Many of these pro—
cedures are complicated with artifacts ( 82,152,204,208 ). Lignin
has also been determined by a newer technique which used sodium
chlorite to oxidize lignin. Lignin is then calculated as a loss

of weight ( 44).

Other constituents. Several other plant cell fractions have

 

been reported. The abundance or scarcity of these constituents has
been found to be dependent upon the plant. These constituents in-
clude: pectins, gums, mucilages, cutin, tannin, cell wall protein
and cell wall minerals.

Pectin has been defined as a partially methoxylated polymer
of galacturonic acid ( 11D. It has been further defined as a group
designation for those complex, colloidal carbohydrate substances
which occur in or are prepared from plants and contain a large pro-
portion of anhydrogalacturonic acid units which are thought to exist
in a chain-like combination ( 59). The sugars found in pectin include

arabinose, galactose, rhamnose, xylose, fucose, glucose, 2—0—methyl—

12

L-fucose and 2-O-methyl-D—xylose (59). Pectin is soluble in water,
formamide, dimethyl sulfoxide, warm glycerols and ammonium oxalate.
Pectin has been regarded as the material soluble in hot solutions
of neutral chelating agents and consists of substituted polygal—
acturonic acid (204). They are part of the non-cellulosic carbo—
hydrates of the cell wall which includes hemicellulosic sugars.
Two structures containing pectic substances have been reported in
sycamore primary cell walls (21). These structures, arabinogal-
actan and rhamnogalacturonan, are zig-zagged shape with a1+2 and
a1+4 glycosidic bonds. It had been assumed that all uronic acid
residues after hydrolysis in acid are present in the hydrolysates
as aldobiouronic acids having D—xylosyl residues. Buchala and
Wilkie (32) demonstrated that the proportion of uronic acids in
the xylans from any one type of tissue decreases as the plant ages.
Waxes in leaf tissue have been examined with gas-liquid
chromatography. Chemically, was consisted of a mixture of hydro-
carbons, ketones, alkyl esters, aldehydes, primary alcohols and
secondary alcohols. Wax in Clarkia elegans leaf tissue occurs in
the form of smooth films, tubes, dendrites or plates depending upon
the growth temperature. An artificial system used for the recry-
stallization of plant waxes has shown that waxes transported as
individual fractions successively to the surface of a porous mem-
brane develop as a composite arrangement of crystals whereas the
same solutions delivered as an homogenous mixture form a uniform
layer with a poorly defined structure. This suggested that indivi—

dual components of waxes are responsible for particular structures

13

and that at the plant surface specific sites may exist for the
exudation of each constituent.

There are many types of gums. These substances of plant
origin which are obtained as exudations from the fruit, trunks or
branches of trees that appear to serve a protective mechanism (99).
They have been classified into three major categories according to
the raw material or origin. They are plant exudates, seaweed ex-
tracts and the seed gums. Gums have described as water-soluble
substances or colloids used as viscosity builders forming colloids
or gels which can have cationic, anionic or non-ionic exchange pro-
perties (117). More specifically, plant gums are the neutral salts
of complex polysaccharide acids composed of hexose residues, pentoses,
uronic acids and methylpentose residues in diverse fashion (99).

They have been distinguished by the fact that glucuronic acid is the
acid component in them all. Mathison (115) has reported that rape—
seed gum consisted of an aqueous emulsion of phospholipids with small-
er amount of triglycerides and non-lipid material. Mangle gum has
been reported to contain highly branched galactose-rhamnose chains
with arabinose, glucuronic and galacturonic acids (160).

Plant mucilages, similar to gums, have been shown to be water—
soluble polysaccharides which form colloids and have been precipi-
tated by ammonium sulfate and sodium chloride. They do not form
jellies like pectin. Jones and Smith (99) showed that if the muc—
ilage is on the outside of the seed coating, extraction with water
was sufficient, however, if the mucilage was in the endosperm or tub-

er, extraction by powdering was necessary.

14

Cutin has been defined as an insoluble biopolyester and a major
component in fruits and vegetables (24.). Gas-liquid chromatography-
mass spectral data has revealed that the major component of the poly-
mer are fatty acids. This polymer has been shown to be fairly resis—
tant to biodegradation (24,208) and has been included as part of a
crude lignin fraction. Tannins are defined as polyphenolic com—
pounds which form insoluble complexes with proteins (€37). Tannins
have also been determined as part of the crude lignin fraction.

Little information has been reported about the secondary cell
wall protein. Lamporte (“17) has reported that there are high levels
of hydroxyproline and disulphide bridges in primary cell walls.
Paradies ( L4» reported that algae cell walls contained 10% to 15%
protein which was high in glycine, alanine, aspartic acid and glu—
tamic acid. Minerals also have been shown to be part of the cell
wall. Plant cell walls that are secondarily thickened contain sil-
icon and other metal cations (juxj. They have appeared to be inte-
gral with polyuronides and carboxyl and phenolic hydroxyl groups.

Grass analyses indicated an accumulation of these minerals (206).

Historical perspective

Fiber analyses. For well over 150 years. the development of

 

techniques for fiber analyses has been difficult. The original
technique compared the nutritive values of different feedstuffs.
This was called hay equivalents and was attributed to both Einhof
and Thaer (190), although this has been highly debated (190).

Neither of these scientists used water, alkali, acid or alcohol in

15

their techniques. The use of these chemicals is part of the crude
fiber (CF) technique which was developed between 1800 to 1820 and
first reported in corn analysis by Gorham in 1820 (83 ). This
technique involved the treatment of a sample with dilute acid for
30 min and dilute alkali for another 30 min (91 ). The definition
and procedure for crude fiber has remained variable at best. Sub-
traction of crude fiber, ash, crude protein and ether extract from
100 yielded a fraction called nitrogen-free extract (NFE). NFE
represented a highly digestible fraction of the feed.

As early as 1907, data was reported upon losses due to chemi-
cal treatments found in the CF technique (89). Haywood (89)
treated pure cotton cellulose with the acid and alkali of the CF
procedure and reported 2.7% and 17.1% loss in weight, respectively,
or 19.8% loss of cellulose.

This was followed by an analysis of the CF residue in 1935 by
Norman (140), who found that 60% to 80% of the cellulose and 4% to
67% of the lignin was recovered in the CF fraction. He determined
that the pentosans are digested during the 30 min acid digestion per—
iod and lignin was extensively removed during the 30 min alkaline
digestion. They suggested an alternative procedure was needed to
replace the acid and alkali because lignin exercises a direct effect
upon the digestibility of the cell walls.

A further complication of the CF-NFE method showed that in many
instances, the NFE of straws and grasses could contain as much as
90% of the hemicellulose, cellulose and lignin. This then made the

NFE appear less digestible than CF in many cases.

16

Williams and Olmsted (224) reported that the crude fiber
fraction contained variable amounts of lignin, hemicellulose and
cellulose and represented the vegetable materials not attacked
by digestive enzymes in the mammalian gut. They described an
enzymatic procedure for crude fiber to decrease the variability
of crude fiber.

A symposium in 1940 (25,149,150,185) showed that the NFE
fraction contained a large amount of the cell wall constituents.
Despite the awareness of inherent difficulties of the CF method,
further modifications have been reported (94,217,220).

In 1946, Matrone e£_§l, ( PHD suggested that cellulose could
be used to evaluate feedstuffs because of its close association with
other polysaccharides in the plant cell wall. This technique did
not consider the importance of hemicellulose and lignin and remain-
ed a procedure in the determination of cellulose.

Paloheimo and Paloheimo in 1949 (11%», described a separation
of plant products into total vegetable membrane substances and en-
closure substances. Total membrane substances represented cellulose,
pentosans, hexopentosans, polyuronides, pectin, lignin, suberins
and cutins, whereas the enclosure substances represented sugars,
dextrins, starch, inulins, proteins, amino acids, amides, amines,
lipids, alcohols, pigments, alkaloids, organic and inorganic salts
and water. These researchers analyzed for membrane substances by
lipid removal in 1:2 v/v ethanolzbenzene, boiling in 0.05 N hydro-
chloric acid, drying and ashing. Protein was determined on each

fraction for back calculation.

17

Walker and Hepburn in 1955 (216) proposed another analysis
for the evaluation of roughages. This was called normal—acid-fi-
ber (NAF) and was based upon their criticisms of the CF technique.
Samples were extracted for 8 hr in 1:2 v/v ethanol:benzene, boiled
in hot 1 N sulfuric acid, filtered and washed with ethanol and ether.
NAF was determined by loss of weight after ashing at 550 C. Ray-
mond e£_al. (153) reported a negative relationship between the
percent NAF in the fecal organic matter and herbage organic matter
digestibility. They also found 80% to 90% and 100% of the NAF
fraction was accounted for as cellulose and lignin in herbage and
feces, respectively. The value of NAF was greater than crude fiber
in all cases examined ( 85). Digestibility of NAF was always lower
than the digestibility of crude fiber ( 213. This was attributed to
the higher concentration of lignin and cellulose. The variability
of NAF due to protein and ash was mentioned. High correlations be—
tween the percent NAF in forages with dry matter digestibility were
reported ( 98). A 3 hr digestion in acid, instead of 1 hr, was
suggested to decrease variability.

A comprehensive analysis of grasses was reported in 1959 by
Waite and Gorrod (213,214). This method involved extraction of the
sample in 1:2 v/v ethanol:benzene, boiling in ammonium oxalate,
digestion in pepsin-hydrochloric acid, oxidation with sodium chlorite,
extraction with water, alkali and acid. Extracts were then analyzed
for sugars by paper chromatography and colorimetry. This procedure
was complicated by increased error through many transfers and lack of

purity in the cellulose fraction. Individual sugars of the hemi-

18

cellulose were reported.

In the 1960's, Van Soest and others (194,195,196,197,198 )
examined the use of detergents in the analysis of fiber in forages.
They suggested that fiber should represent substances resistant to
animal enzymes. Forages were refluxed with 2% hexadecyltrimethyl-
ammonium bromide in 1 N sulfuric acid for 1 hr (194,195) in an
attempt to lower the protein content of the fiber residue. The
fraction retained after filtration was called acid detergent fiber
(ADF) and was composed of cellulose, lignin, minerals and some ni-
trogen (196). Unheated forages retained 2% to 20% of the original
nitrogen in the ADF.

It was also found that sodium lauryl sulfate removed large
amounts of protein in forages (195,20D. This was followed by the
use of sodium lauryl sulfate in determining total plant cell walls.
Samples were refluxed in a buffered solution of 3% sodium lauryl
sulfate containing 1.86% disodium ethylenediaminetetraacetate for
1 hr. This fiber fraction was called cell wall constituents (CWC)
or neutral detergent fiber (NDF). It was composed of hemicellulose,
cellulose, lignin and some protein (197,207). 'NDF minus ADF was
suggested as a calculation for hemicellulose (197).

Lignin was analyzed by digestion of ADF in 72% sulfuric acid
( 198. This was complicated with drying and heating procedures
during the preparation of laboratory samples ( h)». Many digesti-
bilities of lignin were reported using sulfuric acid (18,52,68,71
101, 178 ) which Van Soest attributed to the drying procedures

examined. He reported that drying temperatures greater than 50 C

19

will increase the yield of lignin and fiber due to non—enzymatic
browning ( 198). The nitrogen content of the ADF was suggested as
a sensitive assay for non-enzymatic browning (198). Other com-
plications with this procedure due to artifacts have been reported
(123,204).

In an attempt to overcome this problem, an alternative pro—
cedure with potassium permanganate was suggested (211). In each
forage examined, the permanganate lignin was higher than the
corresponding lignin value obtained by sulfuric acid hydrolysis.
The advantages of potassium permanganate appeared to be: (1)
shorter time, (2) less powerful reagents and (3) fewer reagents.
The complications appeared to be losses of cellulosic carbohydrates
in young grasses and that polyphenolics, tannins, pigments and
proteins in the ADF reacted with permanganate and increased the
value of lignin (211). An alternative procedure was developed in
1973 (65 ). In this method, lignin was determined by measuring
the weight of material removed by heating ADF with a mixture of
triethylene glycol and hydrochloric acid.

The composition of CWC and ADF was examined by Colburn and
Evans (16,38 ). CWC fractions from grasses contained 96.1%, 96.3%
and 90.0% of the ADF, cellulose and lignin, respectively, and al-
falfas contained 89.3%, 83.7% and 97.4%, respectively. An examin-
ation of ADF showed that 92.0% and 88.8% of cellulose was recovered
from grasses and alfalfas, respectively. Hemicellulose values,
as determined by NDF minus ADF, were not reported. Upon calculation,
the values ranged from 5.5% to 30.3%. Calculation of the composition

of their substrates on a dry matter basis showed 0.8% to 8.3% and

20

—O.6% to 1.3% of the hemicellulose value to be due to crude protein
and ash contamination, respectively.

Several investigators examined the ADF fraction further (16,17,
207). Kim.g£_al, ( HM) found that the percent ADF was higher than
CF values in feces, silage and pellets. The ADF contained more
lignin, but less pentosans and cellulose than the CF. Most of the
lignin and cellulose was retained in the feed ADF, however, only
16% to 18% of the pentosans were recovered. Fecal ADF contained
9.7% pentosans.

Bailey and Ulyatt ( 17) prepared neutral and acid detergent
residues from a range of grasses and clovers. He found that NDF
consisted primarily of hemicellulose, cellulose, and lignin, however,
ADF consisted of cellulose, lignin, some hemicellulose and up to
50% of the plant pectin in clovers. He suggested that the extract-
ion time in the acid detergent fiber method be lengthened to 2 hr.

In 1969, Southgate (173,174) presented a more detailed fract-
ionation system for fiber analysis. He proposed that the unavail-
able carbohydrates are those that are not hydrolyzed by any enzymes
secreted into the digestive tract. He suggested that a small amount
of fiber is utilized by the microbes of the digestive tract. Un-
available carbohydrates are measured after extraction in 85% methanol,
treatment with enzymes and hydrolysis in 72% sulfuric acid. Hexoses,
pentoses and uronic acids were analyzed by colorimetry and represent-
ed the sugars from the hemicellulose and cellulose. Available carbo-
hydrates were determined by extraction of the sample in aqueous alco-

hol.

21

In an attempt to examine the structure of the cell wall and its
relationship to ruminant nutrition, Morrison (127,128,129,131,132,
133,134) developed a series of techniques which determined lignin
and lignin-carbohydrate complexes. He stated that different kinds of
fiber have been reported and each one is characterized by its method
of isolation. He suggested that the relevance of the term fiber
is dubious since the chemical composition and ultimately the nutri-
tional significance of different fibers is dependent upon many Var-
iables such as the method of isolation, species of plant and maturity
of the plant. Morrison expressed that the composition of the plant
cell wall ought to be characterized in a more specific way in terms
of its individual components, namely lignin, cellulose, hemicellulose
and pectin. He first developed a spectrophotometric technique to
measure lignin as solubilized in acetyl bromide (127,128). Extraction
with dimethylsulfoxide and alkali yielded fractions he called lignin-
carbohydrate complexes (LCC) and lignin-hemicellulose complexes (LHC),
respectively (128,129,132,133,134). Gas chromatographic analyses of
alditol acetate derivatives of the hydrolyzed cell wall sugars ( 128,
129,132,133,134) were used to analyze the composition of both LCC and
LHC.

Further characterization of plant tissues in foods, feeds and
feedlot wastes through the use of gas—liquid chromatography (GLC)
was proposed by Sloneker (164,170,171,172). He suggested that GLC
had advantages over previous gravimetric and colorimetric procedures
because individual aldoses could be measured directly and aldose

content of different samples were readily visualized. Total neutral

22

carbohydrates were measured in samples after hydrolysis in 72% sul-
furic acid at 30 C. This mixture was then diluted with water to 1 N
and hydrolyzed at 120 C. Hydrolyzed sugars were reduced and acetylated
with pyridine-acetic anhydride for 16 hr at 100 C. Sloneker (171,172
reported that 5% of the aldoses and 20% of the xyloses were degraded
during hydrolysis.

In 1974 , simpler approach to the analyses of fiber was
presented by Hellendoorn g£_al. (90). They proposed that dietary
fiber represented the indigestible residue after treatment in pepsin
and pancreatin and was calculated by subtracting crude protein, fat,
available carbohydrate, ash and water from 100. They stated that the
methods presented by Van Soest (68,194,195,196,197,198,207,211 ) and
Southgate (173,174) were not physiological. Values of 10.4% and
56.0% for crude fiber and indigestible residue in wheat bran were
reported, respectively.

Another method for the determination of fiber was developed
by Elchazly and Thomas in 1976 ( 69). This method was tailored
only for the determination of water-insoluble plant polymers. It
was based on the use of amylolytic and proteolytic enzymes.

In 1977, Furda ( 79) Proposed a fractionation system for both
the water-soluble and insoluble polymers from plant residues which
he termed partially digestible plant polymers (PDPP) or partially
digestible biopolymers (PDB). He suggested that the fractionation
of dietary fiber should provide the distinct fractions of relatively
pure polymers with respect to their chemical structure and solubility.

His method also involved amylolytic and proteolytic enzymes, followed

23

by delignification with sodium chlorite, solubilization in alkali
and precipitation in alcohol. In disagreement with this approach,
Van Soest and Robertson ( 2“) stated that the soluble substances
resistant to animal digestive enzymes should be distinct for ana—
lytical reasons.

The use of chemical procedures prepared in the field of botany
had been avoided. Collings §t_§l, (‘41) used sodium chlorite in
dilute acid to examine the cell wall components further. They a-
dapted the oxidation of lignin with sodium chlorite into a gravimetric
procedure for the determination of lignin. They reported that lignin
as oxidized by sodium chlorite left a white cell wall residue and had
generally higher values when compared to lignin oxidized by potassium
permanganate. Composition of individual cell wall sugars had not
been examined. Collings and Yokoyama ( 44) in.1979 adapted the pro—
cedure of Albersheim et_§l, ( 11) to the hydrolysis of the cell wall
and analyses using GLC. Individual hemicellulosic sugars were hydro-
lyzed in trifluoroacetic acid (T.F.A.A.), reduced in sodium borohy—
dride, acetylated in acetic anhydride and examined by temperature
programming by GLC. Cellulose was determined as the residue remaining
after hydrolysis. Hemicellulose, determined by the detergent system,
was overestimated in most cases, whereas, there was closer agreement
between detergent cellulose and T.F.A.A. cellulose. Examination by
electron microscopy showed partial losses of the cell wall after re—
fluxing in neutral detergent and excessive unspecific attack of the
cell wall after refluxing in acid detergent. It was suggested that

these procedures could offer a more detailed examination of fiber which

24

could be used to determine the mode of action of the different fiber
components.

Gas chromatography. Gunner et.aln( 86) first reported that car-

 

bohydrates, as non-volatile compounds, must be derivitized to a vol-
atile compound before analysis by GLC. Both Gunner 35:31. (EMS) and
Vanden Hevel and Horning,(193) suggested the use of alditol acetates,
a carbohydrate derivative, for GLC analyses. Since then, deriviti-
zation procedures of carbohydrates have consisted of : trimethyl—
silyl ethers (181), trimethylsilyaldonolactones (134), acetyl esters
(170), dimethylsilyl ethers (166), acetylated aldonitriles ( 63) and
trifluoroacetyl esters ( 67).

Sweeley g£_§l, (181) presented a technique for the derivitiza-
tion of carbohydrates to their trimethylsilyl derivatives. They
stated that the anomeric pyranoside and furanoside forms of sugars
were difficult to separate. They determined the optimal proportions
of reagents needed for the maximum yield of each TMS derivative. A
detailed investigation of this reaction on approximately 100 different
carbohydrates demonstrated the utility of the TMS derivative.

In 1965, Sawardeker e£_al, (164) examined the use of alditol
acetate derivatives further for the analysis of carbohydrates. They
suggested that a derivitization method required that a volatile de—
rivative be preparable in quantitative yield from each monosaccharide
and that a mixture of derivatives be resolved completely. Another
problem mentioned was that as many as four glycosides per sugar, re—
sulting from anomeric and ring isomerization, could result in multiple

peaks for each sugar. Sawardeker et al. (164) stated that reduction

25

of monosaccharides to their alditols and then separation of the aldi—
tol derivatives offered better possibilities for their quantitation.
This procedure eliminated multiple peaks because alditols can not
anomerize.

Morrison and Perry (134) also described the many problems of
derivitization techniques of various sugars. They stated that two
glycoses yielded the same glycitol upon alditol acetate derivitiza-
tion. They suggested that the trimethylsilyl derivatives of aldono—
1,4—lactones could be prepared from parent aldonic acids and analyzed
by GLC. They applied this procedure to a mixture of sugars and re—
ported that the method was both accurate and rapid.

Crowell and Burnett ( 49) reported that because water reacted
with reagents during trimethylsilylation, it was necessary to employ
drying procedures and the chromatogram of TMS derivatives was diffi—
cult because of anomerization of the many sugars analyzed. They found
separation with alditol acetates superior to TMS derivatives. This
was also confirmed by Dutton e£_al, ( 62).

Easterwood and Huff ( 63) presented data that prompted the need
for more rapid and facile derivatization methodology. They develOped
a sugar derivative of acetylated aldonitrile and compared it with
alditol acetates. They concluded the procedure of alditol acetates
was a poor choice for the separation of sugar mixtures, however,
their chromatogram indicated a poor alditol acetate derivitization
technique.

Recently, two new procedures for the analysis of di— and tri—

saccharides were reported. Sugars were converted into their methox—

26

imes by a reaction with methoxylamine hydrochloride in pyridine
followed by either esterification with acetic anhydride or by tri-
fluoroacetylation With N—methyl-bis-trifluoroacetamide (166).

In 1968, Sloneker (IND) reviewed the methods of derivitization
for gas chromatography. The choice of which volatile derivative
and of which particular column to use for analysis of sugars depended
upon the complexity of the sugar mixture resolved. The rate at which
free sugars degraded during acid hydrolysis depended upon four fac—
tors: (1) type of acid used, (2) concentration of acid, (3) tempera—
ture of hydrolysis and (4) length of hydrolysis.

The use of GLC techniques in the analysis of plant cell walls
was first described in 1969 by Albersheim et_al, ( 11). They exa—
mined the primary cell wall of pinto bean hypocotyl. Hemicellulosic
sugars were separated by hydrolysis in trifluoroacetic acid at 121 C
for 1 hr. This was followed by reduction, methylation and acetylar
tion of the sugar mixture to their corresponding alditol acetates.
The major advantages of this procedure appeared to be: (1) the
T.F.A.A. used in the hydrolysis of polysaccharides is readily evapor-
ated; (2) the entire procedure may be performed in a single test
tube; (3) acetylation of alditols is catalyzed by sodium acetate,
thus eliminating the need for pyridine; (4) the acetylation mixture
may be injected directly into the gas chromatograph and (5) acetyl-
ation of the alditols was performed in a sealed tube which eliminated
the need for heating under reflux. They also reported that a maximum
1 hr hydrolysis in acid was optimum before sugars, particularly

xylose, would begin to decompose.

27

Also in 1969, Reid and Wilkie (155) reported the analyses of
oat leaves using methylated glycoside derivatives. Hemicellulosic
sugars were hydrolyzed in sulfuric acid at 100 C for 16 hr to 24 hr.
Subsequent to this report, others from the same laboratory have exa-
mined the hemicellulosic sugars of oats (28,30,32,76,77), wheat (31)
and bamboo (223) with alditol acetates.

Sloneker (171,172) developed a technique for complete sugar
analyses in foods, feeds and feedlot wastes. Total neutral carbo-
hydrates were measured in one sample after hydrolysis in sulfuric
acid for 2 hr and derivitization to their corresponding alditol ace-
tates. Cellulose was measured by an extraction procedure described
by Matrone et_al, (116). Total hemicellulose was determined by
difference. He reported that 5% of the aldoses and 20% of the xylose
were degraded during hydrolysis. Avicel, a microcrystalline cellu-
lose, had 0.6% xylose and 1.0% mannose. He concluded that even with
these complications, GLC had advantages over gravimetric and color-
imetric procedures because individual aldoses were measured directly
and because variations in the aldose content of different samples
were readily visualized.

Since then, there has been no agreement upon which GLC technique
should be used for lignified plant tissues. Woolard e£_al, (226)
and Theander and Aman (184) used alditol acetates for sorghum grain
and rapeseed meal respectively, whereas Ericsson gt_§l, (74) used TMS
derivatives for the analysis of soluble carbohydrates in pine needles
and Morrison (133) suggested the use of aldononitriles for poly-

saccharide analysis.

 

28

In 1979, Collings and Yokoyama (1%») described procedures for
the study of fiber components in 15 lignified plant tissues. Tissues
were delignified with sodium chlorite (‘41) and hydrolyzed with T.F.A.
A. The hydrolyzed sugars are dirivatized to their corresponding al—
ditol acetates and analyzed by GLC ( 11). Length of hydrolysis,
chemical additives and other variables were examined. Comparisons
between the detergent fiber analysis system and the gas chromatograph
system were reported. They suggested that this system offered ad—
ditional structural information about the capacity and structure of
fiber or the plant cell wall.

Utilization of fiber by animals and bacteria. The development

 

of fiber component analyses for nutritional studies have depended
upon the particular organism being studied. 'Various approaches have
included the isolation of: (1) the indigestible portion of the
dietary fiber (195), (2) the most indigestible fraction of the plant
cell wall (85,153,192,195,215,216 ), (3) the fractionated plant cell
wall ( LSD, and (4) the soluble and insoluble complex polymers assoc-
iated with the plant cell wall ( 79,173,174).

Many animals have been shown to be able to utilize fiber com-
ponents.- Ruminants, such as cattle and sheep, have a large ferment-
ation system in their rumen which contains bacteria and protozoa.
This flora digests large quanities of the plant cell wall and produces
protein and energy in the form of volatile fatty acids which can be
used by the host animal ( 97). Nutritional studies have shown that
the amount digested within a ruminant depends upon the type and ma—

turity of forage, pelleting, flaking, composition of forage, micro-

 

29

flora, minerals, degree of lignification, degree of Crystallinity,
acetyl groups, particle size, etc. (15,46,174 ).

A comparison of fiber digestibility between sheep_ rats and
swine showed the highest amount digested was by sheep followed by
pigs and rats (104,208. Utilization of fiber by non—ruminants has
appeared to be due to cellulolytic utilizing bacteria in the gastro-
intestinal tract (26,27 ). A cross-species comparison with three
substrates showed that utilization of fiber decreased in the follow—
ing order among species: sheep, swine, horse, voles and rats. The
hemicellulose of the three substrates was more digestible in pigs
and voles (103). Increasing amounts of fiber in the diet did not
affect the digestibility of all detergent fractions in pigs, but
decreased the digestibility of hemicellulose and cellulose in rats
(105). The digestibility of ADF, cellulose and lignin decreased
with increasing amounts of wheat middlings in the diet of pigs ( 39).

Other animals that have been shown to utilize fiber include:
white—tailed deer (191), horses (204), veal calves ( 81), beaver ( 95),
humans ( 93), rabbits ( 36), monkeys (125), and insects such as ter—
mites (114), locusts (124), grasshoppers (124) and shipworms ( 54).

The utilization of fiber in the intestinal tract of a monogastric
animal and in the reticulo—rumen of ruminants has been shown to
involve a close association with cellulolytic bacteria ( 26,27,97 ).
Although fiber has been defined as the plant material resistant to
intestinal enzymes (187,188), reports in many monogastric animals
have shown extensive fiber utilization ( 26,27,39,40, 103,104, 105,

208,212).

 

Fi——— . ... _ _ -. k .. , . nth—V... _‘ ‘A_L‘_
\

30

In 1966, Dehority e£_al: ( 55) showed 60%, 75% and 80% of the
hemicellulose from orchardgrass, alfalfa and timothy, respectively,
was digested in vitro with rumen fluid. Hemicellulose digestibility
was measured as the loss of total pentoses.

MOre specifically, Gaillard et_al. (€M)) found that pasture
plant hemicellulose consisted of three major polysaccharide types:
(1) linear A hemicellulose - a water insoluble heteroxylan containing
uronic acids, but only small amounts of arabinose, (2) linear B hemi—
cellulose — a more soluble heteroxylan containing much more arabinose
and less uronic acids than linear A, and (3) branched B hemicellulose
a water soluble, highly branched polymer which, in addition to pen-
toses, is rich in galactose and uronic acids. They found that linear
B hemicellulose was readily attacked by rumen bacteria in vivo. In
grass—fed animals, grass linear A hemicellulose and branched B hemi-
cellulose hydrolyzed faster than similar clover fractions. In clover—
fed animals, clover and grass linear A hemicellulose and branched B
hemicellulose hydrolyzed at the same rate.

Coen and Dehority ( 37) found that digestibility varied with
maturity and type of plant and bacterial species. They reported that

Bacteroides succinogenes S85 digested greater amounts of bromegrass,

 

alfalfa or fescue hemicellulose when compared to Ruminococcus flave-

 

faciens C94. Hemicellulose was measured as total pentoses.

In 1978, Dehority and Scott ( 56) reported Bacteroides succino-

 

genes S85 was able to digest more cellulose than eight other rumen
bacteria. They found only three bacteria of the eight examined were

able to digest hemicellulose. Ruminococcus flavefaciens C94 digested

 

31

37.2% and 10.3% bromegrass and alfalfa hemicellulose whereas B;

succinogenes S85 digested 13.3% and 0.0%, respectively.

 

Akin and Amos (E3 ) examined forage cell wall after incubation
with rumen contents with scanning electron microscopy (SEM) and trans-
mission electron microscopy (TEM). They found that bacteria attack—
ed the mesophyll and phloem of forages. Latham e£_al. GIMS) report—

ed that B. succinogenes S85 adhered to the cut edges of ryegrass and

 

to the intact mesophyll whereas R. flavefaciens C94 adhered to the cut

 

edges of epidermal cell walls. They suggested that adhesion was simi—
lar, but there were different affinities for each cell wall.

Leatherwood (110) proposed that R. albus may have an affinity
factor which is necessary to hold the hydrolytic factor of cellulase
in position to the insoluble cellulose for multiple attacks to occur.
Such a phenomenon may be required for hydrolysis of the cell walls
more resistant to bacterial degradation where attachment precedes
degradation.

Two modes of bacterial attack were visualized: (1) tunneling
action and (2) erosion of surfaces. Dinsdale gt_al. (58 ) said there
is a serious gap in the knowlege of cell wall digestion. No enzymes
have been isolated and characterized which are capable of attacking
highly ordered cellulose. The bulk of the cellulose in the cell walls
of forages has been shown to have a moderate degree of order and di-
gestion could be high depending upon the order of the cell wall (58 ).

Interest in fiber utilization in monogastrics, especially humans,
has been increasing in recent years (27,158,212 ). Bryant (27 ) has

identified hemicellulose fermenting bacterial species in human feces.

32

Previously to this, he reported significant cellulose utilizers were
found in human feces ( 26). These bacteria have been identified as

species of Bacteroides and Bifidobacterium.

 

 

The importance of the understanding of fiber utilization, struc—
ture and action in humans has increased over the last ten years. The
action of fiber has been implicated in the following: diabetes,
colon cancer, deep vein thrombosis, cardiovascular disease, diver—
ticular disease, obesity, gallstones, appendicitis, hiatus hernia,
hemorrhoids and others (177). Spiller et al. (177) mentioned a need
for techniques to analyze fiber more completely than previous methods

so to examine the mode of action of these diseases.

MATERIALS AND METHODS

Preparation of samples. Twenty feeds and forages were chosen

 

to represent a broad cross-section of substrates (Table 1). Each
was hand collected from controlled University plots, dried at 60 C
for 48 hr, ground in a Wiley mill (1 mm screen) and stored in glass
bottles. A second dry matter (A) on each sample was performed after
grinding to account for water absorption (41,78 ). Fiber components
as determined by the detergent method ( 195,200 ) were completed.
Five to 10 trials with neutral and acid detergent were performed to
collect one to two grams of the neutral detergent fiber and acid
detergent fiber fractions of each substrate for further analyses.
Crude protein (N x 6.25) was determined by the microkjeldahl method
(14).

Delignification of plant samples. The sodium chlorite oxidation

 

procedure as described by Green (84) was modified (41) to determine
the lignin content in samples. A 1.00 g sample was weighed into a
600 ml Berzelius beaker, and 200 ml of a w/v 0.5% ammonium oxalate
solution were added. The suspension was then heated to boiling (5

to 10 min) on a crude fiber reflux condenser. The heat was reduced
to an even boil and refluxedfor 2 hr timed from the onset of boiling.
A sintered-glass crucible (#1 porosity) was preweighed and placed in

a fiber manifold. The oxalate-fiber slurry was poured into the cruc-

33

.umoum memo pomlowmaflm auoo Eoum umnfim mussma pwsmmsn

magmafim>m uocm

 

34

 

 

 

 

 

 

 

 

 

 

 

N.Hm .WNMH.MMM owmaww ouoo .om
m.mm msuoaoowcuoo msuou HHOMmuu uoommpHfim .oH
N.um m>uumm owmuuemz mofimuH< .wu
0.0m mwum> mHHwaopoo coum> cBOMU .NH
m.oN omomumum EDHHomwuH um>oao pom .oH
o.mm wooden 53HHOMHMH uo>oao ocwwmq .mfi
A IIIIII noofiw muons: .qH
m.- muomum muosmopmao mowa< .mH
w.w mHmompono mowoam mopoam .Nfi
0.00 E:>Humom Esowuwpfi omen ummxz .HH
H.0m Es>fiummm Esofiufiua mmCfiprHE umosz .oH
will: E:>Hummm Esowuwue 3muum umosz .m
w.mq mcommn :ou%momw¢ mmmuwxomso .w
m.©q oumumEOwamHH%uomn mmmuwpumnouo .m
m.wq mHEuocfi mHEoum mmmuwoeoum .o
q.mq mwmcoumwm,msusommoa< HfimuxOM boom .m
o.mq moomcfirosnm mammamnm Eooan Haomlmmmuwzumsmo boom .q
n.5m mmomcfiwcspm mwaHmzm wcwuuso pamlmmmuwxumcmo boom .m
m.mm moomowvcsum momwmmm osomwm HHmH .N
N.qq mfimcouomm mom mmmuwwsao axosucmx .#

 

 

 

 

 

 

 

 

 

N .uouumE zpp wamfim

oEm: uwwfiucmwom

mama ooEEoo

 

 

.mmH<mHmm:m mo MMHH<E wan QZ< mmz<z UHhHHzmHUm

Qz< 202200 7. mqm<H

35

ible, and a vacuum suction was applied. The residue was washed twice
with 50 ml of hot distilled water. The crucible plus fiber was then
dried for 48 hr at 60 C in a forced—air oven. The crucible plus fiber
was weighed once after 30 min in the dessicator and once more after
30 min at room temperature. This was needed for measurement of water
absorption of the fiber which was accounted for in later calculations.
The fiber residue was quantitatively scraped from the crucible, weigh-
ed (B), then transferred into a 250 ml Phillips beaker, and 100 ml
of a v/v 1% acetic acid solution were added. This mixture was heated
to 70 C :_1 C; then 1.00 g of sodium chlorite was added. This oper-
ation was performed under a hood because chlorine dioxide generated
during the reaction is both toxic and explosive. Heating continued
for 30 min with frequent stirring. After 30 min, an additional 0.25
g of sodium chlorite was added, and heating continued for an addition—
al 15 min with frequent stirring. At the end of the total oxidation
period (45 min) the solution was cooled, and enough ascorbic acid
was added (up to 0.5 g) to stOp the oxidation reaction. A color
change in the solution from bright yellow to clear was indication
of complete cessation of chlorite oxidation. The solution was pour-
ed into a preweighed sintered-glass crucible (#1 porosity), and the
crucible and contents were dried at 60 C for 48 hr. The crucible was
weighed after cooling for 30 min in a dessicator, and the following
calculations were made. Weights obtained and calculations:

1. (A) Dry matter of ground sample.

2. (B) Oxalate fiber residue.

3. (C) (Wt. of crucible #1 + oxalate fiber) - Wt. of crucible#l=

36

Wt. of oxalate fiber at 0 min.

4. (D) (Wt. of crucible #1 + oxalate fiber) — Wt. of crucible #1 =
Wt. of oxalate fiber at 30 min.

5. (E) (Wt. of crucible #2 + delignified residue) — Wt. of cruc-
ble #2 = Wt. of delignified residue.

6. Wt. of lignin = (BC/D) - E.

7. % Lignin in dry sample = (C) x (Wt. of lignin/(BC/C) x 100)/
(sample wt. x (A)).

Second dry matter of plant samples. Three 1.00 g samples of each

 

plant sample were dried at 60 C for 48 hr and placed in a dessicator
for 30 min. Dry matter was calculated and this value was used in all
subsequent calculations. To demonstrate the absorption of water by

the different fibers, three 1.00 g samples of solkafloc cellulose was
dried at 60 C for 48 hr and placed in a dessicator for 30 min. Weights
of individual samples were taken each minute for 30 min with the sam—
ples at room temperature. Dry matter of each sample was determined

at minute intervals.

 

Preparation of alditol acetates. A750 to 60 mg delignified sam-

ple (F) was transferred to a 18 x 150 mm heavy duty Wheaton tube1

containing 10 ml of 1.0 N trifluoroacetic acid. Myo-inositol (5 to

7 mg) (G) was added to each tube as an internal standard. Each de—
lignified sample was run in triplicate. The tubes were sealed with
slotted stoppers and aluminum seals and hydrolyzed at 120 C for 60 min
in an autoclave. After the hydrolysis, the samples were filtered
through a preweighed sintered-glass crucible (#3 . The filtrate was

then collected and poured into a 50 ml round bottom flask. Total

lWheaton Scientific blueprint no. 37350, Wheaton Scientific, Mill—
ville, N.J. 08332.

37

uronic acids were determined by colorimetric analysis of the ammonium
oxalate and trifluoroacetic acid (T.F.A.A.) filtrates (23). The res-
due was washed with 50 ml deionized distilled water and dried for 24
hr at 60 C. The filtrate was evaporated to dryness under a stream of
filtered air in a 60 C water bath. The hydrolyzed hemicellulosic
sugars in the filtrate were reduced to their respective alditols with
sodium borohydride (1.0 g) in l N ammonia (50 ml) for 1 hr with
occasional swirling. The reduction was ceased with glacial acetic acid
until the reaction stopped. 10 ml of methanol was then added and eva-
porated to dryness under a stream of filtered air in a 60 C water bath.
Five more 10 ml methanOl additions were added ( 11) and evaporated to
dryness as before. Acetic anhydride (4 ml) was then added and the
round bottom flask was sealed with a rubber stopper and wired down.

The alditol mixture was then acetylated to their corresponding al-
ditol acetates by heating at 120 C for 1 hr in an autoclave.

GLC quantification of alditol acetates. A 3 to 5 ul sample of

 

the autoclaved mixture was injected into a gas chromatograph (Varian
model 1800 Aerograph or Hewlett-Packard 5840A) equipped with a hydro-
gen flame ionization detector. A stainless steel column (120 x 0.3
cm) packed with 0.2% poly-ethylene glycol adipate, 0.2% poly—ethy-
lene glycol succinate, and 0.4% silicone XF—1150 on Gas Chrom P

(100 to 120 mesh) was used. Other GLC parameters were: column temp-
erature, programmed between 135 C to 200 C with a 10 min holding at
135 C after injection of the sample, followed by 1 C/min increase in
temperature; helium flow rate of 30 ml/min; injection temperature

of 210 C; detector temperature of 250 C; and attenuation of 32X

38

and range of 1 mvolt. The following formula was used to calculate
the percent hemicellulosic sugars and cellulose:

% cellulose or % sugar = (C) x Wt. of sugar or cellulose
BC/D

Sample wt. x (A)

 

 

x 100

Wt. of cellulose in (E) = JE/F.

Wt. of sugar in (E) = GIE/FH.

Where:
1. (F) Wt. of holocellulose sample (50 mg).
2. (G) Wt. of myo-inositol (5 to 7 mg).
3. (H) Myo-inositol GLC peak area.
4. (I) Sugar GLC peak area.

5. (J) Wt. of cellulose.

Trifluoroacetic acid hydrolysis. To determine the optimum hy-
drolysis time as specified by Albersheim gp_gl. (11 ), samples of
Reed canarygrass (full bloom) delignified tissue was prepared. 50
mg samples plus 5 to 7 mg myo-inositol were hydrolyzed in triplicate
in 10 ml 1.0 N trifluoroacetic acid for 15, 30, 45, 60, 75 and 90
min.

Methanol additions. As shown by Albersheim et al. (11 ), five

 

additions of methanol appeared to be sufficient to permit methylation
of the hemicellulose sugars in sycamore cells. To test this in whole
plants, samples of Reed canarygrass (full bloom) delignified tissue

were prepared. {50 mg samples plus 5 to 7 mg myo-inositol were hy-

\..
\

drolyzed in 10 ml 1.0 N trifluoroacetic acid for 60 min. After

39

collection of the filtrate, evaporation and reduction, one to six
methanol additions (10 ml each) were tested.

Cellulose comppsition. Residual cellulose was collected from

 

Reed canarygrass (full bloom) after hydrolysis in 1.0 N trifluoro—

(Edibles were hydrolyzed by the two-step sul—

/ ).. I ./
I}, '1 1' '3'
‘ . I.

acetic acid. (300 mg
furic acid procedure as described by Adams ( 1 ). Hydrolysates
were dried in a 60 C water bath and 5 to 7 mg myo—inositol was add-
ed. Each hydrolysate was reduced and acetylated as in the previous
procedure and analyzed for the corresponding alditol acetates using
GLC.

Neutral and acid detergent fiber analyses. The neutral deter-

 

gent fiber and acid detergent fiber fractions of all twenty forage
and feeds were treated with ammonium oxalate, sodium chlorite and
trifluoroacetic acid as described before to examine the possible
losses of polysaccharides with detergent treatment (17 ).

Other fiber analyses. The amino acid composition of wheat straw,

 

wheat straw neutral detergent, acid detergent and ammonium oxalate fi-
bers, and the delignified residue of wheat straw were done by ion-ex-
change chromatography. Samples of the ammonium oxalate fiber, delig-
nified residue and T.F.A.A. cellulose were prepared from wheat straw,
Kentucky bluegrass, bromegrass, ladino clover and alfalfa and analyzed
for crude protein (14 ) and aSh (14 ). Calculations of subsequent
losses due to treatment were converted to a dry matter basis.

Pony digestion trial. Three ponies (Chester, Snipper and Elvis)

 

of varying weights (146.1, 161.5 and 129.7 kg, respectively) were

borrowed from the Michigan State University Endocrine Research Unit.

40

The ponies were fed an alfalfa hay-oats ration twice daily (0800 hr
and 1700 hr) and preadjusted 7 days on the ration before a 7 day col-
lection period began. All animals were maintained individually in
183 cm x 213 cm stalls with cement floors and no bedding. Feed con-
tamination of the feces was kept to a minimum by picking any feed
particles out of the feces. No attempt was made to moniter urine
output. Feed and feces were collected three times daily and weighed.
The unconsumed feed was weighed and recorded daily. Dry matter of
the feces was calculated on each daily sampling. At the end of the
collection period, the daily fecal samples were thoroughly mixed

and a dry matter content was determined.

Beef cattle digestion trials. Two digestion trials were con—

 

ducted with Hereford steers to determine the digestibility of fiber
at the Beef Cattle Research Center at Michigan State University.

In trial I, two Hereford steers (approximately 295 kg) were fed a
corn silage—supplemented ration (30.9% D.M. and 13.5% crude protein).
Each animal was preadjusted 7 days on the ration before a 4 day col-

lection period began. Rations were mixed and fed ad libitum once

 

daily. The unconsumed feed was weighed and recorded daily. All
animals were housed in an environmentally controlled metabolism
room and were maintained individually in 91 cm x 244 cm collection
stalls. All steers had free access to water. Plastic bags were
laid immediately behind each steer to collect the feces during the
collection period. Feces were taken daily and 10% of the feces was
collected and refrigerated. Dry matter of the feces was calculated

on each daily sampling. At the end of the collection trial, the daily

 

41

fecal samples were thoroughly mixed and its dry matter content de-
termined.

In trial II, two Hereford steers (approximately 270 kg) were
fed a corn silage-supplemented ration (32.1% D.M. and 13.4% crude
protein) on a restricted intake. Both animals were fed once daily
and preadjusted 7 days on the ration before a 7 day collection
period began. All other parameters were identical to trial I.

Swine digestion trials. Four starting Yorkshire pigs were

 

housed in 60 cm x 80 cm individual cages at the Swine Research Cen-
ter at Michigan State University. Feed in meal form (Table 2), and
water were restricted to two 200 g meals with an equal weight of
water mixed with the feed to form a gruel which the pigs rapidly
consumed. This resulted in minimal contamination of excreta. Six
growing and six finishing pigs were housed in 2.44 x 1.83 m elevated
pens with expanded metal floors. Room temperature was held constant
at 26.7 C with proper ventilation. Six Yorkshire growing pigs were
randomly allotted to two groups of three pigs each with the average
weight being 30 kg for pen I and 51 kg for pen II. Six Yorkshire
finishing pigs were randomly allotted to two groups of three pigs
each with the average weight being 89 kg for pen I and 80 kg for
pen II. Feed in meal form (Table 2), and water were supplied ad
libitum at all times.

Prior to collection, pigs were adjusted to the diet for 4 days.

Collection periods were 4 days in each group of ad libitum-fed pigs

 

and 8 days in individually meal-fed pigs. Chromic oxide (1% of the

diet) was mixed into each diet at the beginning of each test. At

42

TABLE 2. COMPOSITION OF SWINE DIETS.

 

 

 

Item Starter Grower Finisher
% % %

Ground shelled corn 71.15 78.45 84.60
Dehulled soybean meal 25.00 18.00 12.00
Defluorinated phosphate 1.50 1.00 1.10
Calcium carbonate 0.60 0.80 0.70
Salt a 0.50 0.50 0.50
MSU-Vitamin—Trace ‘neral mix 0.50 0.50 0.50
Vitamin E-Se premé 0.50 0.50 0.50
Antibiotic premix 0.25 0.25 0.25

100.00 100.00 100.00

 

aSupplying the following vitamins and trace elements per kilogram of

diet: vitamin A, 3300 IU;
menadione sodium bisulfite, 2.2 mg;
17.6 mg; pantothenic acid,
19.8 ug; zinc, 74.8 mg;

per, 9.9 mg; iron, 59.4 mg.

13.2 mg;

vitamin D, 660 IU;
riboflavin, 3.3 mg;
choline,

110 mg;

n

vitamin E, 5.5 IU;

iacin,

vitamin B
manganese, 37.4 mg; iodine, 2.75 mg; cop-

bSupplying 5.5 IU of vitamin E and 0.1 ppm of selenium to the diet.

CSupplying 110 ppm of chlortetracycline, 110 ppm of sulfamethazine
and 55 ppm of penicillin to the diet of starting and growing pigs.

dSupplying 22 ppm of chlortetracycline to the diet of finishing pigs.

43

the first appearance of the green marker in the feces, the pens were
cleaned and feces were collected daily. Chromic oxide (1% of the in
diet) was fed again 4 days after the first addition of chromic oxide.
At the appearance of this green marker, fecal collections were termin-
ated. Feed intake was recorded. Fecal samples were collected, tho-
roughly mixed and weighed.

Digestion trial samples. Feed and fecal samples were ground

 

through a Wiley mill (1 mm screen) and stored in glass bottles. Each
sample was analyzed for the detergent fiber components (195,200,21D,
crude protein (14 ) and their components as described previously.

Pure culture analyses. Pure cultures of Ruminococcus flavefaciens

 

 

C94 and Bacteroides succinogenes S85 were obtained from M.P. Bryant,

 

University of Illinois. Six fiber samples were chosen to represent

a broad cross-section of fiber types: alfalfa, bromegrass, corn sil—
age, cattle manure fiber, wheat straw and Whatman #1 filter paper.

All substrates were ground through a Wiley mill (0.5 mm screen), wash—
ed three times in distilled water, dried at 60 C for 96 hr and ana—
lyzed for their fiber components as determined by alditol acetates.
Media was prepared in 500 ml round bottom flasks which contained:

0.6 g trypticase, 0.4g yeast extract, 0.2 ml 0.1% Reazurin, 15.0 ml

of 0.6% dipotassium phosphate, 15.0 ml of mineral solution, 0.6 ml
VFA solution, 2.0 ml 0.01% ferric chloride, 2.0 ml 0.01% cobalt chlo—
ride, 0.6 g substrate, 4.0 ml 2.5% cysteine sulfide and 10.0 ml 8.0%
disodium carbonate. The mineral solution contained: 0.6% potassium
phosphate, 1.2% ammonium sulfate, 1.2% sodium chloride, 0.25% magnesium

sulfate and 0.16% calcium chloride. The VFA solution contained:

44

17.0 ml acetic acid, 6.0 ml propionic acid, 4.0 ml n-butyric acid,
1.0 ml isobutyric acid, 1.0 ml DL—methyl—n-butyric acid, 1.0 ml
n—valeric acid, 1.0 ml isovaleric acid and 1.0 g phenylacetic acid.

Each flask was inoculated with R.flavefaciens C94 or B. succinogenes

 

S85 and incubated for 48 hr at 39 C on a stirring plate at low ro—
tational speeds. At the end of the incubation, the pH was taken
immediately and the media was centrifdged at 5000 x g for 20 min.

A minute quantity (10 to 15 mg) of the fiber pellet was transferred
to 5 ml of cold phosphate buffer (6.8), gently swirled and saved for
further analyses. The remaining fiber pellet was washed six times
with c0pius amounts of distilled water and recentrifuged. The final
pellets were dried at 60 C for 96 hr, weighed and then analyzed for
their fiber components as determined by alditol acetates. The per-
cent fermented was determined by loss in weight divided by initial
substrate dry weight.

Electron microscopy. Dried (60 C) wheat straw was ground

 

through a Wiley mill (1 mm screen) and treated with neutral or acid
detergent (2%2), ammonium oxalate, sodium chlorite (41 ) and tri-
fluoroacetic acid (44 ). Each sample was adhered to aluminum stubs
using double stick tape, coated with 200 to 300 A gold in a film-vac
sputter coater, and observed in an ISI-Super III SEM operated at 15
kvolts at the Pesticide Electron Microscopy Laboratory at Michigan
State University. Each sample was fixed in 5% glutaraldehyde in

0.1 M phosphate buffer on ice for 2 hr, followed by 2 hr in 1.0%
osmium tetroxide in the same buffer. Fixed tissues were dehydrated

in a graded ethanol series and embedded in epoxy resin (121).

45

Ultrathin sections were stained with 0.5% uranyl acetate and sub—
sequently stained in 1.0% lead citrate. Sections were examined
in a Phillips 300 transmission electron microscope.

Wet fecal samples were taken from a beef steer in digestion
trial I. One sample was washed through fine screens and dried
at 60 C while another was just dried at 60 C. The dried fecal sam—
ple was analyzed for its neutral detergent, acid detergent and
ammonium oxalate fibers. Each fiber sample was then adhered to an
aluminum stub using double stick tape, coated with 200 to 300 A
gold in a film—vac sputter coater, and observed in an ISI-Super
III SEM operated at 15 kvolts.

Samples from each pure culture incubation were taken and placed
in cold phosphate buffer. Each sample was fixed in 2% glutaralde—
hyde and then with osmium tetroxide. Following fixation, the tissue
was washed in phosphate buffer and dehydrated with ethanol. Samples
were then subjected to critical point drying and mounted on stubs
and coated with gold in a film-vac sputter coater. Each was then
observed in an ISI—Super III SEM operated at 15 kvolts.

Soluble carbohydrates in corn silage. Fresh chOpped corn silage

 

was placed in 11 jug silos, closed and sealed with carbon dioxide.
One jug was opened at the following time periods: 0,6,12,18,24,36,
48,72,96,144 and 312 hr. Three 100 g samples from each jug were
weighed into beakers, washed with 300 ml deionized distilled water
and pressed gently. This slurry was strained through two layer of
cheese cloth and the pH was immediately taken. The liquid was then

freeze—dried for 6 days in a Virtis model 25SRC freeze dryer. The

46

dried samples were then reduced and acetylated to the corresponding
alditol acetates and analyzed by GLC as before.

Statistical analyses. The data within these trials was computed

 

and analyzed on a Hewlett Packard 9825A computer. Correlation coef—
ficients, analyses of variance tables and multiple comparison tests

were computed as outlined by Zar (228 ).

 

RESULTS

Preparation of plant samples. Field dry matters of eight grass—

 

es, five legumes and seven other substrates are given in Table 1.

Of the eight grasses, field dry matter ranged from a low of 33.5% in
tall fescue to a high of 48.5% in bromegrass. Dry matter among the
legumes ranged from a low of 20.7% in red clover to a high of 32.9%
in birdsfoot trefoil. Among all substrates, wheat middlings and
elodea had the highest and lowest dry matter with 90.1% and 8.8%,
respectively.

The fiber fractions as determined by the detergent system of all
substrates are shown in Table 3. Among all grasses, NDF ranged from
57.15% for Reed foxtail to 62.44% for tall fescue with an average of
59.54%. NDF in legumes ranged from 29.62% in red clover to 50.09%
for birdsfoot trefoil with an average of 37.05%. Manure fiber had
the highest NDF and red clover had the lowest NDF with 83.10% and
29.62%, respectively. The values for ADF ranged from 33.81% in Reed
foxtail to 38.70% in orchardgrass in the grasses and from 21.48% in
red clover to 37.70% in birdsfoot trefoil in the legumes. The aver—
age for grasses and legumes was 36.85% and 28.39%, respectively.
Wheat straw and wheat bran had the highest and lowest ADF with 51.75%
and 13.95%, respectively. Hemicellulose was determined by subtracting

ADF from NDF. Hemicellulose ranged from 18.66% in orchardgrass to

47

48

 

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49

24.87% in Reed canarygrass-full bloom in the grasses and from 3.96%
in ladino clover to 12.39% in birdsfoot trefoil in the legumes.
Hemicellulose for all grasses and legumes was 22.69% and 8.66%, re—
spectively. Wheat bran and ladino clover had the most and the least
hemicellulose with 38.30% and 3.96%, respectively. Cellulose was
determined after delignification and ashing. The average for grasses
and legumes was 27.26% and 21.74%, respectively. Cellulose values
ranged from 22.47% in quackgrass to 30.10% in orchardgrass in grasses
and from 16.43% in red clover to 28.94% in birdsfoot trefoil in the
legumes. Manure fiber and wheat bran had the highest and lowest
cellulose values with 40.04% and 9.93%, respectively. Lignin, as
determined by loss in weight with potassium permanganate oxidation,
was highest in wheat straw and lowest in tall fescue with 12.36% and
3.13%, respectively. Silica was determined as the weight recovered
after ashing. This ranged from 1.57% in Reed canarygrass-full bloom
to 10.76% in quackgrass among the grasses and from 0.26% in alfalfa
to 1.62% in crown vetch. The average silica content among grasses
and legumes was 4.77% and 0.90%, respectively. The highest and low-
est value among all substrates was 10.76% and 0.00% for quackgrass
and corn silage, respectively.

The fiber fractions as determined by the proposed method consist
of ammonium oxalate fiber (AOF), holocellulose (cellulose+hemicell—
ulose+chlorite lignin), cellulose, hemicellulose and chlorite lignin.
The values for the 20 substrates are presented in Table 4. AOF re-
presents the cellulose, hemicellulose, lignin, some ash and protein

of the plant cell wall. The AOF values for grasses ranged from

50

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51

66.00% in Reed canarygrass-full bloom to 73.58% in Kentucky blue-
grass and for legumes ranged from 56.57% in ladino clover to 76.41%
in crown vetch. The average among grasses and legumes was 70.01% and
63.05%, respectively. Manure fiber had the most AOF with 91.36% and
corn silage had the least with 47.98%. Holocellulose represents

the structural polysaccharides of the cell wall. This was highest
in wheat straw and lowest in wheat bran with 66.21% and 23.68%, re-
spectively. The values for grasses ranged from 43.50% in bromegrass
to 60.29% in quackgrass and for legumes ranged from 26.05% in red
clover to 54.88% in crown vetch. The average value for grasses and
legumes was 49.14% and 38.30%, respectively. Lignin was determined
by loss in weight after oxidation in sodium chlorite and v/v 1%
acetic acid. Values ranged from 4.94% in Reed canarygrass-2nd cut-
ting to 7.29% in both tall fescue and quackgrass among all grasses
with an average of 6.05%. The values among legumes ranged from
5.66% in alfalfa to 8.02% in red clover with an average of 6.81%.
Algae and elodea had the highest and lowest values of lignin with
15.22% and 3.65%, respectively. Values were, in general, higher
than corresponding permanganate lignin values as shown in Table 3.
With eleven of the samples analyzed, sodium chlorite lignin values
were higher (P<.05) than its permanganate lignin value. There were
no significant differences between the values for the remaining
seven samples. Cellulose was determined by weighing the recovered
substrate after hydrolysis in 1 N trifluoroacetic acid (T.F.A.A.).
In grasses, these values varied from 26.87% in Reed canarygrass—

full bloom to 36.21% in Kentucky bluegrass with an average of 30.30%.

52

The values among legumes varied from 18.95% in ladino clover to
28.79% in birdsfoot trefoil with an average of 27.88%. The highest
and lowest value were found in wheat straw and wheat bran with 46.63%
and 10.36%, respectively. A comparison of T.F.A.A. cellulose
and detergent cellulose (Table 3 and 4) yielded similar results
in most substrates except Kentucky bluegrass, Reed canarygrass-
2nd cutting, Reed foxtail, quackgrass, wheat middlings, wheat straw,
algae and crown vetch which have significantly (P<.05) higher cell-
ulose values as determined by T.F.A.A. hydrolysis. Hemicellulose
as determined in the proposed system was quantitated by gas—liquid
chromatography. The values among grasses ranged from 14.38% in
orchardgrass to 23.04% in quackgrass with an average of 17.93%.
In legumes, the values ranged from 5.79% in red clover to 22.27%
in crown vetch with an average of 13.74%. Wheat middlings contained
the most hemicellulose with 33.29% while algae contained the least
with 3.90%. A statistical comparison could not be done between
T.F.A.A. hemicellulose and detergent hemicellulose (Table 3) because
the detergent hemicellulose represented an approximation as determined
by subtraction of ADP from NDF. However, 16 of the 20 substrates
had higher values when determined by subtraction. Red clover, crown
vetch, alfalfa and birdsfoot trefoil had higher values as determined
by gas-liquid chromatography.

The relationship between NDF and AOF and with ADF were signi-
ficantly correlated (P<.001) as shown in Table A1. AOF and ADF
were significantly correlated (P<.01) as were detergent cellulose

and MSU T.F.A.A. cellulose (P<.001). No relationship existed be-

53

tween detergent and MSU T.F.A.A. hemicellulose.

Hydrolysis of the delignified tissue with trifluoroacetic acid
and derivitization to the corresponding alditol acetate yielded a
pattern of the hemicellulosic sugars (Table 5) which include glucose,
galactose, mannose, xylose, arabinose, rhamnose and possible ribose.
In these calculations, uronic acids, which may include those from
pectins, were included in the hemicellulose. Uronic acids in the
ammonium oxalate and T.F.A.A. filtrates were determined by colorime—
try. Xylose, arabinose and glucose were present in all substrates
examined. Xylose was the highest in concentration in all substrates
with the exceptions of elodea, algae, ladino clover, red clover and
alfalfa. Uronic acids were the predominant hemicellulosic component
in elodea, ladino clover, red clover and alfalfa whereas galactose
was the predominant component of hemicellulose in algae. Mannose
was found in wheat middlings, wheat bran, elodea, manure fiber, la—
dino clover, red clover, crown vetch, alfalfa, birdsfoot trefoil
and corn silage. Rhamnose did not appear to be a common hemicellu—
losic component, however, it was found in Reed foxtail, ladino clover,
crown vetch, alfalfa and birdsfoot trefoil.

The hemicellulosic sugars expressed as a percentage of the total
hemicellulose are presented in Table 6. Uronic acids ranged from
1.86% in wheat middlings to 67.36% in red clover. The average for
grasses and legumes were 12.04% and 37.59%, respectively. The con-
centration of glucose varied from 7.25% in red clover to 27.10% in
wheat bran. The average for all grasses and legumes were 10.13% and

15.89%. Galactose ranged from 0.00% in manure fiber to 31.54% in

54

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56

algae. The average among grasses and legumes were 6.52% and 8.07%,
respectively. Mannose was not found in 10 of the 20 substrates,

most of which were in the Grass family. Rhamnose was found only

in five substrates with the highest amount being in ladino clover.
Correlation analyses showed (Table A2) that hemicellulose content
was strongly correlated with glucose (P<.01), arabinose (P<.001) and
xylose (P<.001) among all substrates. A strong relationship (P<.001)
between arabinose and hemicellulose was observed only in the grasses
(Table A3). Among the grasses, galactose and arabinose, xylose and
galactose, and xylose and arabinose correlated significantly (P<.001).
These relationships were not shown in legumes (Table A3).

A comparison between both fiber systems was made by adding the
corresponding cellulose, hemicellulose and lignin into a total fiber
fraction (Table 7). No specific trends were observed among the sub—
strates. Ten of the detergent values were higher than MSU fiber val-
ues. Grass samples ranged from 50.75% in quackgrass to 58.70% in
Reed canarygrass-full bloom to 61.39% in Kentucky bluegrass for the
MSU fiber system. The average value among all grasses for detergent
and MSU total fiber was 54.89% and 54.27%. Detergent total fiber
in legumes ranged from 28.53% in red clover to 50.06% in birdsfoot
trefoil with an average of 36.30%. The MSU values for legumes ranged
from 34.09% in red clover to 54.88% in crown vetch with an average
of 43.51%.

Ratios between individual fiber fractions are sometimes computed
to predict digestibility. A comparison between the ratios obtained

by the two methods is presented in Table 8. Hemicellulose/cellulose

TABLE 7. COMPARISON OF TOTAL FIBER VALUE§ OF DETERGENT

57

FIBER SYSTEMS.°

FIBER AND MSU

 

 

 

Substrate Detergent MSU
Z %

1. Kentucky bluegrass 56.74 61.39
2. Tall fescue 56.95 59.64
3. Reed canarygrass-2nd cutting 53.41 51.21
4. Reed canarygrass-full bloom 58.70 47.48
5. Reed foxtail 53.72 52.18
6. Bromegrass 55.42 49.04
7. Orchardgrass 53.44 52.95
8. Quackgrass 50.75 60.29
9. Wheat straw 74.55 74.93
10. Wheat middlings 49.93 56.10
11. Wheat bran 52.41 31.41
12. Elodea 30.55 35.10
13. Algae 43.34 45.06
14. Manure fiber 81.80 61.00
15. Ladino clover 28.93 37.65
16. Red clover 28.53 34.09
17. Crown vetch 32.57 54.88
18. Alfalfa 41.42 41.13
19. Birdsfoot trefoil 50.06 49.80
20. Corn silage 63.02 36.17

 

a .
Total fiber represents the cellulose,

fractions.

hemicellulose and lignin

58

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59

ratios calculated from detergent fiber values ranged from 0.62 to
1.04 among grasses and from 0.20 to 0.50 among legumes. The same
ratio calculated from MSU fiber values ranged from 0.45 to 0.77 in
grasses and from 0.29 to 0.90 in legumes. The MSU hemicellulose/
cellulose ratios for grasses were all lower than their corresponding
detergent value. A comparison of ratios in legumes showed that
all MSU values were higher than the corresponding detergent values.
Detergent lignin/cellulose ratios in grasses ranged from 0.10 to 0.22
and in legumes from 0.25 to 0.30. MSU ratios ranged from 0.17 to
0.24 in grasses and from 0.20 to 0.34 in legumes.

Uronic acids as determined by colorimetry are shown in Table 9.
Most of the uronic acids were found in the ammonium oxalate filtrate
with the exceptions of wheat straw and manure fiber. More uronic a-
cids were found in the T.F.A.A. filtrate for these two substrates.
Grass uronic acids ranged from 1.07% to 2.51% whereas legumes were
higher and ranged from 2.69% to 4.34%. The T.F.A.A. filtrates con—
tained fewer uronic acids, but ranged from 0.22% to 0.83% in grasses
nd 0.33% to 0.50% in legumes.

Second dry matter in plant samples. Results of determinations

 

of dry matter of solkafloc cellulose indicate an increase in weight
of 3.4% after samples were dried and then weighed each minute for
30 min at room temperature as shown in Figure 1.

Trifluoroacetic acid hydrolysis. A hydrolysis time of 60 min

 

showed the maximum yield of hemicellulose and decreased with a hy-
drolysis time greater than 60 min as shown in Figure 2. Both ara-

binose and xylose were degraded at hydrolysis times greater than 60

60

TABLE 9. URONIC ACIDS IN AMMONIUM OXALATE AND T.F.A.A. FILTRATES
0F SUBSTRATES.

 

 

 

Ammonium
Substrate oxalate T.F.A.A. Total
% % Z

1. Kentucky bluegrass 1.67 0.83 2.50

2. Tall fescue 2.05 ‘0.22 2.27

3. Reed canarygraSs-an cutting 1.07 0.48 1.55

4. Reed canarygrass-full bloom 1.17 0.49 1.63

5. Reed foxtail 1.61 0.52 2.13

6. Bromegrass 1.28 0.50 1.78

7. Orchardgrass 2.51 0.22 2.73

8. Quackgrass 1.89 0.46 2.35

9. Wheat straw 0.74 0.76 1.50

10. Wheat middlings 0.40 0.22 0.62
11. Wheat bran 2.49 0.36 2.85
2 Elodea 4.57 0.44 5.01

13. Algae 0.66 0.09 0.75
14. Manure fiber 0.15 0.39 0. 4
15. Ladino clover 4.95 0.34 5.29
16. Red clover 3.90 0.37 4.27
17. Crown vetch 4.23 0.41 4.64
18. Alfalfa 4.34 0.50 4.84
19. Birdsfoot trefoil 2.69 0.33 3.02
20. Corn silage 1.15 0.42 1.57

 

a . . . .
Each value represents tne average of tnree determinations.

61

 

100.2
r

FIGURE 1.

I l T I I l I U
<3 ~¢ <3 ~o ml tn -¢ <3
0‘ ox ex «3 a) n. r\ r\
ox ox ox O\ ox ox ox cm

Dry matter, %

CHANGES IN DRY MATTER WITH TIME AT ROOM TEMPERATURE.

I I I r
22 24 26 28 30

I
20

l
18

 

96.6

Time, min

62

Xylose

‘ Arabinose

 

n Glucose

Galactose

 

 

r
O
0\

FIGURE 2.

O
0
O
c
r l I I
Ln 0 Ln 0
r\ \o q- co

Carbohydrate recovered, mg

LENGTH OF HYDROLYSIS TIME AND SUGAR YIELD.

 

Time, min

63

min of hydrolysis at which time cellulose concentration remained
constant.

Methanol additions. The effect of different amounts of meth-

 

anol upon hemicellulosic sugar yield is presented in Figure 3. Max-
imum sugar yield was apparent at two methanol additions and stayed
the same with additional methanol.

Cellulose composition. Samples of delignified Reed canary-

 

grass-full bloom were hydrolyzed in T.F.A.A. These were then hydro—
lyzed in sulfuric acid to analyze for sugar composition by GLC.
Cellulose obtained by T.F.A.A. hydrolysis contained 97% glucose and
3% unidentified.

Neutral and acid detergent fiber analyses. The effect of re-

 

fluxing the 20 substrates in neutral detergent upon cell wall carbo—
hydrates is presented in Table 10. Among the grasses, 82.6% of the
cellulose, 53.3% of the glucose, 35.3% of the galactose, 61.3% of the
arabinose, 67.9% of the xylose, 2.5% of the uronic acids and 55.5% of
the hemicellulose were recovered. Cellulose varied from 73.0% to
93.0% recovered and hemicellulose varied from 14.9% to 79.3% recovered
in the grasses. Among the legumes, 80.0% of the cellulose, 100.4% of
the glucose, 30.4% of the galactose, 38.8% of the arabinose. 55.4% of
the xylose, 4.2% of the uronic acids and 26.5% of the hemicellulose
were recovered. Cellulose varied from 54.5% to 95.1% recovered and
hemicellulose from 11.4% to 37.5% recovered. Among all substrates,
recovery of the individual fiber components was extremely varied.
Cellulose ranged from 70.3% in red clover to 100.0% recovered in

wheat bran. Glucose ranged from 0.0% in Reed canarygrass-full bloom

 

64

 

I
3

Additions of methanol

I
2

 

 

Units of hemicellulose sugars

FIGURE 3. YIELD OF HEMICELLULOSIC SUGARS AS AFFECTED BY METHANOL
ADDITIONS.

.0000.00
0Nm_.cv Lo>o~u to; .Axcr.cv Lo>cdo c:_vm~ .ANNm.cV 2:00: .Ax_m.cv mote—w .ANc_.cv 0:00020 ccmummmgux0ezcc coax :0 to0uo0mc mm3 omcccczu

2.0 0.5 :30.-. .0 .0005

.AN¢_.OV move—m :0 000000;: 3:3 omo:Em;z;

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S

 

IIII.I II IIIII II I I ..t... IIIIII I..I . III I. I u I III IIIII ItIIIIIIlIIIuItIIIIIIIIIIII... III.III. I .I III I.I IIII I II I I I.

ra.m_

20.: v

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00.000 00.0 00.000 00.000 00.0 00.0 0 00.0 00.000 00.0 00.000 00.00 000000 0000
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00.000 00.0 00.0_0 00.0 00.000 00.0 00.000 00.0 00.0 0 00.0 00.000 00.0 00.000 00.00 000000< .00
00.000 00.0 00.0 0 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.00 00000 03000 .00
00.000 00.. 00.0 0 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.0000 00.. 00.000 00.00 t0>000 000 .00
00.000 00.0 00.000 00.0 00.0000 00.. 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.00 000000 000000 .00
00.0000 00.00 00.0 0 00.: 00.0000 00.00 00.0000 00.0 00.0000 00.0 00.0000 00.0 00.000 00.00 L0.0: 0000:: .00
00.000 00.0 00.0 0 00.0 00.0000 00.0 00.000 00.0 00.000 00.0 00.0000 00.. 00.000 00.00 00000 .00
00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.00 000000 .00
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00.000 00.00 Ao.o 0 00.0 00.000 00.0 00.000 00.0 00.000 00.: 00.000 00.0 00.000 00.00 000000005 00002 .00
0_._00 00.00 00.000 00.0 00.0000 00.00 00.000 00.0 00.0000 00.0 00.0000 00.0 00.000 00.00 30000 000;: .0
00.000 00.00 00.0 0 00.0 00.000 00.0 00.000. 00.0 00.0 0 00.0 00.000 00.0 00.000 00.00 0000000000 .0
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00.000 00.00 00.0 0 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.00 0000000000 .0
00.000 00.00 00.0 0 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.00 0000000 0000 .0
00.000 00.0 00.0 0 00.0 00.000 00.0 00.000 00.0 00.0 0 00.0 00.0 0 00.0 00.000 00.00 00000 0000-00000000000 0000 .0
00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.00 0000000 000-000.0000000 0000 .0
00.000 00.00 00.0 0 00.0 00.000 00.00 00.000 00.0 00.0000 00.0 00.0000 00.0 00.000 00.00 000000 0000 .0
00.000 00.0 00.0 0 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.0 00.000 00.00 000000000 00000000 ._
u. ...... nww»umnwnw.nnnnnn»ww- :.-anu:-:1! --n--.z.0 01----IIIIIII-nununnnmnuun.:::::I::;:I.aau-nn.s--| .a-:s-:nua::;-l-;; .
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I In I I.1l.|.II|II.IIII.I I |4l.1II IIIIII IIIII I II IIIIII IIIIIIIII III II l.u.uI\ III.II.IIIIII.III IIIIIII. IIIIIIIII‘IIIIIII III II IIIIIII.IIIIII-IIIII I I I I .Iu I} .II

 

.+ZEZCLZOU :G<: 0C >zm>comz szuzma :z< mmé<zhmm3m zc zchku<z0 arm—L szuzzym: 4<MEDEZ LC m_m>;<z< me<fisv< AOF—Q;< 0: mgm<h

use

66

to 259.3% recovered in red clover. Tall fescue, orchardgrass, wheat
straw, algae and manure fiber had all the hemicellulosic glucose re-
covered in their corresponding neutral detergent fiber fractions.
Alfalfa, corn silage and quackgrass neutral detergent fibers had 0.0%
galactose recovered whereas 100.0% recovered in tall fescue, wheat
straw and manure fiber. Arabinose recovery ranged from 12.6% in
Kentucky bluegrass to 100.0% in quackgrass and manure fiber. Recovery
of xylose was 100.0% in wheat straw, algae, manure fiber and ladino
clover whereas birdsfoot trefoil NDF contained only 6.5% of the orig—
inal xylose. Only seven of the 20 substrates retained any uronic
acids in the NDF. Wheat straw NDF retained the most uronic acids with
50.0% recovered whereas elodea NDF contained the most uronic acids

on a dry matter basis with 1.77%. Hemicellulose recovery in the NDF
ranged from 11.4% in birdsfoot trefoil to 100.0% in manure fiber.

The fiber components of the 20 substrates acid detergent fiber
fractions are presented in Table 11. The grasses ADF fractions con-
tained from 83.1% to 99.0% of the original cellulose and 10.8% to
31.5% of the hemicellulose. Among the grass hemicellulosic sugars,
68.6% of the glucose, 46.3% of the galactose, 0.0% of the arabinose,
15.3% of the xylose and 15.8% of the uronic acids were recovered.
Among all substrates, cellulose recovery ranged from 64.5% in wheat
middlings to 100.0% in alfalfa. Glucose ranged from 0.0% recovered
in bromegrass to 189.1% recovered in elodea. Six substrates of 19
substrates had 100.0% or greater recovered. Ladino clover ADF and
red clover ADF retained 118.4% and 109.5% of the galactose, respective—

ly. Of the 20 substrates, only red clover retained any of the hemi—

67

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68

cellulosic arabinose. Xylose recovery ranged from 0.0% in algae

ADF to 91.3% in red clover ADF. Four substrates contained 0.0%
uronic acids in the ADF. These included Reed canarygrass-full bloom,
quackgrass, manure fiber and crown vetch. Algae ADF contained 108.0%
of the original uronic acids.

Protein and ash composition. The crude protein and ash com-

 

position of wheat straw, bromegrass, alfalfa, Kentucky bluegrass
and ladino clover and their fiber fractions as determined by the
proposed MSU method are presented in Table 12. After refluxing in
ammonium oxalate, 60% to 74% and 16% to 40% of the crude protein and
ash. respectively, were recovered in the AOF fractions. After de-
lignification with sodium chlorite, 39% to 52% and 15% to 38% of
crude protein and ash were recovered, respectively. Of individual
samples, 30% of the wheat straw crude protein was lost after delig—
nification or 1.3% of the dry matter. Of the remaining substrates,
2.1%, 3.5%, 2.1% and 5.2% of the dry matter was lost after oxidation
in sodium chlorite. Sodium chlorite oxidation did not affect ash
content of the fiber residue, with 1% to 2% of the original ash lost.
After trifluoroacetic acid hydrolysis, no crude protein and ash
was found in the recovered cellulose.

Amino acid composition of wheat straw and wheat straw NDF, ADF,
AOF and the delignified residue are presented in Table 13 and 14.
Glutamic acid and methionine were the highest and lowest amino acids
among all fractions. Wheat straw NDF contained a higher amount of
proline and alanine and a lower amount of arginine than intact wheat

straw (Table 13) when expressed on a nitrogen basis. Similarly,

69

TABLE 12. PROTEIN AND ASH CONTENT IN MSU FIBER FRACTIONS.a

 

 

 

Original Ammonium Delignified
Substrate sample oxalate fiber residue Cellulose
Protein % % % 7
Wheat straw 4.19 2.89 (69) 1.65 (39) 0.00
Bromegrass 9.42 6.95 (74) 4.89 (52) 0.00
Alfalfa 16.47 11.86 (72) 8.43 (51) 0.00
Kentucky bluegrass 10.07 6.02 (60) 3.91 (39) 0.00
Ladino clover 25.91 17.92 (70) 12.85 (50) 0.00
Ashb
Wheat straw 8.74 3.48 (40) 3.29 (38) 0.00
Bromegrass 10.10 3.01 (30) 2.97 (29) 0.00
Alfalfa 7.43 2.76 (37) 2.65 (36) 0.00
Kentucky bluegrass 8.24 1.29 (16) 1.26 (15) 0.00
Ladino clover 10.90 4.22 (39) 3.99 (37) 0.00

 

GI

“Value in parentheses is the h retained of protein(N x 6.25) or ash
from a dry sample.

bEach value represents the % ash(920 F).

70

TABLEILL AMINO ACID COMPOSITION IN DETERGENT AND MSU FIBER FRACTIONS
0F WHEAT STRAW (G/16 G N).

 

 

Original Delignified

 

Amino acid sample NDF ADF AOF residue
Aspartic acid 8.83 8.84 8.84 8.67 10.16
Threonine 4.93 4.71 4.44 4.21 6.07
Serine 5.82 5.25 4.97 5.16 8.22
Glutamic acid 11.70 11.47 11.39 10.02 15.62
Proline 5.53 8.55 8.09 5.53 6.57
Glycine 5.74 5.72 5.69 5.16 7.03
Alanine 8.18 9.83 8.12 6.00 7.96
Valine 7.56 7.62 8.01 6.15 8.38
Methionine 0.53 0.46 0.73 0.51 0.26
Isoleucine 4.54 4.77 4.94 4.69 5.56
Leucine 8.80 8.63 6.81 8.73 10.96
Tyrosine 2.45 2.55 2.27 1.90 0.00
Phenylalanine 5.42 5.31 4.75 4.57 7.18
Lysine 9.25 8.86 4.20 7.38 5.21
Histidine 2.64 1.70 1.54 1.81 0.94
Arginine 5.91 4.46 2.22 4.22 5.76

 

TABLE L4. AMINO ACID COMPOSITION AND LOSS IN DETERGENT AND MSU FIBER
FRACTIONS OF WHEAT STRAW (MG/G D.M.).

 

 

 

Original Delignified
Amino acid sample NDF ADF AOF residue
Aspartic acid 2.90 0.73 0.45 2.22 1.13
Threonine 1.62 0.39 0.22 1.08 0.68
Serine 1.91 0.44 0.25 1.32 0.92
Glutamic acid 3.84 0.95 0.57 2.57 1.74
Proline 1.81 0.71 0.40 1.42 0.73
Glycine 1.88 0.48 0.28 1.32 0.78
Alanine 2.68 0.81 0.41 1.54 0.89
Valine 2.49 0.63 0.40 1.58 0.94
Methionine 0.17 0.04 0.04 0.13 0.03
Isoleucine 1.50 0.40 0.25 1.20 0.62
Leucine 2.90 0.72 0.34 2.24 1.22
Tyrosine 0.81 0.22 0.11 0.4 0.00
Phenylalanine 1.79 0.44 0.24 1.17 0.80
Lysine 3.05 0.73 0.21 1.89 0.58
Histidine 0.87 0.14 0.08 0.46 0.11
Arginine 1.95 0.37 0.11 1.09 0.64
Total 31.19 8 21 4.37 21.73 11.81
Recovery, % 26 32 14.01 69.67 37.86

 

71

wheat straw ADF had lower serine, leucine, phenylalanine, lysine,
histidine and arginine. AOF had lower serine, glycine, alanine, va-
line, tyrosine, phenylalanine, lysine, histidine and arginine but
higher proline. After delignification, wheat straw contained high-
er amounts of 10 of 16 amino acids, but had lesser amounts of lysine
and histidine on a nitrogen basis. When amino acids were expressed
on a dry matter basis, it was found that 26.32%, 14.01%, 69.67% and
37.86% of the total amino acids were recovered in the NDF, ADF, AOF
and delignified residue, respectively. Glutamic acid was in the
highest concentration among all fiber fractions. Methionine was the
lowest in all fractions with the exception of the delignified residue
which had no tyrosine.

Electron microscopy of fiber fractions. Scanning and transmis—

 

sion electron microscopy was used in order to examine the effects of
neutral detergent, acid detergent, ammonium oxalate, trifluoroacetic
acid and sodium chlorite treatment of the cell walls of wheat straw.
The surface of intact plant cells is coated with debris (Figure 4A).
Both neutral detergent and ammonium oxalate appear to rid the plant
cell wall of cell cytoplasm and debris (Figure 4B and 5A). Small
frequent disruptions were seen in the cell wall of wheat straw NDF
(Figure 4B). The cell wall of ammonium oxalate treated wheat straw
appeared to have opened cell walls (Figure 5A) with typical flaking
of the cell wall (Figure 5B), but no apparent losses. Acid detergent
has been reported to contain cellulose, lignin and some ash. The
chemical mode of attack appeared to be localized (Figure 6A and 6B)

with many ruptured cell walls. TEM studies showed that hemicellulose

72

 

 

 

 

FIGURE 4. EFFECT OF NEUTRAL DETERGENT TREATMENT ON WHEAT STRAW.
A. Control sample. Surface is coated with debris. No open cell ap—
parent: X1000. B. Section treated with neutral detergent. Open
cells are quite apparent. The wall is intact with small disruptions
(arrows) evident: X3000.

73

 

 

 

 

, ,A . . , \u \——_I B

FIGURE 5. EFFECT OF AMMONIUM OXALATE TREATMENT ON WHEAT STRAW.
A. Section treated with ammonium oxalate. Open cells are quite
apparent: X400. B. Section treated with ammonium oxalate. Cell

wall is smooth and glossy with small typical flaking (arrows):
X3000.

 

 

 

   

 

 

74

 

 

 

FIGURE 6. EFFECT OF ACID DETERGENT TREATMENT ON WHEAT STRAW.
A. Section treated with acid detergent: X1000. B. Close-up
of section A . Ruptured cell walls are apparent (arrows): X3000.

 

 

 

 

75

was disrupted (Figure 7A) and was clearly present in wheat straw ADF
(Figure 7B). Delignification of wheat straw showed no disruptions
and appeared to be evenly extracted (Figure 8B). After trifluoro-
acetic acid hydrolysis, cellulose sheets were clearly evident (Fi-
gure 8B).

Pony digestion trial. Dry matter and crude protein digestibility

 

in three ponies is presented in Table 15. Oat intake was kept con-
stant but hay intake was monitored. The amount of dry matter digest- }
ed was highest in Elvis and lowest in Snippet. The apparent dry mat— Ci
ter digestibility was highest in Elvis with 68.1% digested and lowest '
in Chester with 54.8% digested. Crude protein digestibility was
similar to dry matter digestibility in all three ponies.
The fiber composition of the diets fed to the ponies and feces
are presented in Table 16. Hay, oats and feces contained higher AOF
when compared to the corresponding NDF. Large differences were ob-
served in the hemicellulose content of all samples between the de-
tergent and MSU T.F.A.A. fiber methods. Detergent hemicellulose was
24.26% of the oat dry matter whereas hemicellulose as measured by GLC
was 5.36%. This hemicellulose consisted of arabinose, xylose, gal—
actose , glucose and traces of mannose. GLC examination of the fe-
ces showed similar concentrations of glucose and galactose, but the
concentration of xylose was higher in Elvis' feces when compared to
the other two ponies. No arabinose or mannose was found in the fe-
ces. Lignin, as determined by oxidation in sodium chlorite, was sig—
nificantly higher (P<.05) than all permanganate lignin values.

The digestibility of each fiber fraction in each pony is pre-

76

 

     
 

.. . . . .
~‘ 5 , f': 1.

\'- ‘ ' ”">\n'\- |

’ t . 0..

. '1 '.. .e .
. 2.1.; "1-1. -h v." «at».

 

 

FIGURE 7. TRANSMISSION ELECTRON PHOTOMICROGRAPH OF THE EFFECT OF
ACID DETERGENT ON WHEAT STRAW.
A. Hemicellulose disruption clearly evident (arrows): TEM X40,000.
B. Hemicellulose residue (arrows): TEM X60,000.

77

 

 

 

 

 

_m W g B
FIGURE 8. EFFECT OF SODIUM CHLORITE AND TRIFLUOROACETIC ACID ON
WHEAT STRAW.
A.

Section treated with sodium chlorite. Cell wall very much intact
X10,000. B. Section treated with trifluoroacetic acid. Cellulose
sheets clearly evident (arrows): X1000.

78

TABLEIIL. APPARENT DRY MATTER AND CRUDE PROTEIN DIGESTIBILITY IN

 

 

 

 

THE PONY
Animal

Item Chester Snipper Elvis
Dry matter

Hay intake, g/day 1809.2 1330.6 1384.9

Oat intake, g/day 837.7 840.4 837.7

Total intake, g/day 2646.9 2171;0 2222.6

Dry feces, g/day 1196.0 908.0 708.1

Digested, g/day 1450.9 1263.0 1514.5
Apparent digestibility, % 54.8 58.2 68.1
Crude protein

Hay intake, g/day 201.5 145.6 154.3

Oat intake, g/day 149.6 150.1 149.6

Total intake, g/day 351.1 295.7 303.9

Dry feces, g/day 160.0 106.9 95.1

Digested, g/day 191.1 188.8 206.8
Apparent digestibility, % 54.4 63.9 68.7

 

79

 

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80

sented in Table 17. Digestibility of AOF, NDF and ADF was higher

in Elvis than Chester and Snipper. Digestibility of detergent hemi—
cellulose was 52.7%, 62.8% and 71.5% whereas the digestibility of
T.F.A.A. hemicellulose was 72.0%, 73.6% and 56.7%, for Chester, Snip-
per and Elvis, respectively. Detergent and T.F.A.A. cellulose di—
gestibilities were very similar with the digestibility by Elvis the
highest in both cases. Lignin, a phenylpropane polymer, appeared to
be digested by Elvis using either potassium permanganate or sodium
chlorite to determine lignin. Arabinose digestibility was similar

in Chester and Snipper, but lower in Elvis with 78.6%. Similarly,
xylose digestibility was lower in Elvis than in Chester and Snipper.
Both galactose and glucose digestibility were higher in Elvis with
66.9% and 75.9% digested, respectively. All mannose was digested by
each pony. The digestibility of both holocelluloses were similar

in Chester and Snipper, however, detergent and T.F.A.A. holocellulose
digestibilities of Elvis were 64.3% and 51.8%, respectively. Digest-
ibility of total fiber as determined withchtergent or T.F.A.A. were
similar in Chester and Snipper, but Elvis digested 58.8% and 52.5%

of the detergent and T.F.A.A. holocellulose, respectively. Of all the
digestibility coefficients, there Was a strong significant correlation
(P<.001) between NDF and AOF, AOF and DM and NDF and DM (Table A4).

Beef cattle digestion trials. Within each trial, intake, fecal

 

and digestion of dry matter and crude protein were similar between
animals as indicated by similar digestibilities within trials (Table
18). Dry matter digestibility was higher in Trial I as was crude

protein digestibility when compared to the animals in Trial II.

81

TABLE IJC APPARENT DIGESTIBILITY OF FIBER FRACTIONS IN THE PONY.a

 

 

 

 

Animal
Fraction Chester Snipper Elvis
% % %

Ammonium oxalate fiber 41. 43.4 57.
Neutral detergent fiber 41.1 43.0 60.2
Acid detergent fiber 30.2 22.9 48.8
Detergent hemicellulose 52.7 62.8 71.5
Detergent cellulose 40.9 1 40.3 55.5
T.F.A.A. hemicellulose 72.0 73.6 56.7
T.F.A.A. cellulose 35.0 36.5 49.1
Permanganate lignin -16.6 -6.4 28.7
Chlorite lignin 3.6 0.0 18.3
Hemicellulosic sugars

Arabinose 100.0 100.0 78.6

Xylose 85.9 85.6 34.3

Mannose 100.0 100.0 100.0

Galactose 50.0 55.5 66.9

Glucose 64.8 66.2 75.9
Detergent holocellulose 47.2 52.8 64.3
T.F.A.A. holocellulose 49.3 49.6 51.8
Detergent total fiber 42.3 47.8 58.8
T.F.A.A. total fiber 44.4 45.5 52.5

 

aDiet fed to the ponies.

82

TABLE 18. APPARENT DRY MATTER AND CRUDE PROTEIN DIGESTIBILITY IN BEEF
CATTLE DIGESTION TRIALS.

 

 

Trial I Trial II
Item Steer #153 Steer #187 Steer #528 Steer #992

 

Dry matter

Intake, kg/day 8.28 7.49 4 68 4.49
Feces, kg/day 1.80 1.72 1.47 1.25
Digested, kg/day 6.48 5.77 3.21 3.24
Digestibility, % 78.3 77.0 68.6 72.2
Crude protein
Intake, kg/day 1.12 1.01 0.44 0.42
Feces, kg/day 0.28 0 27 0.19 0.18
Digested, kg/day 0.84 0 74 0.25 0.26
Digestibility, % 75.0 73 3 55.3 56.5

 

83

The fiber composition of feed and feces is presented in Table
19. The feed in Trial I was higher in each fiber component than the
feed in Trial II. NDF was lower than AOF in both feeds and feces as
was the feed T.F.A.A. hemicellulose when compared to feed detergent
hemicellulose. Detergent cellulose was significantly (P<.05) high-
er than T.F.A.A. cellulose in the two feeds, but not in the feces.
Detergent holocellulose and total fiber were higher than the corre-
sponding T.F.A.A. holocellulose and total fiber. The feed in Trial
I had higher arabinose, xylose and galactose than the feed in Trial
II. This corresponded with an increase in arabinose, xylose and
galactose in all fecal samples.

The apparent digestibility of each fiber component was higher
in Trial I than in Trial II with the exception of glucose digest-
ibility which was higher in Trial II (Table 20). AOF and NDF, de-
tergent and T.F.A.A. hemicellulose and detergent and T.F.A.A. di-
gestibilities were similar and each pair were correlated signifi-
cantly (P<.05) as shown in Table A4. Mbre arabinose, xylose and
galactose were digested in Trial I than Trial II. Glucose digest-
ibilities in the two trials were very similar. T.F.A.A. holocell-
ulose and total fiber digestibilities were lower than their corre-
sponding detergent values in both trials.

Feces samples of the animals in Trial I were examined by scan-
ning electron microscopy. Feces were washed and dried at 60 C and
refluxed in neutral and acid detergent and ammonium oxalate. Dried
samples contained debris with some bacteria evident (Figure 9A).

Washing the feces before drying appeared to rid the sample of bac—

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84

 

 

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85

TABLE 20. APPARENT DIGESTIBILITY OF FIBER FRACTIONS IN BEEF CATTLE.

 

 

Trial I Trial II

 

Item Steer #153 Steer #187 Steer #528 Steer #992

 

 

 

- Digestibility, %——

Ammonium oxalate fiber 72.5 72.8 56.3 61.3
Neutral detergent fiber 72.7 72.4 58.3 64.8
Acid detergent fiber 70.5 72.2 51.6 59.4
Detergent hemicellulose 75.1 72.6 65.5 70.1
Detergent cellulose 74.8 77.1 63.3 70.6
T.F.A.A. hemicellulose 74.2 72.7 60.3 65.2
T.F.A.A. cellulose 73.5 73.7 60.9 67.0
Permanganate lignin 67.5 65.3 11.9 16.1
Chlorite lignin 66.7 67.6 46.0 49.2
Hemicellulosic sugars
Arabinose 74.3 77.1 53.3 46.7
Xylose 63.8 61.8 33.2 41.5
Galactose 73.1 66.2 50.0 59.1
Glucose 93.4 94.8 94.4 96.1
Detergent holocellulose 75.0 ‘74.8 64.4 70.3
T.F.A.A. holocellulose 73.8 73.3 60.6 66.4
Detergent total fiber 74.1 73.7 60.9 66.7

T.F.A.A. total fiber 72.1 71.8 56.7 61.8

 

86

 

 

 

FIGURE 9. EFFECT OF WASHING AND DRYING ON FIBER OF BEEF CATTLE FECES.
A. Dried sample (60 C). Bacteria evident in some debris (arrows):
X7000. B. Washed and dried sample (60 C). Absence of bacteria:
X5000.

87

teria (Figure 9B). Neutral detergent fiber from feces was completely
absent of bacteria (Figure 10A). Feces boiled in acid detergent were
absent of bacteria but were fragmented and disrupted (Figure 10B).
Ammonium oxalate treated fecal samples were free of most debris and
bacteria, but not completely (Figure 11A and 11B).

Swine digestion trials. Dry matter and crude protein intake was
identical in starting pigs (Table 21). The amount digested of either
crude protein or dry matter was similar between pigs and is reflected
in similar digestibilities. Dry matter digestibility ranged from
91.0% to 91.8% whereas crude protein digestibility ranged from 89.8%
to 92.9%.

Fiber analysis of feed and feces are presented in Table 22. AOF
was significantly (P<.05) higher than NDF in all samples. Fecal AOF
values ranged from 45.47% in pig 119-13 to 58.86% in pig 119-1. Fe—
cal NDF values were highest in pig 119-13 with 40.21% and lowest in
pig 119-1 with 35.09%. Detergent hemicellulose values for starting
pig feces ranged from 19.34% to 25.63%. T.F.A.A. hemicellulose were
much lower in comparison and ranged from 6.68% to 7.96%. Detergent
cellulose values were very similar between pigs with the exception of
pig 119-10. Values for detergent cellulose ranged from 10.31% to
11.73%. Cellulose as determined with T.F.A.A. was significantly
higher (P<.05) than corresponding detergent cellulose values. Chlor-
ite lignin values were higher (P<.05) than permanganate lignin values
in all samples and were more variable. They ranged from 5.40% to
12.70% whereas permanganate lignin in fecal samples ranged from 1.83%

to 2.55%. Glucose was the highest hemicellulosic sugar in the feed

88

 

If B
FIGURE 10. EFFECT OF NEUTRAL AND ACID DETERGENT TREATMENT ON FIBER
OF BEEF CATTLE FECES.
A. Section treated with neutral detergent. Cell wall free of bact-
eria: X700. B. Section treated with acid detergent. Cell wall
fragmented: X2700.

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B

 

CATTLE FECES.
A. Cell wall smooth with some debris: X2700. B. Cell wall smooth
with bacteria attached (arrows): X2700.

90

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91

TABLE 22. APPARENT DRY MATTER AND CRUDE PROTEIN DIGESTIBILITY IN
STARTER PIGS.

 

 

 

 

Animal #

Item 119-10 119-1 119-13 Y6—1
Dry matter

Intake, g/day 366 366 366 366

Feces, g/day 30 33 31 32

Digested, g/day 336 333 335 334

Digestibility, 7 91.8 91.0 91.5 91.3
Crude protein

Intake, g/day 71.2 71 2 71.2 71.2

Feces, g/day 6.8 5 1 6.4 7.3

Digested, g/day 64.4 65 1 64.8 63.9

Digestibility, % 90.5 92 9 90.9 89.8

 

92

whereas xylose was the highest hemicellulosic sugar in three of four
fecal samples. Galactose and glucose content of all feces were very
similar, however, galactose and glucose were much higher in pig 119-1
than in the other fecal samples. Mannose was found in trace amounts
in feed samples, but was not found in the feces. Detergent holocell-
ulose values for all samples were greater than corresponding T.F.A.A.
holocellulose values. Detergent total fiber was also greater than
T.F.A.A. total fiber in feces, but not in the feed.

The digestibilities of each fiber fraction in starting pigs are
presented in Table 23. The digestibility of AOF ranged from 82.6%
to 87.4% with an average of 85.1% among all four pigs. NDF digesti-
bility was lower than AOF digestibility and ranged from 71.8% to 74.4%
with an average of 72.6%. Acid detergent fiber digestibilities were
lower than corresponding NDF values and was highest in pig 119-10.
NDF digestibility was correlated with both AOF digestibility (P<.05)
and ADF digestibility (P<.001) as shown in Table A4. Detergent hemi-
cellulose digestibility ranged from 73.0% in pig 119-13 to 78.3% in
pig 119-1. T.F.A.A. hemicellulose digestibility ranged from 74.9% in
pig Y6-1 to 79.1% in pig 119-13. The average hemicellulose digestibil-
ity was 75.2% and 77.1% for the detergent and MSU T.F.A.A. methods,
respectively. Cellulose digestibility ranged from 67.9% to 74.3%
and from 63.8% to 73.5% for the detergent and MSU T.F.A.A. methods,
respectively. Cellulose digestibility as determined with detergent
and T.F.A.A. for the four pigs was 70.5% and 70.5%, respectively.
Chlorite lignin digestibilities was higher in each pig than were the

corresponding digestibilities of permanganate lignin. Mannose was

 

TABLE 23. APPARENT DIGESTIBILITY OF FIBER FRACTIONS IN STARTER PIGS.

93

 

 

 

 

 

 

Animal #
Item 119-10 119-1 119-13 Y6—1
Digestibility %

Ammonium oxalate fiber 84.4 82.6 87.4 85.9
Neutral detergent fiber 74.4 71.8 72.0 72.3
Acid detergent fiber 72.6 65.3 69.8 68.9
Detergent hemicellulose 75.3 78.3 73.0 74.0
Detergent cellulose 74.3 67.9 70.4 69.4
T.F.A.A. hemicellulose 78.0 76.4 79.1 74.9
T.F.A.A. cellulose 71.7 63.8 73.5 73.1
Permanganate lignin 68.4 57.9 70.0 56.8
Chlorite lignin 81.6 90.3 91.9 84.9
Hemicellulosic sugars

Arabinose 52.3 80.4 40.2 31.5

Xylose 32.2 71.6 36.1 19.0

Mannose 100.0 100.0 100.0 100.0

Galactose 97.7 36.7 87.8 84.2

Glucose 94.2 86.6 96.7 84.2
Detergent holocellulose 75.0 75.3 72.2 72.7
T.F.A.A. holocellulose 74.3 68.9 76.0 73.8
Detergent total fiber 74.7 74.5 72.1 73.3
T.F.A.A. toral fiber 71.1 70.6 69.0 69.1

 

 

94

found in trace amounts in the feed and none in the feces so its
apparent digestibility was 100.0% in all four pigs. In three of

the four pigs, galactose and glucose were highly digestible, but in
pig 119-1, galactose digestibility was depressed and arabinose

and xylose digestibilities were increased. Xylose digestibility was
lowest in the other three pigs. Total fiber digestibility was 73.8%
and 73.3% as determined by detergent and T.F.A.A., respectively.
Detergent total fiber digestibilities were greater than T.F.A.A. to—
tal fiber digestibilities. Total fiber digestion for all four pigs
was 73.7% and 69.9% for detergent and T.F.A.A., respectively.

Fiber component composition of feed and feces of growing pigs
are presented in Table 24. AOF values for feed and feces are great-
er than corresponding NDF values. Pen I AOF, ADF, detergent cell—
ulose, T.F.A.A. cellulose and chlorite lignin values were higher
than Pen II values. Pen I NDF, detergent hemicellulose, T.F.A.A.
hemicellulose and permanganate lignin values were lower than Pen II
values. Feed samples are high in hemicellulosic glucose. Pen I
fecal samples had higher hemicellulosic glucose than any other sugar
whereas xylose is the highest sugar in Pen II. Detergent holocell-
ulose was higher than T.F.A.A. holocellulose in all samples. Pen II
detergent holocellulose was higher than Pen I, however, Pen I and Pen
II T.F.A.A. holocellulose values were similar. Detergent total fiber
was larger in Pen II, but T.F.A.A. total fiber was higher in Pen I.

Dry matter and crude protein intakes were higher for the growing
pigs in Pen II (Table 25). The pigs in Pen II were heavier (51 kg)

and consumed 2718 g/pig/day of feed compared to 1171 g/pig/day by the

95

TABLE 24. FIBER COMPONENT COMPOSITION OF FEED AND FECES IN GROWER

 

 

 

 

PIGS.a
Feces
Item Feed Pen I Pen II
% % %

Ammonium oxalate fiber 33.40 54.83 52.40
Neutral detergent fiber 15.70 35.90 41.64
Acid detergent fiber 4.41 13.21 12.66
Detergent hemicellulose 11.29 22.69 28.98
Detergent cellulose 3.02 9.70 8.81
T.F.A.A. hemicellulose 6.05 0.81 1.81
T.F.A.A. cellulose 2.46 9.29 8.33
Permanganate lignin 1.37 1.61 2.34
Chlorite lignin 3.90 20.67 13.69
Hemicellulosic sugars

Arabinose 0.16 0.00 0.23

Xylose 0.34 0.11 0.96

Mannose tr 0.00 0.00

Galactose 1.07 0.33 0.24

Glucose 4.48 0.37 0.38
Detergent holocellulos C 14.31 32.39 37.79
T.F.A.A. holocellulose 8.51 10.10 10.14
Detergent total fibere 15.68 34.00 40.13
T.F.A.A. total fiberf 12.41 30.77 23.83

 

a 0
Each value represents the average of three determinations.

b f

- For definition, see Table A7.

 

96

TABLE 25. APPARENT DRY MATTER AND CRUDE PROTEIN DIGESTIBILITY IN
GROWER PIGS.

 

 

 

Item Pen I Pen II
Dry matter
Intake, g/pig/day 1171 2718
Feces, g/pig/day 299 310
Digested,g/pig/day 1472 2408
Digestibility, % 82.6 88.6
Crude protein
Intake, g/pig/day 341.8 524.6
Feces, g/pig/day 72.8 71.2
Digested, g/pig/day 269.0 453.4

Digestibility, % 78.5 86.4

 

97

lighter (30 kg) pigs in Pen I. Dry matter and crude protein
digestibility was also higher in Pen II.

The digestibility of each fiber fraction is presented in
Table 26. The digestibility of AOF, NDF, ADF, detergent hemi-
cellulose, detergent cellulose, T.F.A.A. cellulose and chlorite
lignin were higher in Pen II when compared to Pen I. T.F.A.A.
hemicellulose digestibility and permanganate lignin digestibilities
were similar between pens. AOF digestibility was higher than NDF
digestibility in both pens. Detergent cellulose digestibility was
higher than cellulose digestibility as determined with T.F.A.A. in
both pens. T.F.A.A. hemicellulose was almost completely digested
in both pens with 97.7% and 96.6% for Pen I and II, respectively.

Of the hemicellulosic sugars, arabinose and mannose were completely
digested in Pen I. Digestibility decreased in the following order

in Pen I: glucose, galactose and xylose. Mannose was completely
digested by the pigs in Pen II, however, xylose and arabinose di-
gestibilities were depressed compared to Pen I. In total, holocellu—
lose and total fiber as determined with T.F.A.A. were more digestible
than similar detergent fractions in both pens.

Fiber component composition of feed and feces of finishing pigs
are presented in Table 27. Fecal NDF, ADF, detergent hemicellulose,
detergent cellulose and chlorite lignin were higher in Pen I than Fen
II. Fecal AOF, T.F.A.A. cellulose and permanganate lignin were simi-
lar in both pens. Fecal hemicellulose determined with T.F.A.A. was
higher in Pen II. Of the hemicellulosic sugars, xylose was in the

highest concentration in the feed. Only trace amounts of mannose

98

TABLE 26. APPARENT DIGESTIBILITY OF FIBER FRACTIONS IN GROWER PIGS.

 

 

Item Pen I Pen II

 

N

Ammonium oxalate fiber 72.3 82.1
Neutral detergent fiber 61.3 69.8
Acid detergent fiber 49.4 67.3
Detergent hemicellulose 43.4 70.7
Detergent cellulose 45.8 66.7
T.F.A.A. hemicellulose 97.7 96.6
T.F.A.A. cellulose 36.2 61.4
Permanganate lignin 80.2 80.5
Chlorite lignin 10.5 59.9
Hemicellulosic sugars
Arabinose 100.0 83.7
Xylose 94.5, 67.8
Mannose 100.0 100.0
Galactose 94.8 97.5
Glucose 98.6 99.0
Detergent holocellulose 44.2 69.8
T.F.A.A. holocellulose 80.0 86.4
Detergent total fiber 48.6 70.8
T.F.A.A. total fiber 54.8 78.1

 

TABLE 27. FIBER COMPONENT COMPOSITION OF FEED AND FECES IN FINISHER

99

 

 

 

 

PIGS.a
Feces
Item Feed 'Pen I Pen II
% % %

Ammonium oxalate fiber 23.03 66.92 66.71
Neutral detergent fiber 14.96 55.57 41.98
Acid detergent fiber 4.25 23.20 17.27
Detergent hemicellulose 10.71 32.37 24.71
Detergent cellulose 2.73 8.55 7.92
T.F.A.A. hemicellulose 3.77 6.45 8.94
T.F.A.A. cellulose 3.71 11.45 11.73
Permanganate lignin 1.48 2.48 2.65
Chlorite lignin 4.82 21.60 9.46
Hemicellulosic sugars

Arabinose 0.72 1.07 1.79

Xylose 1.40 3.28 4.09

Mannose tr 0.00 0.00

Galactose 0.59 1.36 ‘1.88

Glucose c 1.06 0.74 1.18
Detergent holocellulose 13.44 40.92 32.63
T.F.A.A. holocellulosed 7.48 17.90 20.67
Detergent total fibere 14.92 43.40 35.28
T.F.A.A. total fiberf 12.30 39.50 30.13

 

a 1 . .
Each value represents the average of three determinations.

b f

- For definition, see Table A7 .

100

were found. Xylose was also the highest hemicellulosic sugar in both
fecal samples. Holocellulose and total fiber as determined with de-
tergent were higher than similar fractions determined with T.F.A.A.
in all fractions.

Dry matter and crude protein intakes for finishing pigs are pre—
sented in Table 28. Pigs in Pen II consumed more than pigs in Pen
I. This was accompanied with an increase in feces and the amount
digested by the pigs in Pen II. Digestibilities of dry matter and
crude protein were similar between pens.

The digestibility of each fraction in finishing pigs is shown in
Table 29. The digestibilities of AOF, detergent cellulose, T.F.A.A.
cellulose and permanganate in Pen I were slightly higher than in Pen
II. NDF, ADF, detergent hemicellulose, T.F.A.A. hemicellulose and
chlorite lignin digestibilities were higher in Pen II pigs. 0f the
hemicellulosic sugars, mannose was completely digested. Individual
sugar digestibilities were always higher in Pen I but decreased in
the following order in both pens: mannose, glucose, arabinose, and
xylose and galactose. T.F.A.A. holocellulose differed from detergent
holocellulose in Pen I with 77.1% to 70.% digested, respectively, and
in Pen II with 69.4% to 73.1% digested, respectively. T.F.A.A. total
fiber digestibility was higher in Pen I but lower in Pen II when com-
pared to corresponding detergent total fiber digestibility.

Pure culture studies. Six substrates were chosen and inoculated

 

with either R. flavefaciens 094 or B. succinogenes 885. In all cases,

 

 

approximately 0.6 g was weighed into each flask. After the 48 hr fer-

mentation period, the substrates were recovered by differential centri-

101

TABLE 28. APPARENT DRY MATTER AND CRUDE PROTEIN DIGESTIBILITY IN
FINISHER PIGS.

 

 

Item Pen I Pen II

 

Dry matter

Intake, g/pig/day 2455 3181
Feces, g/pig/day 235 352
Digested, g/pig/day 2220 2829
Digestibility, % 88.9 90.4
Crude protein
Intake, g/pig/day 356.0 462.3
Feces, g/pig/day 54.4 73.2
Digested, g/pig/day 301.6 389.1

Digestibility, % 84.7 84.2

 

102

TABLE 29. APPARENT DIGESTIBILITY OF FIBER FRACTIONS IN FINISHER PIGS.

 

 

 

Item Pen I Pen II
% %

Ammmonium oxalate fiber 72.2 70.4
Neutral detergent fiber .64.4 69.0
Acid detergent fiber 47.8 55.0
Detergent hemicellulose 71.1 74.5
Detergent cellulose 70.0 67.8
T.F.A.A. hemicellulose 83.6 88.5
T.F.A.A. cellulose 70.5 65.0
Permanganate lignin 84.0 80.2
Chlorite lignin 57.1 78.3
Hemicellulosic sugars

Arabinose 85.8 74.6

Xylose 77.6 67.7

Mannose 100.0 100.0

Galactose 77.9 64.7

Glucose 93.3 87.7
Detergent holocellulose 70.9 73.1
T.F.A.A. holocellulose 77.1 69.4
Detergent total fiber 69.3 79.2

T.F.A.A. tOtal fiber 72.2 73.8

 

103

fugation. Recovery of substrates fermented by R. flavefaciens ranged

 

from 229 mg to 525 mg and by B. succinogenes ranged from 357 mg to

 

595 mg (Table 30). Corn silage was the preferred fermentation sub-

strate by both R. flavefaciens and B. succinogenes over the other

 

 

five substrates. Manure fiber was the least fermented by R.flavefac-

 

iens and filter paper was the least fermented by B. succinogenes.

 

The fiber components of the substrates before and after ferment-
ation are presented in Tables 31 and 32. Whatman filter paper con-
tained the most cellulose with 91.04% and wheat straw contained the
most hemicellulose with 22.33%. Of the hemicellulosic sugars, xylose
was the highest in four substrates. Glucose was the highest in al—
falfa and Whatman filter paper. No mannose was found in Kentucky
bluegrass, wheat straw and Whatman filter paper. Arabinose and gal-
actose were absent in Whatman filter paper and manure fiber, respec—
tively. Of the recovered substrates, filter paper had the most cell-
ulose with both bacteria. Wheat straw had the most hemicellulose a-

mong the substrates fermented by B. succinogenes whereas corn silage

 

had the most among the substrates fermented by R. flavefaciens. Of

 

the substrates fermented by R. flavefaciens, xylose was the highest

 

in Kentucky bluegrass, corn silage and manure fiber. Glucose was
the highest in wheat straw, alfalfa and filter paper. Of the hemi-
cellulosic sugars, xylose was the highest in all substrates fermented

by B. succinogenes. No mannose was recovered in any of the substrates.

 

The percent fermented of the six substrates by each bacteria are
presented in Table 33. Cellulose fermented ranged from a high of

45.2% in corn silage to a low of 21.5% with R. flavefaciens, but

 

104

 

 

 

 

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105

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106

 

 

 

 

 

 

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107

 

 

 

 

o.om N.m 0.00 m.0o «.08 o.om omoahx
1111 0.0 0.0 0.000 o.oo 0.8m omofianmum
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108

ranged from a high of 23.2% in manure fiber to a low of 5.1% 19.3;

succinogenes. In four of the six substrates, R. flavefaciens ferment-

 

 

ed more substrate hemicellulose.than B. succinogenes. Alfalfa hemi-

 

cellulose fermented was higher with B. succinogenes with 72.5% fer-

 

mented compared to R. flavefaciens with 40.9%. Filter paper hemi-

 

cellulose was more fermentable with B. succinogenes compared in R;

 

flavefaciens with 47.0% and 16.5% fermented, respectively. Ferment-

 

ation of hemicellulosic glucose ranged from 0.0% to 50.5% and 0.0%

to 75.4% with R. flavefaciens and B. succinogenes, respectively.

 

 

Galactose fermented ranged from 0.0% to 46.4% in R. flavefaciens com-

 

pared to 0.0% to 100.0% with B. succinogenes. Mannose was completely

 

fermented in all substrates that contained mannose. The fermentation

of arabinose in R. flavefaciens ranged from 0.0% to 100.0% and in

 

B. succinogenes from 0.0% to 100.0%. Fermentation of xylose by R;

 

flavefaciens was higher in all substrates with the exception of al-

 

falfa. Xylose fermented ranged from 14.7% to 97.5% with R. flavefac—

 

iens and ranged from 9.2% to 60.3% with B. succinogenes. Among

 

all substrates fermented by R. flavefaciens, arabinose and galactose

 

content of the cell wall was significantly (P<.05) correlated with
fermentability (Table A6).

Electron microscopy. R. flavefaciens and B. succinogenes attach-

 

 

 

ment to plant cell walls were examined with electron microscopy. _R;

flavefaciens adhered on the surface of wheat straw near exudated de-

 

bris (Figure 12A). Chains of bacteria were found on the surface with
exudated debris (figure 12B). Adherence of R. flavefaciens on alfalfa

appeared near the ends of fibers and surface (Figure 13A). Debris was

109

 

 

FIGURE 12. ATTACHMENT OF RUMINOCOCCUS FLAVEFACIENS C94 ON WHEAT
STRAW.
A. Surface attachments frequently near exudated debris: X10,000.
B. Chains on surface with exudated debris: X10,000.

110

 

 

_._V B

FIGURE 13. ATTACHMENT OF RUMINOCOCCUS FLAVEFACIENS C94 ON ALFALFA.
A. Attachment of bacteria near ends of fibers: X10,000. B. Sur-
face attached with very little debris. Particles are present
(arrows): £10,000.

 

 

111

scarce on alfalfa compared to wheat straw, but particles are present
(Figure 133). Stringlike particles were observed with adherence

of R. flavefaciens to corn silage (Figures 14A and 14B). R. flave-

 

faciens was not found to adhere to filter paper and cattle fiber.

B. succinogenes adhered near the rough surface of alfalfa (Fi-

 

gures 15A and 153). The bacteria mostly attached parallel to the

cell wall (Figure 15A). Adherence of B. succinogenes to filter paper

 

‘was predominately on the surface (Figure 16A) whereas the bacteria
attached to Kentucky bluegrass primarily at the end of the fibers (Fi-

gure 16B). B. succinogenes did not adhere to cattle fiber and wheat

 

straw.

Soluble carbohydrates in corn silag_. The changes in pH, dry

 

matter and soluble carbohydrates in corn silage over time are present-
ed in Table 34. PH decreased from 5.5 to 3.6 at the end of 312 hr.
Dry matter fluctuated between silos and showed no specific change over
time. Xylose increased from 0.06% at 0 hr to 0.29% at 24 hr and then
decreased to 0.04% by 96 hr. Mannose fluctuated between silos, but
was highest at 72 hr and lowest at 312 hr. Glucose appeared to in—
crease with time from 4.71% at 0 hr to 11.37% at 24 hr and then de-
creased to 0.00% at 144 hr. Total sugars also increased and then
decreased with 6.58% at 0 hr to 15.31% at 36 hr and then to 0.71% at

312 hr.

 

1
1

FIGURE 14.

 

112

B

 

ATTACHMENT OF RUMINOCOCCUS FLAVEFACIENS C94 0N CORN SILAGE.
A. Surface attachment upon stringlike particle: X10,000. B. Bac-
teria appeared anchored to stringlike particle: X10,000.

 

113

 

 

 

1 , _,,_,, ,,,__

FIGURE 15. ATTACHMENT 0F BACTEROIDES SUCCINOGENES 385 ON ALFALFA.
A. Mostly parallel attachment evident with exception of one bac—
teria near perpendicular attachment: X8,000. B. Attachment
near rough surface: X8,000.

 

114

 

 

 

B

FIGURE 16. ATTACHMENT OF BACTEROIDES SUCCINOGENES 885 ON WHATMAN FIL-
TER PAPER AND KENTUCKY BLUEGRASS.
A. Whatman filter paper no. 1. Attachment was predominately on sur—
face: X8,900. B. Kentucky bluegrass. Attachment at end of fibers:
X8,900.

 

115

 

 

 

TABLE 34. SOLUBLE SUGARS, PH AND DRY MATTER OF CORN SILAGE OVER TIME.a
Time pH D.M. Xylose Mannose Glucose Total
nr % % % % %

0 5.5 3.7 0.06 1.81 4.71 6.58
6 5.6 36.2 0.17 3.2 5.30 8.67
12 5.6 35.3 0.21 3.70 8.64 12.55
18 5.2 33.3 0.28 1.44 8.28 10.00
24 4.8 33.9 0.29 2.64 11.37 14.30
36 4.3 35.3 0.15 4.32 10.84 15.31
48 3.9 32.2 0.06 4.72 4.46 9.24
72 3.7 32.7 0.05 8.10 2.79 10.94
96 3.7 32.8 0.04 4.62 2.21 6.87
144 3.8 33.9 0.04 1.25 0.00 1.29
312 3.6 34.1 0.04 0.67 0.00 0.71

 

a- . . .
Eacn value is expressed as a percent of the freeze-dried sample.

 

 

DISCUSSION

Fiber analyses. The study of the plant cell is of great import-

 

ance and has long been the object of extensive research in nutrition.
Various authors have expressed the need for definitive nomenclature
in order to better interpret the metabolic role of fiber in nutrition
(177, 186).

Typical values for fibrous components in the 20 feeds and forages
as determined by neutral detergent (NDF), acid detergent (ADF), po-
tassium permanganate or detergent cellulose, detergent hemicellulose
and permanganate lignin were presented in Table 3. These methods
are relatively easy to perform and the values obtained describe the
general physical structure of the various feeds and forages. Neutral
detergent fiber represents the plant cell walls minus pectins (17),
some insoluble ash (203) and possibly some arabinose (19). Acid de-
tergent fiber includes the cellulose, lignin and insoluble ash (82).
ADF has been shown to contain some hemicellulosic sugars (17,106).
The difference between the two detergent fiber fractions is an esti-
mate of hemicellulose which can be either overestimated or underesti—
mated depending on the substrate (203). Oxidation of lignin in the
acid detergent fiber with potassium permanganate and ashing at 493 C
will yield values for lignin, cellulose and insoluble ash.

Studies have been conducted to examine the variability of the

116

117

detergent method between laboratories (202). Results indicated pro-
blems with filtration, uniform weighing procedure, and reliable pre-
paration of asbestos used in the preparation of lignin. The results
in Table 3 were similar to many previous reports (175, 178, 179, 195,
201, 208) with some exceptions. The values reported for cellulose

and hemicellulose for orchardgrass were higher and lower, respectively,
than values reported by Van Soest (202, 206, 208). Red clover NDF

has been reported to be as high as 66% (202) compared to 26.62% in the
sample in Table 3. Ladino clover values were similar to previous
reports (202, 206, 208). The value for alfalfa NDF in Table 3 was
similar to the values reported by Van Soest (199) and Van Soest and
Mertens (209), but they reported 17.9% lignin—cutin in their alfalfa
samples. They also reported a value of 49% NDF in wheat straw com-
pared to our higher value of 79.84%. Grasses have also been shown

to accumulate silica as did the samples in this study, especially
quackgrass (206). In general, most of the values for the detergent
fiber fractions reported in Table 3 were similar to previous reports.
Part of the differences in values could be attributed to chemical
technique, species and variety of the plant examined, maturity of

the plant examined and sieve size of the ground sample.

Determination of fiber in feeds and forages involves the dis-
ruption of the plant cell walls to rid the plant of cytoplasm by
digestion with neutral detergent (82) or with ammonium oxalate (41,
167, 213, 214). Upon comparison of the neutral detergent fiber
(Table 3) and ammonium oxalate fiber values (Table 4) for the 20

feeds and forages, all NDF values were significantly higher (P<.05)

118

than their corresponding NDF values. A significant (P<.001) relat-
ionship between AOF and NDF in substrates was observed (Table A1).
Marked differences were observed in ladino clover and red clover
values. These differences in cell wall values for the substrates
may have been due to losses in pectins and uronic acids (17), insolu-
ble ash (38,204), possible losses of arabinose (19) or might repre-
sent differences in protein and ash content.

After delignification of the cell wall with sodium chlorite (41),
each substrate was hydrolyzed in trifluoroacetic acid (T.F.A.A.). The
percent cellulose in each substrate was determined after collecting
the residue from T.F.A.A. hydrolysis. A comparison of detergent cell-
ulose (Table 3) and T.F.A.A. cellulose (Table 4) yielded similar re-
sults in most substrates except Kentucky bluegrass, Reed canarygrass-
2nd cutting, Reed foxtail, quackgrass, wheat middlings, wheat straw,
algae and crown vetch which have significantly (P<.05) higher cellu—
lose values as determined by T.F.A.A. hydrolysis. These differences
could be due to the possible action of potassium permanganate on
cellulose (66). Sullivan (178) compared 'natural' cellulose (116)
and 'true' cellulose (135) and found similar differences in Kentucky
bluegrass, orchardgrass and alfalfa.

A comparison of permanganate lignin and sodium chlorite lignin
has shown that in general sodium chlorite lignin determinations tend-
ed to be higher in value (41). The differences between the two lignin
values in part may be due to the varied complexity of the structural
components of the different forages and feeds, the nature of the fi—

ber residue after acid detergent or ammonium oxalate treatment, and

119

the mechanism of attack of the two oxidizing agents. Talmadge 33:31.
(182) has described the complexity of the carbohydrate structure in the
hemicellulose sugars. Lignin to carbohydrate bonding also has been
investigated extensively (129, 131, 132, 221). Recently, it has been
shown that the primary cell wall of wood contains bonds between lignin
and hydroxyproline (222). These physical and chemical carbohydrate
interactions are expected to vary among forages and feeds and will con-
tribute to variations with a standardized analytical procedure. The
starting fiber residue, either acid detergent or ammonium oxalate,

also should have considerable influence on lignin. Extraction with
acid detergent will remove hemicellulose leaving behind a fraction con-
taining lignocellulose and insoluble ash (195, 196, 211). The treat-
ment also may result in the loss of lignin-hemicellulose complexes
although this effect has not been examined. Extraction with ammonium
oxalate is a milder process and removes cell wall cytoplasm and some
uronic acids. This leaves the holocellulose fraction virtually in-
tact. Lastly, the mechanism of attack by chlorite and permanganate

on lignin may be different, especially in regards to the starting res—
idue. Permanganate is a stronger oxidizing agent than sodium chlorite,
and its exact reaction on lignin is not known. It is believed also to
effect carbohydrate components (66). Sodium chlorite oxidation of
lignin, although not fully elucidated, has been examined more thorough-
ly (20, 57, 159) and has been used for many years in botany studies
(28, 31). These results may be significant in balance studies for for-
age quality and passage studies with lignin as an internal marker. In-

determinate values for lignin erroneously could suggest high lignin

II.

1.1 {1 41.1.1?! v.54

120

digestibility (39, 40) or apparent breakdown (92).

Hydrolysis of the delignified tissue with trifluoroacetic acid
(T.F.A.A.) and derivatization to the corresponding alditol acetate
yields a pattern of the hemicellulose sugars which include glucose,
galactose, mannose, xylose, arabinose, rhamnose and possibly ribose.
In these calculations, uronic acids, which may include those from
pectins, were included in the hemicellulose. Uronic acids are also
found as a component of the cell wall (32). Xylose, galactose and
glucose were apparent in all substrates examined (Table 5). The value
of wheat bran hemicellulose (Table 5) was higher than previous reports
(161), but much lower than detergent hemicellulose (Table 3). Other
techniques have been used to examine the structure of the plant cell
wall using paper chromatography (179), gas—liquid chromatography (28,
29, 154, 155, 169, 179, 171) and densitometry (22). Binger g£_al.
hydrolyzed the hemicellulose of orchardgrass and reported 15.6% xylose,
10.9% glucose, 4.1% arabinose, 2.4% galactose and 3.1% uronic acids.
This is substantially higher than the value for orchardgrass reported
in Table 5. Sullivan e£_al. (180) used a similar technique and found
similar distributions of hemicellulosic sugars in orchardgrass, Reed
canarygrass, tall fescue and Kentucky bluegrass as presented in Table
6. Orchardgrass hemicellulose was examined by colorimetry after ex-
traction in ethanol:benzene, ammonium oxalate, sodium chlorite, alkali,
and acid (213). Buchala and Wilkie (31, 32) investigated wheat straw
hemicellulose with gas chromatography and reported that as the plant
matures, xylose increased, and arabinose, uronic acids and glucose

decreased. The results for wheat straw (31, 32) and orchardgrass (213)

121

were similar to the hemicellulose values as determined by GLC pre-
sented in Table 5.

The xylosezarabinose ratio has been used as an indicator of
plant maturity, i.e., the ratio increases with age of the plant (31,
31). The similarity in xylose and arabinose content and in the
xylosezarabinose ratio in grasses indicates possible structural sim-
ilarities not apparent in other substrates. The hemicellulosic sugar
composition of three hemicellulose fractions (linear A, linear B and
branched B) were examined in ryegrass and red clover. These studies
enhanced the understanding of in vitro rumen digestibilities of the
same substrates by examining the cell wall more closely (80).

Several investigators have indicated possible losses and compli—
cations in the detergent fiber system (17,19,106,130,152,204). Ana-
lysis of the NDF and ADF fractions of 20 feeds and forages showed
that these fractions were not as exact as was first suggested (200).
55.2% of the grass hemicellulose and 26.5% of the legume hemicellulose
was recovered in the neutral detergent fiber (Table 10). The grass
ADF fractions contained 10.8% to 31.5% of the hemicellulose whereas
the legume ADF fractions contained 7.1% to 50.8% of the original hemi-
cellulose (Table 11). Kim e£_al. (106) compared ADF and CF fractions
in feeds and feces and found 12% to 16% of the original pentosans in
the feed ADF and 9.7% in fecal ADF. ADF has also been criticized for
having more than cellulose and lignin and NDF has been criticized for
having the bulk of the pectic substances removed in clovers (17).
Clover ADF was reported to contain 15% uronic acids (17).

Scanning electron microscopy was used in order to examine the

122

effects of neutral detergent, acid detergent, ammonium oxalate, tri-
fluoroacetic acid and sodium chlorite treatment of cell walls of

wheat straw. Both neutral detergent and ammonium oxalate have been
described to rid the plant cell wall of cell cytoplasm and debris
(Figure 4B and 5A). Neutral detergent ( a solution of sodium lauryl
sulfate, disodium dihydrogen ethylenediaminetetraacetate dihydrate,
disodium hydrogen phosphate, sodium borate dechydrate and ethylene gly-
col) appeared to open the plant cells of wheat straw as was seen in
Figure 43. Small frequent disruptions(arrows) were seen in the cell
wall which may be due to the possible losses of uronic acids (17), in-
soluble ash (38) or arabinose (19). The cell wall of ammonium oxalate
treated wheat straw (Figure 5A and 5B) appeared to have opened cell
walls (arrows) with typical flaking of the cell wall, but no apparent
losses. Both neutral detergent and ammonium oxalate appeared to rid
the cell wall of debris.

The structure of hemicellulose in many plants has been examined
extensively (21,182) and illustrated as cross—linked chains of sugars
evenly distributed over a core of cellulose sheets. Acid detergent
has been reported to remove the hemicellulose leaving cellulose, lig—
nin and some ash. The chemical mode of attack as seen in Figure 6A and
6B appeared to be very localized (arrows) and may not remove all the
hemicellulose. Akin e£_§l. (9) reported acid detergent broke up cells,
removing only non—lignified tissues. They defined cellulose as a lin—
ear array of D-glucopyranose units linked by 8 1—4 glycosidic linkages
with a degree of polymerization of 300 to 2500 units. Cellulose with

a degree of polymerization less than 300 would be removed with meso—

123

phyll, phloem, bundle sheath and epidermal cell walls. They indi-
cated that the removal of large numbers of cell walls by treatment
with acid detergent is not consistent with the cellulose recovery
reported by Colburn and Evans (38). In further studies, Barton

and Akin (19) reported that ADF differed for representative grasses
from warm and cool climates. In bermudagrass, some digestible tis-
sues remained in the ADF. NDF was essentially the cell walls in an
undegraded form in both bermudagrass and tall fescue.

The action of sodium chlorite is to oxidize the lignin of a plant
cell wall without extracting carbohydrates from cellulose and hemi-
cellulose. Figure 8A shows the cell wall of sodium chlorite delig-
nified wheat straw. The cell wall showed no disruptions and appeared
to be evenly extracted. After trifluoroacetic acid hydrolysis, cellu-
lose is collected in a crucible, dried and weighed. Figure 8B
illustrates wheat straw T.F.A.A. cellulose with typical cellulose
sheets (arrows).

The use of detergent in analyzing for fiber in feeds and forages
offered quick, easy and repeatable methodology, however, possible
losses and complications have been described. The chemical action of
acid detergent appears to be unsatisfactory for yielding a fraction of
only cellulose as the major carbohydrate component, however, it does
provide a repeatable measure of fiber in animal rations. The alditol
acetate derivatization system for feeds and forages can be used to
analyze for cellulose, hemicellulose, hemicellulosic sugars and uronic
acids and lignin. It offers additional structural information about

the fiber components in the plant cell wall and compares favorably

124

with the detergent system (Table 7 and 8). Previous investigators
(28,29,31,32,118,170,171,223) have attempted to analyze plant cell
walls for individual components as determined by gas-liquid
chromatography. The preparation of the plant cell wall varied
between investigator, but each used sulfuric acid to hydrolyze the
hemicellulose to individual sugars. In addition to hydrolyzing
hemicellulose, sulfuric acid hydrolyzed cellulose to individual
glucose units, so an additional method was used to account for cellu-
lose in each sample. Meinert and Delmer (118) and Talmadge g£_al.
(182) reported that 97% of the cellulose was resistant to hydrolysis
in trifluoroacetic acid (T.F.A.A.). Our results showed that the cell-
ulose recovered after T.F.A.A. hydrolysis contained 97% glucose, no
ash and no protein. Morrison (129) used trifluoroacetic acid to hydro-
lyze plant cell walls, but the hydrolysis time (16 hr) used appears

to be too long according to Albersheim et al. (11) and our results as
reported in Figure 2. Lignin has been shown to be in close associa-
tion with cellulose, hemicellulose and cell wall protein (88,129,131,
132,221,222). Hydrolysis of the cell wall in sulfuric acid or in
T.F.A.A. before delignification has appeared unsatisfactory (170).
Many investigators have chosen to delignify with sodium chlorite (28,
29,31,32,72,223), although Barton (20) stated that lignin treated with
72% sulfuric acid dissolved readily. Delignification in permanganate
appears to be too harsh due to possible losses in cellulose (66), but
sodium chlorite appears mild enough not to attack plant cell wall
structural carbohydrates (3,4,5). The results presented in Table 5

indicated losses in cell wall carbohydrates with neutral detergent.

125

It then may be important to use a less harsh solution such as ammon-
ium oxalate to remove cell wall cytoplasm. Waite and Gorrod (213)
used ammonium oxalate and sodium chlorite to isolate plant cell walls
however continued their hemicellulosic analysis with a hydrolysis in
sulfuric acid for 2 hr. The use of sodium chlorite for the determin-
ation of lignin would be enhanced if the isolated cell walls were free
of protein. The data in Tables 12, 13 and 14 indicate loss in protein
after treatment with sodium chlorite. Waite and Gorrod (213) suggest-
ed a digestion in pepsin-hydrochloric acid which should be examined
further. Cell wall fractionation methods have involved many transfers
of the plant material (20,135,136,154,1554,171,213) without calcula-
tion of water absorption (Figure 1) by the plant cell wall. This may
be a further complication of some fractionation procedures. Fraction-
ation of plant cell walls using sodium chlorite for delignification
and GLC for sugar analysis appears to have advantages over previous
techniques in time (42,44), cost (Table A8) and overall additional
information about the plant cell wall. Some complications do exist,
however, with further modifications these can be overcome. The deter—
gent fiber system has been modified (165) to overcome filtration pro-
blems and should remain for quick product evaluation, for evaluation
of feeds and forages or for pilot nutritional studies.

Fiber digestibility trials. The digestion of fiber by many ani-

 

mals has been extensively studied. The development of fiber component
analyses for nutritional studies have depended upon the particular
organism being studied. Early studies used the crude fiber (CF)

method (91), but this eventually proved unsatisfactory (25,89,91,113,

126

123,140,149,150,185,224). Cellulose, a major constituent of the
plant cell wall, was next used to predict fiber digestibility (116).
Matrone §£_§l, (116) suggested that cellulose could be used to eval-
uate feedstuffs because of its close association with other polysac—
charides in the plant cell wall. This technique did not consider
the importance of hemicellulose and lignin to the digestibility of
fiber.

Walker and Hepburn (216) first proposed in a series of articles
the use of a fiber fraction they called normal-acid fiber (NAF).

This fraction represented cellulose, lignin, some protein and

some ash. They found NAF digestibility was always lower than the
digestibility of crude fiber (215). High correlations between the
percent NAF in forages with dry matter digestibility were reported
(98). This method failed to acknowledge the importance of hemicellu-
lose as a major constituent of fiber and had a variable composition
of protein and ash.

Van Soest and others (194,195,196,197,198,200,207,209,211) sug-
gested the use of detergents in the analysis of fiber. They proposed
that fiber should represent substances resistant to animal enzymes.
Total fiber was extracted in neutral detergent in a fraction called
neutral detergent fiber (NDF) or cell wall constituents (CWC). An-
other fraction representing lignocellulose called acid detergent
fiber (ADF) was isolated in acid detergent. This fraction represents
a residue which is closely associated with indigestibility (195,196).
Cellulose, lignin and insoluble ash (silica) are determined after

oxidation with potassium permanganate and ashing. Hemicellulose is

127

determined as the difference between the NDF and ADF. This fraction
is just an approximation, but has been used as a quantitative value
in the literature. Several problems exist with each fraction,

which have been mentioned earlier, that suggest that these methods
are not completely satisfactory in fiber evaluation studies. It

does provide quick, easy and repeatable methodology for digestion
trials, however, lignin digestibilities (18,39,40,52,68,71,101,178)
have been reported and the hemicellulose value does not help evaluate
the effects of this fraction upon digestibility.

Fiber as determined by ammonium oxalate significantly correlated
with neutral detergent fiber of all 20 substrates (Table A1). The
digestibility of AOF was also significantly (P<1005) correlated with
NDF digestibility in all species of animals tested (Table A4). Exa-
mination of feed and feces samples in the digestion trials showed that
AOF values were always higher than NDF values. This is probably due to
higher protein and ash content of AOF (Table 14). It may be advisable
to treat the AOF residue with pepsin—hydrochloric acid (73,213) to
decrease the protein content. This remains to be tested.

Large differences were observed in hemicellulose values in all
digestion trials. Hemicellulose as determined by detergent analyses
appeared too high in comparison to the corresponding T.F.A.A hemi-
cellulose value. It might be suggested that T.F.A.A. hemicellulose
underestimates hemicellulose due to improper hydrolysis, methylation,
acetylation, ammonium oxalate digestion or sodium chlorite oxidation.
Examination of these parameters suggest no substantial losses of hemi—

cellulosic sugars. Hydrolysis in trifluoroacetic acid longer than

60 min appears to cause breakdown of sugars (Figure 2) which agrees
with a previous: report (11). The collected residue contains 97%
glucose and appears to represent the cellulose of the cell wall
because very little glucose was found in the hemicellulosic sugars.
This cellulose value was, in general, similar to cellulose as deter—
mined by oxidation in potassium permanganate. Ammonium oxalate has
been used for cell wall cytoplasm extraction because of its mild
action. Uronic acids were not determined in the digestion trials
because of their reported high digestibility (214). Sodium chlorite
was also used because of its mild oxidation of the cell wall. Very
little of the hemicellulosic sugars are removed (19,31,32,57,159).
It appears that the higher hemicellulose values for the detergent
system are due to the suggested inacurracies stated above (8,17,38,
42,43,44,106) which need to be further elucidated. Detergent holo—
cellulose and total fiber, when calculated by summation of fiber
fractions, were always higher than corresponding MSU T.F.A.A. values.
This is largely due to the higher hemicellulose values determined by
these methods.

The digestibility of hemicellulose fractions did not correlate
in all species (Table A4), however, they did correlate in the beef
cattle and swine digestion trials. Hemicellulose digestibility, as
analyzed by GLC, also examines the digestibility of each individual
component. Arabinose digestibility was the highest of all sugars in
the ponies whereas glucose digestibility was highest in the beef cat-
tle. Glucose digestibility varied with age in pigs, but in general

was the highest. All sugars were readily digested by pigs, but in

129

the starter pigs, xylose and arabinose digestibility was lower.
Hemicellulose as a percent digestible versus actual amount digested
was highest in the pigs. This is in agreement with previous re-
search (103) in the examination of fiber digestibility of swine,
voles, sheep, horses and rats. Morris and Bacon (126) followed

the procedures of Sloneker (171) with sulfuric acid and reported
that the digestibilities of hemicellulosic sugars found in grass
decreased in the following order: arabinose, galactose, glucose
and xylose. This is similar to the digestibilities reported in
Table 20, although in corn silage, glucose was the most digesti-
ble. These differences could be due to cell wall structure, animals,
microflora or analysis technique.

Lignin content, as determined by either potassium permanganate
or sodium chlorite, were not satisfactory in digestion trials. Cal-
culated digestibilities for lignin were high in each trial which
suggests the lack of reliability of it use as a marker in passage
studies. This is in agreement with previous studies which have re-
ported high lignin digestibility (18,39,40,52,68,71,101,178). Ammon—
ium oxalate treated with pepsin-hydrochloric acid (213) may prove to
rid the error in chlorite lignin due to protein. Lignin as calculated
by methoxyl number does not appear satisfactory in dogs or cows (50).
Acetyl bromide may prove to be a better alternative to gravimetric
methods.

Fiber has been approached as: (1) the indigestible portion of
the dietary fiber (207), (2) the most indigestible fraction of the

plant cell wall (98,153,207,215,216), (3) the fractionated plant cell

130

wall (157) and (4) the soluble and insoluble complex polymers assoc-
iated with the plant cell wall (19,173,174). It appears unlikely that
any chemical procedure will yield a totally indigestible fraction

and still examine all fiber components. ADF and NAF were supposed to
be highly indigestible, but high digestibilities have been reported

in sheep (104), horses (104,105), swine (39,40,105), vole (103), rats
(105) and the digestion trials described above. Fiber has also

been reported as that which is resistant to gastrointestinal enzymes
(186), however, this ignores the possible utilization of fiber compon-
ents by intestinal bacteria (26,27,158,212). It therefore appears

to be adviseable to fractionate the plant cell wall into chemically
distinct components and examine each of these individually in digest-
ion trials and in vitro studies. Many distinct components could be
examined, but cellulose, hemicellulose, lignin and pectin are of major
importance. Fiber as determined with detergents could remain for
quality control or for pilot nutritional studies whereas GLC compon-
ents as determined with trifluoroacetic acid could be used to deter—
mine the effect of a particular fibrous substrate upon utilization of
nutrients or microbial fermentation in the gastrointestinal tract.

Pure culture. The utilization of fiber in the intestinal tract

 

of a monogastric animal and in the reticulo-rumen of ruminants has
been shown to involve a close association with cellulolytic bacteria
(6,7,26,27,97). The utilization of a particular substrate depends
upon plant cell wall structure (55,56,80). Coen and Dehority (37)
found that digestibility varied with maturity and type of plant and

bacterial species. They reported that B. succinogenes digested

 

131

greater amounts of hemicellulose when compared to R. flavefaciens.
R. flavefaciens digested more hemicellulose in four of the six sub—
strates when compared to B. succinogenes as shown in Table 33. Hemi-
cellulose digested B. succinogenes was greater in filter paper and
alfalfa. Coen and Dehority (37) determined hemicellulose as total
pentosans. In disagreement with their previous report, R. flave-
faciens digested 32.2% and 10.3% of bromegrass and alfalfa hemicell-
ulose whereas B. succinogenes digested 13.3% and 0.0%, respectively
(56). They also reported B. succinogenes was able to digest more
cellulose than eight other rumen bacteria. In disagreement with
these results, cellulose fermented as shown in Table 33 was greater
in R. flavefaciens than B. succinogenes. Manure fiber and filter pap—
er were least digested by R. flavefaciens and B. succinogenes, re-
spectively. Arabinose and galactose content of the cell wall was sig—
nificantly correlated (P<.05) with fermentability in R. flavefaciens
(Table A6). No relationship was found in B. succinogenes (45).
Electron microscopy studies indicated surface adherence of both
bacteria onto the plant cell walls. Two modes of bacterial attack
were visualized by Dinsdale EELEA) (58): (1) tunneling and (2) ero-
sion of surfaces. R. flavefaciens adhered on the surface of wheat
straw near exudated debris (Figure 12A). Chains of bacteria were
found on the surface with exudated debris (Figure 12B). Attachment
of R. flavefaciens on alfalfa appeared near the ends of fibers and
surface (Figure 13A). Stringlike particles were observed with attach—
ment of R. flavefaciens to corn silage (Figure 14B). R. flavefaciens

did not adhere onto filter paper and cattle fiber. B. succinogenes

132

adhered onto the surface of alfalfa (Figure 15A and 15B). Adherence

of B. succinogenes to filter paper was predominately on the surface

 

(Figure 16A) whereas the bacteria adhered to Kentucky bluegrass pri-

marily at the end of the fibers. B. succinogenes did not attack

 

cattle fiber and wheat straw.

Latham et al. (108,109) reported that B. succinogenes adhered to

 

only the cut edges of ryegrass and to intact mesophyll whereas 3;

flavefaciens adhered to cut edges of epidermal cell walls. They Sug-

 

gested that adhesion was similar, but there were different affinities
for each cell wall.

Leatherwood (110) proposed R. albus may have an affinity factor
which is necessary to hold the factor of cellulase in position to
the insoluble cellulose for multiple attacks to occur. Such a pheno-
nomenon may be required for hydrolysis of the walls more resistent to
bacterial degradation where attachment precedes degradation.

The results of the fermentation of R. flavefaciens suggest the

 

use of arabinose and galactose in preference to other hemicellulosic

sugars. It was observed that R. flavefaciens did not attach to filter

 

paper and cattle fiber, two substrates which we found to be low in
arabinose and galactose. It may also be possible that arabinose and

galactose facilitates the attachment of R. flavefaciens, leading to

 

the attack on the cell wall. This may be the affinity factor mention-
ed by Latham §£_§l, (108,109). The appearance of stringlike debris
near to attached bacteria appear similar to waxes seen by electron
microsc0py. Other possibilities include lectins such as in Rhizobia

(53) or by sugars as in E. coli (12, 142). All of these remain to

133

be proven in further studies.
GLC analyses of plant cell walls fermented in pure culture in-
dicate preference of hemicellulosic arabinose and galactose by_R;

flavefaciens. GLC analyses appears to enhance the study of the uti-

 

ization of fiber by rumen cellulolytic bacteria.

Corn silage analyses. GLC analyses of the freeze-dried soluble

 

sugars shown in Table 34 indicated solubilization of sugars up to

24 hr then utilization of these sugars for the remaining fermentation.
PH steadily decreased during fermentation. Variability between sam-
ples makes it difficult to discern trends however most of the sugars
are utilized by the end of the fermentation. GLC analyses of soluble
sugars can be an aid to understanding silage fermentation, but further

studies need to be designed to study this further.

CONCLUSIONS

Within the limits of the experimental conditions and pro-
cedures of the experiments presented herein, the results of this
study have led the auther to make the following conclusions:

1. Gas-liquid chromatographic (GLC) analyses of fiber com-
ponents of 20 feeds and forages was comparable to the fiber compon—
ent values determined by the detergent fiber system with the excep-
tion of hemicellulose.

2. GLC analyses of the detergent fiber fractions of the 20
feeds and forages indicated substantial losses of cell wall compon-
ents treated with neutral detergent and substantial hemicellulosic
sugars recovered in cell walls treated with acid detergent.

3. Lignin, as determined by gravimetric methods, appears to be
unreliable as a marker for digestibility or passage studies. Chlorite
lignin values may prove adequate if samples are first treated with
pepsin-hydrochloric acid. Sodium chlorite is adequate for delignifi—
cation of the cell wall for further structural analyses.

4. GLC analyses of fiber components in digestion studies with
beef cattle, pigs and ponies enhance the examination of fiber and
are comparable with detergent fiber digestibility values.

5. Direct fiber component analyses by GLC appears to be a use-

ful technique for determining the composition of fiber, differentiat-

134

135

ing the availability of fiber components, and characterizing the
specificity of the attacking bacterial species.

6. R. flavefaciens C94 may have a preference for cell walls

 

which contain arabinose and galactose.
7. Examination of freeze-dried soluble carbohydrates of corn
silage indicates solubilization of sugars up to 24 hr of fermentation

and utilization thereafter.

APPENDIX

136

TABLE A1. CORRELATION COEFFICIENTS OF FIBER FRACTIONS OF ALL

 

 

 

SUBSTRATES.
Comparison r P level

NDF x ADF 0.75 0.001
AOF x NDF 0.64 0.01
AOF x ADF 0.74 0.001
Detergent hemicellulose x

MSU hemicellulose 0.31 ns
Detergent cellulose x

MSU cellulose 0.87 0.001

 

TABLE A2. CORRELATION COEFFICIENTS OF MSU T.F.A.A. FIBER FRACTIONS OF
ALL SUBSTRATES.

 

 

 

Comparison r P level
Hemicellulose x glucose 0.58 0.01
Hemicellulose x arabinose 0.83 0.001
Hemicellulose x xylose 0.86 0.001
Glucose x arabinose 0.71 0.001
Rhamnose x uronic acids 0.64 0.01
Rhamnose x mannose 0.55 0.01
Xylose x arabinose 0.67 0.001

 

 

 

 

 

137

TABLE A3. CORRELATION COEFFICIENTS OF MSU T.F.A.A. FIBER FRACTIONS
IN GRASSES AND LEGUMES.

 

 

 

 

Grasses Legumes

Comparison r P level r P level
Hemicellulose x glucose 0.66 0.05 0.83 0.05
Hemicellulose x galactose 0.70 0.05 0.91 0.005
Hemicellulose x arabinose 0.30 ns 0.97 0.001
Hemicellulose x xylose 0.86 0.005 0.97 0.001
Galactose x glucose 0.95 0.001 0.54 ns
Galactose x arabinose -0.35 ns 0.97 0.001
Xylose x glucose 0.21 ns 0.80 0.05
Xylose x galactose 0.30 ns 0.92 0.005
Xylose x arabinose 0.50 ns 0.98 0.001

 

TABLE A4. CORRELATION COEFFICIENTS OF FIBER DIGESTIBILITY COEFFICIENTS.

 

 

 

Comparison Pony Pigs Cattle All species
**** * *** ***
NDF x AOF . 0.80*** 0.99*** 0.92***
NDF x ADF 0.93 0.92*** 0.99*** 0.96***
ADF x AOF 0.92 0.93 0.99 0.87
MSU hemicellulose x * **
Detergent hemicellulose -0.79**** 0.70 0.97*** 0.12***
AOF x DM 0.99**** 0.59** 0.99*** 0.92***
NDF x DM 0.99 0.89 0.99 0 86

 

* **
Interaction of variables significant: (P <.05), (P <.02),

*** ****
(P <.005) and (P< .001).

138

TABLE A5. CORRELATION COEFFICIENTS OF FIBER FRACTIONS FERMENTED IN
PURE CULTURE ANALYSIS.

 

 

 

 

Comparison R. flavefaciens, C-94 B. succinogenes, S—85
r P level r P level
Hemicellulose x arabinose 0.90 0.01 0.53 ns
Glucose x xylose 0.84 0.05 0.13 ns
Arabinose x xylose 0.40 ns 0.96 0.001

 

TABLE A6. CORRELATION COEFFICIENTS BETWEEN FIBER COMPONENTS IN SAMPLE
AND DIGESTIBILITY IN PURE CULTURE ANALYSIS.

 

 

 

Comparison R. flavefaciens, C-94 B. succinogenes, S-85
r P level r P level

 

Cellulose in x cellulose

dig. -0.64 ns -0.59 ns
Hemicellulose in x

hemicellulose dig. 0.90 0.01 -0.40 ns
Glucose in x glucose dig. —0.28 ns 0.32 ns
Arabinose in x arabinose

dig. 0.87 0.05 0.70 ns
Galactose in x galactose

dig. 0.76 0.05 0.52 ns

Xylose in x xylose dig. 0.34 ns 0.19 ns

 

139

TABLE A7. ABBREVIATIONS AND DEFINITIONS.

 

 

uGMwaH

23.
24.

 

ADF ...... 4 ............. Acid detergent fiber

AOF ................... Ammonium oxalate residue or fiber

B;_ .................. . Bacteroides

CF .................. . Crude fiber

CWC...... ...... . ...... Cell wall constituents

CWS ................... Cell wall solubles

Detergent

hemicellulose ......... NDF minus ADF

Detergent

holocellulose .......... Detergent hemicellulose plus detergent
cellulose

Detergent total fiber..Detergent holocellulos plus permanganate

 

lignin
GLC .......... ... ..... ..Gas-liquid chromatography
HPLC ................... High performance liquid chromatography
LCC .................... Lignin-carbohydrate complex
LHC .................... Lignin-hemicellulose complex
NAF .................... Normal acid fiber
NFE.......... .......... Nitrogen—free extract
PDB .................. ..Partially digestible biopolymers
PDPP ................... Partially digestible plant polymers
R; ..................... Ruminococcus
SEM .................... Scanning electron microscopy
TEM .................... Transmission electron microscopy
T.F.A.A .......... ......Trifluoroacetic acid
T.F.A.A. holocellulose.T.F.A.A. hemicellulose plus T.F.A.A.
cellulose

T.F.A.A. total fiber...T.F.A.A. holocellulose plus chlorite lignin
TMS ........ . ........... Trimethylsilyl

 

 

140

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VITA

GEORGE FREDERICK COLLINGS

1952 Born November 11, in Florence, Alabama

1970 Graduated from Summit High School, Summit
New Jersey

1970—1974 Attended Jacksonville University; Majored

. in Biology and Chemistry

1973 Elected into the International Oceanograph—
ic Foundation

1973 Elected into Beta Beta Beta, National Bio-
logical Honor Society

1974 B.A., Jacksonville University

1974—1976 Graduate work in Animal Science, Rutgers
University, New Brunswick, New Jersey

1974-1976 Graduate assistantship, Department of An-
imal Sciences

1976 M.S., Rutgers University

1976—1979 Graduate work in Animal Husbandry, Michigan

State University, East Lansing, Michigan

1976—1979 Graduate assistantship, Department of An-
imal Husbandry

1978 Graduate competition award (First place)
at Midwestern Animal Science Society meet—
ings, Carbondale, Illinois

1978 Married Laurie Lyn Crawford
1978 Candidate for PhD degree in Animal Husbandry
1978 Accepted job offer with Ralston Purina Com—

pany, St. Louis, Missouri

159

 

 

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