....‘._...-...-..--..‘ ‘I

THE RELATION OF CERTAIN END PRODUCTS
OF RUMEN FERMENTATION TO FORAGE
FEEDING VALUE

Thai: for {h Degree of Ph. D.
MICHIGAN sure umvmsm
Richard Warren Rice
1960

This is to certify that the

thesis entitled

The Relation of Certain End Products
of Rumen Fermentation to Forage

Feed i n$r¥§elfl€d by
Richard Warren Rice

has been accepted towards fulfillment
of the requirements for

PhD degree in_l_9;‘_0____

£2,490!on

/_I

‘ Major Blofessor

Date JUIY 8, l960

0-169

LIBRARY

Michigan State
University

 

THE RELATION OF CERTAIN END PRODUCTS
OF RUMEN FBRMENTATION TO
FORAGE FEEDING VALUE

by

Richard Warren Rice

AN ABSTRACT

Submitted to the School for.Advanced Graduate Studies
of Michigan State University of Agriculture and.App1ied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Husbandry
1960

Approved: 9: 4,54 raw/Ax/
V Z)

ABSTRACT

Oat straw and alfalfa-bromegrass hay were compared by
conventional total digestible nutrient determinations, by in
11352 volatile fatty acid production and cellulose digestibility.

The alfalfa-bromegrass hay was found to have an average
of only 2.4% more TDN than oat straw. Four Holstein cows volun-
tarily consumed an average of 23 pounds of alfalfa-bromegrass
hay per head daily; whereas, a similar group of 4 Holstein cows
would consume an average of only 14 pounds of oat straw per head
daily. On in zitrg rumen fermentation alfalfa hay produced
volatile fatty acids at a significantly (P<<.05) faster rate
than oat straw. The acetic to propionic acid ratio was (p.<305)
significantly lower for alfalfa-bromegrass hay for the first
four hours, but not significantly different for the balance of
the 24 hour in 11352 fermentation. The percent cellulose diges-
tion on in 21352 fermentation of alfalfa-bromegrass hay and oat
straw was not significantly different.

Differences in ratio and amount of volatile acids produced
were significant only when innocula for the fermentations were
Obtained from steers fed the same forage as that being used for
ig_zi££2_substrate.

The volatile fatty acid production (amount and ratio) and
cellulose digestion of holocellulose and cellulose prepared from
alfalfa-bromegrass hay and oat straw was not significantly dif-
ferent although early in the fermentation period cellulose di-

gestion and volatile fatty acid production tended to be greater

Richard W. Rice

.ABSTRACT (Continued)

‘when innoculum fermented familiar substrate. Holocellulose and
henicellulose produced a significantly (p<:.05) lower ratio of
acetic to propionic acid than whole forages or cellulose pre-
pared from whole forages. The digestibility of the cellulose
of holocellulose and cellulose prepared from the intact forage
‘was significantly (p<(.05) greater than the digestibility of
cellulose in the intact forage.

Although the in zitrg:method utilized was able to distin-
guish between alfalfa hay and oat straw with respect to quanti-
tative volatile fatty acid production the results on proportions
of volatile fatty acids produced are considered inconclusive.
The proportions of volatile fatty acids produced after 8 hours
or longer of in 31352 fermentation appear to be more character-
istic of the fermentation system than of the substrate being
fermented. A short term in 11153 fermentation of less than 8
hours appears to be necessary for a differentiation between

forages on the basis of proportions of volatile fatty acid.

Richard W. Rice

THE RELATION OF CERTAIN END PRODUCTS
OF RUMEN FERMENTATION TO
FORAGE FEEDING VALUE

by

Richard Warren Rice

A THESIS

Submitted to the School for Advanced Graduate Studies
of Michigan State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Husbandry

1960

Approved: 3¥C?X7JngM/~
—7VI RS

 

’
. {Z} 4‘ {V ..
g«7 if..- {4' 61/ C:

/- . ' .
-’ ‘ ,‘TQ, if" (/4:- 4'?

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation
to Dr. J. A. Hoefer, Dr. R. W. Luecke and Dr. R. L. Salsbury
for their aid, counsel and encouragement during the course
of this study.

Special thanks are due to Dr. R. W. Luecke and the De-
partment of Agricultural Chemistry for the use of facilities
and to Dr. R. L. Salsbury for help and advice when it was
needed most.

Gratitude is due to many others who have been helpful
in numerous ways during this project.

Special recognition is given to the author's wife, Joyce,

and his family for their patience, sacrifice and understanding.

Richard W. Rice
candidate for the degree of

Doctor of Philosophy

Dissertation: The Relation of Certain End Products of
Rumen Fermentation to Forage Feeding Value

Outline of Studies:

Major area: Animal Husbandry (Animal Nutrition).

Minor areas: Biochemistry, Physiology.
Biographical Items:

Born, August 10, 1931, Ainsworth, Nebraska.

Undergraduate Studies: University of Nebraska, 1949-1953.

Graduate Studies: University of Nebraska, 1955-1957
Michigan State University, 1957-1960.

Experience: Member, United States.Air Force, 1953-1955.
Graduate Assistant, University of Nebraska,
1955-1957, Graduate Assistant, Michigan State
University,,l957-l960.

Member: Sigma Xi
Alpha Gamma Sigma

TABLE OF CONTENTS

INTRODUCTION AND HYPOTHESIS
REVIEW OF LITERATURE

Forage Evaluation

The importance of energy . . . . . . . . . . . .
Deficiency of present systems of forage evaluation

£2_Vitro Fermentation

Application to feed evaluation . . .

Source of innoculum. . .

The Importance of the Rumen Fermentation

v

Extent of digestion in the rumen . . .
Importance of volatile fatty acids . .

The Relation of the Diet to the Volatile

The components of the diet . . . .

Components of plants and their relation to

Fatty Acids

VFA production . . . . . . . . . . . . . . .

Other Factors Influencing the Rumen Fermentation

Feed treatment . . . . .
The rate of fermentation
The effect of pH . . . .
Energy relationships . .
PART I. DIGESTION TRIAL

Procedure. . . . . . . . .
Results. 0 O O O O O O O 0

PART II.
BROMEGRASS HAY

Procedure
In Vitro fermentation . .

Cellulose determination .

Results

vzlatile fatty acid determinations.
Holocellulose determination .

Experimental design and reporting of dat

O
O
O
O
a

Volatile fatty acid production. . . . . .
The digestion of cellulose, holocellulose

hemicellulose o e e o e

O

and

THE FERMENTATION OF OAT STRAW.AND ALFALFA-

Page

13
16
17
21
25
27
3O

32
34

.36
37 ,
39
4o
41

43
47

Table of Contents (continued)
Page

PART III. THE FERMENTATION OF ALFALFA AND OAT STRAW
HOLOCELLULOSE, HEMICELLULOSE AND CELLULOSE

Procedure 0 O O O O O O O O O O O O O O O O O O O O O O 52
Results
The production of volatile fatty acids from the
holocellulose of alfalfa hay and oat straw. . . . . 52
‘Cellulose disappearance on the fermentation of
the holocellulose of alfalfa hay and oat straw. . . 55
The production of volatile fatty acids from the
cellulose of alfalfa hay and cat straw. . . . . . . 57
Cellulose disappearance on the fermentation of
the cellulose of alfalfa hay and oat straw. . . 59
The fermentation of hemicellulose. . . . . . . . . . 61

DISCUSSION

Cellulose Degradation of the Whole Plant and
Fractions of the Plant . . . . . . . . .

O O O O O O 64
The Importance of Cellulose to Volatile Fatty

ACid Formation O O O O O O C O O O O O O O O O O O O 65
The Rate of Voluntary Consumption and Fermentation. . . 71
The Ratio of Acetic to Propionic Acid. . . . . . . . .. 72
SourCeOfInnOCillum..o...........o... 73

SUMMARY 0 O O O O O O O O O 0 O O O O O O O O O O O O O O 7 8
LITERATURE CITED. . . . . . . . . o . . . . . . . . . . . 80

-1-
INTRODUCTION.AND HYPOTHESIS

The problem of feed evaluation, especially forages, has
occupied the time of many nutritionists for as long as the
science of nutrition has existed.

Conventional methods of determining TDN, starch equiva-
lent, or net energy values have worked reasonably well in
relation to concentrates but have been less than satisfactory
in the case of forages which are useful largely in the nutri-
tion of ruminants. Ruminants are unique in having a digestive
system which can derive energy from complex plant carbohydrates.
This ability is due to the symbiotic relationship of bacteria
and protozoa with the ruminant. The microflora carried in the
rumen enables ruminants to derive energy from complex plant
carbohydrates since they readily hydrolyze these materials
into compounds which the animal can utilize.

Most methods of feed evaluation depend on the results of
digestion trials which are difficult and expensive. The appli-
cation of the results from a few animals with a few feed samples
to all animals and plants of varying age, species, plane of
nutrition and enviroment leads to considerable error. It would
be useful if a simpler test could be devised which could be
used to evaluate feeds individually for various species of animals
under varying conditions, so that the errors inherent in one
evaluation based on specific conditions when applied to a large
variety of cases could be minimized. The evaluation of a forage

based on its fermentation by rumen microbiota may be more logical

than conventional methods since a large part of the energy made
available from forage is a result of the degradation of ingested
food material by ruminal microflora.

It has been noted for many years that the heat increment
of feeds is higher in ruminants When feeds containing relatively
high proportions of lignin and fiber are consumed. This higher
heat increment is not accounted for by conventional TDN compu-
tations nor by starch equivalent or net energy evaluations
even when a fiber correction as suggested by Kellner (1920) is
used. Recent work by Armstrong, Blaxter and co-workers (1956,
1957a, 1957b) has indicated that the heat increments produced
by the three volatile fatty acids (acetic, propionic and butyric),
'which are the major end products of rumen microbial dissimila-
tion of carbohydrates, may be different.

Many other workers have shown that the type of carbohydrate
fermented influences the relative proportions of volatile fatty
acids which are produced. From the preceding it may be reasoned
that the nutritive value of a forage as a source of energy
depends on the rate, extent and proportions of volatile acids
produced by rumen microbial fermentation.

The objectives of the research reported here were:

(a) To test the hypothesis that the nutritive value
of a forage depends on the rate and extent of and the proportions
of volatile fatty acids produced by rumen microbial fermentation.
(b) To develop a simpler more accurate method for

forage evaluation.

- 3 -
REVIEH'OF LITERATURE

Forage Evaluation

The importance of energy:
McCullough (1959) determined that in the case of forages

the nutrients least likely to be limiting were crude protein
and minerals. He concluded that the limiting factor as far as
forages are concerned was the level of energy. In a summary

of 70 forages based upon analyses taken from the literature,

he found no correlation between crude protein and metabolizable
energy content, while very high correlations occurred between
crude fiber and dry matter digestibility (-.908), crude fiber
and metabolizable energy (-.932) and dry matter digestibility
and metabolizable enrgy (.986). He had found earlier (McCullough,
1956) that dry matter intake and digestibility were highly
correlated.

In a similar series of experiments and calculations
Crampton (1957) concluded that relative to the average TDN
which forages yield, protein, calcium and phosphorus tend to be
present in forages in concentrations equal to or greater than
those indicated by feeding standards to be necessary for cattle
maintenance. Energy was the fundamental limiting factor in
the nutritive value of a forage. In a test using five forages
of varying quality he found that digestible calories and protein
content were significantly correlated. Lignin content was
positively correlated with voluntary consumption. No significant

correlations were found between voluntary consumption and the

-4...

digestibility of dry matter, protein, fiber or calories. How-
ever, the correlations between dry matter consumption and the
digestibility of dry matter, protein and calories were all
nearly significant. The high (.98) positive correlation between
lignin content and dry matter intake was explained on the basis
that the effect of lignin on digestibility is due to its dis-
tribution in the plant and not to its absolute content. The
data illustrate that absolute content of lignin cannot be used
as a measure of forage voluntary consumption. Crampton proposed
that the rate of digestion and forage intake are inhibited by
anything which represses the microfloral activity of the rumen.
‘He suggested that a practical numerical rating of forages could
be evolved by expressing the voluntary daily consumption of a
forage as a percentage of a "normal" or "expected" value of 3.0
lb. (dry wt.) per 100 lb. live weight of animal.

Blaxter (1956) states that shortages of dietary energy
are usually a more important cause of low productivity in farm
animals than dietary deficiencies of other nutrients.

Forbes 53,31. (1928) determined that if a feed fed to cows
was rated on the basis of 1.00 for maintenance, the net energy
utilization was .985 for lactation and only .761 for body in-
crease in energy. Thus, nutritive value depends on the function
for which nutrients are used.

Blaxter (1956) concluded that, with respect to ruminants,
"The key to food evaluation lies in the elucidation of the
chemical, physical and biological factors which control the

bacterial population and the kinetics and energetics of the

-5-

dissociation of complex carbohydrates by ruminal microflora."
Deficiency of present systems 2: forage evaluation:

Reid gtugl. (1959) suggested that present standards or
indices are not very precise in predicting forage value. He
agreed with Crampton (1956) and McCullough (1959) that forage
quality depends largely on the rate of consumption and energy
content of the forage. He also noted that forages, even of the
same species, may be greatly different with respect to the
response they can produce, but that conventional data do not
reflect the magnitude of these differences. He suggested a
rating system for forages based on the voluntary dry matter
intake and energy content of the forage.

Blaxter (1950, 1951) states that the theoretical upper
limit to production occurs at a level of intake which is beyond
the animal! feed capacity. He suggests that the indigestible
residue of lower quality feeds has an effect on feed capacity.
Therefore, for animals on high production, the value of a feed
of low nutritive worth is less than can be inferred from con-
ventional analyses.

Kleiber (1959) concluded that "No single reference unit is
adequate for feed evaluation for growth and milk production."'
He criticizes the use of TDN, net energy, and Scandinavian feed
units on the basis that these evaluate protein as a source of
energy, not as protein 22£.§E"hi°h is best utilized for milk
production or muscle tissue. He proposed a dual reference
substance with two constants, one for energy replacement and

the other for protein replacement value when related to a standard

- 6 -

substance. The standard substances being a combination of common
feeds such as cottonseed meal and barley.

Forbes and Voris (1932) determined that cows receiving
chopped hay transformed 18.3% of the TDN in the feed toimilk,TDN.
When an alfalfa hay-grain ration was fed 22.7% of the feed TDN-
‘was transformed to milk TDN.

Elliot and Loosli (1959) determined that TDN and digestible
energy values overestimate the available energy in roughages and
underestimate the available energy in concentrates when applied
to ruminants.

Huffman and Duncan (1944) determined that an equal TDN
replacement of alfalfa hay with corn or wheat markedly increased
milk production. They theorized that cereals contained a
"lactation factor" which caused increased milk production.

Smith gt_gl. (1945) determined that cows did not produce
as well as expected from the TDN intake of alfalfa hay supple-
mented with salt and phosphorus. Twelve different concentrates
fed alone or with starch or sugar gave superior production
when replacing approximately equal amounts of the TDN of alfalfa
hay. The production from alfalfa was not quite as high as ex-
pected when the adequacy of the ration was calculated on the
basis of net energy.

Saarinen ‘53'21. (1951) recalculated the net energy values
of hay according to Kellner's (1900) method and concluded that
Huffman's (Huffman and Duncan, 1944) grain lactation factor
was simply an energy discrepancy. They fed cows according to

their net energy requirements from 56 to 154 days on alfalfa

- 7 -

alone or in combination with purified carbohydrates or carbo-
hydrates plus fat. No abnormal decrease in milk yield occurred.
‘When corn was used in place of pure substances no efficiency
changes occurred.

Shaw (1959) noted that when good quality roughages are
evaluated using the TDN or digestible energy systems the discre-
pancy between TDN and actual net feeding value is not great.
However, in the case of poor quality roughages, roughage TDN
may have no more than 50% as much available energy as TDN pro-
duced from concentrates or good quality roughages. _

Other workers have noted an over-evaluation of roughages
by the TDN or net energy system using roughages alone versus
roughages plus concentrate on an equal TDN or net energy basis
(Nordfeldt 33 51., 1950; Teichman _e_13 a_1_., 1958: Loosli 31 2.,
1955: Irvin 53,31., 1951).

Moore st 21. (1953) determined that the increased milk
production when concentrates replaced part of an all hay ration
on an equal TDN basis could be explained on the basis of high
net energy content of concentrates. They worked out a series
of equivalences between TDN and net energy. These were: 1 lb.
TDN in corn I 1 Therm net energy, 1 lb. TDN in good hay I .75
Therm net energy and 1 1b. TDN in poor roughage I .50 Therms
of net energy. From these calculations it is apparent that
when poor quality roughages are used their net energies are as
little as 1/2 the amount indicated by the TDN content. Even
TDN from good quality hay supplies only 3/4 as much net energy

as an equivalent amount of TDN from corn. These differences must

- 3 -

be due largely to the extra heat increment and increased amount
of combustible gases produced from the digestion and metabolism
of roughages as compared to concentrates, since these losses

are not considered in TDN and digestible energy calculations.
In Vitro Fermentation

Application _t_o £552 evaluation:

The in 11352 method of investigation into rumen function
has become popular recently due to the difficulties involved
in assessing rumen volumes and absorption of end products of
fermentation. However, a wide variety of techniques have been
developed, many of which have serious limitations (for discussion,
see‘Warner, 1956), the largest one being certainty that conditions
are reasonably representative of those 12,1122. One of the
major difficulties involved is that in 1112 conditions are not
well defined and are very complex. Thus the question of criteria
for validity of in 11352 work is a difficult one to answer.

Louw 55.21. (1949) showed that the accumulation of end
products during in zitrg fermentation slowed the rate. However,
no difference was noted in the proportions of volatile acids
produced compared to in 1312 observations. Gray and Pilgrim
(1950, 1952) and Graycgt‘gl. (1951) noted that the composition
of volatile fatty acids observed in 11352 contained a greater
proportion of propionic acid than rumen fluid. The extent to
which the various carbohydrates were digested was similar to
the rumen breakdown. They hypothesized that the differences

observed in vitro and in vivo were due to the differential

absorption of volatile acids.

Cheng st 31. (1955), using a washed cell technique, in-
vestigated several variables as related to cellulose digestion.
The rate of cellulose digestion was much slower in 11352 than
that observed in 1112, Substrate concentration and pH had a
great influence on rate and extent of cellulose digestion in_
‘11352. Pigden and Bell (1955) describe an in 11352 fermentation
system which, when used with prediction equations developed by
them, gave estimates of TDN and digestible crude protein for
eleven forages which were in close agreement with those derived
from conventional procedures. Barnett (1957) proposes an in
zitrg method for determining the digestibility of cellulose.

His results correlated reasonably well with digestion trials.

Baumgardt gtflgl. (1958) compared several laboratory methods
of TDN estimation with those determined in 1112, Cellulose diges-
tion determined by in litre rumen fermentation was most closely
related to in 1112 digestion. The relationship was not signi-
ficant when results from all the forages tested were compared,
however, a highly significant correlation occurred when only
grass hays were considered. Brown 55.51. (1958) determined
that in vitro digestibilities reflected in 1312 observations
though they were not identical. .A 24 hour fermentation (25
325.2) was suggested by Kamstra at 21. ( 1958) as a measure of
cellulose availability.

Heuter st 11. (1958), using the washed cell technique of

in vitro fermentation, concluded that it was useful in outlining

-10..

or elucidating one or two step reactions but observations in-
volving multi-step reactions were of doubtful significance

unless verified in 1112. Asplund st 21. (1958), employing an

[in vitro method utilizing the dialysing sac technique of

thtanen gtflgl. (1954), showed distinctly different percentages
of dry matter‘were lost and significantly different amounts of
fatty acids produced when feeds of diverse quality were fermented.
There was a high degree of correlation between dry matter diges-
tibility in 1112 and dry matter loss and fatty acid production

Using a different approach Leffel (1958) found that the
relative proportions of rumen volatile fatty acids did not
change though a 20% increase in VFA was noted when rumen con-
tents were incubated in zi££g_for 2 hours. Longer fermentation
periods led to definite discrepancies in proportions of VFA
produced. A similar method was used by Stewart $3.21: (1954)
to estimate rate and amounts of volatile fatty acid production.
His results agreed reasonably well with observations determined
by different techniques.

Quicke gt :1, (1959) obtained good agreement between ig_
.11352 and in 1112 digestibilities with seven grass hays. Howe
ever, results were not consistent with the six legume hays
which were investigated. An inhibitory substance to ig_zit£g
cellulose digestion was reported by Dehority (1957) to be
present in alfalfa extracts which may be related to the discre-
pancies noted when legumes are tested in 11353.

Church and Peterson (1960) investigated the influence of

several variables on in vitro rumen fermentations. They noted

-11-

that a wide variety of in zi££g_systems is in use today, each
'with its characteristic results, which makes interpretation
difficult and between laboratory comparisons of little value.
“However, they concluded that the use of the laboratory fermen-
tation for feed evaluation and as a research tool holds great
promise, pending standardization of technique.

McBee (1953) describes a manometric method for the eval-
uation of rumen microbial activity. Its use is limited since
the proportions of the various volatile fatty acids are not
estimated, total acid production only being determined.

In general the in_zi££g_rumen fermentation technique is
able to yield results on dry matter and cellulose digestibilities
which correlate well with.i£;zi!g observations. The volatile
fatty acid production, however, is not as well correlated with
in 1312 results. The discrepancy may be due to an alteration
of the fermentation due to the experimental conditions or to
the effect of ruminal absorption of acids on the volatile acids
present in the rumen. since there is no real evidence that
differential absorption of volatile fatty acids from the rumen
exists, (most evidence indicates that differential absorption
of the volatile fatty acids does not occur, Annison gt‘gl.,
1957: Pfander and Phillipson, 1953; McCarthy st 21., 1958;
Davis 5tflgl., 1958; Barcroft ££_al., 1943: Conrad 53 31., 1956:
Gray, 1947; Gray, 1948: Stewart gt‘gl., 1958), the tendency
is to believe that the differences observed in volatile fatty
acids present in in 23352 fermentation flasks is due to an

alteration in the fermentation imposed by experimental conditions.

-12-

Source gf innoculum:

Gall 33531. (1949) cultured cattle and sheep rumen bacteria
from animals on various diets. They concluded that the bacteria
present were similar in both species. However, the number of
fast growing organisms increased when high concentrate rations
were fed.

Burroughs'gt‘gl. (1950) reported that the kinds and numbers
of bacteria cultured from rumen contents differed with corn cob-
alfalfa rations with and without starch and with varying amounts
of casein. Fast growing species were more evident when starch
was present in the ration. Hunt st 21. (1952) made a similar
observation. Hungate st 31. (1952) noted rapid changes in
ruminal flora when large amounts of grain were placed in sheep
rumens which had previously been fed an all alfalfa hay ration.

In 1953 Bryant and Burkey compared the microflora from
cows fed a variety of diets. Larger numbers were cultured
on the all-concentrate ration than on other rations. The flora
cultured from a cow receiving wheat straw was least complex,
that from a concentrate fed cow intermediate, and that from a
cow fed alfalfa hay the most complex. The numbers of starch
and cellulose digesters varied directly with the amounts of
starch and cellulose contained in the ration. The level of
feeding of rations did not effect numbers or types of bacteria
cultured.

Maki and Foster (1957) counted directly and cultured
greater numbers of bacteria from rumen fluid of cows receiving

high concentrate rations than in rumen fluid from roughage fed

- 13 -

cows. The number cultured from a high roughage rumen fluid
*was only 3-12% of the total direct count. In contrast the
cultural count of high concentrate rumen fluid was 57-73%
of the direct count. The roughage fermenting bacteria had
much.more fastidious nutritional requirements than concentrate
fermenting bacteria. They grew only when rumen fluid was included
in the culture tubes. They grew better in mixtures, pure isolates
growing poorly or not at all.

These observations would indicate that cultural methods
for counting or isolating roughage type micorflora may lead
to serious errors of estimate. Thus colony counts as an enumer-
ating device should be interpreted cautiously especially when
roughage type microflora are being used. Further, the ration
probably influences the development of a ruminal microflora

characteristic of that ration.
The Importance of the Rumen Fermentation

Extent 25 digestion 32 the rumen:

As long ago as 1883 Tappeiner (cited in.Annison and Lewis,
1959) demonstrated that the fermentation of cellulose in the
rumen of the ox resulted in the formation of large amounts of
volatile fatty acids (VFA's). For many years little nutritional
significance was placed on this observation. The digestion of
carbohydrates in the rumen was believed to be a depolymerization
to simple sugars which were then absorbed and metabolized in
the same manner as in monogastric animals (Annison and Lewis,

1959). It is now established that the VFA's found in the rumen

- 14 -

constitute a major source of energy to the ruminant since little
of the digestible carbohydrate escapes rumen fermentation.

Hale st 31. (1947), using the lignin ratio technique, de-
termined that the rumen digestion of cellulose was 78.9%, dry
matter 84.9%, protein 86.3%, nitrogen-free extract 100.2%, crude
fiber 58.0%, other carbohydrates 101.6% and lignin 14.4% of the
fecal digestion of the above fractions.

Gray (1947) studied the progress of cellulose digestion
along the gut of sheep. An average of 42.7% of dietary cellulose
was degraded by the time digesta had reached the abomasum and,
58.8% by the time the colon was reached. Thus 70% of the di-
gested cellulose was digested in the rumen-reticulo-abomasal
complex, 17% in the caecum and 13% in the colon. Gray 53 31.
(1951b) incubated wheaten and alfalfa hays igflzitgg and found
the yield of volatile acids to be 1/4 of the substrate or 50%
of the digestible matter.

‘Weller and Gray (1954) used the lignin ratio technique
‘with rumen and abomasal fistulae to show that the destruction
of starch in the rumen and cmasum was nearly complete. Starch
passing through the abomasum increased from 1 gram per day
when 3 grams were fed to only 8 grams when 148 grams were fed.

Balch and Rowland (1957) in an extensive investigation of
the effects of several variables on the production of volatile
fatty acids noted that very little carbohydrate which could be
digested in the lower digestive tract escaped rumen fermenta-

tion. Paloheims 55 El: (1955) showed that of the total amount

-15..

of the nitrogen-free, non-lignin organic matter digested in
the whole gut, 76-99% was digested in the reticulo-rumen and
omasum. When Balch (1957) used the lignin ratio technique to
estimate rumen digestion he found that 43% of the dietary dry
matter was digested in the reticulo-rumen. This represented
81% of the fecal digestion of NFE and 54% of the fecal digestion
of crude fiber. Ninety-three to ninety-five per cent of the
starch was digested in the reticulo rumen. Gray gtwgl. (1958)
determined that an average of 40% of the cellulose and 43% of
the pentosans was digested in the stomach of sheep. Kameoka
and Morimato (1959) observed that 61.7 to 85.4% of the diges-
tible organic matter disappeared from the fore-stomach of goats.
Meyer g£_gl. (1959) killed sheep at 0, 1%, 4 and 8 hours after
feeding and found that 89 per cent of the holocellulose was
digested by the time it reached the abomasum. Rogerson (1958)
estimated that 40% cf the dietary dry matter was digested in
the rumen of sheep fed hay compared to 50% on a mixed diet and
75% on a concentrate ration. The total dry matter (fecal)
digestibilities were 49, 62 and 88% respectively.

Consequently, it is apparent that the energy made available
to ruminants is largely the result of rumen fermentation. ‘A
major end product of this fermentation is the volatile fatty
acids. Therefore, any consideration of the energy nutrition of
ruminants involves a consideration of these acids and their

utilization.

- 15 -

Importance 2f volatile fettyflggigg:

The composition of the volatile acids produced on rumen
fermentation of carbohydrates was not known until Elsden 31 El:
(1946) worked out a column chromatographic method for their
separation. iAn average of 329 grams of volatile fatty acids
was found in the reticulo rumen of cattle and 64 grams in sheep.
These acids were about 67% tcetic, 19% propionic and 14% butyric.
Other workers have observed considerable amounts of volatile
fatty acids produced during rumen fermentation (Balch, 1958:
Gray's: 21., 1951b: Phillipson, 1952: El-Shazly, 1952;.Annison,
1954).

Carrol and Hungate (1954) estimate that 66% of the energy
derived from a ration is from volatile fatty acids. It was
shown that 6,000 to 12,000 calories became available in the
rumen of cattle every day. Phillipson and Cuthbertson (1956)
have calculated from the data of Schambye (1951) that at least
600-l,200 calories of energy are absorbed as VFA's from the
sheep rumen every 24 hours. Stewart 53,11. (1958) estimated
from rate determinations-on rumen fluid of steers fed several
rations, that the volatile fatty acids produced in 24 hours
would furnish.from 12,000 - 13,000 calories representing 37.9%
of the digested calories of the ration. This would account
for 63% of the maintenance requirements of the steers. McCarthy
SEMEl° (1957) using a rumen perfusion technique estimated that
rumen VEA would provide 37 to 46% of the energy requirement of

the goat for maintenance. Emery (1956) estimated, from turnover

-17..

curves determined 32 vivo, that a cow would obtain 3-13% of

its energy from the volatile fatty acids.
The Relation of the Diet to the Volatile Fatty.Acids

The components gf the diet:

 

 

The percent of milk fat in the milk of cows is positively
correlated with the proportion of acetic acid in the rumen.
Powell (1938, 1939) first Observed that the fat content of milk
decreased when cows were fed low roughage rations. Not until
10 years later was the depression of milk fat linked with alter-
ations in the proportions of volatile fatty acids occurring in
the rumen.

Stoddard gt :1. (1949) observed that low roughage diets
caused a lowering of the proportion of acetic acid in the rumen
accompanied by a lowered fat test in milk. ‘When acetic acid
was given by stomach tube the milk fat test returned to normal.
Propionic acid administered in the same manner had no effect
on the fat content of milk. These Observations were confirmed
by Tyznick and Allen (1951) and Van Soest and Allen (1959).
Rock and Balch (1959) raised milk fat test in milk from cows
receiving normal rations by acetate infusion. Propionic acid
infusions had no effect on milk fat test.

Gray st 31. (1952) established the presence of formic,
acetic, propionic, n-butyric, isobutyric, valeric, caproic and
probably heptoic acid in rumen fluid. They determined the
molecular proportions of fatty acids in the rumen fluid 1 to

2 hours after feeding. Lucerne hay gave values of 66.9% acetic,

-18..

22.5% propionic, 6.7% butyric, and 3.2% valeric acids. The
averages of 7 Observations from sheep fed wheaten hay were
65.8% acetic, 22.1% propionic, 9.2% butyric and 2.2% valeric
acid.

Card and Shultz (1953) detennined that grain added to hay
or pasture rations caused a decrease in the proportion of acetic
acid. The proportion of propionic acid on the hay plus grain
ration was decreased slightly but a large increase in butyric
acid occurred. lhen grain was fed on pasture there was a fall
in acetic acid proportion accompanied by an increase in butyric
and propionic acids. The lowest proportion of acetic acid
occurred when an all concentrate ration was fed. Roughages
from later dates of cutting tended to cause an increase in
acetic and a decrease in butyric acid. Silage tended to cause
a decrease in acetic and an increase in propionic acid when
compared with key. .Acetic acid proportions were the most
variable with reciprocal changes in butyric acid.

Hibbs‘g§.gl. (1954) fed several diets with various hay-grain
ratios to calves and noted that with higher grain levels the
proportion of butyric was increased with a reciprocal decrease
in the propionic acid proportion of total volatile fatty acids.
Reid (1957) determined that the pre-feeding proportion of pro-
pionic and butyric acids in the rumen of sheep increased as the
level of concentrate in the ration increased. The proportion
of acetic acid in the rumen decreased when Balch and Rowland
(1957) decreased the ratio of bay to concentrate in a diet

fed to cows.

- 19 -

Johns (1955) observed that the proportion of acetic acid
dropped markedly when sheep were changed from hay to pasture.
There was also a marked rise in the butyric acid proportion,
that of propionic remaining fairly constant. A higher pro-
portion of acetic acid was observed when hay of poorer quality
‘was fed. ‘A great consistency of rumen volatile fatty acids
occurred in the rumen.of sheep consuming pasture herbage which
covered a wide range in maturity and chemical composition. In
contrast to Reid (1957), Jamieson (1959) observed, igflzizg,
that sheep grazing normal pasture showed seasonal fluctuations
in the proportions of ruminal volatile fatty acids. Samples
containing low proportions of acetic acid were found in the
spring and fall when lush pastures occurred. The conclusion
‘was that seasonal variation of volatile fatty acids in sheep
on pasture were due to variations in plant composition. When
sheep were dosed with nitrate and nitrite at sub-lethal levels,
decreases in the proportion of acetic with concomittant increases
in the proportion of propionic and butyric acids occurred. This
was hypothesized to be due to a competition between acetate
production and nitrate reduction for hydrogen donors.

Elliot 51.31. (1957) observed 12 1112, increased rumen
acidity and higher total volatile acids with a greater proportion
of propionic accompanied by decreased amounts of butyric and
acetic acids when corn silage as sole roughage was compared to
hay crop silage as sole source of roughage. Rumen acidity was
not increased by either hay-crop or corn silage when they were

fed as a part of the roughage ration.

-20..

The $2 31352 volatile fatty acid production from various
feeds was studied by Stewart and Schultz (1958). Urea con-
sistently increased volatile fatty acid production regardless
of substrate. In a subsequent ig‘zizg experiment urea increased
VRA production only slightly; however, the level of urea achieved
in the rumen was not as great as that in the igflzitgg experiments.
Fresh, hand-clipped legume mixed grass caused a greater VFA
production than legume hay. The grass markedly depressed pro-
pionic acid compared to hay. Molasses decreased acetic acid
production, beet pulp increased acetic acid production and
corn meal increased propionic acid production.

Van Soest and Allen (1959) found no change in rumen acetic
acid on lcw'roughage diets, but an increase in the molar per-
cent of propionic acid occurred. They calculated that a signi-
ficant negative correlation occurred between the fat content
of milk and proportion of propionic acid in the rumen. When
they fed 1 lb. of acetate per day, the depression of milk fat
‘was corrected. Propionate feeding tended to cause a further
decline in milk fat percent.

Elliot and Loosli (1959) determined that the relative
concentration of propionic acid in rumen liquor was highly
correlated with both the gross and net production efficiency.
Negative correlations existed between the acetic/propionic
ratio and gross and net efficiencies. The molar proportions
of acetic and propionic acids were closely related to the
average crude fiber content of the ration (negatively for

propionic and positively for acetic acid.) The net efficiency

- 21 -

of utilization of digestible energy was highly correlated

(negatively) with the crude fiber content of the ration.

Components at; plants _a_n_d_ £13515 relation 2 17.1.39. production:

The wide variety of volatile fatty acid proportions formed
from different feeds by ruminal fermentation has led to the search
for a particular component of these feeds which is responsible
for the production of acetic, propionic and/or butyric acids.

Elsden (1945), in an ip.!i££p_fermentation of glucose, ob-
served that propicnic acid was the predominant acid produced.
When dried grass was fermented, acetic acid was the predominant
acid produced. The ip 11352 fermentation of xylcse and gluc-
uronic acid produced preponderating amounts of acetic acid
(Heald, 1952). Marston (1948) noticed a high proportion of
propionic acid when cellulose was fermented ipflziggg.

Gray and Pilgrim (1952a, 1952b) examined _i_n_ 1233.2, the
volatile fatty acids produced from cellulose and hemicellulose
alone, in combination, in the presence of wheaten hay or in
the presence of a small amount of protein prepared from wheaten
hay. The greatest proportions of acetic and butyric acids and
least proportion of propionic acid occurred when wheaten hay
was the substrate. Cellulose, hemi-cellulose or a combination
of cellulose and hemi-cellulose produced a lower proportion of
acetic and butyric acids and a higher proportion of propionic
acid. lhen 4% wheaten hay protein was added to the cellulose-
hemicellulose mixture the portion of propionic acid decreased
and those of acetic and butyric acids increased over that ob-

served when no protein was added to the mixture. ‘When wheaten

hay was mixed with the pure components, the proportions of acids
produced on ipflzippp fermentation were intermediate between those
of the separate substrates. Casein in the place of wheaten-hay
protein did not affect acetic acid proportions, however, the
butyric acid proportion increased greatly accompanied by a fall
in the proportion of propionic acid.

Phillipson (1952) noted that the proportion of acetic acid
in a fermentation mixture increased with increasing fiber content.
However, in over 400 ipflzizg determinations of the individual
volatile fatty acids of sheep consuming pasture at various stages
of maturity, Johns (1955) observed no marked change in total
volatile fatty acids or in acid proportions. .Although there was
considerable variation in the chemically defined constituents
of the pasture herbage ingested they appeared to bear no definite
relation to the proportions of volatile fatty acids occurring in
the rumen. Jamieson (1959) observed decreases in acetic and in-
creases in propionic and butyric acid proportions when lush pasture
was consumed. However, during the remainder of the season re-
latively small fluctuations occurred in volatile fatty acid
proportions.

Barnett and Reid (1956) determined EEHIEEEE that the amounts
of acetic acid produced diminished in the order: dried grass,
cellulose, glucose and lactic acid. The more easily fermented
the food the less the proportion of acetic acid produced. When
fresh grass was fermented at various stages of maturity the pro-
pionic acid proportion of the volatile fatty acids became greater

as the grass became more mature. In 1957 Barnett and Dowe deter-

- 23 -

that the fermentation of pyruvic acid produced a high proportion
of acetic acid. Propionic acid was the chief acid derived from
lactate. They proposed a metabolic scheme for the formation of
propionic acid via succinic acid and the citric acid cycle.
Barnett and Reid (1957), using an.ig.zit£g technique, studied
the production of volatile fatty acids obtainable from dried grass
and its grass water-soluble and water-insoluble separates. In
contrast to fresh grass, dried grass gave a consistently greater
proportion of propionic acid at all stages of maturity. Aqueous
extracts yielded preponderating amounts of acetic acid while the
residue gave amounts of propionic acid equal to or exceeding the
corresponding production of acetic acid. Commercial cellulose
produced propionic acid in the largest proportion. Following
this up (Barnett and Reid, 1957b) they found that crude fiber
and cellulose, prepared from dried grass, produced proportions
of the volatile fatty acids between those of water extracted dried
grass and commercial cellulose. They concluded that the major
portion of acetic acid comes from the carbohydrate fractions in
whole grass which are simpler than cellulose and that the pro-
portions of acetic acid observed in 1112 were difficult to account
for in regards to in vitro determinations.
Hershberger gthgl. (1956), obtained 32.11352: a preferential
formation of acetate when pyruvate formed 50% of the carbon of
the substrate. Lactate as 50% of the carbon of substrate led to
the preferential formation of propionic acid. Glucose, pyruvate,
lactate and malate increased both acetic and propionic acid pro-

duction while malonate, glutamate and<K4Keto glutaric acid increased

a

- 24 -

acetic acid production. Jayasuriya and Hungate (1959), using
radioactive labeling, determined that lactate was not an im-
portant intermediate in hay fed animals. Lactate ~2-Cl4 was
recovered chiefly as acetic acid when allowed to ferment EEHIEE£2°
Starch yielded lower proportions of propionic and a higher
proportion of valeric, butyric and acetic acids than cellulose
'ig‘zitgg. Xylan and pectin produced the same proportions of
volatile fatty acids as starch (Belasco, 1956). Lewis and
McDonald (1958) found no striking differences in the volatile
fatty acids when they fermented xylan, levan and starch. The
volatile fatty acid production from carbohydrate was enhanced
in the presence of casein in the rumen of sheep.
The importance of protein to roughage digestion when roughage
was fed in the absence of starchy feed was small (Burroughs 53 11.,
1950). When starch was added to a low-protein roughage ration,
the dry matter digestibility of roughage dropped drastically;
however, when starch and protein were added together the decrease
in digestibility due to starch was much less. Grampton (1956)
made a similar observation on the importance of protein to roughage
digestion from which he concluded that with most roughages nitrogen
content was not the first limiting nutrient, the primary limita-
tion to productivity of roughages being energy. Head (1953),
on the basis of experimentation, concluded that if a roughage
has a minimum content of 1% nitrogen, cellulose digestion was
independent of supplementary nitrogen. However, Belasco (1954a,
1954b) determined that 12.!iEES cellulose digestion was best

supported by urea supplied up to the 35% protein equivalent level.

 

- 25 -

Urea as the sole source of nitrogen promoted the production of
higher levels of propionic acid and lower levels of butyric

and valeric acids than did equivalent amounts of nitrogen from
either soybean oil meal, linseed oil meal, cottonseed oil meal
or corn gluten meal. The acetic acid and total volatile fatty
acid levels were unaffected. He postulated that the additional
propionic acid resulted from a high cellulolytic activity in
the artificial rumen in the presence of a readily available
source of nitrogen. .

Annison (1954) noticed that protein induced a greater pro-
duction of butyric plus higher volatile acids. Others have
made similar observatioms (Otagaki gt‘gl., 1955; floodhouse
g 2., 1955; Annison, 1956; Davis _e_t _a_._l_., 1957; Bl-Shazely,
1956).

Other Factors Influencing the Rumen Fermentation

 

£553 treatment:

' It has been found that, in addition to the ration fed,
certain treatments of the ration will affect proportions of
volatile fatty acids in the rumen, milk fat test, and efficiency
of ration energy utilization.

Powell (1939) noted that grinding of hay resulted in de-
pressed milk fat test. These results were confirmed by Balch
(1952, 1958) who also noted an increased proportion of pro-
pionic acid produced by the fermentation. Pelleted alfalfa

hay in lamb diets resulted in increased rate of gain and increased

-26-

feed consumption (Neale, 1953; Cate gt'gl., 1955; Bsplin.3£_gl.,
1957). Blaxter and McGraham (1955, 1956) noted that pelleted
hay fed to cows passed through the digestive tract at a faster
rate, with a lower digestibility and lower metabolizable energy
content than chopped hay, but was equal in net energy value to
chopped hay because of a lower heat increment. In contrast,
Meyer 53 21. (1959), using a different experimental approach,
concluded that pelleted alfalfa hay fed to sheep contained no
greater net energy than long hay and that increased gain noted
with pelleted hay was due to an increased intake.

The form in which the roughage is fed may also influence
relative proportions of volatile fatty acids. Dried grass
produced a greater proportion of acetic acid than the same
grass fed fresh or preserved as silage (Johns, 1955; Card and
Schultz, 1954; Bl-Shazley, 1952). Barnett and Reid (1957)
made the opposite conclusion. From their in zitgg observations,
dried grass gave greater proportions of propionic acid than
fresh grass, however, their in 12352 method was of long duration
(72 hours) and only terminal observations on volatile fatty
acid proportions were made. The results of this type of tech-
nique have been seriously questioned (warmer, 1956).

Balch and codworkers (1954, 1955a, 1955b, 1955c) conducted
a series of experiments in which they determined that the essen-
tial feature of diets which depress milk fat was not simply a
low roughage or crude fiber content per 25. Low roughage rations
did lower fat content, but, the greatest depression of milk fat

was obtained with a high starch intake. In addition, the type

of starch was also instrumental in lowering the fat content of
milk. ‘Hhen flaked maize (heated and rolled corn) was compared
with maize meal or barley and oats, the greatest fat depression
occurred with the flaked maize followed by the maize meal and
the barley and oats ration.

The work of Shaw 53 _a_l_.. (1959) and Ensor .51 21. (1959)
confirms the observations of Balch and codworkers on the effect
of cooked grains on milk fat test and proportions of volatile
fatty acids present in the rumen. Ground, pelleted roughage
alone did not affect the proportions of volatile acids pro-
duced or the milk fat test. However, the effect of cooked
concentrates was enhanced when they were fed with ground hay
instead of long hay.

The efficiency of gain (Ens r 55 21., 1959) and milk pro-
duction (Elliot and Loosli, 1959) may be increased when these

types of rations are fed.

The rate of fermentation:

 

"There is some evidence that the rate of the ruminal fer-
mentation may influence the proportions of volatile acids pro-
duced.

During periods of rapid acid production Gray and Pilgrim
(1951, 1952) found that the ratio of acetic to propionic acid
decreased; whereas, when acid production was slow the ratio
increased. Carroll and Hungate (1954) observed that the in
'11352 rate of fermentation and the proportion of propionic acid

produced was greater when rumen contents were fermented for one

-28-

hour from a grain fed steer than that produced upon incubation
of rumen fluid from an animal fed roughage.

Hershberger 53 21. (1956) using washed cells to ferment
cellulose observed that acetic acid was the major valetile
fatty acid produced during the early part of the fermentation
when the rate was slow. .After 15 hours the rate of fermentation
and the production of propionic acid increased. When Belasco
(1954b) fermented cellulose he noted a more rapid rate of fer-
mentation and the production of a greater proportion of propionic
acid when urea was used as a nitrogen source in place of natural
proteins.

‘When Stewart gt‘al. (1958) fed a mixed diet to cows they
observed from rate curves established 32.11352 that propionic
acid was produced at a faster rate than either butyric or acetic
acids immediately after feeding. {At 10 hours after feeding a
prominent increase in the production of acetic acid occurred.
The rate of production of'volatile fatty acids was greatest
immediately after feeding.

Reid (1957) observed in sheep that the proportion of acetic
acid declined and that of propionic acid increased after feed-
ing. The lowest proportion of acetic acid and highest proportion
of propionic acid coincided with the maximum total VEA levels
in the rumen. .

In contrast Sheppard st 21. (1958) could not find any dif-
ference in the proportions of volatile acids present in the
rumen of sheep fed hay although variations in the amount of

total acids present occurred during 24 hours of fermentation.

The rate of feeding may also affect acetic/propionic acid
ratios. Williams and Christian (1956) noted a higher ratio of
acetic to propionic acids when they fed a limited amount of hay

as compared to liberal feeding.

£22 effect 2: pg:

Balch gtflgl. (1956) found that during rumen fermentation
there was a decline in pH after feeding. This decline was greater
for low roughage diets than for those high in roughage. The
lowest pH occurred at a time when the greatest proportion of
propionic acid was observed. The rate of VFA production was
maximal at this point. Lactic acid was present during periods
of low pH and may have contributed to the pH change. Balch and
Rowland (1957) noted that an acid rumen inhibited the production
of acetic acid. The acidity resulted from the rapid fermenta-
tion of soluble carbohydrates. Elliot gt‘gl. (1957) observed
decreased pH coinciding with increased proportions of propionic
acid when a ration of corn silage was fed as compared to a hay-
crop silage ration.

If the depression in pH is too great, a different result
may occur. .Annison and Lewis (1959) indicate that the normal
range of the rumen pH is 5.5 to 7.0 and that those outside of
this range often lead to rumen dysfunction. Reid glflgl. (1957)
found that when the pH of the rumen fell below 5.2 due to lactic
acid accumulation, the propionic and butyric acid proportions
fell. The pH had lowered to the point where rumen function was

inhibited greatly.

-30-

Energy relationships:

The ruminant animal is recognized as having a higher heat
increment than other classes of livestock. This phenomenon was
noted by Kellner (1920) when he stated that ruminant heat losses
associated with carbohydrate dissimilation were 30 to 70 kilo-
gram calories per 100 kilogram calories of carbohydrate ingested.
This is compared to heat lost in man of only 5 to 7 kilogram
calories per 100 kilogram calories of carbohydrate ingested.
(Lusk, 1928).

The heat of the rumen fermentation has been considered as
a source of the higher heat increment in ruminants. However,
Marston (1948) fermented cellulose in 33.22 and determined
that the heat of fermentation under normal conditions could
account for only 15% of the heat increment. Later Marston (1951)
concluded that the heat loss occurring in the dissimilation of
acetic acid was probably responsible for the relatively high
rate of heat production in the ruminant. McClymont (1952)
postulated that the high heat increment of acetic and butyric
acids could be inferred from the intermediary metabolism of
these acids, their oxidation being thermodynamically wasteful.
He suggested a specific dynamic effect of 50 to 70% for acetic
and butyric acids.

Not until 1956 were there any real attempts to assess the
effect of the volatile fatty acids on ruminant heat increment.
Armstrong and Blaxter (1956, 1957) infused volatile fatty acids
into the rumen of sheep. When only acetic acid was administered

41 to 50% of the energy input appeared as heat. In contrast the

heat increment of propionic acid was 13% and that of butyric

16%. The heat increment of glucose infused into a non-fermenting
rumen was only 6.4%. In a more extensive study.Armstrong ££.El'
(1957) infused various combinations of the volatile acids, acetic,
propionic and butyric, into the rumen of fasting sheep. There
was no difference in heat increment when the infusion mixture
contained from 10 to 90% acetic acid. Some manifestations of
ketosis occurred when 100 and 90% acetic acid mixtures were in-
fused. It appeared that small quantities of propionic acid

(1 molecule of propionic to 15 molecules of acetic acid) were
sufficient to facilitate the oxidation of acetic acid and pro-
tein breakdown to form carbohydrate in the fasting animal.

The conclusion was that sub-maintenance heat increment was not
due to variations in the proportions of volatile acids produced
in the rumen.

‘When volatile fatty acids were infused into the rumen of
sheep receiving super-maintenance diets a different relation-
ship was observed (Armstrong and Blaxter, 1957). The heat
increment of the various acids when fed alone was 67% for acetic,
33% for propionic and 38% for butyric. The mean heat increment
of a mixture containing 75% acetic acid, 15% propionic acid and
10% butyric acid was 68.1%, while that of a 25:45:30 mixture was
41.9%. Any energy retention from the infused acids was assumed
to result in increased fat content of the body. Therefore, for
fat synthesis the mixture containing relatively high amounts of
acetic acid was used much less efficiently than a mixture con-

taining only 25% acetic acid.

- 32 -

PART I. DIGESTION TRIAL

Procedure

Bight dry Holstein cows were used to determine the diges-
tibility of alfalfa-bromegrass hay and oat strawl.

These cows were fed the forages, “ad libitum", for a ten
day preliminary period followed by a 7 day fecal collection
period. The cows receiving the oat-straw were also fed 2 pounds
per head daily of a complex supplement containing protein, vit~
amins and minerals (Table 1). This supplement was considered
necessary in order to keep the cows from going into drastic
nitrogen imbalance during the feeding period. Maynard and
Loosli (1956) state that the actual digestibility of a feed
not sufficient in itself is more truly evaluated when supplements
are fed in much the same manner in which they would be fed in
actual practice. In addition, in this study, the greatest in-
terest was in differences of energy rather than vitamins, min-
erals and protein.

During the ten day preliminary period the cows were adjusted
to a uniform daily intake. During the collection period daily
allowances were kept constant for each cow so that she was con-
suming uniform amounts of the forage each day. All cows readily
consumed the alfalfa-bromegrass hay ration so that several days
prior to and during collection 3 cows were consuming 24 pounds
per day and a fourth (#3) 20 pounds per day. More difficulty was

encountered in getting the oatsstraw fed cows to eat uniform

 

1Provided by Dr. Lassiter of the M.S.U. Dairy Department.

 

 

 

TABLE 1. COMPOSITION OF OAT STRAW SUPPLEMENT
Ingredients Percent
Ground Shelled Corn 30.00
Soybean Oil Meal 32.25
Dried Molasses 10.00
Dehydrated Alfalfa Meal 15.00
Dicalcium Phosphate 5.00
Trace Minerals1 .20
Trace Mineralized Salt 1.50
Vitamin A and D Concentrate .05
Urea 6.00

 

1Trace Mineral Pre-Mix, Calcium Carbonate Company,

Quincy, Illinois.

9,800 I.U. Vit. A/gm.

2

TABLE 2.

1,250 I.U. Vit. D/gm.

PROXIMATE ANALYSIS OF FEEDS

 

 

Crude Ether N-Pree

Ash Fiber Extract water Protein Extract
Alfalfa-
Bromegrass 5.29 29.28 1.51 10.98 14.38 38.56
hay
Oat Straw 5.91 38.76 1.17 10.61 3.82 39.73
Oat Straw
Supplement 9.36 7.50 1.70 11.21 35.56 34.67

 

daily amounts. Three of the four, however, were eating the
straw readily prior to and during fecal collections at a rate
of about 14 pounds per head daily. One of the cows refused
the straw for several days and would not consume uniform daily
amounts.

Fecal collections were weighed daily, mixed thoroughly,
and aliquots, preserved with thymol, stored under refrigeration
in air-tight jars. Conventional proximate analyses were carried

out on the feed and feces (Table 2).

Results
The average TDN content of the alfalfa bromegrass hay

ration was 64.0%, the range from 63.3% to 65.4%, and for the
oat straw ration 57.5% with a range from 45.3% to 62.1% (Table
3). The low value is suspect since this was the value obtained
with the cow which would not consume the cat straw readily. If
this value is eliminated and the average of the other three de-
terminations calculated, the TDN value for the oat straw ration

would be 61.6% with a range from 60.9% to 62.1%.

TABLE 3. TDN VALUES OF RATIONS FED

 

 

 

I Observation
Ration e 1 2 3 4 .Ave.
t
Alfalfa
Bromegrass 65.4 63.5 63.3 63.8 64.0
Oat Straw 1
Ration 61.7 62.1 45.3 60.9 57.5

 

1This value is probably in error. For explanation, see text.

 

- 35 -

This study was conducted to demonstrate that TDN and
forage feeding value are not always in the same relationship.
The results support this contention and led to subsequent ex-

perimental investigations reported in Part II.

- 36 -
PART II. THE FERMENTATION OF OAT STRAW AND ALRALFA-BROMEGRASS HAY
Procedure

In 31:52 fermentation:

Two mature Hereford steers, equipped with permanent plastic
type rumen fistula, were used as sources of innocula in this
study. One steer was maintained on oat straw plus 2 pounds of
supplement (Table 1), the other on alfalfa hay plus 2 pounds
of supplement. Each steer was fed for at least 15 days on their
respective ration before being used as innoculum source for in
32352 fenmentations. This was to insure that the microflora of
the steers had an opportunity to adjust to the type of ration
fed.

On the days when experiments were conducted fluid was
drawn out of the rumen before feeding in the morning and strain-
ed through two layers of cheese cloth into a warmed thermos,
which had been previously gassed with 002. The innocula were
transported to the laboratory where 800 milliters were mixed
with substrate (2% by weight substrate) in 950 m1. bottles,
which were then placed in a 39°C. water bath and bubbled con-
tinuously with C02 during pipetting. The flask was swirled
vigorously before each sac was innoculated to insure mixing
of the substrate. To supply an adequate amount of available
nitrogen 2 m1. of a solution containing 200 mg. urea/m1. was

added to the bottles.

rum'r'aiznm

”“1831? ‘73:?” _'_-‘ PER—Tish ‘-

- 37 -

Twenty m1. of the innocula plus substrate solution were
pipetted, with a wide orifice pipett, into cellophane dialysing
sacs1 which were then placed in 4 ounce, screw cap bottles
containing 80 milliliters of a buffered salt solution warmed
to 39°C. with mineral composition approximating that of ruminant
saliva (Salsbury gt'gl., 1956). The bottles were closed tightly
and transferred to a 39°C. incubator. At predetermined time
intervals representative bottles were removed from the incubator,
the contents of the sac and bottle mixed and transferred quanti-
tatively to a 200 m1. centrifuge bottle. The mixture was cen-
trifuged 20 minutes at 1200 rpm. A 10 ml. aliquot of the super-
natant was made alkaline by adding 1 m1. of normal potassium

hydroxide and evaporated to dryness on a steam bath. The dried

aliquot was stored at room temperature until analysed.

Volatile fatty acid determinations:
For VFA analyses the residue from the dried aliquot of
the fermentation supernatant was dissolved in 2 m1. of 12% by

2 and

volume sulfuric acid, mixed with 2 grams of silicic acid
transferred to a chromatographic column described by Keeney (1955).

The column packing material for the chromatographic columns
was prepared by mixing silicic acid which had been dried over-

night at 175°C. with glycol reagent in proportions of one gram

 

1Made from cellophane tubing 32/32 Visking Corporation, Chicago,
IllinOiSQ

ZMallinckrodt's, stock no. 2847.

-38-

of silicic acid to 0.9 ml. of reagent. The glycol reagent was
made by dissolving 700 mg. of brcm cresol green in 700 ml.
. ethylene glycol on a steam bath. After cooling, 200 m1. of
water were added followed by 40 m1. of 0.1 N. ammonia water.
Additional water was added to make one liter of solution.

Forty grams of the silicic acid-glycol reagent mixture were

slurried with n-hexane1

and poured into a glass chromatographic
column, equipped with a fritted glass disc and tefflon stopcock.
The top of the column packing material was leveled with a flat-
tened glass rod and n-hexane was run through the material under
5 to 10 lbs. of nitrogen gas pressure until the column appeared
packed uniformly, care being taken to prevent the solvent from
sinking below the top of the packing material. The columns
were then ready for the addition of the sample section.

After the sample was added to the top of the column packing
material the yellow bands indicative of acids were developed with
a 1% butanol-hexane solvent which was allowed to run through
the column under atmospheric pressure. When the first (butyric
plus higher acid) fraction had reached the point of elution the
fraction collector was changed. The butyric plus higher acids
fraction was then eluted with the 1% butanol-hexane solution.

The solvent was Changed to 5% butanol-hexane for the elution
of propionic and acetic acids which were collected in separate
flasks. The fractions were titrated with .025 N. alcoholic

potassium hydroxide using thymol blue as the titration end-point

indicator.

 

1Skelly Solve B, Skelly Petroleum Company.

- 39 -

There were three replicate flasks for each time period.

The volatile fatty acids were determined on two of the repli-
cates or three if variation between replicates was greater than
10%. The values reported are the averages of at least two and
sometimes three replicates.

Sets of 12 to 18 columns were run simultaneously. Mixtures
of pure acetic, propionic and butyric acids were used to deter-
mine blank values for the system, appropriate corrections being
made where necessary.

No attempt was made to separate butyric acid from higher
volatile acids since preliminary work had indicated that good
separation of standard mixtures of butyric and valeric acids
‘was not consistently achieved under the conditions in this lab-
oratory. For convenience, in subsequent discussion, the butyric

plus higher acids fraction will be referred to as butyric acid.

Holocellulose determination:

When holocellulose disappearance was determined the proce-
dure of‘Wise‘gt'gl. (1946) was used. The precipitate from the
centrifugation of fermentation flask contents was transferred
to a 200 ml. Berzelius beaker, washing with water, and dried
overnight at 105°C.

The dried material was digested under a hood in 16 m1. of
water plus 0.15 gm. sodium chlorite and one drop of glacial
acetic acid at 70-80°C. in a water bath. ‘After one and two
hours of digestion 0.15 gm. sodium chlorite and one drop of

glacial acetic acid were added to the mixtures. The digestion

-40..

was allowed to continue for a total of three hours.

After the three hour digestion the samples were cooled in
ice water, transferred to a centrifuge bottle and centrifuged
for 20 minutes at 1500 rpm. The supernatent was decanted and
the precipitate collected on a weighed fiber glass filter, washed
with ice water and acetone, dried for 48 hours in a desiccator
and weighed. This weight was taken to be the holocellulose con-
tent of the fermentation flask. The difference between amounts
present in zero hour samples and those which had been incubated
was taken to be the amount of holocellulose digested. The pre-
liminary ether extraction of Wise st 21. (1946) was omitted.
No corrections for ash content were made. Cellulose determi-
nations were carried out on this residue by the Crampton and
Maynard (1938) procedure, with modifications, when both holocell-
ulose and cellulose disappearance was estimated.

Difficulty with filtration and lack of agreement of dupli-
cate samples led to the abandonment of the holocellulose deter-

mination.

Cellulose determination:

For determination of cellulose the precipitate from centri-
fugation of the contents of the fermentation bottle was trans-
ferred quantitatively, washing with water, to a 200 m1. Berzelius
beaker and dried overnight at 105°C. This material was used in
subsequent analyses for cellulose by a modification of the

Crampton and Maynard (1938) procedure (Salsbury st 21., 1956).

- 41 -

Cellulose was determined by adding 15 m1. of 80% (by
volume) acetic acid and 1.5 ml. of concentrated nitric acid
to each beaker. The mixture was refluxed for 20 minutes on a
hot plate. A round-bottom flask filled with cold water served
as a condenser. The samples were then filtered through a selas
porcelain crucible of extra coarse porosity, washing with ab-
solute ethanol.

The filtrate in the crucibles was dried at 105° for 12
hours, cooled in a desiccator and weighed. Then the samples were
ashed at 7000 . for two hours, cooled and weighed a second time.
The difference in weight before and after ashing was taken to
be the cellulose content of the sample. The benzene and ether
washes of the Crampton and.Maynard method were omitted. The
difference between the amounts of cellulose in the zero hour
samples and the amounts in the incubated samples was considered
to be the amount of cellulose digested.

Cellulose determinations were accomplished on three repli-
cate fermentation flasks for each time period of fermentation.

The values reported represent averages of the three determinations.

Experimental design and reporting of data:

The experiment was a 2 x 2 factorial design. There were
four treatments, two with each type of innoculum. These were:
(1) Innoculum from an alfalfa1 fed steer with alfalfa as fer-

mentation substrate (AA),

(2) Alfalfa innoculum with oat straw as substrate (AO),

 

IFor convenience the alfalfa-bromegrass hay will be referred
to as alfalfa hay.

(3) Innoculum from an oat straw fed steer reacting with alfalfa

as substrate (OA), and
(4) ‘With oat straw as substrate (00).
Time periods of fermentation were 2, 4, 8, 12 and 24 hours.
Each experimental treatment was repeated two separate times.
The values reported are the averages of the results of the two
trials.

Statistical tests were conducted using the analysis of
variance technique (Snedecor, 1956).

The values reported for volatile fatty acid production
were obtained by subtracting the micro-equivalents present in
the fermentation flasks at zero time from the amounts present
after fermentation. The amounts of volatile acids produced
due to the substrate carried over in the rumen fluid were measured
on several occasions. After 2 - 4 hours of fermentation the rate
of production had become very slow with little net volatile
fatty acids being produced past the 8th hour of fermentation.
The practice of obtaining rumen fluid approximately 14 hours
after the last feeding is probably responsible for there being
relatively little fermentable substrate carried over in the
rumen fluid innocula. In addition Asplund 35 31. (1958), using
a similar technique, concluded that the fermentation occurring
in the absence of substrate was different from that occurring
in the presence of substrate. Therefore, the volatile fatty
acid values reported represent the difference from zero hour.
They are expressed as micro-equivalents of acid per fermentation

fl‘Sko

- 43 -
Results

Volatile $2552.2232 production:

Table 4 summarizes the cumulative production of the vol-
atile fatty acids on the various treatments. Figure l is a
visual projection of the data for total volatile acid production.
The amounts of volatile fatty acids produced are expressed in
micro-equivalents of acid above that present in the original
sample. Analysis of variance (Snedecor, 1956) indicates that
alfalfa innoculum and substrate produced a greater quantity
(p.<:.05) of total volatile fatty acids than oat straw innoculum
or substrate. Treaiment AA gave the highest volatile acid
production with 00 giving the lowest total volatile fatty acid
production (p<:0.05). The volatile fatty acid production on
treatments.OA and AA were intermediate and not significantly
different.

The results indicate that there was a real difference in
the production of volatile fatty acids when oat straw was com-
pared to alfalfa hay. These differences were most evident when
the substrate tested was fermented with an innoculum obtained
from an animal which had been receiving that substrate as a
major portion of his ration. The volatile fatty acid production
when alfalfa innoculum reacted with cat straw (A0) and cat straw
innoculum reacted with alfalfa substrate (GA) was intermediate
between treatments AA and 00.

The average ratio of acetic to propionic acid produced
during the entire fermentation period was not significantly

different for any treatment. The highest ratio was obtained

- 44

 

.omoe. 6303.. u m ”ounce omcommowm u N ”wowed momma: 1. amuse—5 I H mowuoswma

 

 

 

 

E E E EH .3908
emnH eeo enoH one . n
He.m end am.~ owe em.m oem em.m «mm a
one eeH eeH emH H has em-o
DE EH bbb. E Hench.
oeo see New eon m
am.m mam ee.~ «em em.m _oeH ee.m «OH m
meH NMH we ea H on: «Hue
EH. NHL. E E annoy
was eee can onm m
em.~ end oe.m eeH nm.e om Hm.e on m
eeH we en en H an: hue
pub. E E E H.308
end and eeH eeH n
ea.m eHH em.m me mH.e mm mH.e eH m
as ed on on H was euo
E E E E Haven.
one mHH we eeH m
em.n ee ma.m em w~.e 4H mm.aH e a
em eH «H on , H anemone
oHdee .Ha<_ oHsem .Ha< oHsem .Had oHsee .Ha< H
..<|< dd filo mm a Hmogosum oomwom
umoaasouh n
deo<_sea<m mHHaeHo> do onaosnomm omaH> mm msHaedozao may .4 muses

- 45 -

with the cat straw innoculum-substrate treatment (00) and the
lowest with the alfalfa innoculum-oat straw substrate (AO) treat-
ment. The effect of innoculum source was nearly significant.

The differences in ratio were widest during the early part
of the fermentation. The 00 treatment produced very little pro-
pionic acid during the first two hours of fermentation as is
indicated by the high ratio (17.33) Observed. From the second
to the fourth hour the production of propionic acid became appre-
ciable and the ratio after 4 hours of fermentation was lowered
to 9.13. After 8 hours of fermentation the ratio decreased
further to 4.31.

There was a tendency for the ratio on all treatments to
decline indicating that the production of propionic acid became
relatively more important as the fermentation progressed.

Analysis of variance of the observations for each time
period indicate that alfalfa innoculum produced a significantly
(p .05) lower ratio of acetic to propionic acid up to 4 hours
of fermentation and that after this time there was no signifi-
cant difference in ratio although alfalfa innoculum tended to
produce a lower ratio throughout the fermentation.

A relatively high ratio of acetic to propionic acids was
observed after 2 hours of fermentation on all treatments. This
appears to be characteristic of roughages fermented under this
system. In a preliminary experiment a small amount of concen-
trate was added to the fermentation flasks. After two hours of
fermentation a low ratio of acetic to propionic acid was observed.

This was accompanied by a very rapid rate of fermentation. The

Microequivalents of Acid

-46..

2400 "' 0 AA
GIAO
v'OA
0.00

2000--

1600'P

12001-

 

 

 

 

800--
400+
° = a t t :
2 4 s 12 24
Hours

FIGURE 1. THE CUMULATIVE IN VITRO
PRODUCTION OF VOLATILE FATTY ACIDS

- 47 -

early rapid volatile acid production and low ratio observed when
concentrates were supplied indicate that they were readily attacked
by the rumen microflora. Further, their fermentation led to the
formation of proportionately greater amounts of propionic acid

than roughages.

The digestion of cellulose, holocellulose and hemicellulose:

 

In some cases the precipitate from centrifugation of the
contents of the fermentation flasks was analysed for holocell-
ulose, hemicellulose and cellulose. The results using these
methods were so variable and the method so cumbersome that they
had to be abandoned. It was noted, however, that the digestion
of holocellulose, hemicellulose and cellulose in whole plant
material proceeded at about the same rate.

Table 5 gives the results of the disappearance of holo-
cellulose, hemicellulose, and cellulose during a preliminary
experiment using oat hull holocellulose and alfalfa innoculum.
The disappearance of hemicellulose was determined by difference
so that much of the error involved in holocellulose and hemicell-
ulose determinations appears in this fraction. Little degra-
dation of either substrate appeared to take place before the 8-12
hour fermentation period. The production of volatile fatty acids
was also slow until this period. This would indicate that cat
hull holocellulose was attacked slowly under the conditions of
this experiment. The degradation of cellulose proceeded to a
greater extent (59%) than hemicellulose (46%). Due to the
variation involved in the determinations, this difference could

hardly be considered significant. However, it is apparent that

.pnomoum owoasHHoo one omoHsHHoooHon cookbon oomouommwo so oomms990¢

.oomwom on» mmwnso
oucuoouq owed ommommoum .ooa an owed canoes .ooa may mmmowbmo an oonmsuoo ownsmM

.oomwoo «saw one mmmuso ooosooum owed mo mumon>mnooowomzw
«owed omuoos I m newcommowo u N ”momma: moan commune u H mowuodumH

 

 

 

 

 

 

o.o¢ o.oH o.mm o.n ca. 0.0 eooconsooodumn
oocHsHHoomsom «cocoon
o.on o.om o.HN oo. o.H 0.0 oomdwsoqodoan
owoasHHoo mo «condom
.
no o.on H.hH o.>~ oo. N.e can oo\oomdusommsmma
4 ouoHsHHooOHo: «soouom
.
mm.d nn.H as. on.» III MH.H mofipom
E BF. Eh .mbH an... E Hooch
«do men OMH mo 11: me n .
cod mam voH «H as: 00 N N<m> poz
eon 3H 2. oo 2... ooo H 3:33.338on
.uwm .uw: .mum .uwm .mwm .uwm Hcomucswm
vmto oatmn «Him mic VIN «to

 

GOHmmm ZOHH<H2m2mmm Mm ZOHB<HzmzmMQ mmounaumooaom HAD: H<O .n mAm<H

- 49 -

hemicellulose, when it is in combination with cellulose as
holocellulose, is degraded at about the same rate and to about
the same extent as cellulose.

The greatest amount of hemicellulose (33%) disappeared
during the 8th to the 12th hour of fermentation while cellu-
lose was degraded appreciably during both the 8th to 12th hour
(2r%) and 12-24th hour (30%). During the period when the bulk
of hemicellulose disappeared the relative proportion of pro-
pionic acid produced as indicated by the low ratio (.79) was
greatest. This would indicate that hemicellulose, under the
conditions of this experiment, was fermented with the production
of relatively more propionic acid than was obtained from cell-
ulose. The results on fermentation of the various plant fractions,
discussed in Part III tend to support this conclusion.

The disappearance of cellulose on the various treatments
is summarized in Table 6. The results are expressed as the
weight (milligrams) of cellulose and as the percent of total
cellulose which disappeared during the time period considered.

The cat straw appeared to contain cellulose which was more
resistant to attack.than alfalfa cellulose. During the first
2 hours of fermentation on treatment 00 there was no apparent
disappearance of cellulose while on treatment A0 only 1.7%
of the digestion occurred. This is compared to 4.8% and 2.4%
disappearance on treatments AA and 0A. During the first 8
hours of fermentation an average of only 6.4% of the cellulose

had disappeared when oat straw was the substrate. In contrast

 

 

 

 

 

o.om o.om o.om m.on e.se e.oo ~.oo H.oo woo: eato
«.mH o.~H m.sH H.n~ o.Hn m.en s.o~ o.me doom omamH
e.o m.o o.MH m.o~ o.» o.HH n.» s.MH use: «Hum
o.s o.s o.m s.n e.s a.oH o.~ m.o and: one
-
.0 o.o s.o o.~ o.~ m.o o.o o.~ m.e doom one
5
. o.e o.m s.H n.~ e.m e.m oo oo doom Nuo
uHo s an oz an s mHa H: an s mHn o3 an a uHo o3
o.so o.neH e.eeH m.ooH one: o
Home»: ,Hmaoos suave: Aeneas.”
.3 dd mum .oao. ._ names
a
9:25.33. ”
mnoHemm onaeazmzmma am onemmoHo mmomommmo .o mHmey

'0“-

-51-

17.4% of the cellulose had disappeared after eight hours of
fermentation when alfalfa hay was the substrate. Over the total
fermentation period oat straw innoculum was able to degrade a
greater proportion of the cellulose present (43.8%) than alfalfa
innoculum (38.7%). In every case the total amount of cellulose
digested was greatest from the 12th to the 24th hour of fermen-
tation.

The weight of cellulose digested was appreciably less in
treatment AA than in the other treatments. However, this may
be due to the low (97.6 mg.) level of cellulose contained in
these flasks compared to that on the other treatments. The
amount of digestion late in the fermentation period was less
on this treatment than other treatments which contained greater
total amounts of cellulose. This indicates that the concen-

tration of substrate may have limited the disappearance of

cellulose.

‘ “7" .- vrfis‘ 91:53 9!? flirt-"A In!
I

“a“: a

- 52 -

PART III. THE FERMENTATION OF.ALFALFA AND OAT
STRAW’HOLOCELLULOSE, HEMICELLULOSE AND CELLULOSE
Procedure
There is some reason to believe that there is a difference
in the structure of cellulose from different plant materials

(Baker gl‘gl., 1957). The distribution and amount of lignin

" I?

also has been implicated in differences in forage utilization

 

(Crampton, 1957). Consequently, alfalfa and cat straw holo-
cellulose, hemicellulose and cellulose were prepared by the 3
methods of Wise 33‘31. (1946) to investigate these factors.
These were subjected to the same experimental treatments as the
whole plant in a 2 x 2 factorial design. The production of the
volatile fatty acids and the disappearance of cellulose were
measured.

Results

The production of volatile fatty acids from the holocellulose
of alfalfa hay and cat straw:

 

 

The volatile fatty acid production during each fermentation
period from holocellulose prepared from alfalfa hay and oat
straw is summarized in Table 7. There were no significant
differences in the total amounts of volatile fatty acids pro-
duced nor were there any differences in the ratios of acetic to
propionic acid due to treatment during the 24 hours of fermen-
tation.

There was a tendency for volatile acid production to be
greater when innoculum fermented holocellulose prepared from
the forage to which it was accumstomed (treatments AHA, alfalfa

innoculum-alfalfa holocellulose and OHO, oat straw innoculum-

 

.«coHdbwnoo oHsHm momma Ha OOH mom omos mumoHsbwnoecona so oommowmxo nunsoadw
.oHod omvood In women onoHooum I N Homes Honmmn nnHm oHuausm I H nonosumH

 

 

 

momw mmmw been meow Heoon
nos «ooH one nooH n
Hn.H one oo.n one as.H onn on.H noo n
ooH eon ooH noH H on: emno
me... E ohm E H38
on moo onn no n
oe. oHn oH.n ~Hn Ho. eon nn.o oHn n
on ooH on on H hum enaNH
E mom Won Nob H38.
won ooH «no MoHn n
no.H oHn Hn.H onH mn.n ooH om.n own n
on an oe oo H on: «Hun
mmm new. mmm mmm Hence
. ooH oHn one ooH n
.d on.H onH no.H NHH HH.n no on.n eo n
.o no on on on H has oue
.
.an on“ mph mun Hosea
ooH nnH now «on n
nH.n nn nn.n no on.n oo on.n no n
on oe do on H on: sun
urn bun pHH mwn. Hoods
ooH eon oo on n
on.n ee no.n eo on.~ on oH.n no n .
on .oo on on H on: nuo
oHoum .ua< oHHea .oa<x oHoom .Ha< oHoem ~.»a< ”
III III III II Hmoflvosum oonom
om< <m< emo omo H
umoausowh

.mQOHmmm ZOHH<Hzmzmmm am MH<demDm MMQADHHNUOHQ: zo
ZOHHUDOOmm QHU<.%HH<m mQHHZAO> OmHH> 2H .5 mqmdfi

-54..

oat straw holocellulose).

The greatest amount of volatile acids was produced by the
AHA treatment followed by the OED treatment. The cross~over
treatments produced fewer total acids with the oat straw
innoculum-alfalfa holocellulose (0H0) treatment slightly superior
to the alfalfa innoculum-oat straw holocellulose (AHO) treat- 5
ment. This is an indication that there may be a basic difference
in the holocellulose of alfalfa hay and oat straw.

In contrast to the fermentation of the whole forage the

-___ .__.._..__.__ -V
"' - .lnsv' H"? .~'-t 1"

average ratio of acetic to propionic acid tended to be highest 3
on the AHA treatment which produced the greatest amount of
volatile fatty acids and lowest on the ABC treatment which
produced the least amount of volatile acids. This indicates
that the higher average ratios Observed upon the fermentation
of oat straw as compared to alfalfa hay may be due to factors
other than holocellulose. The ratios of acetic to propionic
acid produced after the first 4 hours of fermentation were
much lower than those Observed when the whole forage was fer-
mented. ‘All except the.AHA treatment were less than 1 during
the last 12 hours of the 24 hour fermentation.

The very low ratios of acetic to propionic acid observed
during the last 12 hours of fermentation on treatments 0H0,
OHA and AHO were accompanied by a slowed rate of fermentation.
However, the.AHA treatment which had a higher (2.18) ratio for
the last 12 hours of fermentation produced a considerably

greater quantity of volatile acids than the other treatments

- 55 -

during this period. It is possible that the slowing of the
fermentation rate on the other three treatments may have had
an influence in causing the low ratios at that time.

The volatile acid production during the early stages of
fermentation was greater for alfalfa innoculum than for oat
straw innoculum as was observed during the fermentation of 5
whole alfalfa hay and oat straw. It was also greater when

innoculum fermented holocellulose prepared from the same forage

sans-J nu
‘ p p. ,

fed to the animal which served as an innoculum donor.

‘2‘.“ 21 .

Cellulose disappearance 22 the fermentation of the holocellulose

of alfalfa hay and oat straw:

The amounts (percent and milligrams) of cellulose degraded
when alfalfa and oat straw holocellulose were fermented are
presented in Table 8. There were no significant differences
in the extent of cellulose degradation due to treatment.

As with the cellulose of whole alfalfa hay and oat straw
there is an indication that oat straw cellulose was more re-
sistant to degradation early in the fermentation than alfalfa
cellulose. Only 2.7% of the cellulose on the OED treatment
and none of the cellulose on the ABC treatment was degraded
during the first two hours of fermentation, while 7.5% of the
alfalfa substrate on the AHA treatment and 8.6% of the cellulose
was degraded on the OHA treatment. This would indicate that
the resistance of oat straw cellulose to degradation observed
early in the fermentation of the whole plant forages is not
due to lignification since most of the lignin is removed during

the preparation of holocellulose.

l...
Mains... ...- 29...; . ... . a . 4.}. finely.

omoasflaooonon wmaomad I aaqsooomw Iowan pace
onOHsHHoooHon soup- uoo I auHsoommw sauna «wow
omOHsHHeooHom audomudsauauoonmm dwdwmn<w
omoHsHHeoonon tempo «sonasasooumm «MHdMH<H

 

o.na n.nn n.on e.enH e.no m.no n.nn n.ns use: enuo
n.nn o.nn n.n¢ o.Hn m.nn n.en m.nn n.nn anon enan
o.oH H.nn o.on n.nn n.oH c.HH n.Hn n.nn use: nHun
. H.»H a.aH o.nH n.nn o.HH n.nH n.nH e.en "so: nun
x n.n n.H n6 o.HH «.0 n6 0; o.n use: in
. o.n o.HH s.n n.e n.n ¢.n o.oo o.oo use: nno

mun fl man #3 “Mn 3 mwn «3 “an fl man #3 mun R man 93

'-'-"'l"' "-‘-' '|--""'-"-'--|'-'-""--' " "g- '--"-"-"-'-""-- :I'-'--':-

 

 

N.HNH N.an c.50H N.oNH use: 0
we we us me a

A on: A on: A can. A vpz.. ooHuum
.III. .IIII _IIII Illl .
vemo nOEO N<m< H024 .
«ooapdowfl H

 

mH<mBmme mmoqagqmuouom .moz<m<mmm<mHn NMQADQAMU om mumdfi

I .l to t
‘ I I )
\l-‘||l'l"l" 14". .l‘ll‘l'llut'lllu ‘lll"!'|llvl '10 .v ‘III III illlllnl'llrl'lll'"|lllll lllll’ls‘l'lll
.II
I I I O D w. 0 9
ll
0 O I an O I 0 .
D O O I l U D O
I]
O O D O I 9 O h
I.
I O O I l O O O .
a o O I O I c 0
I.
.II
I.

- 57 -

Oat straw innoculum tended to degrade more of the cellu-
lose over the entire fermentation period than alfalfa innoculum.
After four hours of fermentation all treatments had begun to
degrade the cellulose extensively so that the extent of cellulose
degradation was greater in every case than when the whole forages

were fermented. P

The production of volatile fatty acids from the cellulose of
alfalfa hay and oat straw: a

 

 

The volatile acid production during each fermentation
period from the fermentation of the cellulose of alfalfa hay
and oat straw is summarized in Table 9._ There were no signifi-
cant differences in the total volatile acid production nor
in the ratios of acetic to propionic acid due to treatment.

The volatile acid production tended to be greater when oat
straw cellulose was fermented. However, this difference may
have been due to the greater amount of cellulose present in
these flasks. Treatment 0C0 (oat straw innoculum-oat straw
cellulose) contained 178.7 mg. and.ACO 175.4 mg. of cellulose
while treatment OCA (oat straw innoculum-alfalfa substrate)
contained 75.6 mg. and treatment.ACA (alfalfa innoculum-alfalfa
substrate) 73.3 mg. of cellulose. The discrepancy in the
amounts of substrate available for fermentation in these
treatments and the greater amounts of volatile acids produced
on treatments which contained greater amounts of cellulose
lead to the conclusion that the quantity of cellulose in the
treatments containing alfalfa cellulose limited the volatile

acid production.

- 53 -

owmonouo\oHuoon mo omwnmm
omen mo nunoHo>HnooonoHa on monmouome
.oHuo oHpooo I m “oHon UHnommouo I N ”nomad noann + oHu>usm I H GOHHouumH

 

 

 

E E E EH H38.
onHH Nob ewe woo m
no.H eon No.N enN eH.N 0Nm Ho.H NNn N
enN on eeH NuH H mum eNuo
wmww mm» bmm mMHH Hence
0mm omm eHm eHh m
ow.H one mn.N NmH No.H eoH mw.H 0mm N
oeH va Nn vb H mum eNnNH
F nob E E H38.
oN NHH N5 N0 m
nN.o no nn.N we 5N.m NN no.N on N
no on wN mm H mum NHuw
mm». .ppH mbH NhH Hanan
eoH we wHH me n
mo.m we mm.N eN oe.N we Hm.H Nn N
we we on VH H mm: wue
E E E E H38.
OOH Ne on cmH m
nN.o 0H oo.N on eo.N wN mw.N we N
Ne 0N oH oN H on: qu
bbl chm MPH .mHl H33
oo emH 00H wH m
oo.o 00 on.» wH on.m mN on.H NH N
co mm wH mH H on: Nno
oHudm .«a<. oHuam .Ha< oHuem .Ha< momaam N.Hn<.Hn0HHoonm o0Huum
coo compounoanom
commandouh lw~

 

GOHmmm ZOHB<Hzmzmmm hm mmogaqam0_3<mkm H<O Qz< (NH<NA<.QO
ZOHH<Hzmzmmm mmH GZHMDD ZOHHUDQOKQ GHU<.NHB<m NAHH<AQ>

.o mqmdfi

The ratio of acetic to propionic acid tended to be higher
when alfalfa cellulose was the substrate. As with holocellulose
this trend is opposite to that observed with the whole plant
material. These observations would lead to the conclusion that

the cellulose and holocellulose of alfalfa hay are not respon-

":7

sible for the tendency of alfalfa hay to produce a lower ratio
of acetic to propionic acid. The fact that the lower ratio
observed when the fermentation of alfalfa hay was compared to

that of oat straw was significantly lower only during the first

w _.-...4‘ .4 “u‘.- Lanna—n.

four hours of fermentation would support this conclusion since
little holocellulose appears to be degraded during the early

part of the fermentation.

Cellulose disappearance 22 the fermentation of the cellulose
of alfalfa hay and oat straw:

The amounts (percent and milligrams) of cellulose degraded
when alfalfa and oat straw cellulose were fermented are pre-
sented in Table 10. There were no significant differences in
the percents of cellulose degraded due to treatment.

The treatments which contained oat straw cellulose (OCO
and.ACO) degraded more cellulose than those containing alfalfa
cellulose (ACA and OCA). However, there were considerably
greater amounts of cellulose present in the treatments contain-
ing oat straw cellulose. The differences in amounts of cell-
ulose degraded were probably influenced by the amounts available
for fermentation.

In the case of the percents of cellulose degraded it is

apparent that oat straw innoculum tended to degrade cellulose

 

onOHsHHoo snuun neonasHsoonnH «MHnMH<e
onOHsHHoo dedemnasHsoounH omHan<n
omOHsHHoo «MHanmuaanoonnH rumen HuON
onOHnHHoo sauna neonaanoonnH rowan udoH

 

 

 

 

n.0n o.enH o.nn n.0n Ho.ne H.Hn n.nn H.eoH .u: enuo
oH.nn n.na H.nn n.nn oH.on o.nH o.o¢ n.Hn .um enunH
on.nH n.nn n.eH o.HH Hn.n o.e n.n H.o .um nH-n
.
o on.o n.HH o.oH n.nH on.n n.n o. e.H .um n-n
6
. on.n n.n H.n n.n o¢.n a.o 0.0 H.HH .um n-n
co. H.H oo. o. oo. o. «o. n. .u: nuo
uHo e an as. uHo n mHn a: mHo n uHa 9: “Ho a nHa a:
e.nnH n.n» n.na n.naH "go: 0
$5 a: $5 a: $5 a: 35 “E ”
III III . . oomuom
<
e8 n<u< neoo Hooo .
Hmoavmoufi H

 

mmOHDAHmU 35am H<O Q72 <mu<mu< mo 20 HH<Hzmzmmm
9.5. 20 mogfimaHn mmOn—Hfi—Qmo .OH mama

- 61 -

less completely than alfalfa innoculum. Treatment OCO degraded
59.3% while treatment OCA degraded 43.6% of the cellulose present
as compared to 76.7% and 75.0% for treatments ACA and ACO respec-
tively.

All treatments attacked the cellulose rather slowly with
negligible amounts of cellulose being degraded during the first
two hours of fermentation. Alfalfa cellulose appeared to be
more readily attacked than oat straw cellulose. After 8 hours
of fermentation treatment ACA had degraded 21.1% and treatment
OCA 11.7% of the cellulose present as compared to 6.9% and
9.5% for treatments 0C0 and ACA respectively. These figures
also show that alfalfa innoculum was more active cellulolyti-
cally early in the fermentation. Oat straw innoculum appeared
to be relatively inactive through the first 12 hours of fermen-
tation. Most of the cellulose degradation took place during
the last 12 hours. The low values observed for total cellulose
degradation by oat straw innoculum suggest that this particular
batch may have been inferior to that used in the fermentation
of holocellulose where the extent of degradation of cellulose

with oat straw innoculum was much greater.

The fermentation of hemicellulose:

This experiment contained only two treatments since the
preparation of hemicellulose yielded small amounts and not enough
was collected for the usual 2 x 2 factorial experiment. A1-
falfa innoculum was used to ferment both alfalfa and oat straw
hemicellulose. The results of this experiment are summarized

in Table 11.

I..- five-I- ‘. A ‘ m."
‘ I

saw“.

-62-

TABLE 11. VOLATILB FATTY ACID PRODUCTION BY
PERIODS FROM THE IN VITRO FERMENTATION OF ALFALFA
AND OAT STRAW HEMICELLULOSE BY.ALFALFA INNOCULUM.

 

 

J ."

 

: Treatment
Fermentation I
Period Practionlt éEé. AEQ
. Amt.2 Ratio3 Amt. Ratio
0-2 Hrs. l 40 28
2 82 2.34 62 2.19
3 192 136
Total 3T1 223
2-4 Hrs. 1 22 20
2 68 1.94 104 2.12
3 132 220
Total 222' an
4-8 Hrs. 1 60 54
2 54 2.56 52 2.19
3 138 114
Total 232 ‘226
8-12 Hrs. l 42 20
2 00 --- 70 2.29
3 34 160
Total 76 236
12-24 Hrs. 1 78 114
2 158 .91 158 1.32
3 144 208
Total 385 185
0-24 Hrs. l 242 236
2 358 1.79 450 ' 1.86
3 640 838
Total 1246 1324

 

_ __., ---.-
‘2." a... . ~

 

1Fraction 1 = butyric plus higher acids; 2 = propionic acid;
3 a acetic acid.

ZMicroequivalents acid.
3Ratio acetic/propionic acid.

- 63 -

The apparent preference of innocula for familiar substrate
is reflected in this fermentation as it was in the case of all
other experiments. The alfalfa innoculum alfalfa substrate
treatment (AKA) produced a greater amount of volatile fatty
acids during the first two hours of fermentation than the al-
falfa innoculum-oat straw hemicellulose combination (AXO).

This lag in fermentation indicates that there may be basic 5
structural differences in the hemicellulose of alfalfa and oat
straw not related to lignin since essentially all of the lignin ;
was removed during the preparation of hemicellulose. i

From the 2nd to the 4th hour the AXO production of volatile
acids surpassed that of the AKA treatment. There was slightly
more total acids produced on the AXO treatment for the entire
fermentation period. The ratios of acetic to propionic acids

produced on these two treatments did not differ greatly.

- 64 -

DISCUSSION

Cellulose Degradation of the Whole
Plant and Fractions of the Plant

The percents of total cellulose degraded for the 24 hour

fermentation period on the various treatments are summarized

in Table 12.

TABLE 12. PERCENT CELLULOSB DEGRADED
DURING THE 24 HOURS OF PBRMENTATION

 

 

Substrate Treatment

00 0A ' A0 AA
Whole Plant 40.3 47.4 38.8 37.8
Holocellulose 89.3 75.9 73.2 62.4
Cellulose 59.3 43.6 76.7 75.0

 

In all cases the purified products were degraded to a
greater extent than the natural materials, except for the oat
straw innoculum-alfalfa cellulose treatment. This is a treat-
ment which had a low cellulose concentration in the fermentation
flask and apparently a relatively inactive innoculum. It is
possible that this value does not represent the normal case.
This reasoning is supported further by the fact that oat straw
innoculum, which was obtained on a different day, degraded the
cellulose of alfalfa holocellulose further than cellulose con-
tained in the whole forage.

Analysis of variance of the cellulose degradation of whole
forage cellulose versus the cellulose degradation of holocellulose
and cellulose prepared from the forage indicates that a signifi-

cantly (p <.05) greater percentage of cellulose was degraded

_65-

in the plant fractions.

Salsbury 33.31. (1956) also obtained increased cellulose
digestion when they compared the in ZiEEE digestion of the in-
tact plant with cellulose prepared from that plant.

The increased degradation Observed in the plant separates

may be attributed to several causes. In preparation of cellulose

‘. Rafi]-

and helocellulose the treatment may partially degrade the cell- a
ulose making it more easily attacked by rumen microflora. Lignin

is removed during the preparation of the holocellulose and cell-

" tr all"; .‘a- In, wwagzr-n I.

ulose. This may also have been a factor in the more complete
degradation of the material.

The fermentation of holocellulose led to the production of
a significantly lower (p<:.05) ratio of acetic to propiOnic
acid than either cellulose or intact forages. Since hemicellulose
and cellulose make up holocellulose the inference is that hemi-
cellulose fermentation would lead to the production of a low
ratio of acetic to propionic acids. This was found to be the
case when hemicellulose was fermented although the ratio was
not less than that observed on the fermentation of holocellulose.

The Importance of Cellulose
to Volatile Fatty Acid Formation

Figure 2 is a visual projection of the microequivalents of
volatile fatty acids produced per milligram of cellulose degraded
for each treatment. This is an expression of the relative im-
portance of the degradation of cellulose to the production of

volatile fatty acids.

-66...

Qmm<mwma mmOQDHHmU m0 dewHHHHE mmm QNUDDOMQ

5.5 no wazmmsrgomomgz

onoHsHHoU

<U< OU<

<00 OUO

onoHsHHoooHom
<m< OE< <m0 030

.N mmDGHm

<<

omeuom oHooz

O<

<0

00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0H

ON

on

em

on

00

Oh

ow

muJSttItw Jed Sinateatnbaoxotw

- 57 -

The first four bars represent the relation between volatile
acid production and cellulose degradation when the whole forages
were fermented. The oat straw innoculum-oat straw substrate com-
bination (00) produced the fewest microequivalents of volatile
fatty acids for each milligram of cellulose fermented followed
by the oat straw innoculum-alfalfa substrate treatment (0A) and
the alfalfa innoculum-oat straw substrate treatment with the
alfalfa innoculum-alfalfa substrate treatment producing the
greatest total amount of volatile fatty acids for each milligram
of cellulose degraded.

Since there are undoubtedly materials other than cellulose
which contribute to the formation of volatile fatty acids, the
lower values are indicative of a greater importance of cellu-
lose to the formation of volatile fatty acids. Therefore, the
cellulose of oat straw was more important than that of alfalfa
in the formation of volatile fatty acids. Oat straw innoculum
also depended on the degradation of cellulose to a greater ex-
tent than alfalfa innoculum. These relationships suggest that
alfalfa contains more materials other than cellulose which con-
tribute to the formation of volatile acids.

It is also apparent that alfalfa innoculum must have pro-
duced more volatile acids from material carried over in the
innoculum since it was able to produce more volatile acids per
mg. of cellulose degraded than oat straw innoculum. The AA
treatment produced more than twice the amount of volatile acids
per milligram of cellulose degraded than any other treatment.

The reason for this is not readily apparent since only a small

- 68 -

increase in volatile acid production can be accounted for by
the difference in substrate. A larger increase may be attributed
to the source of innoculum.

An attempt was made to put a quantitative estimate on the
effect of innoculum and substrate by subtracting the value
for 00 from cross-over treatments. Thus 0A-00 8 7.52 micro-
equivalents of additional microequivalents volatile acids pro-
duced per mg. cellulose degraded due to alfalfa substrate versus
oat straw substrate and A0-00 = 13.05 microequivalents of addi-
tional volatile fatty acids produced due to innoculum. The sum
of these effects equals 20.57 microequivalents of additional
volatile fatty acids which can be attributed to alfalfa innoculum
and alfalfa substrate if no other factors influenced the volatile
acid production. The measured difference between treatments
00 and AA was 51.92 microequivalents. Thus the differences
in volatile acid production due to source of innoculum and
substrate can account for only about half of the actual measured
difference between treatments AA and 00.

'When the substrate was holocellulose a slightly different
relationship was obtained. Treatment 0H0 (oat straw innoculum-
oat straw holocellulose) continued to depend to a greater extent
on the degradation of cellulose for the production of.volati1e
fatty acids than any other treatment. The relative rank of the
cross-over experiments was reversed with AHO being less than
OHA. The alfalfa innoculum-alfalfa holocellulose treatment
appeared to depend to the least extent on the degradation of

cellulose for volatile acid production.

-69...

If the effect of substrate and innoculum are estimated
as before, it is seen that OHA-OHO a 6.42 microequivalents more
volatile fatty acids produced per milligram of cellulose degraded
on the fermentation of alfalfa holocellulose versus oat straw
holocellulose. There were 3.95 additional microequivalents of
volatile fatty acids produced per milligram of cellulose de-
graded when alfalfa innoculum fermented oat straw holocellulose
(0H0) as compared to oat straw innoculum fermenting oat straw
holocellulose (AHO). The sum of these differences (10.37)
represents the additional microequivalents of volatile acids
produced per milligram of cellulose degraded due to the effect
alfalfa innoculum versus oat straw innoculum and alfalfa holo-
cellulose versus oat straw holocellulose. -The measured dif-‘
ference between treatments CEO and AHA was 27.80 microequiva-
lents of volatile fatty acids per milligram of cellulose de-
graded. Thus, differences in the holocellulose of alfalfa and
oat straw and in alfalfa and oat straw innoculum can account
for only 37% of the measured difference in microequivalents of
volatile acids produced per milligram of cellulose degraded.

The relationships when oat straw and alfalfa cellulose was
fermented are different from those Observed on the fermentation
of the whole forage and holocellulose. The oat straw innoculum-
alfalfa cellulose treatment (OCA) produced the most microequi-
valents of volatile fatty acids per milligram of cellulose de-
graded followed by the alfalfa innoculum alfalfa cellulose
treatment (ACA), the oat straw innoculum-oat straw cellulose
treatment (0C0) and the alfalfa innoculum-oat straw cellulose

treatment (ACO).

-70..

The results on the oat straw innoculum treatments (0C0 and
OCA) appear to be in error since the removal of all fermentable
material except cellulose should lower the measured values for
the microequivalents of volatile fatty acids produced per milli-
gram of cellulose degraded. The oat straw innoculum used in
this particular experiment appeared to be less active than innoc- a:
ula procured for previous experiments. There was a small amount

of cellulose degraded and a relatively small amount of volatile

 

acids produced particularly on the OCA treatment. This would

tend to increase the values observed since volatile acids pro-

Tj-rr- -.-. .~

duced from material carried over in the innoculum would be
relatively more important when small amounts of cellulose were
degraded. The values for alfalfa innoculum treatments (ACO
and ACA) decreased as was expected. It was also relatively
more active cellulolytically than oat straw innoculum.

Because of the apparent discrepancy with oat straw innoc-
ulum, comparisons such as that made on previous experiments
cannot be made. However, it appears from the relation on the
ACA and ACO treatments that one milligram of alfalfa cellulose
will produce more microequivalents of volatile fatty acids than
one milligram of oat straw cellulose.

With the exception of the 000 and OCA treatments there was
a decrease in the amount of volatile acids produced as the sub-
strate was simplified.

Apparently, factors other than fermentable substrate and
innoculum source caused the production of greater amounts of

volatile fatty acids on the alfalfa innoculum-substrate treat-

-71..

ment. This may be related to the greater susceptibility of
alfalfa hay and alfalfa holocellulose and cellulose to bacterial
attack. Because of this the.i2.!i££2 fermentation was usually
more active early in the fermentation period. This could have
resulted in maintenance of a greater number and variety of
microflora which were able to degrade cellulose with a greater
efficiency of volatile fatty acid production. I
The Rate of Voluntary

Consumption and Fermentation

The results of these experiments support the contention
of Crampton (1957), McCullough (1959) and Blaxter (1956) that
the worth of a forage depends on its voluntary consumption by
animals. When the alfalfa hay and oat straw rations were fed
29 libitum to cows during a digestion trial (Part I) the 4
cows consuming alfalfa hay ate an average of 23 pounds per
head daily. The cows receiving the oat straw ration would eat
an average of only 14 pounds of straw per day. One of these
straw fed cows would not consume uniform amounts. The actual
measured TDN values were not widely different being 64.0% for
alfalfa hay and 61.6% for oat straw. Consequently, the volun-
tary consumption would be a limiting factor to the productive
value of the oat straw.

The method of in 21332 fermentation used in these experi- '
ments showed that alfalfa hay was fermented at a faster rate
with the production of greater amounts of volatile fatty acids
than oat straw. Crampton (1956) hypothesized that the rate of

digestion and rate of passage of food through the digestive

 

- 72 -

tract could be related to the voluntary consumption of the

food. The recent work of Meyer 53,31. (1959) with pelleted
rations seems to bear this out. These experiments show that
alfalfa hay is fermented at a faster rate and consumed to a

greater extent than oat straw.
The Ratio of Acetic to Propionic Acid

The ratios of acetic to propionic acid produced on the 5

in vitro fermentation of alfalfa hay and oat straw were not

“.1-.. .,
.

significantly different after a 24 hour fermentation period.
This does not necessarily mean that different ratios do not
occur on in_yiyg fermentation of alfalfa hay and oat straw.

When the ratios produced after 2, 4, 8, 12 and 24 hours
of fermentation were compared, it was found that for the first
four hours the fermentation of alfalfa hay resulted in the pro-
duction of a significantly (p<:.05) lower ratio of acetic to
propionic acid. At the next and subsequent observation points
no significant differences in ratio occurred.

It may be significant that the ratios on all treatments
declined as the 22;!i££2 fermentation progressed. The same
observation was made by Gray £3 31. (1952) who also noted that
a similar decline did not occur ig'yiyg.

Leffel (1958) stated that after 2 hours of 32.!iEES fer-
mentation the proportions of volatile fatty acids produced
had changed from those observed in yiyg. Warner (1956) deter-
mined that after 8 hours, EEHZEEEE fermentations conducted

under the best of conditions were no longer representative of

- 73 -

those expected in yiyg. Maki and Foster (1957) experienced
great difficulty in culturing acetic acid producing bacteria
and concluded that they required strictly controlled conditions
which were difficult to produce in the laboratory. Therefore,
the tendency for 32.133£2 rwmen fermentations to produce lower

amounts of acetic acid than expected on the basis of in vivo

 

observations could be due to the reluctance of acetic acid
bacteria to grow under laboratory conditions. The iEHZEEES
decline in acetic acid proportion may be Characteristic of the
fermentation system and not of the substrate being fermented.
From these considerations and from the results of the ex-
periments reported herein it is considered to be entirely
possible that under in yiyg conditions there actually is a
greater proportion of acetic acid produced on the fermentation
of oat straw than on the fermentation of alfalfa hay. It may
be concluded that the in yi££g method of fermentation does not
furnish accurate information about the relative proportions of

volatile fatty acids produced on in vivo rumen fermentation.
Source of Innoculum

Significant differences in the rate, extent and proportions
of volatile acids produced occurred only when forages were
fermented with innoculum obtained from animals which were main-
tained on that forage. This would indicate that the substrate
fermented exerts its influence on the fermentation through a

selection of the types of microflora in predominance.

- 74 -

Asplund s£_al. (1958) also observed the importance of
source of innoculum on the production of volatile fatty acids.
In contrast to the results reported here, innoculum from an
alfalfa fed animal produced a significantly higher ratio of
acetic to propionic acid as compared to innoculum from an oat
straw fed animal after 24 hours of fermentation. They also
obtained the greatest differences in volatile acid production
when innocula fermented familiar substrates.

Other workers have noted variations in innocula obtained
from animals fed different rations. Most were concerned with
the comparison of microflora from concentrate fed ruminants
as compared to that from animals receiving roughages (Gall
‘gg‘gl., 1949; Burroughs £5.2l" 1950; Hunt 53 31., 1952;
Hungate £1.3l" 1952). However, Bryant and Burkey (1953)
reported that the flora from an oat straw fed cow was less
complex than that from an alfalfa fed cow.

These experiments and those of Asplund E£.El° (1958) lead
to the conclusion that important differences exist in the types
of microflora in predominance and the fermentation produced
by the microflora obtained from animals fed different kinds of
forage. Thus, in yiigg comparisons of various forages are
most likely to show significant differences when the innoculum
for in ZiEEE fermentation is obtained from an animal which has
been maintained on that particular forage so that the innoculum

actually represents the forage.

 

- 75 -
Holocellulose, Hemicellulose and Cellulose

The degradation of holocellulose, hemicellulose and cellu-
lose as a part of the whole forage proceeded at about the same
rate and to the same extent. This indicates that these compo-
nents may be in close contact with each other in the plant and
when their structure has become broken down they are utilized
equally well by rumen microflora.

The content of hemicellulose in many forages is nearly as
great as that of cellulose. Hansen 3115;. (1958) reported
an average of 25.2% (range 12.1% to 39.9%) hemicellulose in
common forages. The cellulose content in a similar group of
forages ranged from 27.4 to 52.9% and averaged 32.6%. There-
fore, the hemicellulose fraction could make a large contribution
to the volatile acids produced on rumen fermentation of forages.

The ratios of volatile acids produced on the fermentation
of the isolated plant fractions (cellulose, holocellulose and
hemicellulose) were less than those produced when the whole
forage was fermented. Barnett and Reid (1957a, 1957b) obtained
similar results. They concluded from this that something
other than cellulose or other complex carbohydrates was respon-

sible for the high proportions of acetic acid produced in vivo

 

on forages. Gray and Pilgrim (1952a, 1952b) concluded from
similar observations that the discrepancy between £2.1i352
and in yiyg volatile fatty acid production was due to the
preferential absorption of propionic acid from the rumen.

Neither of these explanations have been entirely satisfactory.

- 75 -

The low ratios of volatile acid production on the fermen-
tation of complex plant carbohydrates have been very difficult
to resolve in view of the consistently high correlation of
the proportion of acetic acid produced with the amount of these
complex carbohydrates contained in forages (Phillipson, 1952;
Johns, 1955; Jamieson, 1959; Balch and Rowland, 1957; Card
and Shultz, 1953; Reid, 1957; Stoddard £3 31., 1949 and
others). Perhaps Barnett and Reid (1957) have indicated the
reason for these inconsistencies when they Observed that the
ratio of acetic to propionic acid produced depended, not so
much upon the particular component considered but upon the ease
of its fermentation, the more easily fermented materials result-
ing in the production of a lower proportion of acetic acid.

The procedures used in isolating complex carbohydrates
from plant material could be responsible for the low ratio
observed on the fermentation of these fractions. These pro-
cedures subject the material to drastic conditions which could
conceivably alter the original structure considerably. As
Simple an operation as grinding hay (Powell, 1939; Balch, 1952,
1959) has been observed to cause a lowering of the proportion’
of acetic acid produced on its rumen fermentation. Cooking or
heating has also been Shown to Change the volatile acid pro-
duction (Balch g: 31., 1955a; Shaw 3: 31., 1959; Ens r‘gt 31.,
1959).

Thus it is not inconceivable that the preparative proce-
dures coupled with the characteristics of the in ZEEES fermen-

tation all combine to cause the in vitro fermentation of purified

- 77 -

plant fractions to result in the formation of relatively greater
proportions of propionic acid than fermentation as part of the
whole forage in yiyg, and that cellulose and hemicellulose as
part of the whole forage could be important precursors of

acetic acid in ruminants.

It was realized that these difficulties were inherent in
the iE.ZiE£2 system of rumen fermentation before the investi-
gation began; however, it was hoped that by using the compar-
ative technique, differences in the volatile acid production
on various feeds could be estimated. It was shown with this
approach that the fennentation of alfalfa hay with innoculum
obtained from an alfalfa fed steer as compared to the fermenta-
tion of oat straw with innoculum from an oat straw fed steer
resulted in a significantly (p<<.05) lower ratio of acetic
to propionic acid only during the first four hours of fermen-
tation, and that at the next time of observation after 8 hours
of fermentation this difference was no longer significant.

If there is a real difference in the proportions of volatile
acids produced from alfalfa versus oat straw'in'yiyg, these
observations tend to agree with those of Harner (1956) who
concluded that after 8 hours the 12.!iEEE fermentation no
longer reflects the in_yiyg fermentation.

A technique such as that suggested by Stewart 53 El.
(1958) which utilizes a series of Short term £2.Zi££2 fer-
mentations to estimate the production of individual volatile
fatty acids may be necessary for accurate prediction of in

vivo volatile fatty acid production.

- 73 -
SUMMARY

Oat straw and alfalfa-bromegrass hay were compared by
conventional total digestible nutrient determinations, by in
yiigg volatile fatty acid production and cellulose digestibility.

The alfalfa-bromegrass hay was found to have an average
of only 2.4% more TDN than oat straw. Pour Holstein cows volun-
tarily consumed an average of 23 pounds of alfalfa-bromegrass
hay per head daily; whereas, a similar group of 4 Holstein cows
would consume an average of only 14 pounds of oat straw per head
daily. On 12.Xi££2 rumen fermentation alfalfa hay produced
volatile fatty acids at a significantly (p<<.05) faster rate
than oat straw. The acetic to propionic acid ratio was (p‘<305)
significantly lower for alfalfa-bromegrass hay for the first
four hours, but not significantly different for the balance of
the 24 hour 12.31352 fermentation. The percent cellulose diges-
tion on EEHZEEES fermentation of alfalfa-bromegrass hay and oat
straw was not significantly different.

Differences were significant only when innocula for the
fermentations were obtained from steers fed the same forage as
that being used for in yiigg substrate.

The volatile fatty acid production (amount and ratio) and
cellulose digestion of holocellulose and cellulose prepared from
alfalfa-bromegrass hay and cat straw was not significantly dif-
ferent although early in the fermentation period cellulose di-
gestion and volatile fatty acid production tended to be greater
when innoculum fermented familiar substrate. Holocellulose and

hemicellulose produced a significantly (p<(.05) lower ratio of

- 79 -

acetic to propionic acid than whole forages or cellulose pre-
pared from whole forages. The digestibility of the cellulose
of holocellulose and cellulose prepared from the intact forage
was significantly (p<:.05) greater than the digestibility of
cellulose in the intact forage.

Although the in 11352 method utilized was able to distin-
guish between alfalfa hay and oat straw with respect to quanti-
tative volatile fatty acid production the results on proportions
of volatile fatty acids produced are considered inconclusive.
The proportions of volatile fatty acids produced after 8 hours
or longer of in yiigg fermentation appear to be more character-
istic of the fermentation system than of the substrate being
fermented. A short term ig_yi££g fermentation of less than
8 hours appears to be necessary for a differentiation between

forages on the basis of proportions of volatile fatty acid.

- 80 _
LITERATURE CITED

Annison, E. F. 1954. Some observation on volatile fatty acids
in the sheep's rumen. Biochem. J. 57:400.

Annison, E. F. ’1956. Nitrogen metabolism in the sheep. Protein
digestion in the rumen. Biochem. J. 64:705.

Annison, E. F., K. J. Hill and D. Lewis. 1957. Studies on the
portal blood of sheep. II. Absorption of volatile fatty acids
from the rumen of the sheep. Biochem. J. 66:592.

Annison, B. F. and D. Lewis. 1959. Metabolism in the Rumen,
New York: John Wiley and Sons, Inc.

Armsby, H. P. 1917. The Nutrition of Farm Animals. New York;
The Macmillan Co.

Armstrong, D. G. and K. L. Blaxter. 1956. Heat increments
of feeding in ruminants. Nature 177:1183.

Armstrong, D. G., K. L. Blaxter and N. McC. Graham. 1957.
The heat increments of steam-volatile fatty acids in fasting
sheep. J. Nutr. 11:393.

Armstrong, D. G. and K. L. Blaxter. 1957. The utilization of
acetic, propionic and butyric acids by fattening sheep.
Brit. J. Nutr. 11:413.

Armstrong, D. G. and K. L. Blaxter. 1957. The heat increment
of steam volatile fatty acids. Brit. J. Nutr. 11:247.

Armstrong, D. G., K. L. Blaxter, N. McC. Graham and F. W. Wainman.
1958. The utilization of the energy of two mixtures of
steam-volatile fatty acids by fattening sheep. Brit. J.

Nutr. 12:177.

Asplund, J. M., R. T. Berg, L. W. McBlroy and W. J. Pigden. 1958.
Dry matter loss and volatile fatty acid production in the
artificial rumen as indexes of forage quality. Can. J.

Animal Sci. 38:171.

Axellson, Joel. 1953. Relationships between contents of
metabolizable energy, total digestible nutrients, Scandina-
vian feed units and starch units in the foodstuffs. Annals
of the Royal College of Sweden 19:145.

Baker, T. I., G. V. Quicke, O. G. Bentley, R. R. Johnson and
A. L. Moxon. 1959. The influence of certain physical pro-
perties of purified celluloses and forage celluloses on their
digestibility by rumen micro-organisms in vitro. J. Animal
Sci. 18:655. "'

- 31 -

Balch, C. C., D..A. Balch, S. Bartlett, C. P. Cox., 3. J. Rowland.
1952. Studies of the secretion of milk of low fat content
by cows on diets low in hay and high in concentrates. I. The
effect of variations in the amount of hay. J. Dairy Res. 19:39.

Balch, C. C., D. A. Balch, S. Bartlett, V. W. Johnson, S. J.
Rowland and ill Turner. 1954. Studies of the secretion of
milk of low at content by cows on diets low in hay and high
in concentrates. IV. The effect of variations in the intake
of digestible nutrients. J. Dairy Res. 20:305.

Balch, C. C., D. A. Balch, S. Bartlett, 2. D. Hasking, V. W.
Johnson, S. J. Rowland, and J. Turner. 1955a. Studies of
the secretion of milk of low fat content by cows on diets
low in hay and high in concentrates. V. The importance of
the type of starch in the concentrates. J. Dairy Res. 22:10.

Balch, C. 0., D. A. Balch, S. Bartlett, M. P. Bartrum, V. M.
Johnson, 8. J. Rowland and J. Turner. 1955b. Studies of the
secretion of milk of low fat content by cows on diets low in
hay and high in concentrates. VI. The effect of the physical
and biochemical process of the reticulo-rumen. J. Dairy
Res. 22:270.

Balch, C. C. 1957. Use of the lignin-ratio technique for de-
termining the extent of digestion in the reticulo-rumen of
the cow. Brit. J. Nutr. 11:213.

Balch, D. A. 1958. An estimate of the weight of VFA produced
in the rumen of lactating cows on a diet of hay and concen-
trates. Brit. J. Nutr. 12:18.

Balch, D. A. and S. J. Rowland. 1957. Volatile fatty acids and
lactic acid in the rumen of dairy cows receiving a variety
of diets. Brit. J. Nutr. 11:288.

Barcroft, J., R. A. McAnally and A. T. Phillipson. 1943. Ab-
sorption of volatile fatty acids from the alimentary tract of
the sheep and other animals. J. Exptl. Biol. 20:120.

Barnett, A. John G. 1957. Studies on the digestibility of the
cellulose fraction of grassland products. J. Agr. Sci. 49:467.

Barnett, A. J. G. and M. G. c. M.'Dow. 1957. Utilization of
carbohydrate metabolites by rumen micro-organisms. Nature
180:548.

Barnett, A. J. G. and R. L. Reid. 1956. Studies on the pro-
duction of VEA from grass by rumen liquor in artificial rumen.
I. The VFA production from fresh grass. J. Agr. Sci. 48.315.

- 32 -

Barnett, A. John G. and R. L. Reid. 1957. The production of
VFA from grass by rumen liquor in an artificial rumen.
II. The VFA production from dried grass. J. Agr. Sci. 49:171.

Barnett, A. John G. and R. L. Reid. 1957. The production of
VFA from grass by rumen liquor in an artificial rumen.
III. A note on VFA production from crude fiber grass cell-
ulose. J. Agr. Sci. 49:180.

Baumgardt, B. R., J. L. Cason and R. A. Markley. 1958. A
comparison of several laboratory methods as used in esti-
mating the nutritive value of forages. J. Animal Sci. 17:1205
(abstr.)

Belasco, I. J. 1954a. Comparison of urea and protein meals as
nitrogen sources for rumen micro-organisms: Urea utilization
and cellulose digestion. J. Animal Sci. 13:739.

Belasco, I. J. 1954b. Comparison of urea and protein meals
as nitrogen sources for rumen micro-organisms. The production
of volatile fatty acids. J. Animal Sci. 13:748.

Belasco, I. J. 1956. The role of CHO in urea utilization, cell-
ulose digestion and fatty acid formation. J. Animal Sci. 15:496.

Blaxter, K. L. 1950-1. Energy feeding standards for dairy cattle.
J. Nutr. 20.1.

Blaxter, K. L. and N. McC Graham. 1955. Calorimetric measure-
ments of the nutritive value for sheep of dried grass prepared
in different ways. Proc. Nutr. Soc. 14:xv.

Blaxter, K. L. 1956. The nutritive value of feed as sources of
energy: A review. J. Dairy Sci. 39:1396.

Blaxter, K. L. and N. McC. Graham. 1956. The effect of grinding
and cubing on the utilization of the energy of dried grass.
Jo Agr. SC]... 47:207.

Bondi, A. H. and H. Meyer. 1943. The chemical nature and diges-
tibility of roughage carbohydrates. J. Agr. Sci. 33:123.

Brown, W. H., B. C. Leffel and S. Lakhsmanan. 1958. The effect
of roughage-concentrate ratios upon production and recycling
of VFA by rumen micro-organisms. J. Animal Sci. 17:1191. (abstr.)

Bryant, M. P. and L. A. Burkey. 1953. Numbers and some predominant
groups of bacteria in the rumen of cows fed different rations.
J. Dairy Sci. 36:218.

Burroughs, W., L. S. Gall, P. Gerlaugh and A. M. Bethke. 1950.
The influence of casein upon roughage digestion in cattle with
rumen bacteriological studies. J. Animal Sci. 9:214.

- 33 -

Card, C. S., L. H. Shultz. 1953. Effect of the ration on
volatile fatty acid production in the rumen. J. Dairy Sci.
36:599. (abstr.)

Carroll, E. J. and R. E. Hungate. 1954. The magnitude of the
microbial fermentation in the bovine rumen. Appl. Microbiol.
2: 2050

Cate, H. A., J. M. Lewis, R. J. Webb, M. E. Mansfield and U. S.
Garrigus. 1955. The effect of pelleting rations of varied
quality on feed utilization by lambs. J. Animal Sci. 14:137.

Cheng, E. W., G. Hall and'W. Burroughs. 1955. A method for the
study of cellulose digestion by washed suspensions of rumen
micro-organisms. J. Dairy Sci. 38:1225.

Church, D. C. and R. G. Peterson. 1960. Effect of several
variables on in vitro rumen fermentation. J. Dairy Sci. 43:81.

Conrad, H. R., J. W. Hibbs and W. D. Pounden. 1956. Absorption
of rumen volatile fatty acids from the forestomachs of young
dairy calves fed high roughage rations. J. Dairy Sci. 39:97.

Crampton, E. W. 1957. Interrelations between digestible nutrient
and energy content, voluntary dry matter intake and the over-
all feeding value of forages. J. Animal Sci. 16:546.

Davis, C. L., R. E. Brown, J. R. Staubus and W. 0. Nelson. 1958.
Volatile fatty acid production in the perfused rumen. J.
Dairy Sci. 41:730. (abstr.)

Davis, R. F., N. S. Woodhouse, M. Keeney and G. H. Beck. 1957.
The effect of various levels of dietary protein upon the
volatile fatty acids in the rumen of the dairy cow. J.
Dairy Sci. 40:75.

Elliot, J. M., E. Bennett and J. G. Archibald. 1957. Effect
of feeding certain silages on the relative concentrations
of rumen volatile fatty acids. J. Dairy Sci. 40:356.

Elliot, J. M. and J. K. Loosli. 1959a. Relationship of milk
production efficiency to the relative proportions of volatile
fatty acids. J. Dairy Sci. 42:1.

Elliot, J. M. and J. K. Loosli. 1959b. Effect of the dietary
ratio of hay to concentrate on milk production, ration diges-
tibility and urinary energy losses. J. Dairy Sci. 42:836.

Elliot, J. M. and J. K. Loosli. 1959c. Relation of milk pro-
duction to the relative proportions of rumen volatile fatty
acids. J. Dairy Sci. 42:843.

Elsden, S. T. 1954. The fermentation of carbohydrates in the
rumen of the sheep. J. Exp. Biol. 22:51.

- 34 -

Elsden, S. T., M. W. S. Hitchcock, R. A. Marshall and A. T.
Phillipson. 1946. Volatile acid in the digesta of ruminants
and other animals. J. Exp. Biol. 22:191.

Ensor, W. L., J. C. Shaw and H. F. Tellechea. 1959. Special
diets for the production of low fat milk and more efficient
gains in body weight. J. Dairy Sci. 42:189.

Esplin, A. L., U. S. Garrigus, E. E. Hatfield and R. M. Forbes.
1957. Some effects of pelleting a ground mixed ration on
feed utilization by fattening lambs. J. Animal Sci. 16:863.

Forbes, R. M. 1950. Protein as an indicator of pasture forage
digestibility. J. Animal Sci. 9:231.

Forbes, E. B., J. A. Fries, W. W. Braman and M. Kriss. 1926.
The relative utilization of feed energy for maintenance,
body increase and milk production. J. Agr. Res. 33:483.

Forbes, R. M. and W. P. Garrigus. 1948. Application of the
lignin ratio technique to the determination of the nutrient
intake of grazing animals. J. Animal Sci. 7:373.

Forbes, E. B., R. W. Swift, L. F. Marcy and Mary Davenport.
1944. Protein intake and heat production. J. Nutr. 28:189.

Forbes, E. B. and L. Voris. 1932. The economy of conversion
of feed energy into milk energy by the dairy cow. J. Nutr.
5:395.

Fraps, G. S. 1931. Productive energy of feeds calculated from
feeding experiments with sheep. Tex. Agr. Exp. Sta. Bull.
436:1.

Fries, J. A.,‘W.‘W. Braman and D. C. Cochrane. 1924. Relative
utilization of energy in milk production and body increase
of dairy cows. U.S.D.A. Bull. 1281:1.

Gall, L. 3., W. Burroughs, P. Gerlaugh and B. H. Edgington.
1949. Rumen bacteria in cattle and sheep on practical farm
rations. J. Animal Sci. 8:441.

Gray, F. V. 1947. The absorption of volatile fatty acids from
the rumen. J. Exp. Biol. 24:1.

Gray, F. V. 1947. The digestion of cellulose by sheep. The
extent of cellulose digestion at successive levels of the
alimentary tract. J. Exp. Biol. 24:15.

Gray, F. V. 1948. The absorption of volatile fatty acids from
the rumen. II. The influence of pH on absorption. J. Exp.
Biol. 25:135.

- 35 -

Gray, F. V. and A. F. Pilgrim. 1950. Fermentation in the rumen
of sheep. Nature 166:478.

Gray, F. V., A. F. Pilgrim and R. A. Weller. 1951. Fermentation
in the rumen of the sheep. I. The production of VFA and
methane during the fermentation of wheaten hay and lucerne
hay in vitro by micro-organisms from the rumen. J. Exp.

BiolT—28?747

Gray, F. V. and A. F. Pilgrim. 1951. Fermentation in the rumen
of the sheep. II. The production and absorption of volatile
fatty acids during the fermentation of wheaten hay in the
rumen. J. Exp. Biol. 28:83.

Gray, F. V. and.A. F. Pilgrim. 1952a. Origins of the volatile
acids in the rumen. Nature 170:375.

Gray, F. V. and A. F. Pilgrim. 1952b. Fermentation in the
rumen of sheep. III. Intermediate stages in the fermenta-
tion of wheaten hay in vitro by micro-organisms. J. Exp.
Bic-1. 29:54.

Gray, F. V., A. F. Pilgrim, H. J. Rodda and R. A. Weller. 1952.
Fermentation in the rumen of the sheep. IV. The nature and
origin of VFA in the rumen of sheep. J. Exp. Biol. 29:57.

Gray, F. V., A. F. Pilgrim and R. A. Weller. 1958. The diges-
tion of food-stuffs in the stomach of the sheep and the
passage of digesta through'its compartments. 1. Cellulose,
pentosans and solids. Brit. J. Nutr. 12:404.

Hale, E. B., C. W. Duncan and C. F. Huffman. 1947. Studies in
the chemistry of rumen digestion. J. Nutr. 34:747.

Hansen, R. G., R. M. Forbes and D. M. Carlson. 1958. A review
of the carbohydrate constituents of roughages. Uni. of I11.
Agr. Exp. Sta. Bull. 634. 47 pp.

Head, M. J. 1953. The effect of quality and quantity of car-
bohydrate and protein. J. Agr. Sci. 43:281.

Heald, P. J. 1951. The assessment of glucose-containing sub-
stances in rumen micro-organisms during a digestion cycle in
sheep. Brit. J. Nutr. 5:75.

Heald, P. J. 1952. The fermentation of pentoses and uronic
acids by bacteria from the rumen contents of sheep. Biochem.
J. 50:503.

Heuter, F. G., R. J. Gibbons, J. C. Shaw and R. N. Doetsch. 1958.
Comparison of in vivo and in vitro techniques in rumenology
studies. J. DaIEy Sci. 41:331. ‘

 

- 86 -

Hershberger, T. V., O. G. Bentley, J. H. Cline and W. J. Tyznik.
1956. Formation of Short-chain fatty acids from cellulose,
starch and metabolic intermediates by ovine and bovine rumen
micro-organisms. J. Agr. Fd. Chem. 4:952.

Henneberg, J. W. J. and F. Stohmann. 1860-1864. Beitrage zur
Begrundung einer rationeller Futterung der Wiederkauer.
Graunschweigh, Hefta 1 and 2.

Hibbs, J. W., H. R. Conrad and W. D. Pounden. 1954. A high
roughage system for raising calves based on the early develop-
ment of rumen function. V. Some effects of feeding aureomycin
with different ratios of hay to grain. J. Dairy Sci. 37:724.

Hilditch, T. P. 1941. The Chemical Constitution of Natural
Fats. New York: Wiley and Sons.

Hinders, R. G. and G. M. Ward. 1958. Effects of various feeds
on volatile fatty acid production in vitro. J. Dairy Sci.
41:730. (abstr.)

Huffman, C. F., C. W. Duncan. 1944. The nutritive value of
alfalfa hay. II. Starch and glucose as supplements to an
all alfalfa hay ration. J. Dairy Sci. 27:821.

Huhtanen, C. N., R. K. Saunders and L. S. Gall. 1954. Fiber
digestion using the miniature artificial rumen. J. Dairy
SCio 37:328.

Hungate, R. E., R. W. Daugherty, M. P. Bryant and R. Cello.
1952. Microbiological and physiological changes associated
with acute indigestion in sheep. Cornell Vet. 42:423.

Hunt, C. H., O. G. Bentley and T. V. Hershberger. 1952. Factors
affecting the synthesis of certain members of the vitamin
B-complex in an artificial rumen. Fed. Proc. 11:233.

Irvin, H. M., J. C. Shaw, P. Saarinen and L. A. Moore. 1951.
Net energy vs. TDN in evaluating the efficiency of an all
alfalfa hay ration for milk production. J. Animal Sci. 10:947.

Jamieson, N. D. 1959. Rumen nitrate metabolism and the changes
occurring in the composition of the rumen volatile fatty
acids of grazing sheep. New Zealand J. Agr. Res. 2:314.

Johns,.A. T. 1955. Pasture quality and ruminant digestion.
II. Levels of volatile acids and ammonia in the rumen of
sheep on a high production pasture. New Zealand J. Sci.
Tech. 37:323.

Kameoka, D. and H. Morimoto. 1959. Extent of digestion in the
rumen-reticulum-omasum of goats. J. Dairy Sci. 42:1187.

r 87 -

Kamstra, L. D., A. L. Moxon and 0. G. Bentley. 1958. The effect
of stage of maturity and lignification on the digestion of
cellulose in farage plants by rumen micro-organisms in vitro.
J. Animal Sci. 17:199. "" —"

Kellner, O. 1920. "Die Ernahrung Landwirtschaftlichen Nutz-
tiere". 9th ed., Parey, Berlin.

Kleiber, M. 1959. Symposium on forage evaluation: 11. Progress
in feed evaluation. Agron. J. 51:217.

Kriss, M. 1931. A comparison of feeding standards for cows
with especial reference to energy requirements: Editorial
Review. J. Nutr. 4:141.

Leffel, E. C. 1958. Rumen study techniques, volatile fatty
acid production and bloat. Proc. U. of Md. 1958 Nutrition
Conf. for Feed Manufacturers.

Lewis, D. and I. W; McDonald. 1958. The inter-relationships
of individual proteins and CH0 during fermentation in the
rumen of sheep. I. Fermentation of casein in the presence
of starch or other carbohydrates. J. Agr. Sci. 51:108.

Lofgreen, G. P. 1951. The use of digestible energy in the
evaluation of feeds. J. Animal Sci. 10:344.

Lofgreen, G. P. and J. H. Meyer. 1956. A method for deter-
mining TDN in grazed forage. J. Dairy Sci. 39:268.

Loosli, J. K., R. F. Davis and R. G. Warner. 1955. Studies
on the productive value of roughages and concentrates for
lactation. J. Dairy Sci. 38:797.

Louw, J. G., H. H. Williams and L. A. Maynard. 1949. A new method
for the study, in vitro, of rumen digestion. Sci. 110:478.

Lusk, G. 1921. The behavior of various intermediary metabolites
upon the heat production. J. Biol. Chem. 49:453.

Lusk, G. 1928. The Elements of the Science of Nutrition. 4th
ed., W. B. Saunders and Co., Philadelphia and London.

Maki, L. R. and E. M. Foster. 1957. Effect of roughage in the
bovine ration on types of bacteria in the rumen. J. Dairy
Sci. 40:905.

Marston, H. R. 1948. The fermentation of cellulose in vitro
by organisms from the rumen of sheep. Biochem. J. 42:554

Marston, H. R. 1951. Energy transactions in homeothermic
animals. J. PIOC. ROY. SOC. N.S. Wales 84:169.

-88..

Maynard, L. A. 1953. Total digestible nutrients as a measure
of feed energy. J. Nutr. 51:15.

McBee, R. H. 1953. Manometric method for the evaluation of
microbial activity of rumen with application to utilization
of cellulose and hemicelluloses. Appl. Microbiol. 1:106.

McCarthy, R. D., J. B. Holter, J. C. Shaw, F. G. Hueter and
J. McCarthy. 1957. Rumen perfusion: A new technique for
the rumenologist. Md; Agr. Exp. Sta. Misc. Pub. 291:33.

McCarthy, R. E., J. C. Shaw, Jeanne McCarthy and S. Lakshmanan.
1958. Metabolism and absorption of butyrate-l-C14 by the
perfused rumen. J. Dairy Sci. 41:730. (Abstr.)

McClymont, G. L. 1952. Specific dynamic action of acetic acid
and heat increment of feeding in ruminants. Australian J.
sci. Res. B, 5:374.

McCullough, M. E. 1956. A study of techniques for measuring
differences in forage quality using dairy cows. Georgia Agr.
Exp. Sta. Tech. Bull. N.s.4.

McCullough, M. E. 1958. Conditions influencing forage accep-
tability and rate of intake. Paper. Joint Symposium, North
Carolina State College, June 17-18, 1958.

McCullough, M. E. 1959. Symposium on forage evaluation.
III. The significance of and techniques used to measure
forage intake and digestibility. Agron. J. 51:219.

Meyer, J. H., R. L. Gaskill, G. S. Stoewsand and W. C. Weir.
1959. Influence of pelleting on the utilization of alfalfa
hay. J. Animal Sci. 18:336.

Meyer, J. H. and G. P. Lofgreen. 1956. The estimation of the
total digestible nutrients in alfalfa from its lignin and
crude fiber content. J. Animal Sci. 15:543.

Mitchell, H. H. 1942. The evaluation of feeds on the basis
of digestible and metabolizable nutrients. J. Animal Sci. 1:159.

Moir, R. J. and M. Somers. 1957. Ruminal flora studies.
VIII. The influence of rate and method of feeding a ration
upon its digestibility, upon ruminal function and upon the
ruminal population. Australian J. Agr. Res. 8:253.

Moore, L. A., H. M. Irvin and J. C. Shaw. 1953. Relationship
between TDN and energy values of feeds. J. Dairy Sci. 36:93.

Moxon, A. L., G. Gastler, G. E. Staples and R. M. Jordan. 1951.
Grass hay at its best. S. D. Agr. Exp. Sta. Bull. 405.

-89..

Moxon, A. L. and O. G. Bentley. 1953. Some discrepancies in
AOAC crude fiber and NFE values in roughages. J. Animal
Sci. 12:925.

Neale, P. E. 1953. Alfalfa cubes for fattening lambs and
wethers. N. Mex. Agr. Exp. Sta. Bull. 398.

Nordfeldt, S., I. Iwanaga, K. Morita, L. A. Henke and Annie
K. S. Tom. 1950. Influence of crude fiber in the ration
on efficiency of feed utilization by dairy cows. J. Dairy
Sci. 33:473.

Nordfeldt, S., Q. Svanberg and O. Claesson. 1949. Studies re-
garding the analysis of crude fiber. Acta Agr. Suecana 3:135.

Norman, A. G. 1935. The hemicelluloses. I. Alcoholic sodium
hydroxide as a pretreatment to extraction. Biochem. J. 29:945.

Otagaki, K. K., A. L. Black, H. Goss and M. Kleiber. 1955.
In vitro studies with rumen micro-organisms using carbon-14-
TEbeIea casein, glutamic acid, leucine and carbonate. Agric.
Fd. Chem. 3:948.

Paloheims, L., A. Makela and M. L. Salo. 1955. Maataloust,
Aikakausk. 27:70. (cited in Balch, C. C. 1957) Br. J. Nutr.
11:213.

Pfander, W. H. and.A. T. Phillipson. 1953. The rates of ab-
sorption of acetic, propionic, and N-butyric acids. J.
Physiol. 122:102.

PhillipsOn, A. T. 1952. The fatty acids present in the rumen
of lambs fed on a flaked maize ration. Br. J. Nutr. 6:190.

Phillipson, A. T. and R. A. McAnally. 1942. Studies of the
fate of carbohydrates in the rumen of sheep. J. Exp. Biol.
19:199.

Pigden, W. J. and J. M. Bell. 1955. The artificial rumen as
a procedure for evaluating forage quality. J. Animal Sci.
14:1239.

Powell, E. B. 1939. Some relations of the roughage intake to
the composition of milk. J. Dairy Sci. 22:453.

Powell, E. B. 1938. One cause of fat variation in milk. Proc.
Am. Soc. An. Prod. 40:1938.

Quicke, G. V., O. G. Bentley, H. W. Scott and A. L. Moxon. 1959.
Cellulose digestion in vitro as a measure of the digestibility
of forage cellulose IE ruminants. J. Animal Sci. 18:275.

Quicke, G. V. and O. G. Bentley. 1959. Lignin and methoxyl
groups as related to the decreased digestibility of mature
forages. J. Animal Sci. 18:365.

Raymond, W. F. 1951 The problem of measuring the nutritive
value of herbage. Brit. Grassl. Soc. 6:139.

Reid, R. L., J. P. Hogan and P. K. Briggs. 1957. The effect
of diet on the individual VFA in the rumen of sheep, with
particular reference to the effect of low rumen pH and
adaptation on high starch diets. Australian J. Agr. Res.
8:691. ’

Reid, J. T., w. K. Kennedy, K. L. Turk, s. 'r. Slack, G. w.
Trimberger and R. P. Murphy. 1959. Symposium on forage
evaluation. Agron. J. 51:213.

Reid, J. T., P. G. Woolfolk, W. A. Hardison, C. M. Martin,
A. L. Brundage and R. W. Kaufmann. 1952. .A procedure for
measuring the digestibility of pasture forage under grazing
conditions. J. Nutr. 46:255.

Ritzman, E. G. and F. G. Benedict. 1938. Nutritional physiology
of the adult ruminant. Publ. Carneg. Inst. 494:200.

Richards, C. R., H. G. Weaver and J. D. Connolly. 1958. Com-
parison of methoxyl, lignin, crude fiber and crude protein
as indirect indicators of dry matter digestibility. J. Dairy
Sci. 41:956.

Rogerson, A. 1958. Diet and partial digestion in sections of
the alimentary tract of the sheep. Brit. J. Nutr. 12:164.

Rook, J. A. F. and C. C. Balch. 1959. The effects of intra-
ruminal infusions of acetic and propionic acids on the yield
and composition of the milk of the cow. Proc. Nutr. Soc. 18:34.

Saarinen, P., M. A. Sami, J. C. Shaw and L. A. Moore. 1951.
The adequacy of an all alfalfa ration for milk secretion.
J. Dairy Sci. 34:287.

Salsbury, R. L., A. L. Vander Kolk, B. V. Baltzer and R. W.
Luecke. 1958. The rates of digestion of the cellulose of
some plant fractions by rumen micro-organisms in vitro. J.
Animal SCi. 17: 293 .

Schneider, B. H., H. L. Lucas, H. M. Pavlech anan. A. Cipolloni.
1951. Estimation of the digestibility of feeds from their
proximate composition. J. Animal Sci. 10:706.

Schneider, B. H. and H. M. Pavlech. 1950 Proximate composi-
tion, TDN and digestible energy of feeds. J. Animal Sci.
9:667. (Abstr.)

Shaw, J. C. 1959. Symposium on forage evaluation. VIII. Re-
lation of digestion end-products to the energy economy of
animals. Agron. J. 51:242.

-91..

Shaw, J. C., S. Lakshmanan, A. C. Chung, E. C. Leffel and
R. N. Doetsch. 1958. Intermediary metabolism in the rumi-
nant including studies on rumen, liver and lactating udder.
Proc. Intern. Cong. Peaceful Uses.Atomic Energy.

Shaw, J. C., R. R. Robinson, M. E. Senger, S. Lakshmanan and
T. R. Lewis. 1959. Production of low fat milk. I. Effect
of quality and Quantity of concentrate on the VFA of the
rumen and on the composition of milk. J. Nutr. 69:235.

Sheppard, A. J., R. M. Forbes and B. Connor Johnson. 1958.
Twenty-four hour pattern of rumen volatile fatty acid
accumulation in sheep fed an all-hay ration with additional
data involving the use of trimethyl-alkyl-ammonium-stearate.
J. Animal Sci. 17:1206. (Abstr.)

Smith, V. R., I. R. Jones and J. R. Haag. 1945. Alfalfa with
and without concentrates for milk production. J. Dairy Sci.
28:343.

Snedecor, G. W. 1956. Statistical Methods. 5th Ed. The Iowa
State College Press, Ames, Iowa.

Stewart, W. E. and L. H. Schultz. 1958. In vitro VFA production
from various feeds by bovine rumen micro-organisms. J.
Animal Sci. 17:737.

Stewart, W. E., D. G. Stewart and L. H. Schultz. 1958. Rates
of volatile fatty acid production in the bovine rumen. J.
Animal Sci. 17:723.

Stoddard, G. E., N. N. Allen and‘W. H. Peterson. 1949. Some
effects of a low noughage, high concentrate ration on the fat
of cows milk. J..Animal Sci. 8:630. (Abstr.)

Swift, R. W., R. L. Cowan, R. H. Ingram, H. K. Maddy, G. P.
Barron, E. C. Grose and J. B.‘Washko. 1950. The relative
nutritive value of Kentucky bluegrass, timothy, bromegrass,
orchard grass and alfalfa. J. Animal Sci. 9:363.

Teichman, R., H. D. Eaton, G. Beall, R. E. Johnson, H. L. Lucas
and L..A. Moore. 1958. Effect of previous ration on lac-
tation response to substituting corn for alfalfa according
to net energy and total disgestible nutrient systems of
estimation. J. Dairy Sci. 41:129.

Tyznik, W. and N. N. Allen. 1951. The relation of roughage
intake to the fat content of milk and the level of fatty
acids in the rumen. J. Dairy Sci. 34:493. (Abstr.)

Ward, G.‘W., G. Vidacs and W. H. Palmer. 1959. Low fat milk
production from a high level feeding of dried beet pulp. J.
Dairy Sci. 42:396. (Abstr.)

Weller, R..A. and F. V. Gray. 1954. The passage of starch
through the stomach of the sheep. J. Exp. Biol. 131:40.

-92..

Van Soest and N. N. Allen. 1959. Studies on the relationships
between rumen acids and fat metabolism of ruminants fed on
restricted roughage diets. J. Dairy Sci. 42:1977.

Williams, V. J. and K. R. Christian, 1956. Rumen studies in
sheep, III. The effect of feed intake on the concentrations of

microbial end products. New Zealand J. Sci. Tech. 38A:403.

Woodhouse, N. S., R. F. Davis and G. H. Beck. 1955. The effect
of protein level on rumen volatile fatty acids. J. Dairy
Sci. 38:605. (Abstr.)

talul‘

.....

 

1
I

I“
|
“I
“II
n
n
V."

3196 6090