INFLUENCE OF AUREOMYCIN ON SYNTHESIS AND DIGESTION IN
THE RUMEN OF CATTLE FED NATURAL AND PURIFIED RATIONS

By
CHARLES MARION CHANCE

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Dairy

1952

ProQuest Number: 10008221

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A CKNQWLEDGEMENTS
The author wishes to express his sincere appreciation to
Dr. C. F. Huffman, Research Professor of Dairying, for his
interest and timely suggestions throughout this investigation
and for his assistance in the preparation of this manuscript;
to C. W. Duncan, Associate Professor (Research), Department of
Agricultural Chemistry, for his helpful suggestions and con­
structive criticism throughout the course of the investigation
and also for his critical reading of this manuscript.
Gratitude is likewise expressed to Dr. R. W. Luecke and
Dr. E. J. Benne, Professors

(Research), Department of Agricul­

tural Chemistry, and their associates for technical assistance
and facilities for conducting the chemical determinations
required in this study.
The writer is indebted to Dr. C. K. Smith and Mrs. Carol L.
Frank, Department of Bacteriology and Public Health, for the
bacteriological analyses reported herein; to Dr. Earl Weaver,
Professor of Dairying,for the award of the Graduate Assistantship and for the provision of the facilities necessary to make
this study possible.

INFLUENCE OF AUREOMYCIN ON SYNTHESIS AND DIGESTION IN
THE RUMEN OF CATTLE FED NATURAL AND PURIFIED RATIONS

By
Charles Marion Chance

AN ABSTRACT
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Dairy

Year

Approved_

<?■

1952

Charles 5,1. Chance

ABSTRACT
Two steers, each with a rumen fistula fitted with a
plastic plug, were used to investigate the influence of aureo
mycin on the synthesis and digestion in the rumen when the
steers were fed natural and purified rations.

The animals

were fed a natural ration of 15 pounds of second-cutting
alfalfa-brome hay and four pounds of corn once daily through­
out the feeding trials.

Aureomycin was fed at each level

(0, 0.5 and 1.0 gram per day) for 15 days before changing to
the next highest level.

The period in which the steers re­

ceived no aureomycin served as the control period for com­
parison with those in which aureomycin was fed.
The samples of the rumen contents were collected by com­
pletely evacuating the rumen before feeding, 0-hour and at 6and 12-hours after feeding the ration.

Determinations for

dry matter, crude fiber, crude protein and ether extract as
well as nitrogen-free extract were made on both the rumen
contents and the ration.

Microbiological methods of assay we

used for the determination of the 10 essential amino acids,
riboflavin, nicotinic acid and pantothenic acid.
Neither of the steers showed any signs of anorexia or
diarrhea at any time during aureomycin supplementation when
the natural ration was fed.

Steer 714 went off-feed just

prior to the addition of 0.5 gram of aureomycin to the puri-

Charles M. Chance

fied ration and did not regain its appetite during the period
that aureomycin was included in the ration.
The pH declined for about six hours after feeding with
only a slight increase toward alkalinity at the end of 12 hours.
7hen the aureomycin was included in the natural ration, the
pH did not become as acid as that observed when no aureomycin
was fed; however, the response was in the same direction in
each case.
A definite increase in the total bacterial count of the
rumen contents and the feces occurred when aureomycin was in­
cluded in the ration.

The number of rumen streptococci de­

creased when aureomycin was fed, while the number of coliform
organisms in the rumen remained approximately the same in one
animal and increased in the other.
The rate of removal of dry matter, crude fiber, crude
protein, nitrogen-free extract, non-protein nitrogen, the 10
essential amino acids and riboflavin from the rumen was the
highest when 0.5 gram of aureomycin was fed.

There was an

accumulation of ether extract, nicotinic acid and pantothenic
acid in the rumen when aureomycin was included in the ration.
There was an accumulation of dry matter and crude fiber
in the rumen at the 0-hour when 1,0 gram of aureomycin was fed
which indicated that a slight depression of digestibility of
these constituents may have occurred.

Charles M. Chance

The pH and the total bacterial count were approximately
the same for the periods when no aureomycin and 0.5 gram of
aureomycin was added to the purified ration.

The synthesis

of the amino acids was lower when 0.5 gram of aureomycin was
included in the purified ration.

A definite decrease in the

synthesis of riboflavin, nicotinic acid and pantothenic acid
also occurred when 0.5 gram of aureomycin was added to the
purified ration.

TABLE OF CONTENTS
INTRODUCTION ............................................

Page
1

REVIEW OF LITERATURE ....................................

2

I n t r o d u c t i o n .............................

2

Antibiotics in Poultry Nutrition . . .

3

.............

Antibiotics in Swine Nutrition .....................

7

................

11

Antibiotics in Other NutritionalStudies ...........

15

Effect of Antibiotics on Bacteria

18

Antibiotics in Ruminant Nutrition

Theories Proposed for the Action of

.................
Antibiotics

..

21

pH of Rumen C o n t e n t s ................................

25

Rate of Passage of Materials Through the Digestive
T r a c t ..............................................

29

Microorganisms in the R u m e n ................... .. .

31

Numbers and t y p e s .............................
Effect of Various Rations on Rumen Microorganisms
Functions of Rumen or Intestinal Microorganisms

31
.

33

. .

39

Formation of Protein in the R u m e n ................
B-Complex Vitamin Synthesis in the Ruminant

42

. . . .

47

Thiamine synthesis .............................
Riboflavin synthesis ...........................
Nicotinic acid synthesis .......................
Pantothenic acid synthesis .....................
Synthesis of other B-vitamins
.................

48
49
51
52
52

Summary of the Review of Literature . . . . . . . .

53

EXPERIMENTAL PROCEDURE ..................................

Page
56

Animals Used in E x p e r i m e n t ............................ 56
Rations U s e d ...........................................56
Method of F e e d i n g .........................
Sampling Procedure . . . . .

. . . .

57

. ♦

58

Chemical analysis
. . . . . ...................
58
Bacterial and pH d e t e r m i n a t i o n s ..............
59
..............................
60
Chemical analyses
Bacteriological analyses .......................
61
Amino acid analyses
..........................63
B-vitamin analyses ..............................
65
R E S U L T S ................................................... 70
Health of the A n i m a l s .............................

70

Weight and Composition of the Rumen Contents from
Cattle Fed a Natural and a Purified Ration
. . . .

71

Influence of Aureomycin on the Rate of Removal of
Some of the Ration Constituents from the Rumen . .

74

Amino Acid A n a l y s e s .................................. 81
B-Vitamin Analyses ................................
pH and Bacteriological Analyses

.

...................

86
87

D I S C U S S I O N ................................................ 101
Health of the A n i m a l s ............................... 101
pH and Bacteriological Analyses

...................

103

Passage of Some of the Ration -Constituents from the
R u m e n ..........

108

Amino Acid A n a l y s e s ..............

112

B-Vitamin Analyses . . . .

115

.........................

R i b o f l a v i n ........................................115
Nicotinic a c i d .......................
116
Pantothenic a c i d ................................. 118

S U M M A R Y .................................................

Page
119

......................................

122

A P P E N D I X ..............................................

143

LITERATURE CITED

INTRODUCTION
The use of antibiotics in animal nutrition was incidental
to the development of vitamin Bq2 concentrates from by-products
of antibiotic manufacture.

Some of the by-products were more

effective in stimulating growth than others and this extra
effect was traced to the residual antibiotic.
Many investigations with swine and poultry have shown that
various antibiotics were beneficial in promoting growth, less
digestive disturbances and an increased feed efficiency.

Re­

cent reports have shown favorable growth responses when aureo­
mycin was fed to young calves.

However, several investigators

reported that aureomycin produced adverse effects, such as
anorexia and diarrhea, when fed to steers and lambs.

Also,

there was a decreased digestibility of dry matter and crude
fiber by the steers and the lambs lost weight when aureomycin
was included in the ration.
This investigation was undertaken to study the influence
of aureomycin on the digestion and synthesis of certain ration
nutrients in the rumen, the synthesis, if any, of the essential
amino acids and some of the B vitamins in the rumen.

The in­

fluence of aureomycin on the bacterial population of the rumen
and the feces also was determined in an attempt to correlate
these findings with the digestion studies.

REVIEW OF LITERATURE
Introduction
The term antibiotic is defined in pharmaceutical and
medical practice as a metabolic product of one microorganism
that is detrimental to the life activities of other micro­
organisms*

Antibiotics have been known, if not in pure form,

at least by their effects for centuries; but have only re­
cently assumed the dominating position in clinical medicine
and in the pharmaceutical industry that they now occupy.
Most of the research and development in the field of anti­
biotics has developed following Fleming's observation of the
action of Penicillium sp. on Staphylococcus aureus in 1929,
During the early years of World War II, sulfa drugs were the
only chemotherapeutic agents available for the treatment of
bacterial infections and these drugs had several undesirable
characteristics.

Therefore, there was need for new anti­

bacterial agents that might not have these undesirable pro­
perties.
By definition, sulfa drugs cannot be called antibiotics;
however, their action is similar to that of an antibiotic.
An occasional reference is made to some sulfa compound merely
to show some of the earlier attempts to arrive at a solution
of the same problem that antibiotics are being used for at
the present time.

- 3 -

Some of the literature concerning the use of antibiotics
with poultry, swine and other animals is included in this
review because most of the antibiotic research has been done
with those animals.
Antibiotics in Poultry Nutrition
Moore at a l . (1946) fed streptomycin with an experimental
purified diet which stimulated growth of chickens.

These

workers were interested in finding a drug or combination of
drugs that would completely inactivate all bacteria in the
intestinal tract in order to study vitamin requirements un­
complicated by vitamins or toxic substances.

Sulfasuxidine

and streptomycin, singly or in combination, increased the
growth of chicks supplemented with adequate amounts of folic
acid.
Oleson and coworkers
factor

(1950) found that animal protein

(APF)-depleted chicks showed no response to aureomycin

in the absence of

in

diet; however, there was a

marked response to the antibiotic when Bqg was present in
suboptimal or optimal amounts.
Berg at al. (1950) observed a cessation of the accelerated
growth response when APF was removed from the ration.

When

this material was added to a ration for chicks which had not
t#he
received any during first 4.5 weeks of age, there was an im­
mediate acceleration of growth.

The same effect was observed

- 4 -

when crystalline aureomycin was used.

They concluded that

the mode of action of the antibiotic in promoting growth is
dependent on the continuous presence of the material in the
diet.
Similar results in growth were obtained by Groschke and
Evans

(1950) who reported that the best gains occurred when

synthetic vitamins or vitamin B]_g were fed in combination with
streptomycin or aureomycin.

These workers also injected aureo­

mycin but failed to get an increase in growth.
Stokstad and Jukes (1951) studied the effect of graded
levels of vitamin B^g on growth in chicks in the presence
and absence of aureomycin.

A growth-promoting effect for

aureomycin was noted both in the presence and absence of vita­
min B^g.

some of their trials, a "sparing effect" of aureo­

mycin upon vitamin B^g was observed; but in others, no effect
was observed.

The mortality of deficient chicks on the diet

containing no added vitamin B^g was markedly reduced by aureo­
mycin.
Biely and March (1951) have observed considerable dif­
ferences in response of chicks to aureomycin.

When they fed

rations which contained vitamins above levels recommended by
the National Research Council, no increase in growth occurred
following the addition of aureomycin to the experimental
ration.

If riboflavin, nicotinic acid and folic acid were

omitted individually, a significant decrease in growth re­

- 5 -

salted.

The addition of aureomycin to these vitamin defi­

cient rations stimulated growth in each case so that the
weights attained were similar to those fed the complete basal
ration.
Kratzer et. al.. (1951) fed droppings from month-old chicks
which had been receiving a normal diet or a diet supplemented
with aureomycin to day-old chicks for three days.
pings

The drop­

(fresh or autoclaved) from chicks fed aureomycin showed

less growth depression than droppings from chicks receiving
the normal ration.
While most investigators were concerned mostly with
growth, Slinger and coworkers

(1951) reported a condition in

turkeys known as "white feathers".

This condition occurred

when the turkeys received rations containing sunflower seed
oil meal but was not shown when they received soybean oil
meal.

The addition of APF to the sunflower oil meal ration

increased the severity of the white feather condition.
same authors

These

(1950) had shown that one per cent lysine was

sufficient to prevent white feathers with a ration containing
20 per cent protein.

When the ration containing 20 per cent

protein and 1.2 per cent lysine was supplemented with peni­
cillin, aureomycin or APF concentrates, a very high incidence
of white feathers resulted.

These results indicate that the

antibiotics and APF concentrates increased the lysine re­
quirement of the poult for normal feather pigmentation.

The

- 6 -

authors suggested that the requirement of lysine for growth
takes precedence over the requirement for feather pigmenta­
tion because an increased growth occurred simultaneously with
an increase of white feathers.
Couch (1951), Halick and Couch (1951) and Elam, Gee and
Couch (1951a) conducted several experiments using antibiotics
and vitamin

to study the effect on the life cycle of the

chick, egg production, and hatchability of the

eggs.

The

feeding of antibiotics alone assisted in the depletion of
the birds of vitamin B]_g and possibly the factor in liver "L".
Elam ejt a l . (1951a) observed that birds injected with peni­
cillin after 14 weeks of age grew very well but no live chicks
were obtained from pullets injected with penicillin.

In this

study it was observed that the vitamin B]_g content of liver,
kidney, and egg yolk was directly related to the injection of
this vitamin into the birds.

However, Halick and Couch (1951)

fed a practical all-mash ration and found that the hatchability
of eggs from birds on this ration was higher than from birds
fed the experimental ration and injected with vitamin B^g even
though the vitamin B-^g content of the eggs was

lower than those

from birds injected with vitamin B^2 »
Using another antibiotic, Sieburth (1951) found that
terramycin stimulated an increase in the body weight of tur­
keys .

- 7 -

Antibiotics in Swine Nutrition
Using a corn and peanut meal ration, Cunha et al.

(1949a)

showed that Lederle's APF and vitamins were different in their
response in the pig.

The addition of the APF supplement had

considerable effect, especially with lighter pigs, while the
addition of vitamin
even when doubled.

concentrate was of no apparent help,
Burnside ejt al. (1949) observed that

Lederle 's APF increased the feeding value of peanut meal and
soybean oil meal so that these plant supplements were similar
in feeding value to fish meal.

Again, with the same corn and

peanut meal ration, Cunha and coworkers

(1949b) found that

Lederle's APF and vitamin B]_g spared the methionine needs of
the pig.

This latter study also indicated that Lederle’s APF

contains vitamin B^g plus some other factor.
Luecke and coworkers

(1950a) observed that streptomycin

increased growth in pigs fed a ration of corn and soybean oil
meal.

Later, however, they reported that streptomycin pro­

duced no increase in growth while aureomycin and penicillin
produced a significant response over the B vitamin supple­
mented basal ration (1950b).

These same authors (1950c)

studied the effect of the addition of low levels of antibiotics
on the stimulation of growth when the corn and soybean oil meal
ration contained inadequate amounts of niacin, pantothenic acid,
and riboflavin.

Again, streptomycin without added B vitamins

did not significantly increase the rate of growth of young

- 8 -

pigs; however, there was some increase when B vitamins were fed
with streptomycin.
Jukes et al. (1950), using a corn and peanut meal ration,
and Carpenter

(1950), using a corn and soybean oil meal ration,

demonstrated that the growth stimulation obtained by feeding
Lederle 's APF supplements to pigs was due to the aureomycin
they contained.

Carpenter (1951) found that aureomycin, terra-

mycin, penicillin, streptomycin, and Chloromycetin were effec­
tive in stimulating growth in pigs fed a corn and soybean oil
meal ration.

Pigs supplemented with aureomycin, terramycin,

and penicillin made the greatest growth response even though
streptomycin and Chloromycetin caused some increase in growth
over the pigs fed the basal ration.

Similar results were ob­

served by Brown and Luther (1950) who used the same antibiotics,
except for Chloromycetin, as Carpenter.

Cunha et al.

(1950a,

1951) reported that penicillin was of no benefit in stimulating
growth of swine fed a ration of corn and peanut meal and that
streptomycin was not as effective as a growth stimulant as
aureomycin.

These authors stated that the growth-promoting

effect of various antibiotics varied with the species of
animal and the nature of the basal ration.
Edwards and coworkers

(1951) observed that vitamin

gave the greatest growth response in pigs that had been depleted
of this vitamin for six weeks.

They treated Lederle's APF with

1.0 N NaOH for 30 minutes to destroy vitamin Big and most of

- 9 -

the antibiotic properties of aureomycin and fed some of the
remaining material to pigs.

The data indicate that the

alkali-treated material contained a factor (s) which increased
the rate of growth of pigs.

The factor from the alkali-

treated APF promoted growth and may have had a sparing effect
upon the vitamin B^g needs of the pig.
Wahlstrom and Johnson (1951a) found that aureomycin caused
an increase in daily gain over the basal group when synthetic
rations were fed; however, the addition of aureomycin to the
ration of pigs not getting vitamin B ± q did not prevent vita­
min B-j_g deficiency symptoms.

The injection of vitamin B^g to

these pigs after 49 days resulted in an increase in average
daily gain and feed efficiency.
Shefchik et_ a l . (1950) and Nesheim et^ al . (1950) observed
that aureomycin and streptomycin stimulated growth in two-day
old pigs fed a "synthetic" milk.

Shefchik stated that a com­

bination of streptomycin and aureomycin gave the best results.
Nesheim e^t al^. reported that streptomycin stimulated growth
when alpha-protein was used in the "synthetic" milk; but when
vitamin-free casein was used, penicillin, aureomycin or strep­
tomycin did not stimulate growth significantly above the basal
ration.
Speer et^ a l . (1950) reported an experiment in which the
addition of aureomycin (5 or 10 milligrams per pound) had no
effect on the growth rate.

The authors explain this lack of

-10-

re sponse to aureomycin by the "disease level" theory in which
healthy pigs do not respond to aureomycin as well as unthrifty
pigs in unsanitary surroundings.

The pens had been cleaned

daily to reduce copraphagy.
Cunha et^ a l . (1950b) report that pigs fed a 19.6 per cent
protein ration without B vitamins did not gain as rapidly as
pigs fed 12.2 per cent protein ration plus APF and B vitamins.
They suggested that the accepted values for protein require­
ments of swine may need to be re-evaluated by using adequate
amounts of vitamin B^g irl

ration as well as other factors

present in Lederle’s APF supplement.
Miller and coworkers (1951) obtained about the same growth
response by feeding sulfathalidine as was obtained from a com­
bination of vitamin B]_g and streptomycin.

Growth in both

cases was much higher than that obtained with pigs by the
addition of vitamin B^g alone.
Wahlstrom and Johnson (1951b) reported that the addition
of aureomycin to an alpha-protein "synthetic milk" diet, which
was used to produce vitamin B^g deficiency, did not give a
beneficial response, either before or after the deficiency
existed.

The injection of 20 milligrams of cortisone daily

into vitamin Bj_g deficient pigs enhanced the deficiency state
rather than having any curative effect.

Antibiotics in Ruminant Nutrition
Sulfa drugs are used for treating various ailments in
the ruminants and are usually given orally.
Hignett

Stableforth and

(1942) observed with dairy cows that sulphanilamide

caused sleepiness, anorexia, some incoordination, diarrhea
and a loss of milk production, if the dosage level was greater
than one gram per ten pounds of body weight.

Oyaert, Quin

and Clark (1951) used various amounts of sulphanilamide in
in vitro and in iin vivo studies with sheep and ruminal ingesta.
The in_ vivo work confirmed the iji vitro studies by showing
that sulphanilamide depressed cellulose digestion and this
in turn was reflected in decreased appetite.

The cotton thread

technique was used for digestion of cellulose i_n vitro and
sulphanilamide caused retardation of cellulose digestion al­
most linear to the amount of sulphanilamide added.

Extra

glucose exerted an antagonistic action to that of sulphanila­
mide.

Johnson et al.

(1947) observed that nicotinic acid was

excreted in the urine of two newborn calves fed a nicotinic
acid-free diet.

The addition of one per cent sulfathalidine

had no effect on the growth of the animals or excretion of
nicotinic acid over a 2.5-week period even though the bacterial
count of the feces decreased about 80 per cent.

Teeri ejt a l .

(1950) used the same drug and noted a slight decrease in the
fecal excretion of thiamine but the total excretions of thiamine
and pantothenic acid were greater than the dietary intakes,
dicating rumen or intestinal synthesis of these vitamins.

in­

- 12 -

Antibiotics and APF supplements have proved to be very
successful in swine and poultry nutrition.

Consequently, in­

vestigations have been conducted or are in progress to deter­
mine the growth-stimulating properties of these materials in
calves and older cattle.

Rusoff and Haq (1950, 1951a) did

not observe an increase in growth when APF (Merck) was added
to the ration of dairy calves weaned at 28 days of age.

Simi­

lar observations were made by Williams and Knodt (1951) with
slightly younger calves and by Hibbs and Pounden (1950) who
fed APF to calves which had been rumen-inoculated with cud
material.
Loosli and Wallace

(1950) obtained an increase in growth

when Lederle 's APF or crystalline aureomycin was
the diet of young dairy calves.

included in

The incidence and severity

of diarrhea also was reduced in the calves.

The authors sug­

gested that the responses may be largely the result of an
antibiotic effect of aureomycin.

Loosli et al.

(1951) ob­

served that the Lederle antibiotic supplement fed to young
calves at the rate of two per cent of the ration caused an
increase in growth over that obtained with the controls.

The

calves getting the supplement used 40 per cent more feed but
required less total digestible nutrients to make one pound of
gain.

Studies of the rumen microflora failed to reveal any

striking difference in the total count or morphological types.
Rusoff

(1951b) fed Lederle's APF at two per cent level to

14-week old calves and obtained a 35 per cent increase in

- 13 -

growth over the controls for the next six weeks, after which
the daily gains for both groups were the same.
Murley et al ♦ (1951a) and Rusoff et_ al_. (1951c, 1951d)
fed crystalline aureomycin to newborn calves.

There was an

increase in the rate of gain and feed utilization in the
supplemented groups over that of the controls.

Rusoff et al.

(1951c, 1951d) observed that the Jersey calves receiving
either Lederle's APF or crystalline aureomycin gained 25 per
cent more than the controls while the Holstein calves only
showed a 15 per cent increase over the controls.
Jacobson et al. (1951) and Bartley et_ al_. (1951) observed
an increase in the growth of calves when aureomycin was in­
cluded in the ration.

Bartley et_ al. fed one 16-week old

calf 2,500 milligrams of aureomycin daily for four weeks with­
out any deleterious effects on feed consumption, rumination
or growth.
Voelker and Cason (1951) obtained increases in the rate
of growth of experimental calves over control groups with
both terramycin or aureomycin (Aurofac).
Neumann and coworkers (1951) did not observe any bene­
ficial effect when aureomycin, either in crystalline form or
as Aurofac, was fed to yearling beef heifers for 150 days.
There was a slight reduction in the appetite for a few days.
Murley e_t a l . (1951b) found no difference in the urinary
content of reducing sugars and nitrogen or the fecal content

- 14 -

of dry matter, nitrogen, reducing sugars, ether extract and
ash of calves fed aureomycin.
Bell et a l , (1951) fed 0,6 grams of crystalline aureo­
mycin daily to yearling steers and observed a marked anorexia
and diarrhea within 43 to 72 hours after feeding.

This con­

dition persisted for several days after the aureomycin was
discontinued.

Some of the steers showed these symptoms even

when the amount fed daily was reduced to 0,2 grams.

The

authors conducted a digestion trial and found that the in­
gestion of 0,2 grams of aureomycin daily apparently appeared
to decrease the digestibility of dry matter 15 per cent and
crude fiber 50 per cent.
Using aureomycin, penicillin, and streptomycin in feeding
trials with fattening lambs, Colby et al.

(1950) found that

lambs fed 100 milligrams of aureomycin daily went almost
entirely off-feed and lost weight.

The lambs fed penicillin

went off-feed and had diarrhea for a week but gradually re­
covered.

Streptomycin was the least severe of the antibiotics;

however, all groups fed antibiotics gained less than the con­
trols.

Jordan and Bell (1951) also fed aureomycin to suckling

and fattening lambs but could not confirm the results of Colby
et a l .

The lambs receiving the aureomycin made faster and

more efficient gains than the controls.
Wasserman et_ a l . (1952) made an in. vitro study of cellu­
lose digestion with rumen microorganisms in the presence of
penicillin, streptomycin, neomycin or Chloromycetin.

In the

- 15 -

concentrations used, penicillin stimulated cellulolytic rumen
microorganisms at the lower concentrations, neomycin was
stimulatory in all concentrations, streptomycin was slightly
stimulatory in the lowest concentration and Chloromycetin
adversely affected the microorganisms.

As the cellulolytic

activity increased, a corresponding increase in the bacterial
count was observed*
Antibiotics in Other Nutritional Studies
Emerson and Smith (1945) produced a biotin deficiency in
rats by the oral administration of streptomycin.
responded to biotin therapy.

The rats

Miller (1945) observed signs

of nutritional deficiency in rats which could be corrected by
feeding biotin and folic acid.

He used a purified diet with

all the known growth factors added and then added succinylsulfathiazole or phthalylsulfathiazole to the diet.

Lower levels

of biotin, folic acid and pantothenic acid were excreted in
feces of rats fed the diets containing the drugs than were
excreted by the rats on the control diet.

Grundy ejt al. (1947)

found a decreased fecal excretion of the L. casei factor and
biotin from human beings fed sulfathalidine.

In the above

studies a decrease in the number of coliform organisms occurred
simultaneously with the nutritional deficiency or lowered
excretion of the vitamin studied.
Waisman and coworkers

(1951) found that 5 to 10 milligrams

of aureomycin per 100 grams of purified diet were effective in

- 16 -

overcoming toxic signs of folic acid deficiency produced by
including aminopterin in the diet.

Aureomycin had no effect

on this deficiency when it was caused by the use of sulfaphthalidine in the diet.

These workers found that the citro-

vorum-factor concentrates were more efficient than folic acid
in overcoming the deficiency caused by aminopterin or sulfa
drugs.

Schwarz (1951) noted that cecal contents from rats on

the basal diet had a higher proportion of the citrovorum
factor in the inactive form than in the active form; however,
when aureomycin was added to the diet, only the active citro­
vorum factor was found.

The same relationship was observed

in the liver on both diets as was observed in the cecal con­
tents.
According to Linkswiler et al. (1951), aureomycin caused
a marked increase in growth of rats fed pyridoxal or pyridoxamine, in fact, growth was approximately the same for the three
forms of vitamin Bg when fed in presence of aureomycin.
Swick, Lih and Baumann (1951) and Lih and Baumann (1951)
obtained an increase in the growth of rats receiving limited
amounts of thiamine, riboflavin or pantothenic acid by the
addition of penicillin, aureomycin or streptomycin to the diet.
The antibiotics were most effective in diets which contained
only enough vitamins for one-half maximum growth and were about
equal to the growth obtained when the vitamin content was
doubled or trebled in the absence of the antibiotic.

The

- 17 -

effectiveness of the antibiotic varied with the vitamin defi­
ciency:

penicillin gave the best response in thiamine defi­

ciency, penicillin and aureomycin gave the same results in
riboflavin deficiency, while aureomycin and streptomycin were
superior to penicillin in pantothenic acid deficiency.
In the microbiological assay for nicotinic acid with
Lactobacillus arabinosus, Teeri and Josselyn (1949) and Teply
et al. (1943) found that the results were not affected by
sulfa drugs if the medium contains jo-amino benzoic acid.

In

the case of higher concentration of the sulfa drugs, the
effect of the drugs could be prevented by increasing the
amount of jD-amino benzoic acid in the basal medium.
Marx and Wainfan (1951) observed that approximately 15
per cent of the cholesterol fed to mice on a high cholesterol
diet was destroyed or modified.

When one per cent sulfasuxi-

dine and 0.04 per cent streptomycin were added to the diet,
the total cholesterol ingested was recovered within limits of
error of the method.

Results indicate that microorganisms

in the intestine are primarily responsible for the destruction
or modification of cholesterol.
Davis and Chow (1951) using Co^O in the diet of rats
found that aureomycin caused an increase in the radioactivity
and microbiological activity or the vitamin

content of

the feces.
Meites and Ogle

(1951) studied the effect of penicillin,

neomycin and streptomycin in counteracting the retardation

- 18 -

of growth caused by feeding Protomone in thyrotoxic amounts.
Penicillin and neomycin, but not streptomycin, were effective
in overcoming the growth inhibition when the ration was ade­
quate in vitamin B^ 2 ‘
> however, all three antibiotics were
only partially effective when the ration was deficient in
vitamin B ^ .

Protomone increased basal oxygen consumption

74 per cent, whereas the antibiotics were ineffective.
Sutherland and coworkers (1951) observed that the in­
gestion of therapeutic levels of aureomycin by normal dogs
and rats did not cause fatty liver.

Histological studies

were made from needle biopsy in dogs and necropsy from rats.
They also fed aureomycin to three healthy human beings and
did not find any liver abnormality as shown by liver function
tests.
Effect of Antibiotics on Bacteria
In 1945, Miller observed that either succinylsulfathiazole
or phthalylsulfathiazole caused a noticeable drop in the number
of coliform microorganisms in the feces of rats; although,
there was not a significant decrease in the total bacterial
population.

Johnson and coworkers

(1947) noted that the

bacterial count of feces from calves decreased about 80 per
cent when one per cent sulfathalidine was added to the ration.
The sulfathalidine added to the ration had no effect on the
growth of the animal or on the excretion of nicotinic acid.
Grundy et_ a l . (1947) also observed a marked and immediate

- 19 -

decrease in the number of coliform microorganisms in human
beings when sulfathalidine was included in the diet.

Along

with the decrease in the number of coliform microorganisms,
there was a decreased bacterial synthesis of folic acid and
biotin.
Moore et_ al. (1946) produced a marked reduction of coli­
form bacteria in the feces of chickens by the oral administra­
tion of streptomycin.

Emerson and Smith (1945) observed a

similar effect in their studies with rats; however, the
coliform organisms increased gradually to normal within a
few days, indicating a development of fastness to the drug.
Spaulding et^ al.

(1949) found that bacteria readily developed

resistance to streptomycin, even in presence of sulfathalidine.
Kane and Foley (1947) observed that E_. coli could be eliminated
from the stool of human beings after two days by the oral ad­
ministration of streptomycin; also, it could be kept free of
these microorganisms as long as streptomycin was present.

The

microorganisms would reappear as soon as the administration
of the antibiotic ceased.

A similar observation of this latter

fact was made by Hamburger and Berman (1950).
Bierman and Jawetz (1951) found that aureomycin caused
a prompt disappearance of coliform microorganisms from the
human stool with a marked reduction in the usual flora, but
a normal flora returned within £4 to 48 hours after discon­
tinuing the antibiotic.

Williams et al.

(1951), using chickens,

observed that the total numbers of coliform and lactic acid

- 20 -

bacteria were not significantly changed but the total numbers
of anaerobic bacteria were reduced 150- to 200-fold by aureo­
mycin.

Hemolytic clostridia were eliminated almost completely

from the intestinal contents and feces of chicks by feeding
aureomycin.

McVay and coworkers

(1951), using patients with

cecostornies and transverse or sigmoid colostomies, found that
aureomycin appeared rapidly in the intestine and maintained
a significant intestinal bacterial flora.

Only Streptococcus

faecalis was not significantly reduced by the administration
of aureomycin.

This fact also was reported by Metzger and

Shapse

Bartley et al.

(1950).

(1951) fed aureomycin to calves

but could not find any consistent microscopic differences
between the rumen microflora in the control and aureomycin-fed
calves.

Neumann and coworkers

(1951) noted that the total

counts were about the same for the control as for the aureomycin-fed heifers, but the types found in the heifers receiving
aureomycin were much less diverse, suggesting that the normal
rumen flora had been disturbed.

Wahlstrom and Johnson (1951)

observed that chloramphenicol decreased the number of coliform
bacteria in feces from pigs; but had no apparent effect after
three weeks.

They also noted that aureomycin, streptomycin,

and penicillin appeared to have no effect on the coliform,
lactobacilli or yeast cell counts.

Sborov et_ al . (1951) found

that aureomycin or terramycin were effective in inhibiting
urobilinogen formation in the bile, urine and feces of human

- 21

beings.

This inhibition was associated with a disappearance

of coliform microorganisms and a reduction or disappearance of
Clostridia in the feces; consequently, the data lend support
to the bacterial concept of urobilinogen formation.
Elam et al.

(1951a) and Couch and coworkers

(1951) ob­

served an increase in the total number of intestinal micro­
organisms when penicillin was included in the diet of chicks.
Jawetz et_ al. (1950) made some i_n vitro studies of the combined
action of penicillin with streptomycin or Chloromycetin on
enterococci.

They observed that streptomycin failed to inhibit

the growth of enterococci, while penicillin decreased the
growth of enterococci for 48 hours and then gradually increased
but stayed below the controls for the drug.

Combination of

streptomycin and penicillin produced complete sterilization of
the medium which suggests there may be a synergism of the two
drugs; that is, increase in rate of bactericidal action beyond
the optimum obtained with penicillin alone.

A penicillin and

Chloromycetin combination was not as effective as the combina­
tion of penicillin and streptomycin.
Theories Proposed for the Action of Antibiotics
Many investigations have been carried on during the past
several years using antibiotics.

The investigators observed

and commented on their results, but they did not have a real
explanation to what actually happened.

In 1949, Waksman, one

- 22 -

of the pioneers in the antibiotic field, proposed several
theories to explain the action of antibiotics:

first, anti­

biotics may interfere with some of the metabolic processes of
the bacterial cell by substituting for one of its essential
nutrients; second, may interfere with various enzymatic sys­
tems, especially the respiratory mechanism of the bacterial
cell; third, may prevent the synthesis of some essential
metabolite by the bacterial cell; and fourth, may act as a
detergent and affect the surface tension of the bacterial
cells.
Since these theories were first proposed by Waksman,
reports have appeared in the literature, which confirm to some
extent the theories he proposed.

In fact, Moore et al.

(1946)

suggested that sulfasuxidine and streptomycin increased growth
in chicks by inhibiting the intestinal bacteria that were
either producing toxic materials or were rendering certain
dietary vitamins unavailable for utilization,
coworkers

Sieburth and

(1951) also stated that antibiotics promote growth

by inhibiting or preventing the production of bacterial toxins
in the intestine.

Their data supported this theory by showing

a suppressed growth of Clostridia perfringens which has been
shown to cause enterotoxemia in sheep,
Swick et al.

Biely and March (1951),

(1951), Linkswiler et al. (1951), and Bratzler

and Black (1951) postulated that antibiotics exert their growth
promoting effect by reducing the bacterial flora which might
compete with the host for nutrients, thereby making more nu­

- 23 -

trients available for utilization by the host,

Biely and

March (1951) also stated that the antibiotic at proper levels
may permit the proliferation of microorganisms which synthe­
size the vitamins required by the animal.
(1950) and Slinger et al.

Groschke and Evans

(1951) suggested that antibiotics

may aid in the establishment of more favorable types of
bacteria which stimulate growth of the host as a result of
intestinal synthesis of unidentified factors required by the
chick.
The inhibition of the formation of certain adaptive en­
zymes in E. coli was reported by Hahn and Wisseman (1951) who
also thought this effect probably pointed toward a general
interference with protein metabolism of the organism.

Loomis

(1950) suggested that aureomycin may exert its antibacterial
activity through an inhibition of aerobic phosphorylation.
He observed that aureomycin depressed phosphorylation without
inhibiting respiration while penicillin, Chloromycetin, and
sulfadiazine were inactive when tested in a similar fashion,
Weinberg (1952) observed excellent growth of bacteria when
phosphate was included in the medium and that terramycin,
aureomycin, chloramphenicol and streptomycin inhibited growth
while penicillin and bacitracin were inactive in the presence
of phosphate.

When phosphate was omitted from the medium,

growth of the bacteria was moderate and aureomycin became in­
active while streptomycin and chloramphenicol remained anta­
gonistic to the bacteria.

This suggested that the aureomycin

- 24 -

exerted its antibiotic effect by acting on the phosphorylation
system of the bacteria,
Lichstein and Gilfillan (1951) found that streptomycin
markedly inhibited growth of S_. fragilis when it was growing
in a synthetic medium containing I3-alanine as the precursor
for pantothenate, but no effect on growth was observed when
pantothenic acid was included in the medium.

From this fact,

they postulated that streptomycin may inhibit the coupling
of I3-alanine and pantoyl lactone to form pantothenic acid.
Ely (1951) found certain surfactants would produce an
increased growth response in chicks similar to that observed
when antibiotics are used.

Luecke e_t a l . (1952) observed that

Ethomid C/15, a surface active agent, stimulated growth in
swine equal to that obtained when aureomycin was included in
the ration.
Berg et^ a l . (1950) stated that the effect of the anti­
biotic in promoting growth is dependent on the material con­
tinuously present in the diet.

He observed a cessation of the

accelerated growth response when aureomycin was removed from
the ration of the chicks.

Cunha and coworkers

(1951) reported

that the growth-promoting effect of various antibiotics will
vary with the species of animal and the nature of the basal
ration.
Elam et a l . (1951b) surmised that the antibiotic molecule
or a fragment of it might act as a metabolite within the body
of the chick, since parenteral administration of antibiotics

- 25 -

and autoclaved penicillin increased the rate of growth and had
little effect on the fecal microflora count.
pH of Rumen Contents
Phillipson (1942a) found that the rate of production of
volatile acids in the rumen of sheep depended on the diet*
Changes in pH reflected the fluctuations in the quantities of
organic acids that accumulate in the ingesta as a result of
fermentation of the food in the rumen.

The greatest fluctua­

tions were observed when the diet was high in soluble carbo­
hydrates and least when the diet was fibrous.

Gall et^ a l .

(1949) observed that the type of ration had some effect on
the pH of the rumen contents in cattle.

The pH values ob­

tained for a fattening ration were 6.3 to 6.7, a breeding
ration 7.0 to 7.3, and a dairy ration 6.8 to 7.3.
(1938)

Kick et_ al .

reported that the pH of rumen ingesta varied from 5.5

to 7.7 depending on the ration.

The ingesta was most alkaline

when alfalfa was fed alone and the pH decreased as the amount
of corn was increased in the ration.

Similar results were

noted by Hunt and coworkers (1943).

Smith (1941) also ob­

tained lower pH values when beet pulp was fed with alfalfa
hay than when the hay was fed alone.

When the ratio of starch

to roughage was increased in the ration, Burroughs et. al,. (1949)
found lower rumen pH values in a shorter time following feed
consumption.

Kick et. al^. (1938), Monroe and Perkins

(1939),

Wegner at al.

(1941), Smith (1941) and Myburgh and Quin (1943)

- 26 -

reported that the pH declined for about four hours with a
gradual increase toward alkalinity following the decline.
This cycle is repeated after each feeding.
Jacobson e_t al.

(1942) working with cattle and Phillipson

(1942a) working with sheep observed that the rumen contents
were more acid when the animals were grazing than when they
were stall-fed.

Gall e_t al.

(1949) did not notice any dif­

ference in the pH when the animals were on pasture or were on
the winter ration.
Roine and Elvehjem (1950) stated that the pH was not
determined by the food but by the kind of microflora which
developed in the digestive tract.

Gall et^ al. (1949) ob­

served that the fast-growing organisms usually ferment glu­
cose with the production of high acidity and turbidity while
the slow-growing cultures seldom exhibit much turbidity or
lowered pH.

Huhtanen and coworkers (1951) found the most

common types of organisms in calves' rumens were fast-growing
lactic acid forming bacteria which produce a low pH and heavy
turbidity.

The older cattle had types which grew more slowly

with little turbidity.
Coop (1949) working with sheep and Stone

(1949) working

with cattle observed that during fasting, the pH and the
production of the volatile fatty acids in the rumen ingesta
decreased.

Coop also reported that it took from 12 to 24 hours

for sheep to recover to normal ruminal activity following
starvation for several days.

Jacobson et^ a l . (1942) using an

- 27 -

In vitro method observed that the rumen contents of cattle
were very alkaline and gas production was low when the feed
was withheld for 24 hours.
Meites et al.

(1951) while studying _iri vitro digestion

of cellulose by rumen microorganisms found that the optimum
pH lies between 4.53 and 7,35.

Above pH 7.35, digestion

dropped sharply.

(1951), Clark et al.

Oyaert et al.

(1951a)

and Clark and Lombard (1951a) observed a decrease in the
motility of the sheep’s rumen as the alkalinity of the ruminal
ingesta increased.

When the pH reached 7.5 to 7.8, a complete

rumen paralysis resulted which could be relieved by adminis­
tering dilute acetic acid either orally or by intravenous
injection.

Clark et_ a l . (1951a) also observed a sharp rise

in pH and an increase in ammonia when the sheep were dosed
with urea.
Monroe and Perkins

(1939) and Smith (1941) noted that

the pH values varied in different regions of the rumen, es­
pecially soon after feeding.

Clark and Lombard (1951b) found

that the pH values of samples taken through the fistula were
lower than those taken by stomach tube.
Phillipson and McAnally (1942b) observed that volatile
fatty acids were the result of fermentation of carbohydrates
in the rumen.

Glucose, fructose and cane sugar fermented

more rapidly than maltose, lactose and galactose, while the
fermentation of starch and cellulose was much slower and the
production of volatile fatty acids was prolonged.

Elsden e_t a l .

- 28 -

(1946) observed that volatile fatty acids (acetic, propionic
and butyric acids) occur in the rumens or large intestines of
seven species of animals.

Gray (1948) found that both acetic

and propionic acids are absorbed readily from the rumen at an
acid reaction.

Acetic acid was not absorbed from an "isolated"

rumen when the reaction was made slightly alkaline by a solu­
tion of sodium acetate.
Barcroft et^ a l . (1944) stated that the volatile fatty
acids were absorbed through the rumen wall in order of acetic y
propionicbutyric•

Johnson (1951) showed the rate of absorp­

tion of these acids to be in the order of butyric > propionic >
acetic.
Myburgh and Quin (1943) noted that the rumen material was
well buffered between pH 6.8 to 7.8 against N_ HC1 or N_ NaOH,
whereas beyond this range, both on alkaline and acid side, the
efficiency of the buffering action was distinctly reduced.
Reid and Huffman (1949) found that the pH of saliva was 8.53
to 8.71.

They believed that this secretion is responsible

for the maintenance of a medium which appears to be optimal
for microbial activity and chemical changes in the rumen.
Clark and Lombard

(1951b) stated that the normal regulation

of pH of ruminal ingesta depends on the interaction between
organic acids from microbial activity of carbohydrate and
sodium bicarbonate of saliva or by selective absorption of
volatile fatty acids from the rumen.

- 29 -

Rate of Passage of Materials Through the Digestive Tract
The length of time a feed remains in the rumen would appear
to influence its eventual utilization.
been made to study this factor*

Various attempts have

Fish (1923) and Reed e_t al*

(1928), in studies with cattle, found that Sudan III dye first
appeared in the feces within 15 to 17 hours and that 50 to 60
hours

were required for complete passage through the tract*

Balch

(1951a) using stained particles noted that the marker

first occurred in the feces 12 to 24 hours after feeding.
Approximately 80 per cent of the stained residues appeared
within 70 to 90 hours while it took seven to ten days to com­
plete the excretion of all of the stained particles.

Iron

oxide and rubber disks were used by Moore and Winter (1934)
who observed that most of these materials had passed through
the tract within 36 to 40 hours while it took five to six days
to complete the elimination of the materials.
noted

Hoelzel

(1930)

that the rate of passage was found tobe more or less

proportional to the

specific gravity of the test materials, the

heavier materials required more time than the lighter materials.
Also, the rate of passage varied considerably with different
species and individuals.

Amadon (1926) stated that the weight

of the feed determined the route to be followed; in that, the
light food went to the back part of the rumen while a portion
of the heavy food went directly into the reticulum.
et al.

Mitchell

(1928) suggested that the rate of feeding may be an

- 30 -

important factor in determining the length of time required
for food to pass through the digestive tract*
Burroughs et_ a l . (1946) studied two methods for measuring
the rate of passage of food through the rumen of cattle.

The

first method was a mathematical approach which was based upon
the nutrient composition of the feeds ingested and the total
weights of various nutrients found in the rumen at a given
time.

The second method consisted of the physical separation

of the dried rumen contents by fanning or by floatation with
water.
Phillipson (1948) using sheep with a duodenal cannulae
observed from quantities of digesta collected for 1.5 hours
at different intervals throughout the day that there is no
simple relation between total dry matter of the food and the
flow of the digesta.
Quin and Van der Wath (1938) found that after three to
four days of complete starvation of well nourished animals, a
complete cessation of ruminal movement may take place at any
time.

On subsequent feeding of such starved animals, rumen

motility usually reappeared after considerable delay, as the
appetite itself may be seriously disturbed after prolonged
fasting.

Coop (1949) observed that the type of ration had an

influence on the rate of activity in the rumen.

- 31

Microorganisms in the Rumen
The ruminant is unique from other animals in that it has
four stomachs.

It relies mainly upon its fermentation vat

for the nutrients which are made available by bacterial fer­
mentation and decomposition of feedstuffs in this chamber.
Numbers and types.

Baker (1942a) and Gall et al.

(1947)

stated that the number of bacterial cells in each milliliter
of rumen fluid is expressed in billions.

These values were

obtained by the microscopic examination of formalized rumenfluid samples.

Baker

(1947) stated that both pre-cultural

and direct microscopic methods of evaluating bacterial counts
were superior to the plate method which gave an unreliable
estimate of the kinds and numbers of organisms concerned.
Gall et a l . (1947) and Moir and Williams (1950) observed that
samples of rumen juice obtained by use of the stomach tube
gave consistently lower bacterial counts than samples taken
directly from the rumen through a rumen fistula.

Moir and

Williams stated that the method was constant and compares
favorably with the fistula method.
Henneberg (1922) was the first investigator to apply
direct microscopy to detect the microorganisms concerned in
the digestion of structural cellulose.

He stained the organ­

isms with iodine and called the organisms that gave a blue
color, iodophiles.

Of the iodophilic organisms, Baker (1942a,

- 32 -

1943) has distinquished at least five species by their large
size:
1*

Oscillospira guillermondi - A colorless spore-forming
oscillarian.

2.

A giant Spirillum - Divided by transverse septa into
spherical or ovoidal compartments.

3.

Large Sarcina Packets.

4.

An unidentified navicular organism forming rosette­
shaped oscillations of 5 to 30 units.

5.

Coccoid chains of 2 to 8 units.

In more recent investigations Bryant

(1951) found 50 to

60 different kinds of bacteria in rumen contents.

The mor­

phological types included various rods, cocci, oval-shaped
and spirochetes.

There was considerable variability in the

frequency of occurrence of the different kinds of bacteria but
about one-fourth were found with some regularity.

Gall and

Huhtanen (1951) designed some criteria for determining a true
rumen organism.

These are:

(a) anaerobiosis,

(b) presence

in numbers of one million or more per gram of fresh rumen con­
tents,

(c) isolation of a similar type bacterium at least ten

times from at least two animals,

(d) isolation from animals in

at least two geographical locations, and (e) production by the
organisms of end-products found in the rumen from substrates
found in the rumen.

They also observed that at least 99 per

cent of the organisms isolated from the rumen were anaerobes.
Huhtanen et a l . (1951) found nine organisms characteristic of
calves' rumens that almost never occur in the rumen of healthy

- 33 -

adult cattle maintained on a balanced practical ration.

The

11 organisms usually found in the rumen of adult cattle begin
to appear in the rumen of calves as early as two months of
age, depending on the ration, and become more prominent as the
calf approaches maturity.

The most common types found in the

calves ' rumens were fast-growing lactic acid-forming bacteria
which produce low pH and heavy turbidity while the adult types
grew more slowly with little turbidity and a higher pH.
Baker

(1942a, 1943), Hoflund and Hedstrom (1948), Johnson

et a l . (1944) and Uzzell et al . (1949) found that protozoa are
another group of organisms that are normal inhabitants of the
rumen.
Effect of Various Rations on Rumen Microorganisms
Bortree et al. (1946) found by taking samples every two
hours for 10 to 12 hours that an increase in the numbers of
bacteria occurred about two hours after feeding and remained
high for several hours after which there was a gradual return
to normal.

The addition of glucose to the ration caused the

counts to increase about 100 per cent over those on hay alone.
These workers only counted iodophiles, therefore, the changes
do not reflect changes in the total population.

The addition

of grain or a readily fermentable carbohydrate decreased the
time required to reach the peak of the bacterial population
in the rumen.

In 1948, Bortree et_ aj. found that an iodine

staining "giant spirillum11 occurred in the rumen when methion-

- 34 -

ine was added to a ration consisting of corn, starch, glucose
and minerals.
Pounden and Hibbs

(1948a, 1948b, 1949, 1950b) were suc­

cessful in establishing certain microorganisms in the rumen
of young calves by inoculating them with cud material obtained
from mature animals.

The inoculations assisted in the estab­

lishment of protozoa in the rumens of calves eating hay alone
or both hay and grain.

No protozoa or hay organisms were

present in calves on grain alone.

Very small gram-negative

organisms and a moderate number of protozoa were prevalent
in calves on rations containing hay alone or high proportions
of hay.

Small gram-positive short rods or cocci were observed

in increasing proportions after the addition of grain to rations
of hay.

Rations high in grain or low in roughage depressed the

numbers of rumen microorganisms which were characteristically
associated with relatively high roughage ingestion.

They also

found that the rumen microorganisms can be established in young
calves on pasture if grain is not fed in excessive amounts.
Conrad et_ al.

(1950) observed that inoculated calves digested

a significantly higher percentage of cellulose and dry matter
than the calves not inoculated.
Gall

(1949a) and Gall et a l . (1949c) noted that animals

on pasture had higher bacterial counts than animals fed in the
barn.

Gall et al.

(1949c) postulated that the fast-growing

organisms which break down glucose use starch and other soluble

- 35 -

carbohydrates in the rumen; while slower growing bacteria,
which clump on cotton, might be cellulose digesters.

Gall

(1949a) found that cobalt-deficient sheep showed a simpler flora
and lower bacterial count than sheep on the same ration plus
cobalt.

Gall et al.

(1951) studied the effect of purified

diets upon the rumen flora and found that sheep fed the ureawithout-sulphur ration supported a rumen bacterial population
consisting almost entirely of facultative anaerobes in place
of the obligate anaerobes usually found in the rumen.

Different

physiological types of bacteria were found in the rumen of sheep
fed casein than in sheep fed urea plus sulphur.
Burroughs et_ aj.
Arias et al.

(1950a, 1950d, 1950e, 1951a, 1951b) and

(1951) used an artificial rumen to study the

factors affecting cellulose digestion by rumen microorganisms.
They

(1950a) found that a complex salt solution, ash of alfalfa

extract, autoclaved rumen liquid and an autoclaved water ex­
tract of manure were beneficial in aiding rumen microorganisms
to digest cellulose.

There was a decrease in bacterial popu­

lation and a decrease in individual size of bacteria without
any noticeable change in predominating types in the flasks
promoting poor cellulose digestion.

In recovery experiments

in which any one of the above substances was added to the flask,
cellulose digestion progressively improved; the size and the
numbers of bacteria increased but the types of bacteria were
the same.

Burroughs e_t al.

(1950d) found that protozoa were

always present in the flasks showing some cellulose digestion.

- 36 -

Burroughs et a l . (1950d, 1951a) stated that rumen microorganisms
had three general nutrient requirements; first, related to
energy which is the motivating force for rumen bacteria to
digest compounds like cellulose; second, related to protein or
elements such as nitrogen which are eventually synthesized to
protein; and third, inorganic nutrients which are involved in
enzymes or enzyme systems of the organism.

Burroughs et_ a l .

(1951b) found that phosphorus and iron were effective in sti­
mulating urea utilization and cellulose digestion by rumen
microorganisms.

Arias at a l . (1951), using six different

carbohydrate sources for energy, observed that small amounts
of a readily available carbohydrate aided in cellulose digestion
which in turn increased urea utilization; whereas, large amounts
of such materials inhibited cellulose digestion.
Bentley et a l . (1951) used the artificial rumen technique,
similar to that devised by Burroughs, to study the effect of
feeding poor quality hay on the biochemical functions of the
rumen microorganisms.

They found that the activity of the

rumen microorganisms from a steer receiving a poor hay ration
had decreased ammonia utilization by 85 per cent at the end of
one week and the rate of cellulose digestion had dropped 90 per
cent at the end of four weeks.

Volatile fatty acid production

dropped 30 per cent and riboflavin synthesis was reduced 14
per cent.

When the steer was allowed free choice of a mineral

mixture consisting of two parts of steam bonemeal, two parts
of ground limestone and one part of salt, the activity of the

- 37 -

microorganisms was similar to that observed when alfalfa hay
was fed.

When the mineral mixture was removed from the ration,

the activity of the microorganisms was lowered again.

Pre­

liminary evidence indicated that the low phosphorus content
of the poor quality hay was a limiting factor in cellulose
digestion.

Burroughs et a l . (1950b), from their study with

corncobs, suggested that the quality of roughages as applied
to animal feeding may be dependent to a large extent upon the
mineral makeup of the roughage supplying nutrients for the
rumen microorganisms involved directly in roughage digestion.
Burroughs et al.

(1950c) found a decided decrease in the

bacterial counts when starch was added to the roughage, but
these counts increased when casein was added to the ration.
These findings lend support to their theory that protein aids
roughage digestion by furnishing an essential nutrient for
the bacteria concerned directly in roughage digestion.
McNaught et^ ad.

(1950a) observed that 1,000 parts per

million of iron, 25 parts per million of cobalt, 1,000 parts
per million of copper and 2,000 parts per million of moly­
bdenum definitely inhibited growth of rumen bacteria.

It was

shown that one to two parts per million of iron in rumen liquid
produced good bacterial growth.
Depending on concentration used, //einstein and McDonald
(1945) found that both urea and urethane exert a bacteriostatic
and bactericidal action on gram-negative bacteria and to a
lesser degree on gram-positive organisms.

- 38 -

Thomas et_ al_. (1951) observed that lambs on a sulfurdeficient ration gradually lost appetite, declined in body
weight and finally died.

There were marked changes in the

types and numbers of rumen flora in the sulfur-deficient lambs.
Reed et_ a l . (1949) found that sheep fed dry feed had
coccal and oval cells and some giant iodophiles, while lambs
receiving green feed had a greater proportion of rod-forms
and an increase in spiral-forms.

Moir and Williams

(1950)

observed a very high correlation between the levels of protein
intake and the number of microorganisms in the rumen.
Quin (1943) and Quin et_ al_. (1951) found that fasting
caused a marked decrease in the ability of the rumen microflora to ferment glucose.

Quin et al.

(1951) observed that

starvation caused a decrease in cellulose digestion; also, it
affected the free organisms in the rumen liquid before those
embedded in the plant particles were affected.

They also ob­

served that sheep automatically regulate the intake of protein
to correspond with the ability of the flora to metabolize it.
Balch and Johnson (1951b) observed that the rate of
breakdown of cellulose in the rumen of cattle was much faster
in the ventral sac than the dorsal sac.

They also noted that

a low dry matter content of the ration favored the rapid break­
down of cellulose.

- 39 -

Functions of Rumen or Intestinal Microorganisms
In 1939, Baker described a process of bacterial decom­
position by formation of zones of erosion around the respon­
sible microorganisms.

The zones were studied by double re­

fraction and the loss of double refraction indicated the
region had been used up.

Baker (1942b) made a direct micro­

scopic examination in polarized light to study the decomposi­
tion of cellulose by iodophilic organisms on the surfaces and
in the interstices of vegetable fragments or, in the case of
starch, the organisms concerned would be lodged on the sur­
face of the starch granule.
Hungate has made some very good studies concerning the
ability of the rumen microorganisms to digest cellulose
1943, 1944, 1946, 1947, 1950).

He

(1942,

(1944, 1946) postulated

that the bacteria secreted an enzyme, cellulase, which diffused
into the substrate, attacked the substrate to produce sugars
which diffused back to the bacterial cell.

A clear area de­

veloped around each colony which indicated that the cellulase
was not free to diffuse until the substrate immediately adja­
cent was completely dissolved.

Hungate

(1947, 1950) was

successful in isolating some microorganisms found in the rumen
in pure culture.

He isolated a cocci which was important in

the decomposition of crude fiber in the rumen.
lated several of the rod-type organisms.

He also iso­

One of these, a non­

spore-forming rod, actively fermented cellulose with the forma­

- 40 -

tion of acetic and succinic acids*

Another rod attacked many

carbohydrates, including hemicelluloses, to form butyric acid.
He also observed that some of the rods did not show an iodophilic reaction and stated that this feature alone was not an
adequate index of the cellulose digesting ability of the
microorganisms.

Hungate

(1942, 1943) found that the Diplodin-

ium protozoa secreted cellulase and were capable of digesting
cellulose while the Entodinlum, Qsatricha. Dasytricha and
Biitschlia do not.

Burroughs et al.

(1950d) using the arti­

ficial rumen technique always found protozoa present in the
flasks showing some cellulose digestion.
The protozoa digest the bacteria in order to synthesize
their own protein (Baker 1942b, 1942c, 1943).
(1944)

Johnson et al.

observed a symbiotic relationship between bacteria and

protozoa in the rumen.

The greatest number of bacteria and

fewest number of protozoa were present one hour after feeding,
whereas the bacterial population gradually decreased and the
protozoan population increased for about 16 hours.

Their data

are in agreement with the hypothesis that the nitrogen of the
food is first synthesized into bacterial protein; then the
protozoa use bacterial protein for their own use; and finally,
the host digests protozoan protein and the remaining bacterial
protein.

Pounden et_ al^. (1950a) examined contents from various

parts of the digestive tract of cattle for the presence of
four types of bacteria normally present in the rumen.

They ob­

- 41 -

served that the large coccoids were found in all parts of the
digestive tract, although fewer in the large intestine; the
cigar-shaped organisms disappeared in the abomasum; the large
or small rods disintegrated gradually as they reached the
posterior part of the digestive tract; and protozoa were des­
troyed in the abomasum.

Uzzell et_ al. (1949) observed that

the stomach and small intestine of calves were devoid of pro­
tozoa at birth.

They found 18 species of protozoa in 12

animals and agree with Baker and Pounden as to the fate of the
protozoa in the abomasum.
Van der Wath

(1948) observed that bacterial disintegration

of starch began within five hours after feeding and was com­
pleted in 18 to 20 hours when sheep received starch regularly,
but it took seven hours for disintegration to begin and 8 to
10 hours longer for disintegration to be completed in sheep
not used to receiving starch.

He also stated that the rate

of breakdown of different carbohydrates depend upon the com­
plexity of the molecule.

Baker et al.

(1950) observed that

corn starch was broken down more readily than potato starch
and in a shorter time in ruminants.
The ability of the ruminant to synthesize some of the
vitamin B complex was first noted by Bechdel and Honeywell
(1927).

Hoflund and Kedstrom (1948) stated that the fungi,

another type of organism in the rumen, synthesized the B vita­
mins and had the ability to synthesize amino acids.

They also

stated that the bacteria in the rumen were concerned primarily

- 42 -

with carbohydrate decomposition, particularly cellulose; while
the infusoria (protozoa) assimilated vegetable protein and con­
verted it into animal protein more suitable for the host.
More complete information on the role of microorganisms
in the rumen may be obtained from the reviews by Hastings (1944),
Slsden and Phillipson (1948) and Johansson and Sarles

(1949).

Formation of Protein in the Rumen
Zuntz

(1891) first suggested that non-protein nitrogen

might be converted to protein by the bacteria, which in turn
was used by animals.

Armsby (1911) concluded that non-protein

nitrogen could serve as a partial substitute for protein for
maintenance, milk production and growth when the level of
protein in the ration was low and the other conditions were
favorable.

He stated, however, that non-protein nitrogenous

substances were inferior in nutritive value to protein of an
equivalent nitrogen content.
In 1939, a series of experiments using urea as the prin­
cipal source of non-protein nitrogen were begun by Hart e_t al .
and continued through the war years by VVegner e_t al.. (1940a)
and Mills et. a l . (1942).
(1939)

Using growing calves, Hart et al.

found that urea and ammonium bicarbonate could be used

as a partial supply of nitrogen in the ration.

They also ob­

served that the most efficient utilization came when some
soluble sugar, such as corn molasses, was included in the

- 43 -

ration.

Wegner et_ al_. (1940a) using an in vitro technique

presented evidence to show that rumen bacteria convert in­
organic nitrogen to protein.

They noted that the extent of

disappearance of inorganic nitrogen depended on the amount of
carbohydrate in the medium and not the source, except cellu­
lose; also the decrease in ammonia could be accounted for by
an increase in protein nitrogen.
Wegner et a l . (1941a, 1941b) observed that the ureanitrogen would always be hydrolyzed to ammonia within one hour
after feeding.

These workers used an animal with a rumen fis­

tula from which the samples could be removed whenever desired.
Wegner e_t a l . (1941b) noted that the rate of conversion of
urea-nitrogen to protein decreased whenever the protein level
of the rumen ingesta became greater than 12 per cent.

Mills

et a l . (1942) observed that the hydrolysis of urea to ammonia
was delayed when urea was fed with hay and about one-half of
the urea was found in the rumen six hours after feeding.

When

starch was added to the hay and urea ration, the urea was hy­
drolyzed within one hour after feeding and the ammonia formed
from the hydrolysis would disappear from the rumen in six
hours.

As the ammonia level decreased, the protein level of

the rumen increased.

Mills et a l . (1944) found that starch was

superior to molasses in promoting protein synthesis in the
rumen.

The starch was less soluble and remained in the rumen

longer than the molasses.

Bell et_ a l . (1951), using different

- 44 -

carbohydrate feeds in digestion studies with steers receiving
urea nitrogen, found that nitrogen retention in the steers
was greater when corn was added than when molasses was added
to the ration.
Harris and Mitchell

(1941a, 1941b) observed that urea

added to the low nitrogen ration improved the digestibility
of cellulose and itself was digested to the extent of 88 per
cent.

On calculation, they found that the biological value

of urea nitrogen was 62, while the value for casein nitrogen
was 79.

Harris and Mitchell

(1941b) found that as the amount

of urea was increased to produce rations of higher protein
equivalent, the average biological value decreased.

Harris

et a l . (1943) observed that more true protein was found in
the rumen of steers receiving urea than in those subsisting on
the same low protein ration without urea.

Johnson ejb^ a l . (1942)

noted that the protein is digested by the ruminant as it passes
through the abomasum in the same manner as any other preformed
protein.

In this manner, the ruminant would actually digest

approximately the same type or quality of protein irrespective
of the sources of nitrogen, provided the total nitrogen con­
sumed did not exceed the maximum amount which the microorganisms
could utilize.

They also stated that the biological value of

nitrogen in rations containing 10 to 12 per cent protein is
about 60.

Moir and Williams

(1950) found that about 50 per

cent of the ingested protein was converted to bacterial protein.

- 45 -

They also noted that as the amount of protein in the ration
increased, a definite decrease in biological value occurred.
Loosli and Harris

(1945) obtained an increase in the

rate of gain and nitrogen stored when urea was added to the
basal ration.

The addition of methionine to the urea ration

increased the rate of gain and nitrogen retention to the same
level as the linseed oil meal ration.

They stated that it

appeared likely that the protein formed in the rumen by bac­
terial action was deficient in methionine or that the diet
was deficient, which could limit the quantity of protein syn­
thesized.

Reed et_ a l . (1949) separated bacterial cells from

rumen juice by use of a Sharpies centrifuge.

The rumen samples

were obtained from sheep that were fed either dry or green
feed.

Both samples had about the same cysteine content but

the lambs fed the green feed had a higher level of methionine.
They concluded that the rumen bacterial protein must be re­
garded as low in digestibility, relatively high in biological
value, but mildly deficient in methionine.

Thomas et al.

(1951)

observed that lambs fed a sulfur-deficient ration containing
urea lost appetite and body weight, became emaciated and
finally died; also, the urea nitrogen was apparently not
utilized, since the deficient lambs were consistently in nega­
tive nitrogen and sulfur balance.

Block and Stekol

Block et al_. (1951) fed radioactive sodium sulfate
a cow and a goat.

(1950) and
(S35 ) to

They observed that both cystine and methion­

- 46 -

ine found in the cow's milk contained radioactive sulfur in
appreciable amounts.

In the case of the study with the goat,

the radioactivity was the same for methionine and cystine in
the milk, serum albumin and rumen.

The results indicated that

methionine and cystine were synthesized in the rumen at approxi­
mately the same rate and were used by the tissues to make new
protein in quantities needed.
Loosli e ^ a l . (1949) working with sheep and Agrawala

(1950)

using steers found that the animals can synthesize considerable
quantities of the essential amino acids in the rumen.

In both

instances a purified ration was used as the basal ration with
urea as the sole source of nitrogen.

Loosli et_ al . (1949) also

reported that animals on a purified ration containing glycine
as the only source of nitrogen synthesized the amino acids at
a lower level.
Since the rumen contents are part of a moving system,
heterogenous in character, and therefore difficult to sample,
some investigators have developed _in_ vitro techniques for con­
ducting studies of rumen problems.

Pearson and Smith (1943a)

observed that the total nitrogen content of the rumen ingesta
varied as much as 20 per cent when taken from four different
locations in the rumen; while there was 16 per cent difference
when the samples were taken at different depths.

Pearson and

Smith (1943a, 1943b, 1943c) filtered the rumen contents through
muslin to remove the coarse particles and the resulting liquid
was incubated at 39°C. for three to four hours under conditions

- 47 -

similar to those in the rumen.

When the temperature was

increased to 50° or 60°C. , hydrolysis of the protein occurred.
It was believed, therefore, that the changes which occurred
in the incubated material during the first few hours after
removal from the rumen closely resembled those which occur in
vivo,

They also stated that the synthesis of protein was

microbiological as shown with toxic substances such as boric
acid, quinone and sodium fluoride,

Tlhen the concentration of

these increased, hydrolysis of the protein occurred.

McNaught

et a l . (1951b) separating bacterial cells in a Sharpies cen­
trifuge observed that 58 per cent of the bacterial protein was
present in the liquid when it was removed from the rumen.
other 42 per cent was synthesized during incubation.
(1945)

The

Smith

stated that the greatest part of synthesis occurred

within the first two hours of incubation.

Pearson and Smith

(1943b) stated that the urease activity of rumen ingesta was
so great that all the urea ever likely to be fed would be
readily converted to ammonia in one hour.
actually been observed by Wegner

This fact has

(1941a, 1941b) in their

studies with the rumen fistula animal.
B-Complex Vitamin Synthesis in the Ruminant

Bechdel and Honeywell

(1927) observed that cows maintained

on a ration deficient in the vitamin B complex from growth
through completion of the first lactation produced milk with
vitamin B potency equal to that of the herd milk from cows

- 48 -

receiving a good winter ration.

Bechdel et al.

(1928) stated

that the vitamin B complex was produced in the rumen by bac­
terial fermentation.

Gall et a l . (1951) have been successful

in isolating some organisms responsible for the synthesis of
some of the B-vitamin complex.

Type RO-575 was the predominat­

ing organism and was found to be able to synthesize several
members of the B-vitamin complex.

This type was found mostly

in urea plus sulphur ration and occasionally in the other
rations.

Type RO-T also was present in the sheep fed urea

plus sulphur but it required several of the B vitamins for
growth rather than synthesizing any.

Type R0-C8 was found in

animals receiving the urea plus sulphur and casein ration and
was capable of synthesizing several of the B vitamins.
Thiamine synthesis.

Hunt et al.

(1941, 1943) observed a

slight increase in the quantity of thiamine in the rumen about
four hours after feeding but a decrease 16 hours after feeding.
By increasing the amount of corn in the ration, a slight in­
crease in thiamine in the rumen could be observed.

Lardinois

et a l . (1944) could not find evidence for the synthesis of
thiamine in the rumen.

McElroy and Goss

(1939, 1941a) found

that thiamine was synthesized in the rumen of the sheep, but
no thiamine could be detected in two cows with rumen fistulas
fed the same deficient ration as the sheep.

However, they did

detect thiamine in the rumen contents of an intact cow and
suggested that an artificial opening into the rumen may alter

- 49 -

the conditions in such a way as to make it unfavorable for the
growth of rumen organisms capable of synthesizing thiamine.
Johnson et a l . (1941) observed that the rumen contents from
goats, sheep and calves fed the same thiamine-deficient ration
contained some thiamine.

Teeri et, al.

(1950, 1951a, 1951b)

noted that the thiamine excretion of heifers was always greater
than the dietary intake.

Kesler and Knodt

(1950, 1951a, 1951b,

1951c) working with young dairy calves found that the concen­
tration of thiamine in the digestive tract, especially the
rumen, was greater than the feed consumed on a dry matter basis.
After the calves were eight days old, the concentration of the
vitamin in the digestive tract did not change with age.

Also,

no effect could be observed between the time of the last feed­
ing and time of slaughter following the feeding.

Wegner .et al .

(1940b) observed that thiamine added to the rumen was not des­
troyed and that there was an apparent stimulation of the other
factors when thiamine was added.
Riboflavin synthesis.

McElroy and Goss (1939, 1940a)

found that the riboflavin content of the rumen ingesta in
sheep increased 100-fold over that of the feed.

Further evi­

dence of riboflavin synthesis was presented by Wegner et_ a l .
(1940b, 1941c) and Hunt e_t al. (1941).

Wegner et al.

(1941c)

observed that the riboflavin values of rumen contents de­
creased as the nitrogen level of the ration increased.
et al.

Hunt

(1941) observed no increase in riboflavin when steers

- 50 -

were fed hay alone, but that riboflavin increased when corn
was included in the ration*

However, Lardinois et al . (1944)

found that the addition of urea to a ration containing a
readily fermentable carbohydrate definitely increased the
synthesis of riboflavin in the rumen.

Teeri e_t al.

(1951b)

found that cows fed a low quality of late-cut hay had a de­
creased excretion of riboflavin.

Also, the excretion values

of riboflavin indicated that silage and cane molasses favor
the rumen or intestinal synthesis of this vitamin.
and Knodt

Kesler

(1950, 1951a, 1951c) observed that in the case of

calves the riboflavin concentration was highest in the small
intestine.
Loosli and McCay (1943) observed that the riboflavin
content of the organs and edible meat was not increased by
feeding supplements of B vitamins to calves.
Several investigations have been conducted to study the
influence of ration on the riboflavin content of milk.

V/hit-

nah e_t a l . (1938) noted a breed difference in the riboflavin
content of milk; Jersey milk was the highest and Ayrshire
was the lowest.

Also, the riboflavin content of milk was

fairly constant between 15 days and 10 months after freshening.
Kramer e_t al.. (1938) found that colostrum was richer in ribo­
flavin than milk produced later in the lactation.
et al.

Whitnah

(1938), Kramer et. al.. (1939) and Hand and Sharp (1939)

observed that cows on pasture produced milk higher in ribo­

- 51 -

flavin than cows on a winter ration.

Johnson et, a l . (1941)

found that when cows were changed from pasture to a ration
low in riboflavin the riboflavin content of the milk decreased
25 per cent.

They also observed that goats fed purified

rations continued to secrete large amounts of riboflavin in
the milk; which indicated that riboflavin was not a dietary
essential for lactation in the goat.

Agrawala (1950) observed

a very high synthesis of riboflavin in the rumen of steers
fed a purified ration.
Nicotinic acid synthesis.

Wegner et, al . (1940b) obtained

first proof of synthesis of nicotinic acid.

These workers

found a three- to four-fold increase in the concentration of
this vitamin in the dried rumen contents as compared with the
feed.

Lardinois et^ a l . (1944) observed that urea plus a

readily fermentable carbohydrate increased the level of nico­
tinic acid in the rumen.

Johnson et^ al . (1947) found that

nicotinic acid excretion remained normal throughout the experi­
ment while the calves were on a nicotinic acid-free ration,
even though one per cent sulfathalidine was included in the
ration.

They stated that the nicotinic acid was synthesized

in the body tissue rather than in the rumen or intestinal
tract of these animals.

Kesler and Knodt (1950, 1951a, 1951c)

observed that nicotinic acid, like riboflavin, was in the
highest concentration in the small intestine.

Teeri et, al .

(1951a) found that wood molasses and cane molasses were com­
parable with respect to nicotinic acid synthesis in the rumen.

- 52 -

Lindahl and Pearson (1951) observed that the excretion of
niacin from sheep getting a ration containing casein was
higher than on a low protein ration.

They believed that

tryptophan was converted to niacin by sheep.
of riboflavin, Agrawala

As in the case

(1950) observed a very high synthesis

of this vitamin in the rumen of steers fed a purified ration.
Pantothenic acid synthesis.

Ruminal synthesis of panto­

thenic acid was demonstrated in sheep by McElroy and Goss
(1939).

These same authors (1941b) observed that rumen con­

tents from sheep and cows receiving a low B complex vitamin
ration had a 25-fold increase of pantothenic acid over the
ration fed.

Wegner et al.

(1941c) and Lardinois et al.

(1944)

have observed that pantothenic acid was synthesized in the
rumen.

Teeri et^ a l . (1950, 1951a) observed that the excretion

of pantothenic acid was greater than the dietary intake, es­
pecially when molasses was included in the ration.

Agrawala

(1950) observed that there was appreciable synthesis of panto­
thenic acid in the rumen when the synthetic ration was fed,
but not as great as that when the steers received a normal
ration.
Synthesis of other 3-vitamins.
1940b) and Lardinois et_ al.

McElroy and Goss (1939,

(1944) have reported the synthesis

of pyridoxine in the rumen of sheep and cattle.

Lardinois

et a l . (1944) stated that pyridoxine showed a definite in­
crease when a fermentable carbohydrate was fed with urea.
The same authors

(1944) found folic acid in the rumen of a

- 53 -

cow receiving a ration which contained little or none of this
vitamin,
McElroy and Jukes

(1940c) observed that biotin was syn­

thesized in the rumen of a cow.

Within four hours after feed­

ing, Wegner _et a l . (1940b) found a five-fold increase in biotin
of the rumen contents as compared with the feed,
Abelson and Darby (1949) feeding radioactive cobalt to
sheep noted that large amounts of vitamin B^g occurred in the
sheep feces,

Lindahl and Pearson (1951) observed an increase

in the synthesis of vitamin B]_g i*1 sheep receiving an all-hay
ration as compared to a purified ration.
Summary of the Review of Literature
Many investigators have reported that antibiotics produce
increased growth, less digestive disturbances and an increase
in feed efficiency in swine and poultry.

The antibiotics used

in these studies were, namely; aureomycin, penicillin, strepto­
mycin and terramycin.

Favorable growth responses have been

obtained when aureomycin was fed to young calves; however,
adverse effects have been reported by several investigators
when this antibiotic was fed to beef steers, heifers and lambs.
There are conflicting reports in the literature concerning
the effect of the antibiotics on the intestinal microorganisms;
nevertheless, whether the antibiotic caused an increase or de­
crease in the number of microorganisms, the effect of the

54 -

antibiotic appeared to be dependent on the concentration used,
the type of ration and the species of animal.
Most of the theories which have been proposed for the
action of antibiotics are as follows:

Antibiotics reduce the

bacterial flora which may compete with the host for nutrients;
antibiotics inhibit intestinal microorganisms that are either
producing toxic materials or are rendering certain dietary
essentials unavailable for utilization; antibiotics at proper
levels permit proliferation of microorganisms which synthesize
vitamins and unidentified factors required by the animal.

The

inhibition of bacterial growth by antibiotics is probably the
result of some interference with the metabolic processes of the
bacterial cell, or the interference with various bacterial en­
zymatic systems, or by preventing the synthesis of some essen­
tial metabolite by the bacterial cell, or may act as a deter­
gent and affect the surface tension of the bacterial cell.
As many as 50 to 60 different types of bacteria have been
observed in the rumen contents.

The total number of the micro­

organisms was very large and was usually expressed in billions
per milliliter of rumen fluid.
The type of ration that the animal received had a definite
influence on the type of organisms present in the rumen and
also on the number and size of the bacteria.

The pH of the

rumen contents was dependent upon the type of ration fed and
the stage of activity of the microorganism in the rumen.

55 -

Bacterial synthesis of amino acids and B vitamins on
natural and purified rations has been demonstrated ijn vitro
and i_n_ vivo.

Most of the investigations concerned with the

formation of protein have used urea as the non-protein nitrogen
supplement.

EXPERIMENTAL PROCEDURE
Animals Used in Experiment
Two-year old steers, 707-a Guernsey and 714-a Holstein,
which were fitted with plastic rumen fistula plugs were used
in these experiments.

The plugs were easily removed when

samples of rumen contents were desired.

Rations Used
The steers were fed a natural ration which consisted of
79 per cent second-cutting, alfalfa-brome hay and 21 per cent
corn.

The chemical composition of these feeds is shown in

Table 1.

The composition of the purified ration fed to 714

is shown in Table 2.
TABLE 1
CHEMICAL COMPOSITION OF THE CORN AND ALFALFA-BROME HAY
USED IN THE NATURAL RATION

Dry
matter

Crude
protein

Crude
fiber

Ether
extract
i

Nitrogen- Non­
free
protein
extract nitrogen
%

%

%

Corn

87.76

9.90

3.13

4.36

80.33

0.107

Alfalfabrome hay

89.72

14.18

34.38

1.84

43.52

0.628

- 57 -

TABLE 2
COMPOSITION OF THE PURIFIED RATION 1 *2

Corn starch

42#

Cellulose^

Glucose

24#

Lard

4#

Hay

1%

Urea

4#

Mineral mixture

5#

CaHP0 4

11.0#

FeC 6H 50 7 *5H20

2.5#

CaC0 3

16.0$

MnS0 4 *4H20

0.7#

k 2hpo 4

27.0#

KI

0.08#

MgS0 4 *7H20

15.0#

ZnClg

0 .02#

5.0#

CuS0 4 -5H20

0.03#

22 .6#

CoS0 4 *7H20

0.07#

NagS0 4
NaCl

20#

1 Steers received 400,000 I.U. of vitamin A and 5,000,000
I.U. of vitamin D every two weeks.
^ Protein equivalent

(N x 6.25) = 12.3#.

3 Solka - Floe, cellulose product, Brown Co., Berlin, N.H.
Method of Feeding
The steers received the total ration of four pounds of
corn and 15 pounds of hay once daily.

The corn was fed first

and the hay was placed before the steers by 8 A.M. each day.
The animals consumed the corn in about 10 minutes and the hay
in about three hours.
Crystalline

Water was available in a drinking cup.

aureomycin was added to the ration for 15 days at

0.5 gram level and then was increased to 1.0 gram for the next

- 58 -

15 days.

The aureomycin, when fed, was mixed with the corn

before feeding.
In the case of the purified ration, the steers were
gradually changed to the new ration over a five-day period.
They were given 14 pounds once daily.

Steer 714 ate the

ration well, while 707 refused to eat it at all.

The portion

refused was put into the rumen through the fistula opening.
After two weeks of this treatment, 707 was removed from the
experiment,
Steer 714 went off-feed just prior to the addition of 0.5
gram of aureomycin to the purified ration and did not regain
its appetite during the period that aureomycin was included
in the ration.

The animal was fed through the fistula for

the following two weeks.
Sampling Procedure
Chemical analysis.

The rumen contents were removed through

the fistula opening after removing the plastic plug.

The solid

material was removed by hand, while the liquid portion was re­
moved by use of a beaker.

The contents were weighed and thor­

oughly mixed; after which, approximately a 550-gram representa­
tive sample of liquid and solid material was taken for chemical
analysis.

Approximately one hour was required to perform the

above operations, which were completed rapidly so as to prevent
the ingesta from becoming too cool.

The material that was

- 59 -

taken for chemical analysis was ground in a food grinder and
after mixing thoroughly, exactly 500 grams were placed in a
brown glass bottle.

The samples were frozen and stored in a

deep freeze until completion of collections for all of the
experiments.

The rumen contents that were removed before

feeding were designated as the 0 -hour samples, while those
collected at six and 12 hours after feeding were called 6-hour
and 12 -hour samples, respectively.
Bacterial and pH determinations.

At each collection of

rumen material a five milliliter sample of rumen fluid was
preserved in 10 milliliters of a 10 per cent formalin solution
for total rumen bacterial count.

This made a 1 ;3 dilution.

A pH determination was made at each collection of rumen
material.
Additional samples were taken for bacterial and pH deter­
minations for each level of aureomycin feeding.

The samples

were collected at 2-hour intervals for 12 hours for the first
three days of each level of aureomycin intake.
sample was taken before feeding each day.

The first

The rumen fluid was

drawn into a pipette connected to a large rubber bulb.

At

each of these collections a pH determination was made and a
five-milliliter portion was preserved for total bacterial count.
Cultural studies for the presence of rumen streptococci and
coliform organisms were made on another portion of fresh rurnen
fluid.

The rumen fluid was transported in a screw cap tube

- 60 -

filled to capacity in order to maintain anaerobic conditions.
In addition to the three-day collection periods, samples of
the rumen fluid were collected for total bacterial counts and
cultural studies before feeding and six hours after feeding
on Monday, Wednesday and Friday throughout the period concerned
for each level of aureomycin intake.
Bacterial studies were made on the feces from the steers
for all levels of aureomycin intake.

The fecal samples were

collected once per day (before feeding), three times per week
throughout the period for each level of aureomycin intake.
The fecal material was collected directly from the steer into
a sterile Petri dish.

Immediately after collection, a 10-gram

sample of the fresh fecal material was placed in a sterile
90-milliliter blank and taken directly to the laboratory where
the total volume was made up to 100 milliliters.

The sample

was agitated by shaking in the blank for a short time, then
a 10 -milliliter portion was preserved in five milliliters of
40 per cent formalin for total bacterial count.
1:15 dilution.

This gave a

The remaining portion was used for the cul­

tural determinations for the presence of fecal streptococci
and coliform organisms in the feces.
Chemical analyses.

The samples of rumen contents taken

for chemical analysis were dried in a forced-hot air oven at
60°C.

The dried samples were ground in a Wiley mill to pass

a 20-mesh sieve and stored in brown glass containers until

- 61 -

analyzed.

The standard procedures for feed analysis according

to the A.O.A.C.

(1950) were followed for the determination of

moisture, total nitrogen, crude fiber, ether extract and ash*
Non-protein nitrogen in the dried rumen contents was
determined by precipitating the protein in a 10-gram sample
by mixing with 175 milliliters of 5 per cent trichloroacetic
acid in a Waring blender for 10 minutes.
centrifuged at 2000 r.p.m.

The mixture was

The nitrogen in a 50-milliliter

aliquot was determined by the Kjeldahl method as outlined in
the A.O.A.C*

(1950).

In the case of the purified ration, a

smaller sample had to be used because of the limited amount
of the original sample.
The pH of the rumen

fluid was determined by the use of

the Beckman pH meter.
Bacteriological analyses.

The bacteriological analyses

of the rumen and fecal material were made in the Dairy Bac­
teriology laboratory.

The microscopic method developed by

Bortree et_ a l . (1948) was used for the total count.

The stain

was prepared by saturating 10 milliliters of ethanol with
crystal violet

(gentian violet).

One milliliter of the ethanol

solution was added to 49 milliliters of distilled water, mixed
thoroughly and filtered.

One milliliter of a 1:15 formalin

solution of fecal material was placed in seven milliliters of
distilled water to produce a 1:120 dilution.

Three milliliters

of a 1:3 formalin solution of rumen material were placed in 22
milliliters of distilled

water to produce a 1:25 dilution.

milliliter of a 1:120 dilution

One

of fecal material or one millili­

- 62 -

ter of a 1:25 dilution of rumen material and one milliliter of
the stain were transferred into eight milliliters of distilled
water in a test tube and shaken well.

This resulted in a final

dilution of 1:250 for the rumen samples and 1:1200 for the
fecal samples.

The test tube was heated over a low flame until

the solution bumped gently.

Before cooling, a blood pipette

was filled with the bacterial suspension and a drop was placed
on a clean Petroff-Hausser counting chamber.

The bacteria in

200 small squares were counted for each sample.

The number of

bacteria present was computed by the following formula:
Number of bacteria per milliliter =
Bacterial count x Dilution x 20 x 20 x 50 x 1000
100

100 = average number of small squares counted
20 x 20 * side of small square or 20 millimeter
50 = millimeter depth of material with coverslip on
material

1000 = conversion factor to change millimeters to
milliliters
The rumen and fecal samples which were collected for cul­
tural studies were tkaen care of immediately on arrival at the
laboratory.

One milliliter of the fresh rumen fluid was placed

in a sterile 99-milliliter blank to produce a 1:100 dilution.
The remaining dilutions necessary for the analysis were made
from this dilution.

In the case of the fecal samples, one

milliliter of the original 1:10 dilution was added to a sterile

99-milliliter blank to yield a 1:1000 dilution.

Also, a 1:100

dilution was made by placing 10 milliliters of the 1:10 origi­
nal dilution in a sterile 90-milliliter blank.

Again, the

- 63 -

dilutions necessary for the analysis were made from these
dilutions.

Lauryl tryptose broth prepared according to the

formula of Mallmann and Darby (1941) was used to determine
the coliform organisms, while dextrose azide broth was used
for determining the presence of rumen and fecal streptococci
(Mallmann and Seligmann, 1950),

The highest dilution in which

turbidity could be observed was used to represent the number
of rumen or fecal streptococci present.

The highest dilution

showing gas formation was used to indicate the number of coli­
form organisms present.
Amino acid analyses.

The samples were ether extracted

before analysis for the amino acids.

Hydrolyzates for the

determination of arginine, histidine, isoleucine, leucine, ly­
sine, methionine, phenylalanine, threonine and valine were pre­
pared according to the procedure of Stokes et_ ^L.

(1945).

One

gram of the material was dispersed in 25 milliliters of 6 N
HC1 and autoclaved for eight hours at 15 pounds pressure.

The

hydrolyzates were cooled, neutralized to pH 6 .6-6.8 with 18 N
NaOH, made up to 100 milliliters, filtered, covered with a few
drops of toluene and stored in the refrigerator until analyzed.
The concentration of the stock solution was 10 milligrams per
milliliter.

In the case of tryptophan, one-half gram of sample

was hydrolyzed in 16 milliliters of 4 N NaOH by autoclaving for
eight hours at 15 pounds pressure according to the procedure of
Kuiken et a l . (1947).

The hydrolyzate was cooled, neutralized

- 64 -

to pH 7.0 with 12 N HC1, made up to 100 milliliters, filtered
with the aid of Super-Cel, covered with a few drops of toluene
and stored in the refrigerator until analyzed.

This gave a

final five milligrams per milliliter concentration for the
stock solution.

In all cases, the assays were run after the

proper dilutions had been made.
The microbiological method was used to determine the
amino acids.

The composition of the media used for determining

the amino acids is given in Table 5.

Medium I, Schweigert

et al. (1944), was used for Lactobacillus arabinosus 17-5
(8014) to assay for isoleucine, leucine, phenylalanine and
valine.

Kuiken et_ a l . (1943) included tomato eluate in the

media for isoleucine and valine to overcome the lag in growth
of L. arabinosus in the tubes containing the lower concentra­
tions of the standard.

Medium II, Greenhut et al.

used for Streptococcus faecalis

(1946), was

(9790) to analyze for arginine,

histidine and threonine; while Medium III, McMahan and Snell
(1944), was used for Leuconostoc mesentroides P-SQ (8042) to
determine lysine.

Medium IV, Krehl et^ al.

(1943), worked best

with L. arabinosus for tryptophan; while Medium V, Lyman ejt a l .
(1946) was used for L. mesentroides for the determination of
methionine•
In the assay of the various amino acids, DL-configurations
of isoleucine, leucine, methionine, phenylalanine, threonine,
tryptophan and valine were used to prepare standards for these

- 65 -

amino acids, while L-configurations were used for the prepara­
tion of the arginine, histidine and lysine standards.

A

standard curve was determined by adding in triplicate at 0.5
milliliter increments from 0 to five milliliters a known quan­
tity of the amino acid to be studied.

The samples to be

assayed were diluted and pipetted in duplicate so that the
tubes contained 1.0, 2,0 and 3.0 milliliters of the amino acid
hydrolyzate.

Five milliliters of the basal medium, from which

the amino acid to be assayed was omitted, was added to each
tube of the standard and samples.

The total volume in all of

the tubes was brought to 10 milliliters with water.

The tubes

were capped and sterilized by autoclaving at 15 pounds pressure
for 10 minutes.

Each tube was inoculated aseptically with one

drop of the appropriate organism suspended in 0.9 per cent
saline solution.

The tubes were incubated 72 hours at 37°C.

to permit the development of lactic acid.

The lactic acid

produced in each tube was titrated electrometrically with
0.1 N NaOH to pH 7.0.
B-vitamin analyses.

As in the case of the amino acids,

the samples were ether extracted before analysis for the Bvitamins.

The hydrolytic procedure outlined by Snell and

Strong (1939) was used for riboflavin.

One gram of the

material was suspended in 50 milliliters of 0.1 N HC1 and
autoclaved for 30 minutes at 15 pounds pressure.

The sample

was centrifuged after cooling and 25 milliliters of the supernatent was adjusted to pH 4.6, diluted to 50 milliliters and

- 66 -

filtered.

In. the case of nicotinic acid, the samples were

prepared in the same manner as for riboflavin except that
1.0 N HC1 was used to suspend the material

(Krehl et_ al_. 1943).

The procedure designed by Buskirk et ai,
which was modified by Skeggs and Wright

(1942, 1948)

(1944) was used for

the preparation of the material for the pantothenic acid
assay.

One gram of the material and one gram of mylase-P

were dispersed in 10 milliliters of two per cent acetic acid
and one milliliter of 1.0 N NaOH (to buffer the reaction at
pH 4.2 to 4.5).

The mixture was incubated at 37°C. for three

hours, after which it was centrifuged, diluted to 100 milli­
liters and filtered.

The concentration of the stock solution

for each of the vitamins was 10 milligrams per milliliter.
The B-vitamins were determined microbiologically with
Lactobacillus casei

(7469) for riboflavin and L. arabinosus

for nicotinic acid and pantothenic acid.
these assays are given in Table 4.
determined for each B-vitamin.

The media used in

A standard curve was

The procedure was similar to

that used for the amino acids; however, the increments were
varied so that only eight levels were required to make the
curve.

Also, the procedure for diluting and pipetting the

samples, making the volume up to 10 milliliters, sterilizing,
inoculating and titrating was the same as that given for the
amino acids.

- 67

TABLE 3
COMPOSITION OF THE MEDIA USED IN AMINO ACID ASSAY 1
(Per 500 milliliters of double-strength medium)

Composition
Casein hydrolyzate

(gm)

HgOg treated peptone
D L (-)-Alanine

(gm)

(mg)

L (/)-Arginine-HC1

(mg)

I

II

III

IV

--

—

—

—

—

—

200

100

200

—

—

50

50

100

—

—
——

5.0

V
-7

—

L-Asparagine

(mg)

200

200

200

—

L(-)-Cystine

(mg)

100

200

200

200

100

400

400

400

—

—

20

20

100

—

—

50

50

100

—

—

200

200

200

—

—

200

200

200

—

—

L(/)-Lysine*HCl*H20 (mg)

200

200

200

—

—

DL-Methionine

100

100

200

—

—

100

100

100

—

--

50

50

50

—

—

50

50

200

—

—

200

200

200

—

--

DL-Tryptophan (mg)

50

100

100

..

L(-)-Tyrosine

50

100

100

200

200

200

20

20

20

L (/)-Glutamic acid (mg)
Glycine

(mg)

L(/)-Histidine*HCl*H 20 (mg)
DL-Isoleucine
DL-Leucine

(mg)

(mg)

(mg)

DL-Phenylalanine
L(-)-Proline
DL-Serine

Glucose

(mg)

(mg)

DL-Threonine

DL-Valine

(mg)

(mg)

(mg)

(gm)

(mg)

100
100

—

—

20

20

TABLE 3 (concluded)

Composition
Na acetate

I
(anhyd.)(gm)

II

20
25

Na citrate«HgO (gm)

III

IV

V

20

20

12

—

—

£
O

NH 4CI (gm)
500

500

500

5000

500

500

500

200

200

200

200

200

FeS0 4 '7H20 (mg)

10

10

10

10

10

MnS0 4 *4H20 (mg)

10

10

10

10

10

NaCl

10

10

10

10

10

Adenine S04 *2H 20 (rag)

10

10

10

10

10

Guanine HC 1 *2H 20 (mg)

10

10

10

10

10

Uracil

10

10

10

10

10

10

10

10

K H 2P0 4 (mg)

500

KgHP0 4 (mg)

500

MgS0 4 *7H20 (mg)

(mg)

(mg)

Xanthine

(mg)

Thiamine *HC1

(mg)

Pyridoxine»HC1

(rag)

DL-Ca pantothenate

(mg)

Riboflavin (mg)
Nicotinic acid

(mg)

£-Aminobenzoic acid
Biotin

( ug)

Folic acid

(mg)

(mg)

- -

0,5

0.5

0.5

0.1

1.0

1.0

1.0

1.0

0.1

2.0

0.5

0.5

0.5

0.1

2.0

0.5

0.5

0.5

0.2

2.0

1.0

1.0

1.0

0.4

2.0

0.1

0.1

0.1

0.1

0.01

1*0

1.0

1.0

0.01

0.01

0.01

200

5.0
0.0015

- 69 -

TABLE 4
COMPOSITION OF THE MEDIA USED IN THE ASSAY
FOR SOME OF THE B VITAMINS
(Per 500 milliliters of double-strength medium)
Composition
Riboflavin
Nicotinic
Pantothenic
____________________________________ acid_________ acid
Peptone

(NaOH treated)(gm)

L-cystine

(mg)

5
100

Yeast supplement

(gm)

Hydrolyzed casein

(gm)

200

100

5

5

2.0
—

DL-Tryptophan (mg)

--

200

200

Adenine S04 *2Hg0 (mg)

--

10

5

Guanine HCl*2HgO (mg)

--

10

5

Uracil

—

10

5

—

—

5

—

0.1

0,1

(mg)

Xanthine

(mg)

jD-Aminobenzoic acid

(mg)

Nicotinic acid

(mg)

—

—

1.0

Pyridoxine*HC1

(mg)

--

0.1

2.0

Riboflavin (mg)

—

0.2

1.0

Thiamine*HCl

--

0.1

2.0

—

0.1

Biotin ( ug)

--

0.2

12.5

Na acetate

3-

20

20

10

20

20

(ml)

5

5

5

Solution B^ (ml)

5

5

5

(mg)

Ca pantothenate

Glucose

(mg)

(gm)

(gm)

Inorganic salts
Solution Al

pH before autoclaving________ 6 .8__________ 6 .8____________ 6.8
1 Salts A contain 100 mg K 2HPO 4 and 100 mg KH 2PO 4 per ml.
2 Salts B contain 40 mg MgS 04 *7H 20 , 2.0 mg NaCl, 2.0 mg
FeS 0 4 *7H 20 and 2.0 mg MnS 04 *4H 20 per ml.

RESULTS
Health of the Animals
The steers used in the experiment with the natural ration
did not show any signs of digestive disturbances at any time.
The addition of aureomycin at 0.5 and 1.0 gram levels did not
cause any decrease in appetite in either case.

The steers con­

tinued to gain in body weight throughout the entire experimental
period even though the ration was fed at approximately the
maintenance level.
When the purified ration was fed, the steers did not con­
sume it very readily at first.

In fact, 707 continued to re­

fuse to eat it for two weeks and was removed from the experi­
ment at that time.

Steer 714 received the purified ration for

25 days before the first collection of rumen contents was made.
Simultaneously with the feeding of 0.5 gram of aureomycin, 714
began to refuse to eat the ration and was fed through the side
for the remainder of the experiment.

Approximately one week

before the end of the aureomycin feeding period, the steer
began to act sluggish and the rumen contents appeared to be
separated into two layers, with the top layer consisting mostly
of the coarse material while the bottom layer was very watery.
The rumen contents were very greasy.

Two days before the end

of the experimental period the steer had severe diarrhea, a
mucus discharge from the nostrils and an increase in body tem-

- 71 -

perature.

The paper pulp which was used as the cellulose source

in the ration settled to the bottom of the rumen.

The steer

died two days after the rumen samples were collected for the
aureomycin trial.
autopsy:

The following conditions were found on

fat necrosis of the omentum, hemorrhagic pancreatitis,

extensive fatty degeneration of the kidneys and liver, petechial
and ecchymatic hemorrhages at the base of the heart and on the
epicardium, slight degenerative changes in the heart muscle and
the heart was somewhat flabby, and the lungs were congested and
edematous with beginning of bronchopneumonia.

The final diag­

nosis was toxemia due to degenerative changes of the liver,
kidney and heart, while the septicemia present probably origi­
nated from the early pneumonia.
Weight and Composition of the Rumen Contents from
Cattle Fed a Natural and a Purified Ration
The data pertaining to the weights of the rumen contents
obtained at the 0 -, 6 - and 12-hour collections along with the
percentages of dry matter, crude protein, crude fiber, ether
extract, nitrogen-free extract and non-protein nitrogen are
presented in Table 5 for the natural ration and Table 6 for
the purified ration.

The data are arranged by the hour of

collection for each level of aureomycin fed so that comparisons
could be made to determine the influence of each level of
aureomycin intake on the different ration constituents.

As

one would expect, due to the fact that the ration fed, espec-

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CONTENTS
RUMEN

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40.39
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16.19
14.38
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14.72
15.40

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AND COMPOSITION OF THE
A NATURAL RATION

O in in
n cm o

54,026
59,474
62,879

•h

o 01 tO

XI

THE WEIGHT
CATTLE FED
ON
FROM
OF AUREOMYCIN

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INFLUENCE

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3.51
35.02
0.487
6.01
8,275
4.06
33.04
0.582
6.20
11,775
3.51
33.74
0.467
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ially the natural ration, was high in each constituent con­
sidered and that it took the steer 3 several hours to consume
the ration, the values obtained for each constituent at the

6 -hour collections were higher than the 0 - or 12-hour collec­
tions.

The pH and total bacterial count were taken at each

collection of rumen material.

However, one must keep in mind

that steer 714 was not in normal health when 0.5 gram of
aureomycin was added to the purified ration, but the informa­
tion was included merely to serve as a comparison to a normal
condition.
Influence of Aureomycin on the Rate of Removal of Some
of the Ration Constituents from the Rumen
With the aid of the data presented in Tables 1 and 5, the
quantity of each ration constituent present at the 0 -, 6 - and

12 -hour collections for each animal, and the amount of each
constituent fed to the animals can be calculated.

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amount present and that fed, calculations were made for the
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rumen or which had been removed over a particular 6- or 12-hour
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crude fiber, crude protein, ether extract, nitrogen-free ex­
tract and non-protein nitrogen which had accumulated or had been
removed from the rumen in 6- and 12-hours in relation to the
0-hour.

The rate of disappearance of the ration constituent at

the 6- and 12-hour collections was based on the intake of the
particular constituent in question.

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Calculations also were made for the second 6-hour period
and are designated as the 6-12 hour samples.

In the latter

case, the amount present at the 6 -hour collection was used as
the base to calculate the change between the 6- and the 12hour collection.

In nearly every case, the rate of removal

of a ration constituent from the rumen was definitely faster
when aureomycin was included in the ration, especially at the
0.5 gram level, than when no aureomycin was fed.

Ether ex­

tract and non-protein nitrogen are an exception; in that,
there was an accumulation during the first 6 hours and the
amount present at the O-hour was much larger when aureomycin
was fed.
Amino Acid Analyses
Only the 10 essential amino acids were determined in this
study.

The amino acid composition of the rations fed is pre­

sented in Table 13, while Table 14 gives the amino acid com­
position of the rumen contents from cattle fed the natural
ration.

Again, the data are arranged by the hour of collection

for each level of aureomycin intake.

In general, when aureo­

mycin was included in the ration, there was a decline in the
percentage of the amino acids present at each collection of the
rumen material in comparison to when no aureomycin was fed.
The rate of removal of each amino acid from the rumen was cal­
culated for the 6- and 12 -hour collections based on the intake

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- 86 -

of each amino acid; also the rate of removal from six to 12
hours was calculated for each amino acid.

The data for the

removal of the amino acids are summarized for each time of
collection and for each level of aureomycin intake in Table 15.
The detailed data for each amino acid in Table 15 are found
in Tables 23 through 32 (appendix).

The data indicate that

the rate of removal of the amino acids was more rapid when
0.5 gram of aureomycin was included in the ration.

In the

case of the purified ration (Table 16) the data indicate that
synthesis of the amino acids does occur in the rumen.

The

amino acid composition of the rumen contents from the steer
fed the purified ration is found in Table 33 (appendix).
B-Vitamin Analyses
The rations fed and the rumen contents were assayed for
riboflavin, nicotinic acid and pantothenic acid.

The 8 -vitamin

composition of the rations fed is given in Table 17.
TABLE 17
B-VITAMIN COMPOSITION OF THE RATIONS FED

Ration

Riboflavin
/gm

Nicotinic
acid
/gm

Pantothenic
acid
/gm

Corn

1.65

23.24

2.02

Alfalfa-brome hay

6.48

25.89

22.44

Purified

0.49

0.58

0

- 87 -

Table IS gives the riboflavin, nicotinic acid and panto­
thenic acid content of the rumen contents from cattle fed the
natural ration at each time of collection for each level of
aureomycin fed.

As in the case of the amino acids, the amounts

of these B-vitamins that were synthesized in or removed from
the rumen were calculated.

The detailed data for these cal­

culations are presented for riboflavin, nicotinic acid and
pantothenic acid in Tables 19, 20 and 21, respectively.

The

data for the synthesis of the B-vitamins in the case of the
purified ration are presented in Table 22.

Table 34 (appendix)

gives the B-vitamin composition of the rumen contents when the
purified ration was fed.
pH and Bacteriological Analyses
Samples of rumen fluid were preserved for total bacterial
count for three consecutive days at the beginning of each level
of aureomycin intake.

A pH determination was made at the same

time that a sample was collected for the bacteriological analy­
sis.

Figure 1 shows graphically the influence of aureomycin

on the pH and the total bacterial count of rumen contents from
cattle fed the natural ration.
was offered for a 15-day period.

Each level of aureomycin intake
Figure 2 presents the in­

fluence of the aureomycin on the total bacterial count of the
rumen contents from cattle fed the natural ration for each of
these 15-day periods.

There was a definite increase in the

- 88 -

total bacterial count when 0.5 gram of aureomycin was included
in the ration and not much additional change when the aureo­
mycin was increased to 1.0 gram.

Detailed data for these

figures are found in Tables 35, 36 and 37 (appendix).
Figures 3 and 4 show the results of the cultural study
for the rumen streptococci and the coliform groups present in
the rumen of cattle fed the natural ration when aureomycin was
included in the daily ration.

The average for the first three

days of each level of aureomycin intake is shown in Figure 3,
while the average for each 15-day period is shown in Figure 4.
Detailed data for these figures are presented in Table 38
(appendix).
Samples of fecal material were collected periodically for
each level of aureomycin fed while the cattle were on the natu­
ral ration.

Determinations for the total bacterial count,

fecal streptococci and the coliform groups were conducted on
these samples.

Figure 5 shows the bacterial picture for the

total count, while the results of the cultural studies for the
fecal streptococci and the coliform groups are shown in Figure

6.

Detailed data for these figures are presented in Tables

39 and 40 (appendix).
When steer 714 was fed the purified ration, the same pro­
cedure was followed in collecting the samples for pH and bac­
teriological studies.

Figure 7 presents the results for the

pH and the total bacterial count when no aureomycin and when
0.5 gram of aureomycin was fed.

Detailed data for Figure 7

are presented in Tables 41 and 42 (appendix).

- 89 -

TABLE 18
INFLUENCE OF AUREOMYCIN ON THE B-VITAMIN COMPOSITION OF RUMEN
CONTENTS FROM CATTLE FED A NATURAL RATION
Animal
Aureo­
no • Time mycin
hr
gm

Riboflavin
*/gm

707

0
0
0

0
0.5
1.0

7.25
6.57
4.00

12.22
38.99
11.31

11.91
29.67
17.54

707

6
6
6

0
0.5
1.0

8.01
7.21
5.15

24.21
36.29
26.73

51.94
52.60
40.58

707

12
12
12

0
0.5
1.0

5.81
5.43
3.88

20.96
28.12
18.35

41.51
69.2-2
42.63

714

0
0
0

0
0.5
1.0

7.38
7. 80
4.08

10.30
27.03
14.04

10.65
40.83
26.72

714

6
6
6

0
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1.0

8.42
5.85
4.19

26.54
25.07
22.84

46.25
41.33
44.17

714

12
12
12

0
0.5
1.0

7.99
5.36
3.79

29.21
26.50

65.69
60.30
50.92

Pantothenic
Acid
r/g m

22.68

Nicotinic
Acid
f/gm

- 90 -

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- 91 -

TABLE 20
INFLUENCE OF AUREOMYCIN ON THE AMOUNT OF NICOTINIC ACID
ACCUMULATED OR REMOVED FROM THE RUMEN OF CATTLE FED A
NATURAL RATION IN 6 AND 12 HOURS

Nicotinic Acid
Animal Aureo- In
no •
mycin rumen Intake Total
gm
mg
mg
mg

Nicotinic Acid
ReIn
rumen Diff. Accuraul. moved
mg
mg
%
%

0'-hr. samples

0-6 hr. samples#

707

0
0.5
1.0

58.3
137.6
109.0

218.5
218.5
218.5

276.8
356.1
327.5

442.8
396.8
376.9

166.0
40.7
49.4

76.0
18.6
22.6

0
0.5
1.0

58.5
257.0
197.7

218.5
218.5
218.5

277.0
475.5
416.2

470.4
429.7
545.2

193.4
45.8
129.0

88.5

714

Nicotinic Acid

Nicotinic Acid
Animal Aureo- In
mvcin rumen
no.
mg
gm

Accumul.
mg
%

Diff.
mg

707

714

0
0.5
1.0

300.0
400.0
327.6

10.6
20.1
0

142.8
3.2
49.3

0
0.5
1.0

565.7 288.7 132.1
527.9 52.4 24.0
493.1 76.9 35.2

95.3
98.2
52.1

* Based on nicotinic acid intake.

Accumul.

Removed

%

£

6-12 hr. samples

0-12 hr. samples#
23.2
43.9
0

21.0
59.0

32.2

0.8
13.0
20.3
22.9
9.6

- 92 -

TABLE 21
INFLUENCE OF AUREOMYCIN ON THE AMOUNT OF PANTOTHENIC ACID
ACCUMULATED OR REMOVED FROM THE RUMEN OF CATTLE FED A
NATURAL RATION IN 6 AND 12 HOURS

Pantotheni c Acid
Animal AureoIn
no •
mycin rumen Fed Total
mg
gm
mg
mg

Pantothenic Acid
In
rumen
mg

0-6 hr. samples &

O-hr'. samples
707

714

ReDiff. Accumul. moved
mg
%
%

0
0.5
1.0

59.9
180.8
70.3

156.5
156.5
156.5

216.4
337.3
226.8

206.4
273.7
248.3

10.0
63.6
21.5

0
0.5
1.0

56 .6
170.2
103.9

156.5
156.5
156.5

213.1
326.7
260.4

269.9
260.7
281.9

56.8
66.0
21.5

Animal Aureo- In
no •
ravcin rumen
mg
gm

6.4
40.6
13.7
36.3
42.2
13.7

Pantothenic Acid_________ Pantothenic Acid
Re­
Diff. Accumul. moved
Removed
mg
mg
i
fo
%

0 - 12 hr,. samples *
707

0
0.5
1.0

151.5
162.5
141.0

64.9
174.8
85.8

714

0
0.5
1.0

251.6
232.0
219.6

38.5
94.7
40. 8

—

24.6
—

* Based on pantothenic acid intake.

6-12 hr. samples

41.5
111.7
54.8
-»->

60.5
26.1

54.9
111.2
107.3

26.6
40.6
43.2

18.3
28.7
62.3

6.8
11.0
22.1

- 93 -

TABLE 22
INFLUENCE OF AUREOMYCIN ON THE SYNTHESIS OR REMOVAL OF
B-VITAMINS IN OR FROM THE RUMEN OF 714
FED A PURIFIED RATION

Animal Aureono •
mycin
gm

714

714

Time
hr

Riboflavin

Nicotinic
Acid

%

i

Pantothenic
Acid
%

0

0-6

4243.8

41286.3

4152.2

0

0-12

4220.5

41542.4

4481.1

0

6-12

- 6.8

418.5

4206.8

0.5

0-6

420.8

-67.7

-63.4

0.5

0-12

428.2

-35.3

445.6

0.5

6-12

46.1

4100.5

4296.6

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Influence of aureomycin on the pH and total bacterial count
contents from cattle fed a natural ration (av, first 3 days
period)e

FEEDING

CO

1.

o:
AFTER

co

Figure

in
HRS

of rumen
of 15-day

- 94 -

- 95 -

0 .Oo.
AUREOftYCIW

0.5*0.

1. 0 $.

AUREOMYCIN

a u r e o m y c iw

22

BILLIONS

PER

ML

20

707 —
714 - - \

0

6

12

0

6

12

0

6

12

0

6

HRS AFTER FEEDING
Figure 2.

Influence of aureomycin on the total bacterial
count of rumen contents from cattle fed a
natural ration (av* of 15 days)*

-

0.0$. AUREOMTCIN

96 -

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7 0 7 -----714 - -

HRS AFTER FEEDING

Figure 3.

Influence of aureomycin on the rumen strep­
tococci and the coliform group In the rumen
of cattle fed a natural ration (av, first
3 days of 15-day period).

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97 -

Figure

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BILLIONS

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60

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Figure 5.

6

8

10

12 14 16
NO. OF DAYS

20

22

24

Influence of aureomycin on the total bac­
terial count of feces from cattle fed a
natural ration,,

-

GRAMS

99

AUREOMYCIN

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707
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Figure 6.

6

8

10

12 14 16
NO. OF DAYS

18 20 22

24

Influence of aureomycin on the fecal strepto­
cocci and the coliform group in the feces
from cattle fed a natural ration*

-

-

0. 5$. A U R E O M Y C I N

BILLIONS

PER ML

0.09. A U R E O M Y C I N

100

6.5

6.0
* 5.5
5.0
4.5-

HRS AFTER FEEDING
Figure 7.

Influence of aureomycin on the pH and total bac­
terial count of the rumen contents from steer 714
fed a purified ration.

DISCUSSION
Health of the Animals
In this investigation, neither of the steers showed any
signs of anorexia or diarrhea that had been reported by Bell
et_ a l . (1951) with steers and by Colby et_ al . (1950) with
fattening lambs when crystalline aureomycin was included in
the ration.

Also, the steers used in this study continued to

gain in body weight when the aureomycin was fed.

Colby et_ a l .

observed that the fattening lambs receiving aureomycin lost 0.2
pounds per day while the lambs on the basal ration gained 0.52
pounds per day.
The rations used by both of these investigators contained
a high proportion of grain to roughage; in fact, 50 per cent
of the total daily ration that Bell e_t al. used in their diges­
tion trial came from grain.

In the study reported here, the

daily ration consisted of 79 per cent hay and 21 per cent ground
corn.

Pounden and Hibbs

(1948a, 1948b) reported that grain

feeding was responsible for inhibiting the establishment of the
varieties of flora which were associated with hay ingestion.
Gall et^ al_. (194 9c) observed that as the amount of grain in the
ration increased, there was a corresponding increase in the
number of fast-growing bacteria.

Consequently, from the observa­

tions of Pounden and Hibbs and Gall et_ al_. as well as those of

- 102 -

Bell et_ al_. and Colby £t_ a l . , it is possible that the rumen
bacteria adapted to a low cellulose-high energy ration were
more sensitive to aureomycin; whereas in the present study the
bacteria were adapted to a high cellulose-low energy ration
and were not as sensitive to aureomycin, at least at the levels
fed.
The illness and subsequent death of steer 714 cannot be
attributed to a particular cause; however, several investigators
have reported toxic effects from urea in the case of cattle and
sheep.

Hart ej^ aj^. (1939) slaughtered a steer that had been

receiving a ration which contained 4.3 per cent urea and found
necrosis in some areas of the liver and badly damaged kidneys.
Clark et_ al_. (1951a) observed severe degeneration of the kidney
and the liver in sheep dosed with urea*

However, they also

found that the toxicity of urea was dependent on the activity
of the ruminal flora as determined by the basal ration and the
presence of available carbohydrate.

A greater tolerance for

urea was observed when the bacterial flora was conditioned to
a high plane of nitrogen metabolism by including casein in the
ration.

Likewise, the readily available carbohydrate would be

mixed with the urea and would help to keep the urea from being
converted to ammonia too rapidly which could cause toxic symptoms
if the concentration in the blood should become too high.
and Mitchell

Harris

(1941b) fed rations containing up to 3.16 per cent

urea, on the dry basis, for 110 days to lambs without any his­
tological evidence of kidney damage.

Work ejt al_. (1943) did

- 103 -

not observe any liver or kidney damage in steers fed urea at
the rate of 2*29 per cent of the dry matter for 244 days,
pH and Bacteriological Analyses
From the data presented in Figure 1, the pH of the rumen
contents from 707 for each level of aureomycin intake never
went as low as that observed for 714 for the same periods when
the animals received the natural ration.

In both animals, the

pH would decrease after feeding and reach the lowest point in
about six or eight hours.

However, there was only a slight in­

crease toward alkalinity 12 hours after feeding.

This fact is

not entirely in agreement with the observations of Kick et_ a l .
(1938), Monroe and Perkins (1939), Wegner et al.

(1941), Smith

(1941) and Myburgh and Q.uin (1943) who reported that the pH
declines for about four hours with a gradual increase toward
alkalinity following the decline.

A possible explanation for

the differences observed in the investigation reported here may
be that the animals were fed once daily, whereas the other
workers fed their animals twice daily.

Consequently, in the

present study, the bacteria were conditioned to being fed once
daily and performed their fermentation processes at a slower
rate than those that were fed twice daily.

It is also possible

that the cellulose digesting bacteria were still producing acids
which would keep the pH down.

Roine and Elvehjem (1950) stated

that the pH was not determined by the food but by the kind of
microflora which developed in the digestive tract.

- 104 -

In the case of the purified ration, the pH was definitely
lower when no aureomycin was fed as compared to the period when
0*5 gram of aureomycin was fed as shown in Figure 7.

However,

steer 714 was off-feed during the aureomycin trial and most
likely the bacteria were not as functional as those during the
control period.

As in the case of the natural ration, the pH

increased slightly toward alkalinity at 12 -hours after feeding,
possibly for the same reasons as mentioned in the discussion of
the pH when the natural ration was fed.
There was a definite increase in the total bacterial count
of the rumen contents when 0.5 gram of aureomycin was added to
the natural ration.

The total bacterial count remained at

approximately the same level when the aureomycin was increased
to 1,0 gram per day, possibly because the bacteria may have
developed some resistance to the antibiotic.

It is possible

that the increased removal of dry matter, crude fiber, crude
protein, nitrogen-free extract and the amino acids when 0.5
gram of aureomycin was fed was the result of the activity of
the increased bacterial population.

Since there were more

bacteria to digest the feed, it is logical to assume that they
should perform a more complete digestion of the nutrients.
Elam e_t al_* (1951a) and Couch et_ a U

(1951) observed an increase

in the total number of intestinal microorganisms when penicillin
was included in the ration of chicks.

Burroughs et_ al_. (1950,

1950d, 1950e, 1951a, 1951b) observed that a complex salt solu­

- 105 -

tion, alfalfa ash extract, autoclaved rumen liquid and an
autoclaved water extract of manure were beneficial in aiding
rumen microorganisms to digest cellulose.

They also observed

that an increase in the size and number of bacteria present
and an improvement in cellulose digestion occurred when any
of these materials were added to a flask in which poor cellu­
lose digestion was found.

It is possible that the aureomycin

used in the present study either stimulated the bacteria directly
or was responsible for the releasing of some factor(s) in the
rumen which caused the bacterial population to increase.
As observed in Figures 3 and 4 there was a reduction in
the number of rumen streptococci for both animals when aureo­
mycin was included in the ration.

In the case of the coliforra

group, the number present in the rumen of 707 six hours after
feeding was always higher than observed at the 0 -hour; whereas,
in 714, the number was lower at the 6-hour collection.

Also,

the number of coliforra bacteria observed in 707 was consistently
higher than in 714.

The increase in the coliform group when

0.5 gram of aureomycin was fed may be of some significance be­
cause it was at this concentration that the highest removal of
the various nutrients from the rumen was observed.

Also, in

the case of 714, it was interesting to note that the rate of
removal of the various nutrients from the rumen was lower than
that observed in 707 and that this coincided with a lower num­
ber of coliforra organisms and total bacterial count than ob­
served in 707.

However, over-all, the rate of removal of the

- 106 -

nutrients from the rumen was the highest in both animals when
0.5 gram of aureomycin was fed, although the highest percentage
of removal occurred in 707.
Bartley et a l . (1951) did not observe any consistent
microscopic differences of the rumen microflora between the
control and aureomycin-fed calves.

Neumann et^ al.

(1951) noted

that the total bacterial count was about the same for the con­
trol as for the aureomycin-fed heifers, but the types found in
the heifers fed aureomycin were much less diverse, which sug­
gested that the normal rumen flora had been disturbed.

Neumann

et al. also reported some reduction in appetite for a few days
after the addition of aureomycin to the ration and then fol­
lowed by a partial recovery.

It was interesting to note that

the ration Neumann ejt al_. fed to their beef heifers consisted
of a high proportion of grain and that they had some reduction
in appetite when aureomycin was added to the ration.
al.

Bell et_

(1951) reported more severe results, but they also fed a

ration high in grain and fed a higher concentration of aureo­
mycin.

As mentioned before, it is possible that the rumen

flora adapted to a high proportion of grain may be sensitive
to aureomycin, while the flora which developed on the type of
ration fed in the present study can withstand a higher concen­
tration of aureomycin.

In the present study, the amount of

aureomycin fed daily was much higher than that fed by Bell et
al. and Neumann et al.

- 107 -

An increase of the total bacterial count of the fecal
material was observed when aureomycin was fed to animals re­
ceiving the natural ration.

The decrease in the total bac­

terial count just before the aureomycin was fed cannot be
explained.

The total bacterial count of the feces was about

four times higher than that observed for the rumen contents.
The rumen contents were higher in moisture which may account
for a lower total bacterial count due to a greater dilution.
There was not much difference between the number of strepto­
cocci in the rumen and fecal material.

However, the number

of coliform organisms in the feces were higher than that ob­
served in the rumen.
Gall et a l . (1951) observed that the types of bacteria in
the rumen of animals receiving a purified ration were different
from those when the animals were fed casein or urea plus sul­
phur.

In the study reported here, the total bacterial count

of the rumen contents when 714 was fed the purified ration
was lower than that observed with the natural ration.

When

0.5 gram of aureomycin was added to the ration, the total bac­
terial count remained about the same as the control period.
Some change in the types of bacteria must have occurred when
the aureomycin was added because there was a definite decrease
in the synthesis of the amino acids and B-vitamins.

The fact

that the animal was off-feed may be closely related to the
types of bacteria and their functions under certain conditions.

- 108 -

Passage of Some of the Ration Constituents
from the Rumen
It is very difficult to measure the rate of passage of the
various constituents from the rumen because one is concerned
with a moving system.

However, the amount of the various con­

stituents present at each collection of rumen contents could
be accounted for while the part unaccounted for was assumed to
have passed from the rumen either by absorption through the
rumen wall or by passing on to the remainder of the digestive
tract.

It can be observed from Tables 7 through 12 that there

are distinct differences in the rate of removal of the various
constituents from

the rumens of steers 707 and 714.

of removal of the

different nutrients from the rumen of 714

in six hours was considerably lower than 707.

The rate

However, in the

case of ether extract, there was a greater accumulation in the
rumen of 714 than in 707 in six hours.

Even though the rate of

removal of the nutrients from the rumen of 707 was greater than
in 714, the trend

in 714 was similar to that in 707.

In most

cases the rate of

removal of the nutrients from the rumen was

faster when 0.5 gram of aureomycin was included in the ration.
When 1.0 gram of aureomycin was included in the ration, the
data for 714 indicated a delayed digestion during the first

6-hour period but an increased rate of removal during the
second 6-hour period for dry matter, crude fiber, crude protein
and nitrogen-free extract.

In the case of 707, when 1.0 gram

109 -

of aureomycin was added to the ration, the rate of removal of
dry matter, crude fiber, crude protein and nitrogen-free ex­
tract from the rumen for both the first and second 6-hour per­
iods was slightly less than when 0.5 gram of aureomycin was
fed, but usually a little more than when no aureomycin was fed.
An apparent accumulation of dry matter

(Table 7) in the

rumen of both 707 and 714, at the 0-hour, resulted when 1.0
gram of aureomycin was fed.

This was probably due to the in­

creased crude fiber content of the dry matter as shown in
Table 8 and also the weight of the rumen contents was the
heavier at this time.

A corresponding accumulation of crude

protein and nitrogen-free extract is shown in Tables 9 and 11.
It is possible that 1.0 gram of aureomycin produced a concen­
tration great enough to have a slight inhibitory effect on the
cellulose digesting bacteria; therefore, the crude protein and
nitrogen-free extract would be bound by the cellulose so that
it could not be utilized by the bacteria or removed from the
rumen.

Bell et_ al_. (1951) observed that 0.2 gram of aureomycin

caused a decrease in the digestibility of dry matter and crude
fiber as much as 15 per cent and 50 per cent, respectively,
when fed to steers during a digestion trial.

Wasserman et_ a l .

(1952) using an in vitro technique observed that penicillin
stimulated cellulolytic rumen microorganisms at the lower con­
centrations, neomycin was stimulatory in all concentrations,
streptomycin was slightly stimulatory in the lowest concentra­
tion and Chloromycetin adversely affected the microorganisms.

110 -

Several investigators have used animals with rumen fistulas
as a means to study the digestion of various nutrients in the
rumen.

Silver

(1935) studied the digestion and absorption of

alfalfa hay by removing the rumen contents at the time of feed­
ing and taking "grab" samples at 2-hour intervals thereafter.
He compared the percentage composition of the various samples
of rumen contents as the period of digestion progressed.

Hale

et a l . (1947) used the lignin ratio technique to study the
quantitative digestion in the rumen.

This method was useful

in studying the significance of rumen digestion as related to
the subsequent digestion in the remainder of the digestive
tract.

The rumen digestion coefficients obtained by Hale et^

al. for dry matter, crude fiber, crude protein and nitrogenfree extract for the first 6-hour period after feeding were
22.1, 0, 33.3 and 45.1 per cent, respectively.

When the same

method of calculation that was used in this investigation was
applied to the data reported by Hale et al. the values 55.7,

47 .1 , 59.1 and 66.7 per cent were obtained for dry matter,
crude fiber, crude protein and nitrogen-free extract, respec­
tively.

Except for crude protein these values are in good

agreement with those obtained from 707 when no aureomycin was
fed.
In the study reported here approximately 80 per cent of
the dry matter had passed from the rumen within 12 hours after
feeding.

Crude fiber passed out of the rumen at about the same

rate as the dry matter while the protein disappeared at a lower

- Ill -

rate and nitrogen-free extract at a slightly higher rate.

Hale

ftl» (1947) observed that the rumen digestion was practically
completed 12 to 14 hours after feeding.

They suggested that the

lignin in the plant material probably imposed a "ceiling" on
rumen digestion.

Hale et al,

(1940) did not observe any dif­

ferences in the extent of rumen digestion of alfalfa hay when
fed at levels varying from 10 to 30 pounds per day.
noted that there was not much difference in

They also

the rumen fill for

these varying levels of hay intake.
There was an accumulation of ether extract in the rumen
during the first 6-hour period as shown in Table 10.

This fact

was observed for both 707 and 714; however, 714 showed a greater
accumulation for the first 6-hour period and greater passage
from the rumen for the second 6-hour

period than 707. The

amount present in the rumens of both

steers at the 0 -hour period

when aureomycin was fed was considerably higher than when no
aureomycin was fed.

The accumulation of ether extract was the

higher when 1.0 gram of aureomycin was fed.

A possible explana­

tion of this accumulation of ether extract at 0 -hour may be
that there was a delayed synthesis of the fat from carbohydrate
in the rumen or a decreased absorption of fat from the rumen.
Hale e_t al_. (1940, 1947) also observed a definite increase of
the ether extractive substances in the rumen six hours after
feeding.
Table 12 shows an accumulation of non-protein nitrogen in
the rumen for the first 6—hour period after feeding and a re—

- 112 -

moval for the second 6-hour period.

The rate of removal from

the rumen was the faster when 1.0 gram of aureomycin was in­
cluded in the ration.
An increase in the number of bacteria in the rumen was ob­
served when 0.5 gram of aureomycin was fed as shown in Figures
1 and 2.

Since the most rapid removal of dry matter, crude

fiber, crude protein and nitrogen-free extract occurred when
this level of aureomycin was included in the ration, it is
possible that the aureomycin stimulated bacterial action in
the rumen and also may have caused a change in the wall of the
rumen which would facilitate faster absorption.

The total

bacterial count in the rumen remained approximately the same
when 1.0 gram of aureomycin was fed as observed for 0.5 gram
of aureomycin.

There was a decrease in the amount of nutrients

removed from the rumen when 1.0 gram of aureomycin was fed as
compared to the 0.5 gram level.

It is possible that the in­

creased aureomycin concentration may have inhibited some of the
cellulose-digesting bacteria but was not high enough to affect
the total bacterial count to any great extent.
Amino Acid Analyses
There was a definite decrease in the percentage of the
amino acids present at each collection of rumen material as
the concentration of aureomycin was increased

(Table 14).

A

possible explanation of this could be that there was a higher
percentage of each amino acid removed from the rumen when 0.5

- 113 -

gram of aureomycin was included in the ration as compared to
no aureomycin and 1.0 gram of aureomycin (Table 15).

Another

possible reason could be that since there is an accumulation
of dry matter and crude fiber in the rumen at the 0-hour collec­
tion, the amino acids present may be diluted by being distri­
buted in a larger volume; therefore when the sample was taken
for an assay, the sample contained less of the amino acids in
question.
Synthesis of the amino acids could not be observed when
the natural ration was fed because the ration itself contained
a large quantity of each amino acid.

Consequently, only the

rate of removal of these amino acids could be determined when
the natural ration was fed.

No doubt there was some synthesis

of the amino acids when the natural ration was fed but there
was no method to determine this synthesis in a moving system
and especially since the ration already contained such large
quantities of the amino acids.
The type of ration may have some influence on the amount
of the amino acids that are found in the rumen just as the
ration influences the type of bacterial flora present in the
rumen.

Reed ejt al.

(1949) observed that the bacterial protein

obtained from sheep receiving either dry or green feed contained
about the same level of cysteine, but the sheep fed the green
feed had a higher level of methionine.
and Block et al.

Block and Stekol

(1950)

(1951) using radioactive sodium sulfate (S3 5 )

- 114 -

observed that methionine and cystine were synthesized in the
rumen at approximately the same rate and were used by the
tissues to make new protein in the quantities needed.

McNaught

<3t_ al_. (1951b), separating bacterial cells in a Sharpies cen­
trifuge, found that 58 per cent of the bacterial protein was
present in the liquid when it was removed from the rumen while
the remaining 42 per cent was synthesized during incubation.
Synthesis of the 10 essential amino acids has been shown
by Loosli et_ a^l. (1949) in sheep and by Agrawala (1950) in
steers with a purified ration which had urea as the sole source
of nitrogen.

Loosli et_ a l . also reported that sheep on a

purified ration containing glycine as the only source of nitro­
gen synthesized the amino acids at a lower level when compared
with urea.
In the investigation reported here, a purified ration
similar to that used by Loosli et al . (1949) and Agrawala (1950)
except that a different cellulose source was fed to steer 714.
Data in Table 16 indicate that the amino acids are synthesized
in the rumen and these values agree with the lower values re­
ported by Agrawala (1950).

He observed an increase in arginine

from 52 to 218 per cent, histidine from 35 to 210 per cent, iso­
leucine from 26 to 190 per cent, leucine from 40 to 178 per
cent, lysine from 46 to 192 per cent, methionine from 22 to
260 per cent, phenylalanine from 44 to 194 per cent, threonine
from 42 to 150 per cent, tryptophan from 72 to 200 per cent
and valine from 26 to 204 per cent in six hours after feeding.

- 115 -

In the investigation reported here, there was an apparent
delayed synthesis of lysine in the first 6-hour sample when
no aureomycin was fed.

Only lysine showed any accumulation

in the rumen in the second 6-hour period.

7/hen 0.5 gram of

aureomycin was included in the ration, only arginine, histi­
dine, isoleucine, leucine, threonine and valine were synthe­
sized in the first 6-hour period.

The concentration of lysine,

methionine, phenylalanine and tryptophan were lower at six
hours after feeding than at the 0-hour,

There was an accumu­

lation of all of the amino acids in the second 6-hour period
which would indicate that the aureomycin may exert some de­
pressing effect on the bacteria during the first 6-hour period.
However, one must keep in mind that steer 714 was off-feed
during the aureomycin trial and was fed through the fistula;
also, as shown in Table 6 , the values for non-protein nitrogen
when 0.5 gram of aureomycin was fed were considerably higher
than when no aureomycin was fed, which would indicate a de­
layed utilization of the urea in the ration.
B-Vitamin Analyses
Riboflavin.

McElroy and Goss

(1939, 1940a) observed that

the riboflavin content of the rumen ingesta in sheep increased
100-fold over that of the feed.

Hunt e_t al.

(1941) found no

increase in riboflavin when steers were fed hay alone, but that
riboflavin increased when corn was included in the ration.

- 116 -

Teeri e t a l . (1951b) found from analysis of the feces that cows
fed a low quality of late-cut hay had a decreased excretion of
riboflavin.

Kesler and Knodt

(1950, 1951a, 1951c) observed

that the riboflavin concentration was highest in the small
intestine in the case of calves.
No evidence of synthesis of riboflavin could be detected
when the steers were fed the natural ration; even though there
must have been some synthesis, there was such a large quantity
in the feed which could mask any synthesis.

As in the case of

the amino acids, only the rate of removal could be determined.
The rate of removal of riboflavin from the rumen was the high­
est when 0.5 gram of aureoraycin was fed as observed in Table 19.
Riboflavin synthesis

(Table 22) was very evident when the

purified ration was fed to 714 without any aureoraycin.

Agrawala

(1950) observed an increase in riboflavin from 162 to 382 per
cent.

With no aureomycin in the ration, a 243.8 per cent in­

crease in riboflavin was obtained; however, when 0.5 gram of
aureomycin was added to the ration a large decrease in ribo­
flavin synthesis occurred.

The aureomycin may have had a de­

pressing effect on the rumen bacteria that synthesize ribo­
flavin, also 714 was off-feed at the same time.

The combina­

tion of these two factors may have been responsible for the
results obtained here.
Nicotinic acid.

Wegner e_t a l . (1940b) observed a three-

to four-fold increase in the concentration of nicotinic acid
in the dried rumen contents as compared with the feed.

Kesler

- 117 -

and Knodt

(1950, 1951a, 1951c) found that nicotinic acid, like

riboflavin, was the highest in the small intestine of calves.
Lindahl and Pearson (1951) noted that the excretion of nico­
tinic acid was higher when sheep received a ration containing
casein than on a low protein ration.
Synthesis of nicotinic acid was observed in this investi­
gation for both the natural and purified rations.

The highest

synthesis occurred in the case of the natural ration when no
aureomycin was fed.

It is possible that the rate of removal

of nicotinic acid from the rumen was higher when 0.5 gram of
aureomycin was fed just as in the case of the riboflavin, dry
matter, crude fiber, crude protein and nitrogen-free extract.
Therefore , it appeared that there was a decreased synthesis of
this vitamin when aureomycin was included in the ration.

The

concentration of nicotinic acid at the 0 -hour was definitely
higher when aureomycin was included in the ration as compared
to no aureomycin.

This may be due to delayed synthesis of the

vitamin by the rumen bacteria or to decreased absorption from
the rumen.
There was tremendous synthesis of nicotinic acid in the
case of the purified ration when no aureomycin was fed (Table 22).
Agrawala (1950) reported a 93.1 to 490.1 per cent increase in
the synthesis of nicotinic acid six hours after feeding.

A

definite decrease in the synthesis of nicotinic acid occurred
when aureomycin was added to the ration.

A possible explanation

- 118 -

for the decreased synthesis was given in the riboflavin dis­
cussion.
Pantothenic acid.

Ruminal synthesis of pantothenic acid

has been observed by McElroy and Goss (1939) in sheep and in
cattle

(1941b), and by Wegner et_ al_. (1941c) and Lardinois

e^ aJ_. (1944) in cattle.

Teeri et, al.

(1950, 1951a) observed

that the excretion of pantothenic acid from cattle was greater
than the dietary intake.

Synthesis of pantothenic acid occurred

in the rumen when both the natural and purified rations were
fed, although not at a very high rate for the natural ration.
Accumulation of pantothenic acid occurred during the first

6 -hour period when 1.0 gram of aureomycin was fed.

The rate

of removal of this vitamin was highest when 0.5 gram of aureo­
mycin was included in the ration.

The quantity present at the

0-hour was the highest when 0.5 gram of aureomycin was fed
which may be as a result of delayed synthesis or due to de­
creased absorption from the rumen.
Agrawala

(1950) observed a 5.4 to 562.2 per cent increase

in the pantothenic acid concentration in the rumen contents
when the animals received a purified ration.

In the investiga­

tion reported here, the increase in the pantothenic acid was
the highest when no aureomycin was fed.

A delayed synthesis

resulted when 0.5 gram of aureomycin was added to the ration
(Table 22).

SUMMARY
Two steers, each with a rumen fistula fitted with a
plastic plug, were used to investigate the influence of aureomycin on the synthesis and digestion in the rumen when the
steers were fed natural and purified rations.

The animals

were fed a natural ration of 15 pounds of second-cutting
alfalfa-brome hay and four pounds of corn once daily through­
out the feeding trials.

Aureomycin was fed at each level

(0, 0.5 and 1.0 gram per day) for 15 days before changing to
the next highest level.

The period in which the steers re­

ceived no aureomycin served as the control period for com­
parison with those in which aureomycin was fed.
The samples of the rumen contents were collected by com­
pletely evacuating the rumen before feeding, 0 -hour and at 6 and 12-hours after feeding the ration.

Determinations for

dry matter, crude fiber, crude protein and ether extract as
well as nitrogen-free extract were made on both the rumen
contents and the ration.

Microbiological methods of assay were

used for the determination of the 10 essential amino acids,
riboflavin, nicotinic acid and pantothenic acid.
Neither of the steers showed any signs of anorexia or
diarrhea at any time during aureomycin supplementation when
the natural ration was fed.

Steer 714 went off-feed just

prior to the addition of 0.5 gram of aureomycin to the puri­
fied ration and did not regain its appetite during the period
that aureoraycin was included in the ration.

- 120 -

The pH declined for about six hours after feeding with
only a slight increase toward alkalinity at the end of 12 hours.
When the aureomycin was included in the natural ration, the
pH did not become as acid as that observed when no aureomycin
was fed; however, the response was in the same direction in
each case.
A definite increase in the total bacterial count of the
rumen contents and the feces occurred when aureomycin was in­
cluded in the ration.

The number of rumen streptococci de­

creased when aureomycin was fed, while the number of coliform
organisms in the rumen remained approximately the same in one
animal and increased in the other.
The rate of removal of dry matter, crude fiber, crude
protein, nitrogen-free extract, non-protein nitrogen, the 10
essential amino acids and riboflavin from the rumen was the
highest when 0,5 gram of aureomycin was fed.

There was an

accumulation of ether extract, nicotinic acid and pantothenic
acid in the rumen when aureomycin was included in the ration.
There was an accumulation of dry matter and crude fiber
in the rumen at the 0 -hour when 1.0 gram of aureomycin was fed
which indicated that a slight depression of digestibility of
these constituents may have occurred.
The pH and the total bacterial count were approximately
the same for the periods when no aureomycin and 0,5 gram of
aureomycin was added to the purified ration.

The synthesis

121 -

of the amino acids was lower when 0.5 gram of aureomycin was
included in the purified ration.

A definite decrease in the

synthesis of riboflavin, nicotinic acid and pantothenic acid
also occurred when 0.5 gram of aureomycin was added to the
purified ration.

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APPENDIX

144 -

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- 155 -

TABLE 34
INFLUENCE OF AUREOMYCIN ON THE B-VITAMIN COMPOSITION OF RUMEN
CONTENTS FROM 714 FED A PURIFIED RATION

Animal
Aureono. Time raycin
hr
gm

Riboflavin
f/ gm

0

0

10.81

23.72

17.47

0

0.5

3.10

2.02

15.53

6

0

21.31

31.47

135.83

6

0.5

2.45

0.40

2.81

25.01

91.22

202.53

3.21

1.94

6.98

Pantothenic
acid
f/gm

Nicotinic
ac id
x/ m

714

714

12

0

12

0.5

714

- 156 -

TABLE 3 5
INFLUENCE OF AUREOMYCIN ON THE TOTAL RUMEN BACTERIAL COUNT
FOR 707 WHILE RECEIVING A NATURAL RATION
(Billions per milliliter)

Aureornycin
gm

Days ________________Hours After Feeding
samp­
ling
0
2
4
6
8

0
0
0
0
A v . , 15
days

3
2
1
1

0.5
0.5
0.5
0.5
0.5
A v . , 15
days

3
3
2
1
2

1.0
1.0
1.0
1.0
1.0
Av. , 15
days

3
2
2
1
1

0
0
Av., 10
days

2
3

12,510 12,710
9,234
10,025
6,375
9,536
9,968
14,910
18,180
19,875
21.090
16,805

6,781

8,074
7 ,02 8
11,200
6,100

8,567

8,101
8,776 13,920 13,110 18,560
15,110
16,230
14,325
20,080
15,771

19,970 21,090 19,540 17,180 15,590
15,380
19,960
14,220
20,410
20,350
23,000
16 ,686
22,950
21,950

16,686

16,890
15,300

16,600
13,510

16,095

15,055

10

12

11,750 11,150
5,462
4,450
- -

7,021
14,310 12,030
—

19,725
—

l b ,87£
18,180 18,480
—

13 ,77 5
14,327
14,327

- 157 -

TABLE 36
INFLUENCE OF AUREOMYCIN ON THE TOTAL RUMEN BACTERIAL COUNT
FOR 714 WHILE RECEIVING A NATURAL RATION
(Billions per milliliter)

Aureo­
mycin
gm

Days
samp'
ling

0
0
0
0
A v , , 15
days

3
2
1
1

0.5
0.5
0.5
0.5
0.5
Av., 15
days

3
3
2
1
2

1.0
1.0
1.0
1.0
1.0
A v . , 15
days

3
2
2
1
1

0
0
Av., 10
days

2
3

Hours After Feeding
0

2

8,869 10,570
7 ,600
9,428
7 ,776
8,418

4

6

8

10

12

6,814

7,958
7,075
7,925
6,275

6,803

8,831

7,663
8,275
5,729
4,825

7,308

6,623

6,988 14,400 12,910 8,904 12,490 10,620 13,930
14,600
16,970
13,340
24,290
il ,775
12,250
25,875
—
18,750
11 .250
17,075

13,569

12,853

20,630 22,080 16,200 15,480 16,380 16,840 17,800
—
14 ,340
11,920
—
13,380
14,470
7 ,150
19,250
17,775
15,025
17.800
18.100
16,579

16,050

14,760
16,360

14,450
14,670

15,560

14,560

13,325

- 158 -

TABLE 37
INFLUENCE OF AUREOMYCIN ON THE pH OF RUMEN CONTENTS FROM
CATTLE RECEIVING A NATURAL RATION
(Av. first 3 days of 15-day period)

Animal Aureo­
no.
mycin
gm

707

714

0

2

Hours After Feeding
4
6
8

10

12

0

6.90

6.61

6.35

6.21

6.12

6.28

6.08

0.5

6.90

6.62

6.57

6.60

6.52

6.62

6.39

1.0

7.07

6.50

6.46

6.46

6.48

6.36

6.35

0

6.78

6.61

6.24

5.70

5.73

5.54

5.66

0.5

6.56

6.42

6.20

5.90

5.98

6.00

5.76

1.0

6.88

6.48

6.10

5.91

6.00

6.13

6.03

- 159 -

TABLE 38
INFLUENCE OF AUREOMYCIN ON THE RUMEN STREPTOCOCCI AND THE COLIFORM
GROUP IN THE RUMEN OF CATTLE FED A NATURAL RATION
(Values in logarithms)
Days
Aureo­ samp­
mycin ling
0
gm
0
3
6.97
5.97
0
3
6.30
0
1
0
1
6.30
Av. , 15 days 6.38

707
2

714

Hours After Feeding
4
6
8
0
2
4
S t r e p t o c o c c i
6.63 6.63 6.97
6.80
6.63
7.30
6.30
6.30
6.30
6.72
6.41

6

8
6.30
6.30
6.30
6.47

0.5
3
0.5
3
2
0.5
2
0.5
Av. , 15 days

5.97
7.63
6.80
4.80
6.30

5.97 5.63 5.63
6.97
6.30
5.80
5.30
5.30
5.80
5.93
5.84

5.97
5.30
5.80
5.59

1.0
3
2
1.0
2
1.0
1
1.0
Av. , 15 days

5.97
5.30
5.80
4.30
5.34

5.63 5.63 5.97
6.30
6.30
5. 80
6.30
6.30
5.30
6.09
5. 88

4.80
6.30
5.50
5.59

5.80
5.97
5.88

5.30
5. 97
5.63

G r o u p
2.63
3.30
4.30
5.80
4.01

4.30
4.30
4.30
3.88

2
4.80
0
6.63
3
0
Av. , 10 days 5.72

4.80
5.30
5.05

C o l ii ff oo rr m m
4.30 4.30 3.30
3.63
2.97
0
3
3.97
4.65
3
0
5.30
3.30
1
0
4.30
6.30
1
0
4.47
Av. , 15 days 4.31
0.5
3
3
0.5
2
0.5
2
0.5
Av. , 15 days

3.63 4.97 4.30 4.97 4.97 1.97
3.30
4.63
4.63
2.30
5.30
4.80
4.80
4.80
5.30
3.09
4.93
4.59

3.63
1.80
2.30
2.68

3
1.0
2
1.0
2
1.0
1
1.0
Av. , 15 days

3.97 4.30 4.63 4.63 3.63 2.97
2.80
3.30
3.30
4.80
3.80
3.80
2.30
3.30
2.30
3.22
3.76
3.34

2.80
3.30
3.30
3.01

4.30
2.30
3.30

3.30
2.65
2.97

2
2.80
3
3.63
AV. , 10 days 3.22
0
0

3.80
3.63
3.72

- 160 -

TABLE 39
INFLUENCE OF AUREOMYCIN ON THE TOTAL BACTERIAL COUNT OF FECES
FROM CATTLE FED A NATURAL RATION
(Billions per milliliter)

Aureo­
mycin
gm

Collection
No.

Animal
707

714

0

1

44,750

61,650

0

2

30,000

22,450

0

3

21,550

16,750

0.5

4

36,550

29,900

0.5

5

62,800

49,000

0.5

6

50,150

71,150

1.0

7

54,150

76,300

1.0

8

82,400

69,450

1.0

9

45,800

57,850

0

10

51,600

56,850

0

11

68,750

67,300

0

12

68,300

64,450

- 161 -

TABLE 40
INFLUENCE OF AUREOMYCIN ON THE FECAL STREPTOCOCCI
AND THE COLIFORM GROUP IN THE FECES
FROM CATTLE FED A NATURAL RATION
(Values in logarithms)

Aureomycin
gm

Fecal
Coliform
Collection
Streptococci______
Group
No.________ 707_______ 714____________ 707______ 714

0

1

4.30

5.80

4.80

5.80

0

2

5.80

5.80

5.30

5.30

0

3

5.80

4.80

5.80

6.30

0.5

4

5.80

5.30

5.80

5.80

0.5

5

7.30

5.80

6.30

5.80

0.5

6

7.80

6.80

5.80

6.30

1.0

7

6.80

5.80

4.80

6.30

1.0

8

5.30

5.30

4.80

6.30

1.0

9

5.80

5.80

5.30

5.30

0

10

6.30

6.80

5.80

6.80

0

11

5.80

6.30

4.80

5.30

0

12

6.30

5.80

6.30

4.80

- 162 -

TABLE 41
INFLUENCE OF AUREOMYCIN ON THE TOTAL RUMEN BACTERIAL COUNT
FOR 714 WHILE RECEIVING A PURIFIED RATION
(Billions per milliliter)

Animal Aureono.
mvcin
gm
714

0

714

0.5

Hours After Feeding
4
6
8

0

2

9,842

6,825

4,633

6,883

4,592

8,375 6,983

12,750

8,475

7,567

8,542

8,775

5,525 7,175

12

10

TABLE 42
INFLUENCE OF AUREOMYCIN ON THE pH OF RUMEN CONTENTS FROM
STEER 714 FED A PURIFIED RATION
(Av. first 3 days of 15-day period)

Animal Aureono.
mvcin
gm

0

2

Hours After Feeding
4
6
8

10

12

714

0

6.65

5.43

4.87

4.75

4.97

4.91

5.00

714

0.5

6.33

5.99

5.66

5.80

5.75

5.42

5.23