1
T HESTQ

Mie‘é‘afiflw f-‘z‘j,3';;§;~,3 F
Univemte" i
/.

 

 

7.“. arv vv (IV we VT

This is to certify that the

thesis entitled

Physiological State of Bacteria
in the Rumen

presented by

Joy Ann Gillett

has been accepted towards fulfillment
of the requirements for

MS degree in Animal Science

 

 

Werner G. Bergen

Major professor

Date May 2. 1983

0.7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

MSU

LIBRARIES
m

‘1

 

 

RETURNING MATERIALS:
Place in book drop to
remove this checkout from
your record. FINES wiil
be charged if book is
returned after the date
stamped beIow.

 

 

 

 

 

PHYSIOLOGICAL STATE OF BACTERIA
IN THE RUMEN

BY
Joy Ann Gillett

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1983

/37- 2251/

ABSTRACT
PHYSIOLOGICAL STATE OF BACTERIA IN THE RUMEN
BY
Joy Ann Gillett

Four mature wethers were fed isonitrogenous (12.2% CP)
diets (I, II, III) every 12 hours containing 3.03 (I), 3.46
(II), 3.91 (III) Neal DE/kg. Free bacteria and bacteria
bound to feed particles (estimated by colony counts x
log/gm) were determined 0, 2, 5, 8, 11 hours after
feeding. RNA/protein for both bacterial fractions was
determined every other hour (0 hr. - 12 hr.). Higher free
and bound counts were observed on III and exhibited
variation over sampling times. Free counts were: (initial
and maximum values) I, 2.96 and 4.87: II, 6.03 and 8.34;
III, 13.24 and 22.96. Bound counts were: I, 4.65 and 7.03;
II, 7.01 and 8.13: III, 18.00. Free bacterial RNA/protein
increased after feeding for all diets (initial and maximum
values: I, .251 and .323; II, .245 and .344; III, .384 and
.516) and were highest on III indicating free bacteria
respond physiologically to changes in available sub-
strates. Bound bacterial RNA/protein were lower than free
bacterial RNA/protein and were relatively stable over
sampling times (average values: I, .228; II, .147: III,

.216) indicating that bound bacteria are growing slowly.

ACKNOWLEDGEMENTS

I would like to express my sincere thanks (and
appreciation) to Dr. Werner G. Bergen for his guidance and
endless encouragement throughout my graduate program and
during preparation of this manuscript. Special appreciation
is also extended to Drs. M. T. Yokoyama, J. T. Huber and
J. C. Waller for their careful review of this manuscript and
assistance in my graduate work. Gratitude is expressed to
Dr. W. T. Magee for his help in the statistical analysis of
data.

I wish to thank Dr. R. R. Nelson, chairman of the
Department of Animal Science, for providing the facilities
and financial support which allowed this research to be
undertaken. I also would like to thank the following people
for their assistance: Donna Jo Cox for her help in the
statistical analysis of data, Elizabeth Rimpau for
laboratory assistance, and Mary Gillett and Scott Barao for
the preparation of graphs and slides. A very special thank
you is extended to Kris Johnson for her unselfish willing-
ness to help in any and every way possible. These people
took time out from hectic schedules to help me so that I
could complete my work in time to leave for a new
position. I cannot thank them enough.

My sincere appreciation is also expressed to Douglas B.
Bates. His extensive knowledge and technical expertise in
the area of rumen microbiology were invaluable to me.
Without his help this research would not have been possible.

Finally, I would like to thank my parents, Dolly and
Jack Gillett, for their unceasing support throughout :my
academic career. I would especially like to thank my mother
for the many long nights she spent carefully typing this
manuscript and for her love and understanding.

ii

TABLE OF CONTENTS

Page
LIST OF TABLESOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOV

LIST OF FIGURES. O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O OVii
I 0 INTRODUCTION 0 O O O O O O O O O O O O O I O I O O O O O O O O O O O O O O O I 1
I I 0 LITERATURE REVIEW. 0 O O O O O O O O O O O O O O O O O O O O O O O O O O 3

Rumen Fermentation...........................3
The Relationship Between Bacterial RNA and
Protein Content, and Growth Rate...........7
The Use of Microbial RNA as a Marker for
Rumen Digesta Passage Studies.............11
Cultivation of Rumen Bacteria...............l4
Factors Affecting Bacterial Numbers in the
Rumen.....................................20

III. MATERIALS AND METHODS

General Design of Experiment................28
Treatments..................................30
Sample Collection and Preparation...........30
Determination of Viable Counts in Rumen
Contents..................................34
Volatile Fatty Acids Determination..........35
Rumen Ammonia Determination.................37
RNA-Protein Ratio Determination in Ruminal
Bacteria..................................37
Statistical Analysis........................39

IV. RESULTS

Rumen pH, and Volatile Fatty Acid and Ammonia
Concentrations............................40
Free Bacterial Colony Counts................45
Bound Bacterial Colony Counts...............50
Variation of RNA-Protein Ratios with Time for
Free Ruminal Bacteria.....................56
Variation of RNA-Protein Ratios with Time for
Bound Ruminal Bacteria....................6l

iii

VI.

VII.

VIII.

TABLE OF CONTENTS (Continued)

Page

DISCUSSION

General Rumen Fermentation Parameters.......68

Rumen Bacterial Colony Counts...............69

RNA-Protein Ratios for Free and Bound Ruminal
BacteriaOOOOOO0.00.0.0.0...0.0.0.00000000071

GENERAL CONCLUSIONSOOOOOOOOOO0.00.00.00.000077

LIST OF REFERENCESOOOO00.00.000.00000000000078

APPENDIX.0.0.0....000O0.00.00.00.0000000000087

iv

Table

A1

A2

A3

A4

A5

A6

LIST OF TABLES

Page

Composition of Experimental Diets..............29
Composition of Rumen Fluid Media...............36

Rumen Fluid pH, and Volatile Fatty Acid and Ammonia
Concentrations for Sheep Fed Diet 1..........41

Rumen Fluid pH, and Volatile Fatty Acid and Ammonia
Concentrations for Sheep Fed Diet 2..........42

Rumen Fluid pH, and Volatile Fatty Acid and Ammonia
Concentrations for Sheep Fed Diet 3..........44

Free Bacterial Colony Counts Per Gram of Rumen
contentSOOOOO00......OOOOCOOOOOOOOO0000......46

Bound Bacterial Colony Counts Per Gram of Rumen
contentSOOOOOOOO0......00.0.0000000000000000051

RNA-Protein Ratios For Free Rumen Bacteria.....57
RNA-Protein Ratios For Bound Rumen Bacteria....62

Free Bacterial Colony Counts Per Gram of Rumen
contents-Sheep 905....OOOOOOOOOOOOOOOOOOOO.87

Bound Bacterial Colony Counts Per Gram of Rumen
contents-Sheep 9050......0.0.0.00000000000087

Free Bacterial Colony Counts Per Gram of Rumen
contents-Sheep 913.00000000000000000000000.88

Bound Bacterial Colony Counts Per Gram of Rumen
contents-Sheep 913.000.000.00...0.0.00.0...88

RNA-Protein Ratios For Free Rumen Bacteria
-Sheep 905.00.000.00.0000000000000000000000.89

RNA-Protein Ratios For Bound Rumen Bacteria
-Sheep 905.00.000.00.000.00.00.000000000000089

A7

A8

A9

A10

A11

A12

LIST OF TABLES (Continued)

Page

RNA-Protein Ratios For Free Rumen Bacteria
-Sheep 898.000......0.0.0.0....0.00.00.00.0090

RNA-Protein Ratios For Bound Rumen Bacteria
-Sheep 898.00.00.000I.0.0.0.000000000000000090

RNA-Protein Ratios For Free Rumen Bacteria
-Sheep913.00.000.00.000.0.0000000000000000091

RNA-Protein Ratios For Bound Rumen Bacteria
-Sheep 913.0.0...0.0.0.00.00.00.00000000000091

RNA-Protein Ratios For Free Rumen Bacteria
—Sheep 9000......0.000000000COOOOOOOOOOOOCO.92

RNA—Protein Ratios For Bound Rumen Bacteria
-Sheep 900.00.000.00...0.0.0.00000000000000092

vi

LIST OF FIGURES

Figure Page

1 Flow Chart of Sampling Procedures and Laboratory
Analyses for Bacterial RNA and Protein.......33

2 Free Bacterial Colony Counts Per Gram of Rumen
Contents for Two Sheep Fed Diet 1............47

3 Free Bacterial Colony Counts Per Gram of Rumen
Contents for Two Sheep Fed Diet 2............48

4 Free Bacterial Colony Counts Per Gram of Rumen
Contents for Two Sheep Fed Diet 3............49

5 Bound Bacterial Colony Counts Per Gram of Rumen
Contents for Two Sheep Fed Diet l............53

6 Bound Bacterial Colony Counts Per Gram of Rumen
Contents for Two Sheep Fed Diet 2............54

7 Bound Bacterial Colony Counts Per Gram of Rumen
Contents for Two Sheep Fed Diet 3............55

8 RNA-Protein Ratios for Free Bacteria From Sheep
Fed Diet 1.0.0.0000...OOOOOOOOOOOOIOOOOOO0.0.58

9 RNA—Protein Ratios for Free Bacteria From Sheep
Fed Diet 2.0...OOOOOOOOOOOOI0.0.00.000000000059

10 RNA-Protein Ratios for Free Bacteria From Sheep
Fed Diet 3......OOOOOOOOOOOOOOO0.0.0.00000000060

11 RNA-Protein Ratios for Bound Bacteria From Sheep
Fed Diet 1.0...0.0.0.0000...0.0.00.000000000064

12 RNA-Protein Ratios for Bound Bacteria From Sheep
Fed Diet 2.0.0.00000000000000.00.00.00.00000065

13. RNA-Protein Ratios for Bound Bacteria From Sheep
Fed Diet 3.00......OOOOOOOOOOOOIOOOOOIOOOIO0.66

vii

INTRODUCTION

The importance of rumen bacteria to the nutrition of
ruminants is well documented. Rumen microorganisms
degrade B-linked polysacchar’ides (cellulose) into products
that the host animal can either digest or absorb. Dietary
nitrogen, whether protein or non-protein in origin, is
converted to microbial protein of relatively high biological
value. This conversion is advantageous when low quality
protein or non-protein nitrogen sources are fed, but a
disadvantage when the ration contains high quality protein.

Chemically modifying high quality protein in order to
protect it from ruminal degradation has attracted great
interest. In order to determine the amount of dietary
protein passing from the rumen to the duodenum, it is
necessary to estimate the fraction of the digesta protein
which is of microbial origin. Bacterial ribonucleic acid
(RNA) has been used to estimate the microbial nitrogen in
digesta samples by relating the RNA content in the digesta
to the ratio of bacterial RNA nitrogen - total cellular
nitrogen. It is assumed that the bacterial RNA-N-total N
ratio does not vary over time or between rumen bacterial
species.

Growth rate and rate of removal determines the size of
the rumen bacterial population. As the dilution rate of the

1

liquid and solid fractions of the rumen digesta are
different, the growth rate of a microorganism would depend
mainly on whether it was ”free” in the rumen liquor or bound
to feed particles. Since the RNA-protein ratio is directly
proportional to bacterial growth or dilution rate, it would
appear that the free and bound rumen bacteria should have
different RNA-protein ratios. In addition, as the dilution
rate of the rumen varies with time after feeding it seems
that the bacterial RNA-protein ratio must vary also.

The present study was designed to determine the numbers
of free and bound bacteria in the rumen at various times
after feeding and the RNA-protein ratios of these bacteria

for sheep fed corn silage and high grain rations.

LITERATURE REVIEW

Rumen Fermentation

 

The rumen may be compared to "a continuous microbial
culture system with a more or less continuous substrate and
buffer supply and fermentation end product removal system”
(Bergen et a1., 1982). The pH is usually about 6 to 7 and
is maintained by the buffering action of the saliva which
contains sodium and potassium bicarbonate and urea, by
absorption of volatile fatty acids (VFA) through the rumen
wall and by ammonia production by the rumen microorganisms
(Bryant, 1970).

There are three distinct bacterial populations in the
rumen: l) bacteria which are free floating in the rumen
fluid, 2) bacteria which are attached to feed particles and_
3) bacteria which adhere to the rumen epithelium (Cheng and
Costerton, 1980).. Rumen bacteria anaerobically ferment
carbohydrates such as starch, cellulose, pectin and xylan
(Allison, 1965a). The polysaccharides are hydrolyzed to
soluble oligosaccharides and sugars by polysaccharide-
hydrolyzing bacteria. These organisms and iother sugar-
fermenting microbes ferment the extracellular intermediates

to yield characteristic ruminal fermentation end

products: acetate, propionate and butyrate (VFA) and carbon
dioxide (Bryant, 1970). Rumen methanogenic bacteria reduce
C02 to methane. Many rumen microorganisms form products
such as hydrogen, succinate, formate, lactate and ethanol
which are metabolized to the final end products by other

species. Ruminococcus flavefaciens and Bacteroides

 

 

 

succinogenes produce succinate which is subsequently

decarboxylated to propionate by Selenomonas ruminantium

 

 

(Blackburn and Hungate, 1963). Other microbes produce
formate which is rapidly degraded to C02 and hydrogen. The
methanogens utilize the hydrogen to form methane (Wol in,

1974). Selenomonas ruminantium produced lactate when grown

 

in pure culture but when this organism was grown in the
presence of a methanogen there was a marked decrease in
lactate production and an increase in acetate production
with the hydrogen being used to reduce C02 (Chen and Wolin,
1977). Iannotti et a1. (1973) co-cultured lRuminococcus
‘glbgg and Vibrio succinogenes. The hydrogen produced by R,
£1331: was used by l. succinogenes to reduce fumarate to
succinate. No ethanol production was detected but acetate
production increased relative to the mono-culture.

These interspecies hydrogen transfer systems are
energetically favorable. The shifts in fermentation caused
by the Hz-utilizing organisms results in a decrease in the
flow of carbon atoms into single culture fermentation
products such as ethanol and lactate. Acetate formation

from pyruvate produces adenosine triphosphate (ATP) whereas

lactate or ethanol production from pyruvate yields no ATP.
More energy is therefore made available for bacterial
growth.

The nitrogen entering the rumen consists of protein and
non-protein nitrogen sources such as peptides, amino acids,
purines, pyrolles, choline, urea, ammonia, nitrates and
nitrites (McDonald, 1952). The amino compounds and
nitrogenous bases may be deaminated by the rumen
microorganisms (McDonald, 1952) while the nitrate and
nitrite are reduced to ammonia (Lewis, 1951). Bacterial
proteolysis of dietary protein produces peptides and amino
acids which may be deaminated to form ammonia, C02 and
VFA. The concentration of free amino acids in the rumen is
low and there is little absorption of the amino acids
through the rumen epithelium (Leibholz and Hartmann,
1972). Rumen microorganisms exhibit a high urease activity,
degrading urea to ammonia and C02. Ammonia is absorbed from
the rumen into the portal blood stream and is converted to
urea by the liver (McDonald, 1952). The urea may be
excreted in the urine or return to the rumen via the saliva
or diffusion from the blood (Wolin, 1981).

The ammonia produced from the degradation of protein
and non-protein compounds is probably the most important
source of nitrogen for rumen bacteria. Up to 78% of
bacterial nitrogen may be derived from ammonia depending on
the ration fed (Smith et al., 1980). It is necessary to

supply an adequate amount of both nitrogen and energy in a

ration in order to maintain a balanced rumen fermentation
(Wallace, 1979). If energy intake is insufficient, there
will be an increase in both ammonia concentration in the
rumen and ammonia absorption into the bloodstream. This
situation is most prevalent when the diet is deficient in
readily available carbohydrate such as starch. Cellulose is
of little benefit in enabling the bacteria to utilize free
ammonia (Smith, 1969). Many rumen microorganisms synthesize
cellular amino acids using ammonia even when an exogenous
supply of amino acids are available (Allison, 1965a).
Apparently, some rumen bacteria lack a transport systen for
amino acids or this mechanism is functioning at a very low
activity (Allison, 1969). Bacteroides ruminicola is able to
make use of oligopeptide nitrogen as the sole source of cell
nitrogen, but not in preference to ammonia nitrogen (Pittman
et al., 1967).

Carbon for amino acid biosynthesis is iderived from
fermentation end products. Isovalerate, isobutyrate and 2-
methylbutyrate are carboxylated and aminated to produce
leucine, valine and isoleucine, respectively. This type of
reaction is not limited to the biosynthesis of branched-
chain amino lacids, as phenylacetic acid carbon is
incorporated into phenylalanine (Allison, 1965b) and indole-
3-acetic acid is incorporated into tryptophan (Allison and
Robinson, 1967). The carbon skeletons of isobutyrate,
isovalerate and 2-methy1butyrate are also incorporated into

higher fatty acids (Garton, 1965). Ruminococcus

flavefaciens produced branched-chain acids consisting of 15
and 17 carbon atoms from isovalerate and 3. 3133s used
isobutyrate to form C14 and C16 branched—chain fatty acids
(Garton, 1965).

The Relationship Between Bacterial RNA and Protein Content,
and Growth Rate

Bacterial growth may be defined as the orderly increase
of all chemical constituents (Stanier et al., 1976). The
chemical composition and size of a microorganism varies
depending on the rate of growth which is attainable in a
given environment (Schaechter et al., 1958). However, an
increase in mass may be due to the synthesis of storage
products such as polysaccharides, polyphosphates and
poly-B -hydroxybutyrate. Production of these storage
molecules is not indicative of true growth.

When bacteria are cultured in a nutritionally adequate
medium, a state of balanced growth eventually will be
achieved. This steady state of growth is reached when all
cellular components increase at the same rate over a given
period of time (Schaechter, 1973). The growth rate of a
microbial population with a constant chemical composition
may be determined by measuring the increase of any cell
constituent. Bacterial ribonucleic acid (RNA), deoxy-
ribonucleic acid (DNA) and protein content increase with
increasing growth rate (Schaechter et al., 1958). It has

been observed that the ratios of RNA/protein and RNA/DNA are

linear functions of the growth rate while the ratio of
DNA/protein is independent of growth rate (Rosset et al.,
1966).

Ribosomal efficiency has been described as the average
protein synthesis rate per ribosome and is determined by the
fraction of the total number of ribosomes which are engaged
in protein synthesis (active ribosomes) and. the ;peptide
chain elongation rate (Dennis and Bremer, 1974a). Several
workers reported a constant number of protein molecules
synthesized per unit of RNA at various growth rates for
Salmonella typhimurium (Kjeldgaard, 1961; Ecker and Kokaisl,
1969). This implied a constant efficiency of the ribosomes
in the synthesis of proteins. Leick (1968) observed a
similar ribosomal efficiency in several different types of
microorganisms with growth rates varying from u=0.5/hour
to u=2.33/hour. Microorganisms shifted from a nutritionally
poor to a nutritionally rich medium (shift up) exhibited an
immediate increase in RNA production followed by a rise in
protein synthesis (Kjeldgaard, 1961). During both steady
state and shift up conditions, a constant ratio existed
between ribosomes and protein synthesized. However, this
does not indicate a constant efficiency in the synthesis of
specific proteins as ribosomal proteins comprised one third
of the total proteins synthesized during the shift up.

Other workers have disagreed with the constant
ribosomal efficiency theory. Rosset et a1. (1966) reported

that the efficiency of ribosomal RNA (rRNA) increased with

increasing growth rates for Escherichia coli grown atu>0.4

 

doublings/hour. Dennis and Bremer (1974a) concluded that
the ribosomal efficiency for g, ggli_strain B/r is constant
for u>1.2 doublings/hour and gradually decreases for 1J<l.2
doublings/hour. Recent work has shown that the efficiency
of ribosomes in protein synthesis for _E_. 2911 grown at
moderate rates (43 and 65 minutes doubling times) was less
than that for cultures growing rapidly with a doubling time
of 17.4 minutes (Koch, 1980). A decrease occurs. in the
peptide chain elongation rate for slowly growing 3;. £211,
but at high growth rates this factor is constant
(Forchhammer and Lindahl, 1971; Dalbow and Young, 1975).
The lower efficiency of ribosomes at decreasing growth rates
might also partially be explained by decreased numbers of
transfer RNA (tRNA) molecules and a low ratio of tRNA to
active ribosomes (Skjold et al., 1973). Harvey (1970)
observed no detectable change in polypeptide chain
elongation rate at low growth rates, but found an increase
in the fraction of ribosomes in polyribosomes after a
nutritional shift up. This resulted in an increase in
ribosomal efficiency with increasing growth rates.
Kjeldgaard (1961) observed that during shift up
experiments the RNA increased in a diphasic manner. He
attributed this rise to an increased production of ribosomal
particles. However, immediately after a nutritional shift

up, there is a slow change in total RNA synthesis (Nierlich,

10

1972) with RNA chain elongation rates and initiation
frequency remaining relatively' constant, (Mowbray' and
Nierlich, 1975). Even though there was little change in
total RNA. production, rRNA. and tRNA. synthesis increased
relative to mRNA synthesis (Nierlich, 1972). This increase
in stable RNA was due to a shift in the functioning RNA
polymerase molecules from transcribing messenger RNA genes
to ribosomal and transfer RNA genes (Dennis and Bremer,
1974b). The second increase in RNA synthesis which
Kjeldgaard (1961) observed was due to an increase in the
number of functioning RNA polymerase molecules (Bremer and
Dennis, 1975; Mowbray and Nierlich, 1975).

Maal¢e and Kjeldgaard (1966) concluded that new RNA
must be synthesized in order to achieve the increase in
protein synthesis observed after a shift up transition.
Conversely, the synthesis of new ribosomes requires the
production of new ribosomal proteins in excess of the amount
formed during the pre-shift growth conditions (Champney,
1977). Further investigations involving organisms
exhibiting balanced growth at low growth rates revealed the
synthesis of ribosomes and tRNA which were not functional at
low growth rates (Koch, 1970). This extra RNA can quickly
become functionable under more favorable environmental
conditions (Koch, 1970). In addition, microorganisms have a
reserve supply of RNA polymerase and enzymes for purine and
pyrimidine synthesis which are not being used to full

capacity at low growth rates (Koch and Deppe, 1971).

11

Therefore, the total rate of protein synthesis is not
limited by the number of ribosomes in a bacterial cell

although it is proportional to it (Harvey, 1970).

The Use of Microbial RNA as a Marker for Rumen Digesta
Passage Studies

 

In ruminants, the non-ammonia nitrogen (NAN) reaching
the duodenum consists of dietary protein, microbial protein
and nucleic acids, and a small amount of free amino acids
and peptides (Bergen, 1978). Determining the nitrogen in
the dietary and microbial fractions has attracted great
interest. Microbial nitrogen has been estimated by
measuring the concentration of some microbial component
(marker) in the digesta and relating this value to the
marker concentration in the actual microorganisms (Ling and
Buttery, 1978).

Various microbial constituents, such as diaminopimelic
acid (DAPA), D-alanine, DNA and RNA have been proposed as
markers for bacterial cell mass. DAPA is not found in
protozoa nor is it found in all bacteria. D-alanine is
present in bacteria but only in small levels. Nucleic acids
comprise a large fraction of bacterial organic matter and
since early work indicated that RNA content was quite
constant in microbial matter, RNA has been used extensively
as a marker. From a RNA analysis and knowledge of the ratio
of bacterial RNA to cellular N, microbial N in digesta could

be estimated.

12

McAllan and Smith (1969) developed a procedure for the
determination of RNA in digesta samples. This method has
been utilized by other investigators (Cole et al., 1976;
Prigge et al., 1978) to determine microbial NAN in digesta
contents. The use of bacterial RNA as a marker relies on
the assumption that dietary RNA is not a contributing factor
to the total RNA leaving the rumen. Pure nucleic acids were
shown to be rapidly degraded in the rumen (Smith and
McAllan, 1970; and McAllan and Smith, 1973) however,
significant amounts of dietary material often escape rumen
degradation. The RNA associated with this undegraded
dietary fraction may contribute a sizeable amount of RNA to
the duodenal digesta RNA (Coelho da Silva et al., 1972). To
determine if an over estimation of microbial RNA was being
made, Smith et a1. (1978) observed 32p incorporation into

rumen bacterial RNA. 32P-

labelled RNA nucleotides comprised
85% of the nucleotides based on total duodenal RNA
measurements. The difference was attributed to total RNA
values being inflated by the presence of small amounts of
dietary RNA. To correct for this error duodenal RNA values
were multiplied by 0.85 (adjusted RNA).

Using bacterial RNA as a marker also assumes that RNA-
N/total N is the same for both bacteria and protozoa. This
assumption is not valid as RNA-N/total N for protozoa is
lower than that of bacteria (Ling and Buttery, 1978). With

various feeding regimes, protozoa may make up a significant

preportion of the microbial population leaving the rumen.

13

Under these conditions, using a value derived from bacterial

RNA-N/total N would underestimate the duodenal microbial N.
RNA nitrogen - total nitrogen ratios have been found to

be fairly constant in several strains of pure cultures of

rumen bacteria. values varied from 0.10 for Streptococcus

 

bovis $839 to 0.13 for Str. bovis 597 (Smith, 1969). The

 

RNA-N/total N in mixed bacteria separated from rumen
contents of animals receiving roughage diets varied slightly
from 0.08 to 0.15 (Smith, 1969). Isichei (1980) observed a
constant RNA-N/total N of 0.155 - 0.156 for mixed bacteria
isolated from rumen contents of steers fed high grain and
high roughage rations, while Cole et a1. (1976) and Prigge
et a1. (1978) obtained a bacterial RNA-N/total N value of
0.1 for steers fed roughage and concentrate rations. Ling
and Buttery (1978) found considerable variation existed in
bacterial RNA-N/total N values, but these differences did
not appear to be related to the effects of animals, diets,
or periods of sampling.

It has been suggested that the diet may have an effect
on bacterial RNA-N/total N. Washed cell suspensions of
rumen bacteria were incubated for 12 hours in media
containing 100% urea-N or 75% urea-N plus 25% amino acid-
N. While no significant time variation was observed during
the incubation period, the bacterial RNA-N/total N. in the
urea plus amino acids media was slightly higher (ave. 0.148)
as compared to the value obtained for the media with urea

(ave. 0.135) (Maeng and Baldwin, 1976a). Little variation

14

was seen in the RNA-N/microbial N for a cow sampled 0, 1, 2,
4, 6, 8 and 10 hours after feeding a purified diet in which
urea was the sole nitrogen source (Maeng and Baldwin,
1976b). This is in contrast to the findings of Smith and
McAllan (1974) who observed a significantly lower bacterial
RNA-N/total N before feeding cows a hay and concentrate diet
as compared to 4 and 6 hours after feeding.

From the above results it appears that the bacterial
RNA-protein ratio was fairly constant regardless of diet fed
or time of sampling. This does not coincide with what is
known of bacterial RNA. RNA is more sensitive to changes in
nutritive sources than DNA or protein (Neidhardt and
Magasanik, 1960) and consequently shows a greater response
to changes in growth rate. This results in the RNA-protein
ratio being directly proportional to growth or dilution
rate. Since the dilution rate of the rumen varies with
different types of rations and with time after feeding, it
would seem that the bacterial RNA-protein ratio should
reflect this change. Bates (1981) cultured several strains
of rumen bacteria and found that the RNA-protein ratio
varied with specific growth rates. Additional work needs to
be done to determine if similar results are attainable under

in vivo conditions.

Cultivation of Rumen Bacteria
The enumeration of rumen bacteria has been accomplished

by microscopic observation (direct counts) and by culturing

15

the bacteria in suitable media (viable counts). Direct
counts have been obtained by using Petroff—Hauser or Helber
counting chambers and by stained smears similar to the Breed
milk smear (Warner, 1962a). Rumen bacteria have been
cultured in anaerobic roll tubes and plates. Direct counts
are more reliable than viable counts, but the former method
does not distinguish living from. dead cells (Chung and
Hungate, 1976). Conversely, culture media does not support
the growth of all rumen microorganisms and consequently,
viable counts underestimate the number of bacteria in the
rumen.

Maki and Foster (1957) compared direct and viable
counts from rumen samples of cows fed roughage and
concentrate diets. Colony counts from cows fed a
concentrate ration were 57 to 73% of the microscopic counts
while viable counts from roughage fed cows were only three
to 12% of the direct counts. This difference in percentages
may partly be explained by the fastidious nutritional
requirements of rumen bacteria from roughage fed animals.
Many of these organisms were unable to grow in the culture
media provided. Possibly a greater proportion of dead
bacteria were also present in the rumen of the roughage fed
cows. Dry forage is retained longer in the rumen and
soluble carbohydrates are liberated slowly, thus increasing
the proportion of dead bacteria (Chung and Hungate, 1976).

Viable bacterial numbers also differ depending on the

type of cultural technique that is employed. Leedle and

16

Hespell (1980) obtained 20 to 50% higher cell counts on
culture plates than in roll tubes, regardless of media
used. The higher plate counts were probably due to the
differences between the two culture methods. During
inoculation of roll tubes the bacteria in the inoculate are
exposed to molten agar (45 - 50°C) and to rapid cooling.
Although no loss in viability was observed due to the cold
stress involved in roll tube preparation, a five to 20% loss
in viability did occur when bacteria were exposed to a
temperature of 47°C for 0.5 minute. The difficulty in
detecting colonies in roll tubes might also account for the
lower bacterial numbers. Surface colonies are easier to see
on the flat surface of a culture plate. With roll tubes it
is necessary to look through the agar medium to detect
colonies. It is especially difficult to distinguish
colonies in roll tubes containing cellulose or xylan media,
as this media is opaque in color (Leedle and Hespell, 1980).

Rumen bacteria have been cultured in different types of
rumen fluid - containing media and more defined media in
which the rumen fluid has been replaced by Trypticase, yeast
extract, hemin solution and volatile fatty acids (Leedle and
Hespell, 1980). Bryant and Burkey (1953a) developed a media
which contained 44% rumen fluid, .2% glucose, .2% cellobiose
and 2% agar. This media (RGCA) grew about 8.4% of the
bacteria which were observed via direct counts for animals
fed roughage and roughage-concentrate rations. Bryant and

Robinson (1961) observed spreading of colonies and gas

17

formation because of the high level of carbohydrate in RGCA
media. In addition, it was difficult to detect colonies
because the media had unclarified rumen fluid which often
contains particulate matter. The RGCA media was modified by
clarifying the rumen fluid by centrifugation, reducing the
concentration of glucose and cellobiose: to 0.025% each,
adding 0.05% soluble starch and changing the reducing agent
from cysteine to a combination of cysteine and sodium
sulfide. About twice as many colonies grew on this media
(98-5) as compared to RGCA media.

Some investigators felt a media was needed which
contained more standardized ingredients. Rumen fluid,
collected from different animals or from the same animal on
different days, varies in composition and is not always
available to workers (Hungate et al., 1964). Caldwell and
Bryant (1966) compared colony counts with a rumen fluid
medium (RFM) and a medium without rumen fluid (medium 10).
The RFM was similar to medium 98-5 except it contained 0.05%
each of glucose, cellobioses and starch. The medium 10 was‘
identical to the RFM except the rumen fluid was replaced by
yeast extract, Trypticase, hemin and a mixture of vOlatile
fatty acids. Similar viable counts were obtained with both
media from rumen contents of cattle fed roughage and grain
rations. Thorley et a1. (1968) reported higher colony
counts with medium 10 (VFA mixture added at a level of 0.31%
rather than 0.45% added by Caldwell and Bryant, 1966) as

compared to medium 98-5 for cattle fed dried grass and

18

pelleted grass rations.

Grubb and Dehority (1976) compared counts obtained in a
40% rumen fluid, 0.3% substrate medium with clarified or
standard rumen fluid. Contrary to the results of Bryant and
Robinson (1961) who found no significant difference in total
counts with clarified or standard rumen fluid, Grubb and
Dehority (1976) observed 24 to 32% less colonies in media
with clarified rumen fluid. In (order to :minimize the
problems mentioned earlier with unclarified rumen fluid, the
volume of media was reduced from 9 ml to 4 ml per tube. The
media (RGCSA) used consisted of 0.025% each of glucose and
cellobiose and 0.05% soluble starch with 4 ml per tube.
RGCSA media supported the growth of more rumen bacteria then
either RFM or medium 10 (9 ml of each per tube) from sheep
fed roughage and concentrate rations.

Lower viable counts were still obtained from rumen
contents of animals fed roughage rations as compared to
animals fed concentrate diets. Chung and Hungate (1976)
modified medium 98-5 to contain 33% rumen fluid and added an
alkali treated alfalfa fiber substrate consisting of a
hemicellulose (H) and cellulose (C) fraction. ‘It was
anticipated that this H + C fraction would partially satisfy
the growth requirements of bacteria prevalent in the rumen
of animals fed roughage rations. Higher counts were
obtained with this media, but were still only 20 to 40% of
the direct counts. Henning and van der Walt (1978) used a

medium which contained 40% clarified rumen fluid, 0.033%

19

each of glucose and cellobiose and 0.067% each of soluble
starch and xylan. Higher colony counts were obtained with
this media than with medium 98-5 or the media used by Chung
and Hungate (1976). From these results it appeared that it
is necessary to supply a source of pentose to achieve
maximal viable counts.

It is still advisable to use media containing rumen
fluid when possible because certain strains of rumen
bacteria have unknown growth factors which might not be
supplied in a media such as medium 10 (Caldwell and Bryant,
1966). However, the advantages of reproducibility and
reliability of a more defined medium cannot be overlooked.
Leedle and Hespell (1980) used a media which consisted of
only 16% rumen fluid, but also contained Trypticase, yeast
extract, hemin solution, VFA solution and 0.05% each of
glucose, cellulose, cellobiose, maltose, pectin, soluble
starch, xylan and xylose. This media was considered to be
more reproducible than other rumen fluid media and grew at
least as many colonies as medium 98-5 and medium 10.

It is difficult to compare the effectiveness of
different types of media because of variations fin the
composition of rumen fluid, differences in culture methods
and feeding regimes, and individual animal differences.
However, in order to reproduce the rumen environment in a
culture media, more information is needed concerning the

specific nutritional requirements of the rumen bacteria.

Factors Affecting Bacterial Numbers in the Rumen

Total viable numbers of bacteria per unit weight or
volume of rumen fluid vary and are affected by the type of
ration fed, method of feeding, time interval since feeding
and presence or absence of protozoa (Hungate, 1966).

Bryant and Robinson (1968) fed heifers hay and hay-
grain rations every twelve hours and found that the
bacterial colony counts were lowest at one hour after
feeding and increased significantly between 1, 2.5 and 5.5
hours. A slight but significant decrease occurred between
5.5 and 10 hours after feeding. Only when the pelleted
ration was fed were the numbers at one hour not
significantly lower than those 10 hours after feeding.
Dilution of the bacteria by increased saliva secretion on a
long hay ration compared to a pelleted ration (Moore, 1964)
could account for the lower numbers obtained at one hour on
the hay and hay-grain diets. In addition, only at one hour
after feeding were the colony counts for the pelleted ration
significantly higher than those for the hay and hay-grain
diets. A pelleted ration, as compared to a long hay ration,
contains feed particles which are finely divided and easily
wetted with a more rapid solution of feed nutrients. This
results in a more rapid bacterial fermentation and
production of organic acids (Moore, 1964). Thus, the
pelleted diet provided a readily available source of energy
to the bacteria and a faster bacterial growth rate was

obtained in the first hour. Higher mean bacterial counts

20

21

were observed for heifers on the hay crop silage ration.
The reason for this was unknown, but the authors suggested
that the lower counts obtained on the hay-grain ration may
be attributed to increased numbers {of ciliate protozoa.
Caldwell and Bryant (1966) also observed large numbers of
ciliate protozoa from cows fed alfalfa hay-grain rations.
Bryant and Robinson (1968) also noted that at any given
time, lower numbers of bacteria were cultured from the
ventral portion of the rumen as compared to the dorsal
area. The dry matter of the dorsal sac is higher for
animals on a variety of rations (Hungate, 1966) and the
rumen bacteria tend to be associated with the solid material
in the rumen (Cheng et al., 1977). Munch-Petersen and
Boundy (1963) noticed differences in numbers of viable
bacteria from different parts of the rumen, but found no
consistent pattern. These workers found no "site-to-site"
differences for bacterial numbers in the sheep rumen.

. Shehata (1958) reported diurnal changes in direct
counts of bacteria from sheep fed once a day. The diet
consisted of wheat straw with various concentrates added.
Individual differences between sheep were noticed in both
initial and maximum bacterial counts obtained after
feeding. Diet also affected the bacterial numbers as well
as the time after feeding that highest counts were
reached. When berseem (Trifolium alexandricum), cotton-seed
cake or linseed oil meal was added to the diet, a rapid

increase in colony counts was followed by a rapid decrease

22

in numbers. A rapid increase for two to three hours
followed by a maintenance of this high count for several
hours was seen when dried beans (Vicia faba) or dried beans
plus starch was used. Bacterial numbers rapidly increased
after feeding fish meal or meat meal and this rise was
followed by a decrease and another rapid increase. These
differences in diurnal variation may be caused by the
chemical and physical properties of the diet fed (Shehata,
1958). These properties can affect the rate of availability
of nutrients to the rumen bacteria and consequently
influence the diurnal changes. Nutrients which are readily
available to the bacteria allow for rapid increases in
growth and numbers and are then followed by a reduction in
counts. When only part of the nutrients are readily
accessible to the microorganisms with the rest becoming
available after degradation, then the early rapid increase
in bacterial numbers may be sustained for several hours.
This type of diurnal variation was seen with sheep fed a
lucerne and wheaten chaff diet once a day (Warner, 1962b).
Total bacterial counts decreased for about two hours after
feeding and then increased and plateaued for several hours
before a slight decrease brought the bacterial numbers to
the pre-feeding level.

Altering the physical state of a feed changes the
characteristics of the rumen microbial population. Cows fed
artificially dried grass had significantly lower colony

counts than cows fed the same grass ground and pelleted

23

(Thorley et al., 1968). In addition, the distribution of
microbial species changed when the pelleted ration was
fed. Decreases were seen in isolates of butyrivibrio
species and Bacteroides ruminicola. While increases in
lactobacilli suggested lower pH values and more rapid
fermentation of readily available carbohydrates, there was
not a corresponding decrease in cellulolytic bacteria. Cows
fed a concentrate diet had as many cellulolytic bacteria as
those fed roughage diets (Bryant and Burkey, 1953b). These
workers and Slyter et a1. (1971) did observe an increase in
the proportion of cellulolytic bacteria however, as the
amount of fiber in the diet increased. A higher percentage
of cellobiose fermenting bacteria and a lower percentage of
starch digesters were seen in sheep fed hay compared to
concentrate-hay diets (Dehority and Grubb, 1976).

Bryant and Robinson (1961) observed diurnal changes in
the microbial population from a cow fed an alfalfa hay diet
and a grain diet. A decrease in counts occurred at the time
of first water intake of the day. A further reduction
occurred one to two hours after feeding. This was probably
due to a dilution of the rumen contents with feed and
saliva. The bacterial counts then increased for two to four
hours followed by a decline (for the grain ration) or a
plateau, (for the hay ration), to pre-feeding and watering
levels. The grain diet also supported higher colony counts
than the hay diet. Maki and Foster (1957) obtained two to

three times higher direct bacterial counts from cows fed a

24

concentrate diet compared to cows fed a roughage diet.
Caldwell and Bryant (1966) observed higher colony counts
from the rumen contents of grain fed cattle than from cattle
fed alfalfa hay-grain diets. Differences in total numbers
of colony counts were noted in two animals fed the same
grain ration (Bryant and Robinson, 1961). The animal with
the higher bacterial counts also had a lower pH and less
protozoa. Diets which contain only grain are characterized
by a low number or absence of protozoa. Addition of
roughage to an all grain diet is usually necessary to
establish a prominent protozoal population (Eadie et al.,
1970).

The effect of individual animal differences on colony
counts has been observed with dried grass and pelleted diets
(Thorley et al., 1968), orchardgrass hay and 60% corn-40%
orchardgrass hay diets (Dehority and Grubb, 1976); alfalfa
hay and grain diets (Bryant and Robinson, 1968): and a
lucerne and wheaten chaff diet (Warner, 1962b). Warner
(1962b) also noted daily variation in microbial numbers in
one lamb sampled at the same time each day.

Varying the quantity of lucerne and wheaten chaff fed
to lambs from 300 grams to 1200 grams per day had little
effect on the concentration of rumen microorganisms, but
prolonged under-nutrition drastically reduced numbers of
some organisms (Warner, 1962b). Numbers and types of
bacteria showed little variation in cows fed an alfalfa hay-

concentrate diet at or above maintenance level (Bryant and

25

Burkey, 1953b). Greatly restricting the intake of an all
concentrate diet (barley cubes) caused drastic changes in
the microbial population (Eadie et al., 1970). A large
ciliate protozoal population. developed (on the restricted
diet with a corresponding increase in butyric acid relative
to propionic acid in the rumen liquor. Total bacterial
counts for this restricted level of feeding were similar to
counts obtained from animals on roughage rations. Total
bacterial counts increased with ad libitum feeding of the
concentrate. This increase in bacteria corresponded with a
decrease in ciliate numbers.

Warner (1966a) gave sheep small amounts of lucerne and
wheaten chaff every three hours. Total bacterial counts
remained relatively stable at 0.5 hours, 1.5 hours and 2.5
hours after feeding, but the selenomonads did show a slight
increase 0.5 hours after feeding. Although colony counts
remained stable, immediately following feeding there was an
increase in metabolic activity as evidenced by an increase
in ammonia and volatile fatty acid concentrations and a
decrease in pH. Sheep fed mixed pasture or wheaten chaff ad
libitum consumed 60% to 80% of the day's feed in six to nine
hours (Warner, 1966b). The diurnal changes in the microbial
population were similar to those observed for sheep fed once
daily, rather than patterns found in frequently fed
animals. Warner (1966b) suggested that this type of diurnal
pattern will be exhibited by the rumen microorganisms any

time the ration is consumed in a fairly continuous period

26

followed by long periods during which little or no feed is
consumed.

When an animal is changed from one diet to another, a
period of microbial adaption occurs which is defined by
Grubb and Dehority (1975) as the ”time interval required for
the rumen microbial population to stabilize". Sheep
abruptly changed from an orchardgrass hay diet to a diet of
60% cracked corn plus 40% orchardgrass hay exhibited
significant increases in rumen bacterial colony counts
(Grubb and Dehority, 1975). This increase in colony counts
was due to an increase in available energy substrates. The
numbers of cellulose digesters decreased immediately after
the diet change. In one sheep numbers decreased for five
days after the change in diets and then increased back to
nearly the same level as obtained on the all roughage
diet. For the other two sheep the numbers of cellulose
digesters decreased immediately after the diet changed, then
increased until day five and then decreased to a lower level
than that reached on the hay diet. Bacterial numbers also
did not appear to have stabilized within 21 days after a
diet change. This is in contrast to the findings of Warner
(1962b) who reported that only 10 days were needed to
stabilize the rumen microbial population when sheep were
switched from a hay diet to a hay-concentrate diet.

It is debatable as to whether rumen bacterial colony
counts are indicative of the actual number of bacteria

occurring in the rumen. Higher colony counts obtained from

27

animals fed grain diets compared to animals fed roughage may
be due to an increase in the number of rumen bacteria in
grain fed animals and/or an increase in the number of
bacteria which are able to grow in the media provided. The
anaerobic roll tube method is a very efficient technique for
the cultivation of rumen bacteria, but it may be selecting
for bacteria which are able to tolerate heat stress. In
addition, there are factors affecting viable counts which
can't be controlled, such as individual animal
differences. Although the culture media and techniques used
to enumerate rumen bacteria are not perfect, much

information still may be gained from their use.

MATERIALS AND METHODS

1. General Design of Experiment

Four Rambouillet-Targhee crossbred wethers weighing
approximately 63.5 kg each were fitted with rumen
cannulas. The sheep were housed in individual pens
(approximately 1.8 m x 0.9 m) in a protected metabolism room
(temperature range 10°C to 18°C). The sheep were fed three
different diets (Table l) in three consecutive periods and
all four wethers were fed the same diet during each
period. Thus, with this feeding regime there was no
randomization of diets and animals with time. Sheep were
individually fed twice daily at 12 hour intervals with free
access to water. Diets were mixed immediately before
feeding and fed at a level. which allowed the sheepi to
consume most of the ration before the next feeding.
Adaptation to each diet was at least 21 days, after which
rumen sampling commenced at various times following
feeding. Viable counts of free bacteria, bacteria attached
to feed particles and the RNA-protein ratios of both
bacterial fractions were determined. Other parameters
measured included rumen fluid pH and rumen VFA and ammonia

concentrations.

28

29

 

 

 

TABLE 1. COMPOSITION OF EXPERIMENTAL DIETS
Diet No.

International I II III
Ingredient Reference No. %DM %DM %DM
Corn, aerial pt,
w-ears, w-husks,
ensiled, well-eared 3-08-153 88.1 50.0 10.0
mx 50% mn 30%
dry matter
Corn, dent, yellow 4-02-931 40.0 81.7
grain, gr 2 US
Soybean, seeds, meal 5-04-604 9.9 8.0 6.3
solv-exted
Dicalcium Phosphate, .5
21% Ca, 18% P
Limestone, grnd, .5 1.0 1.0
38% Ca
Trace Mineral Salt 1.0 1.0 1.0
Crude protein %a 12.2 12.2 12.2
DE, Meal/kga 3.03 3.46 3.91

 

Anchor Injectable Vitamin A-D-500 given every 3 months.

aCalculated protein and energy values (NRC, 1975).

2. Treatments

For all three periods, the sheep were fed equal
portions of their daily allotment at 12 hour intervals. The
three diets were isonitrogennous, but varied in roughage to
concentrate ratios.

During period one, sheep 898 was fed 1106.8 9 of corn
silage (all feed ingredients given on dry matter basis) and
145.4 g of supp1ement daily. Sheep 900, 905 and 913 were
fed 1161.2 9 of corn silage and 146.0 g of supp1ement per
day. The supp1ement contained soybean meal (SBM), limestone
and high zinc trace mineral salt (TMS).

During period two, each wether was fed 900.0 g of corn
silage and 907.2 g of supp1ement daily. This supplement
consisted of corn, SBM, limestone and TMS.

During period three, the sheep were fed 145.2 g of corn
silage and 1360.0 9 of supp1ement per day. This supplement
contained the same ingredients as the supplement fed during
period two, but had a higher percentage of corn and lower

proportion of SBM.

3. Sample Collection and Preparation

The diets used during this experiment.*were typical
examples of rations often fed to cattle in feedlots. These
types. of diets however, have not generally been fed to
sheep. The sheep used during these feeding trials
occasionally went off feed and this necessitated lengthening
adaptation and collection periods to enable the animals to

30

31

get back on feed.

Sheep ‘were fed the first diet starting October 1,
1980. Collections for the determination of rumen pH,
ammonia and VFA were done on November 15, 1980; RNA-protein
ratios on December 18, 1980; and viable cell counts on
February 16, 1981.

The second diet was fed beginning February 18, 1981.
Rumen fluid collections for the determination of rumen pH,
ammonia and VFA were done on March 24, 1981; RNA-protein
ratios on April 6 and 7, 1981; and viable cell counts for
sheep 905 and 900 on April 11 and 13, 1981, respectively.

The third diet was fed starting on April 14, 1981.
Rumen fluid collections to determine viable cell counts for
sheep 905 and 900 were on May 5 and 6, 1981, respectively;
RNA-protein ratios on May 8 and 9, 1981; and rumen pH,
ammonia and VFA on May 13, 1981.

Rumen fluid collections were done with a wide bore
glass tube. Zero and 12 hours designate samples taken
immediately before the morning and evening feedings,
respectively.

Rumen fluid collections for the determination of rumen
pH, ammonia and VFA were done 0, 2, 4, 6, 8 and 12 hours
after feeding. Rumen contents were immediately placed on
ice and quickly analyzed for pH level with a Corning
Scientific Instruments Model 7 pH meter. Rumen contents
were then squeezed through two layers of cheesecloth to

obtain the rumen liquor. Nineteen milliliters of the rumen

32

liquor were mixed with 1.:m1 of saturated mercuric chloride
solution and then stored at 3°C until further analysis.
Collections for the determination of bacterial RNA-
protein ratios were done on two consecutive days (except
during period one). On the first day, collections were
taken 0, 4, 8 and 12 hours after feeding. On the following
day, samples were taken 2, 6 and 10 hours after feeding.
Rumen fluid was collected in vacuum bottles and quickly
transported to the laboratory for further processing. About
35 m1 of rumen contents were squeezed through two layers of
cheesecloth. The rumen solids were resuspended in the same
amount of anaerobic dilution solution (Holdeman et al.,
1977) and homogenized on ice using a Polytron homogenizer.
The homogenate was then strained through cheesecloth and the
solids discarded. The two liquors were centrifuged at 121 x
g for five minutes to remove large feed particles and
protozoa. The supernatants were centrifuged at 1,085 x g
for 10 minutes to remove additional feed residues and
protozoa. The supernatants were then centrifuged at 45,900
x g for 20 minutes and the supernatants discarded. The
pellets (containing bacterial cells) were resuspended in .9%
NaCl and centrifuged at 45,900 x g. The residues were then
dispersed in 10% TCA and placed in ice for 30 minutes. The
samples were then centrifuged at 45,900 x g for 20 minutes
and the pellets frozen until further processing. The above

procedure is shown schematically in Figure l.

33

Collection of Rumen Contents - - 0, 2, 4, 6, 8, 10 and
12 hours after feeding

squeeze through cheesecloth

 

Rumen Liquor Rumen Solids

resuspend in ADS,
homogenize and squeeze
through cheesecloth

 

Rumen Liquor

J

centrifuge 121 x g for
5 minutes

 

Supernatant

centrifuge 1,085 x g for
10 minutes

Supernatant

centrifuge 45,900 x g for
20 minutes

Pellet
resuspend in .9% NaCl and

centrifuge 45,900 x g for
20 minutes

Pellet
resuspend in 10% TCA,
place in ice for 30 minutes

and centrifuge 45,900 x g
for 20 minutes

Pellet

Freeze —-—-> RNA, Protein

Figure 1. Flow Chart of Sampling Procedure and Laboratory
Analyses for Bacterial RNA and Protein

4. Determination of Viable Counts in Rumen Contents

The anaerobic technique used to culture the rumen
bacteria was based on procedures developed by Hungate
(1950). Rumen fluid for the preparation of media was
obtained from a fistulated Holstein steer fed a corn silage
diet.

Rumen fluid from sheep 900 and 905 was used to
determine numbers of free and bound bacteria. Samples were
taken 0, 2, 5, 8 and 11 hours after feeding. Rumen fluid
was collected in vacuum bottles and transported to the
laboratory for further processing. Approximately 20 g of
rumen contents (care was taken to obtain a uniform mixture
of solids and liquid) were weighed and squeezed through two
layers of cheesecloth. The solids were placed in ice and
the rumen liquor was placed in. a Waring blender which
contained 90 ml of anaerobic dilution solution (ADS). A
stream of C02 was passed into the blender. The carbon
dioxide used to maintain anaerobic conditions was initially
passed through a column of hot, reduced copper filings to
remove trace amounts of oxygen. The rumen liquor was mixed
vigorously for three minutes. Five milliliters of this
dilution was pipetted into a dilution bottle containing 45
m1 ADS. One milliliter was then pipetted into another
dilution bottle containing 100 ml ADS. This process was
repeated two more times to achieve a final dilution of
10‘8. Each dilution bottle had a stream of co2 bubbled

through the ADS and each bottle was shaken 30 times between

34

35

transfers. One milliliter of the final dilution was
inoculated into 10 ml of agar media (Table 2) which had been
melted previously and maintained at 50°C in a water bath.
The test tube was gently shaken tat mix the 'media and
inoculate, and then quickly rolled on ice to harden and
distribute the media on the inside surface of the test tube.

The rumen solids were then blended, diluted and
inoculated following the same procedure used for the rumen
liquor.

The roll tubes were incubated at 37°C for six days and
colony counts were determined from an average of four or

more of the roll tube cultures.

5. Volatile Fatty Acids Determination

Five milliliters of the rumen liquor-mercuric chloride
sample was mixed with 1 ml of 25% metaphosphoric acid and
centrifuged at 12,100 x g for 10 minutes. The supernatant
was used for the determination of rumen VFA concentrations
by gas liquid chromatography. Samples were run on a Hewlett
Packard G.L.C. Model 5840A. The column (six feet long and 2
mm internal diameter, stainless steel tubing) was packed
with 10% SP-1200/1% H3PO4 on 80/100 Chromosorb W AW, the
flow rate was 40 ml/minute nitrogen and the column
temperature was 130°C (Anonymous, 1975). A standard
solution of volatile fatty acids was prepared and used for
the identification and quantification of the VFA in the

samples.

36

 

 

TABLE 2. COMPOSITION OF RUMEN FLUID MEDIAa

L _Ingredient % in medium
Carbohydrateb (wt/vol) 0.2
Trypticase (wt/vol) 0.2
Yeast extract (wt/vol) 0.05
Mineral onec 3.5
Mineral twoc 3.5
Hemin solutionc .l
Volatile fatty acid solutionc .1
Resazurin (0.1%) .05
Clarified rumen fluid 22.2
Agar (wt/vol) 2.0
Distilled water 66.7
Cysteine sulfidec 2.0
Na2C03 solution (8%) 4.4

 

aPrepared under C02; volume/volume unless otherwise specified.

bGlucose and cellobiose, 0.033% (wt/vol) each; starch and xylan,
0.067% (wt/vol) each.

cHoldeman, Cato and Moore, 1977.

37

6. Rumen Ammonia Determination

An additional .2 ml of saturated mercuric chloride was
added to 5.3 m1 of the rumen liquor-mercuric chloride sample
in order to have the same concentration of mercuric chloride
in the samples as in the blank and standards. The samples
then had .5 ml 9N H2804 added and were centrifuged at 43,500
x g for 15 minutes. The residue was discarded and the
supernatant was analyzed for rumen ammonia nitrogen with a
Technicon Auto Analyzer II. With this system a color
reaction is produced by the mixing of the sample with sodium
hypochlorite and salicylate-nitroprusside.

Standards were prepared by dissolving 4.8 g (NH4)ZSO4
in one liter of water to yield a concentration of 100 mg
N/100 m1. This standard was diluted to give 5, 10 and 20 mg
N/100 ml. To each 5 ml quantity of standard, .5 m1 9N H2804
and .5 ml of saturated mercuric chloride were added. The
blank consisted of 5 ml H20, .5 ml mercuric chloride, and .5

7. RNA-Protein Ratio Determination in Ruminal Bacteria

Ten milliliters of 5% TCA was added to frozen samples
and hand homogenized. Samples were then placed in a 95°C
water bath for 30 minutes and centrifuged at 48,200 x g for
20 minutes. The RNA was determined in the supernatant and
the pellet was analyzed for protein.

For the RNA analysis, 2 ml of 1% orcinol reagent

solution (1 g orcinol in 100 m1 of 0.1% FeCl3'6H20 in

38

concentrated HCl) was added to test tubes containing 2 ml of
sample, 2 m1 5% TCA (blank) or 2 ml of standard and placed
in a boiling water bath for 30 minutes. Samples were cooled
in running water and read at room temperature on a Gilford
Spectrophotometer at 680 nm.

Standards were prepared by dissolving 5 mg of Bakers
Yeast RNA in 100 ml of 5% TCA. This represented a
concentration of 50 ug/ml which was further diluted to yield
concentrations of 12.5, 20.0 and 37.5 Ug/ml. Standards were
run with each batch of samples and samples were
appropriately diluted to allow readings within the range of
standard values. Samples were prepared and analyzed at
least three times.

The protein was determined by resuspending the pellet
in 5 m1 1N NaOH and placing in a 90°C water bath for 15
minutes. A gelling affect was noticed and for diet 2, 20 ml
of l N NaOH was added and samples were placed in a 95°C
water bath for 30 minutes. An incomplete solubilization of
the protein still occurred and for diet 3, samples were
mixed with 10 m1 of l N NaOH, heated to 95°C in a water bath
for 30 minutes and then placed in a 50°C water bath (until
further dilution) in order to prevent solidification of the
sample.

The Hartree (1972) modification of the Lowry (1951)
method for determination of protein was used. Reagent A
consisted of 2 g KNaC4H406°4H20 and 100 g Na2CO3 dissolved

in 500 m1 1N NaOH and diluted with water to one liter.

39

Reagent B contained 2 g KNaC4H406°4H20 and 1 g CuSO4‘5H20
dissolved in 90 ml H20 and 10 ml 1N NaOH. Phenol reagent
was prepared by diluting one part Phenol Reagent, Folin, &
Ciocalteu (Harleco) in ten parts water. A water blank was
used and standards were prepared by gently heating 100 mg of
bovine serum albumen (BSA) in 500 ml H20 until dissolved.
This standard concentration of 0.2 mg/ml was diluted to give
0.15, 0.1 and 0.05 mg/ml of BSA.

One milliliter of samples, standards and blank were
placed in test tubes containing 0.9 ml of reagent A.
Samples were then placed in a 50°C water bath for 10
minutes, cooled to room temperature and then 0.1 ml of
reagent B was added. Samples were left at room temperature
for 10 minutes. Three milliliters of phenol reagent was
added and then the samples were placed in a 50°C water bath
for 10 minutes. Test tubes were cooled to room temperature
and read at 650 nm on a Gilford SpectrOphotometer.
Standards were run with each batch of samples and samples

were appropriately diluted and run at least three times.

8. Statistical Analysis
Colony count and RNA-protein ratio data were analyzed
for treatment differences by the split-plot analysis (of

variance method on the Hewlett Packard 9825A computer.

RESULTS

Rumenng, and Volatile Fatty Acid and Ammonia
Concentrations

Rumen pH, volatile fatty acid and ammonia
concentrations were measured to characterize the ruminal
fermentation in the sheep fed the three different diets.

Rumen fluid pH, volatile fatty acids and ammonia
concentrations for sheep fed diet 1 are presented in Table
3. Rumen pH values decreased to a minimum at 2 hours post
feeding and increased through the 12 hour sampling time.
Ammonia concentration increased to a maximum at 2 hours post
feeding, decreased to a minimum at 6 hours after feeding and
then increased to the 12 hour sampling time. Total VFA and
acetate concentrations increased between 0 and 8 hour
sampling times and decreased at 12 hours after feeding.
Propionate concentration increased between 0 and 2 hours
post feeding, remained relatively stable through 8 hours
after feeding and then decreased at 12 hours post feeding.
Butyrate concentration increased between both 0 and 2 hour
sampling times, and 4 and 8 hour sampling times and
decreased at 12 hours after feeding.

Rumen fluid pH, and volatile fatty acid and ammonia

concentrations for sheep fed diet 2 are given in Table 4.

40

41

.ooosu v uOu coauo~>oo unaccoun can scorn

 

afiva w ac.co
vot— n out"

as...” .+. up;

SYN "YTNH
ant" u v0.5
am.~ wocéd
91m “Neon

oat—n u 97”:

do...” « nm.~

nm.m « oa.m

ca; « mafia

.3..— unqa

oN.o «mien
«c8.— .+. vo.om

avid wao.¢a
cN.c 2.0.."

No.0 n ¢~.~

oo.~ “an...”

ho.N a 6N5

no.m «hvéu
am.c “cog;

vat: “avg;
mN.o ”.24

2..o “hm;

vain «co.N.—

so; w 2.6

mv.~ «hwéu
°N.o «23"

«Hod «vu.oo
nH.o “vs!"

90.9 «hm.~

o~.n «ha.NH

2.2—” a '06

nc.v « muffin
.34. w 20...:

o~.n~ "he.ee 1:5. sauce
oe.~ "ce.~ .xe. ouuuoda>
.25.
Ha.e. "a." oueuauenaaruoza~
u ouuuoao>0un

3.. e 3.2 2.5 333.6

ma.«» cm.m 1:5. ounesuanoun
Ho.n.eov.ma Axe. ouucoanoum

aim « $9.2” :95 0969001

 

 

 

mm.~ edd.na om.n”«~o.a ~e.~“.o~.e ca." “ma.m~ au.~ “em.w~ m..~ “no.a~ .Hoxue. umcoea<

36 womé 2.3.26 2.336 3.336 2.32.6 8.32.6 an
eeuH

«A a u e N c mcscoem nouua meson

ca same can manna mom monecmazuozoo anzozza oz: ano¢ space ug~e¢an> azc .am oases zuzam .n mamas

42

.moocn . uOu couuou>oo unaccouu one cause

 

 

 

2.2 2...: ...3 29.3 3.2 .223 2.3 3.63 2.3» 2.2 8.2 e 3.2 :3. 230...
2.. “2.2 a... 3.2 22 “2.... m... «2.2 8.2 22 o... a 2.2 :3. 333.5
.2:.
3.2 “2.. 2... “2.2 22 2:... 2.2 1.2 2.2“ 32 S... 3.. 333233.22
I ouauoauboun
a... w ...2 m... “8.2 3.. “3.2 3.. fl ...2 2... «2.3 3.. e 2.... :3. 333:.
m... «2.. ...2 2.. 2... .2... 3.2 2.... 2.2 3.. 2... m... E... 332303
3.2 “2.3 2.2 “2.2 ...~e 8.2 22 H 3.2 3.. w 2.2 2... e 2.3 :3. 32838..
.... « ...... n... e 2... 8.2 a 2... 2.. . 2.2. 2.... 2.... 2... e .o... is. 9.3.2
3." «.22 22 .2... 2.2 2.... 2.2 “.32 2... “3.3 a... «2... Saxon. 3:35..
3.. “a... 2... “3.. 3... 2:... 2.. e2... .2... 3... 3.. 2... an
no.”
2 . . . ~ . .52.... 3...... 3.6..

 

6N EHO Duh mum—a «Oh mZOHBEZfiUZOU tuna—Oi 02¢ an“: Edh Hawking 02¢ 33 OHDJh zgafl .v ”405—.

43

Rumen pH values decreased from 0 hours after feeding to a
minimum at 6 hours post feeding and then increased to a
maximum value at 12 hours after feeding. Rumen ammonia
concentration increased to a maximum at 2 hours after
feeding and decreased to a minimum at 6 hours post
feeding. Total VFA concentration remained stable between 0
and 2 hour sampling times, then increased to a maximum at 8
hours post feeding and then decreased to a minimum at 12
hours. Acetate and propionate concentrations increased
between 0 and 8 hours post feeding and then decreased to
minimum levels at 12 hours after feeding. Butyrate
concentration increased between 0 and 4 hours, remained
relatively constant between 4 and 8 hours after feeding and
then decreased to a minimum at 12 hours post feeding.

Rumen fluid pH, and volatile fatty acid and ammonia
concentrations for sheep fed diet 3 are shown in Table 5.
Rumen pH values decreased from a maximum at 0 hours post
feeding to a minimum value at the 2 hour sampling time,
remained relatively constant through 8 hours after feeding
and then increased at 12 hours post feeding. Ammonia
concentration increased from 0 hours after feeding to a
maximum at 2 hours post feeding, decreased to a minimum at 6
hours after feeding and then it increased to 12 hours post
feeding. Total VFA concentration increased from a minimum
at 0 hours after feeding to a maximum at 4 hours post
feeding. Acetate and propionate concentrations increased

from minimum values at 0 hours post feeding to maximum

44

.moosu . new couuo0>oc unaccouu can cumin

 

vu.o~ “vo.~m nv.ma «oc.cm

H¢.H «ON.H nn.d «mo.n
hr.d wom.~ aa.d «an.N
«c.v uoH.o vN.v «on.o
o~.~ «mm.N on.a «cm.N

oo.oa «cm.mu H¢.NHM ma.ha

NN.¢ ao~.nN «o.n «mo.mN

Nm.vd whc.hm MN.0H uhh.nw

w~.H woo.n ca.c «c~.~
mm.H “Hv.N wh.H woN.N
mN.n umm.h av.v «no.0
¢H.H mam.N ma.o nou.~

mm.NHu ch.hH vm.nH who.°N

no.0 nom.vN hm.N «va.hu

cm.aH wvn.Nw
oo.H “av.”

No.uw vw.n

on.v “av.od

mv.au uc.~

hc.HHw vo.od
aw.Nn Nh.m~

.a... "~n.o. .za. deuce
2..” “.2.2 .2:. ououo2a>
.25.
...H .2... oueuauan2aruuxn~
I ounuoau>0un
n.... c... .2:. ouauauan

6N.H« wH.N all. ououauanoun

o... a....2 .xe. ouuc02n00m

mn.v nov.HN ASE. cucum0¢

 

no.o “a... o~.. woo.o w... “mo.h .n.m «.o.h .m.hu vc.HA mm.h wow... .~O\oe. oucoeat

n~.o we... an.c «an.» on.o “an.m av.o w-.. .m.o .o~.o .N.o «om.w an
.mmww

«A o w v N o mcavoom nouut mason

 

an BNHO 0mm 00000 000 MZOH9<¢EZEUZOU (H20224 02¢ 0HU< rbh<h NQHB<00> 02¢ .20 0H00h 20:00

.m 00019

45

levels at 4 hours after feeding. Butyrate concentration
increased from a minimum at 0 hours post feeding and reached

a maximum at 2 hours after feeding.

Free Bacterial Colony Counts

Free bacterial colony counts for sheep 905 and 913 fed
diets l, 2 and 3 are shown in Table 6. Diet effects (P<-25)
were noted with diet 3 consistently having higher colony
counts for each collection time. Colony counts decreased
between 0 and 2 hours after feeding for diets l and 2, but
remained relatively stable for diet 3. Colony counts
increased between 2 and 8 hours post feeding for diet 1 and
between 2 and 11 hours after feeding for diets 2 and 3.
Maximum counts were obtained at 8 hours post feeding for
diet 1 and at 11 hours after feeding for diets 2 and 3.

Variation of free colony counts with time after feeding
for individual sheep fed diets l, 2 and 3 are given in
Figures 2, 3 and 4, respectively. For diet 1, both sheep
exhibited a decrease in colony counts between 0 and 2 hours
after feeding, reaching minimum values of 1.21 x 108/9 for
sheep 905 and 0.83 x 103/9 for sheep 913. Colony counts
from sheep 905 increased from 2 hours post feeding to a
maximum value of 5.99 x 108/9 at 11 hours after feeding.
Colony counts for sheep 913 increased from 2 hours after

feeding, reaching a maximum level of 6.00 x 103/9 at 8 hours

after feeding, and then decreased at 12 hours. For diet 2,

free colony counts for both sheep decreased between 0 and 2

46

TABLE 6. FREE BACTERIAL COLONY COUNTS PER GRAM OF RUMEN

 

 

 

 

CONTENTSa
Hours After Feeding Diet 1 Diet 2 Diet 3
(108/9)
0 2.9610.67 6.03:0.07 13.2410.80
2 1.02:0.19 2.4810.06 l3.60:1.40
5 2.70:0.52 5.14:2.35 17.0024.89
8 4.8711.13 7.1411.51 20.90i10.58
11 4.6811.31 8.3420.68 22.96ill.76
Meanb 3.24:0.55 5.8310.79 17.5412.80

 

aMean and standard error for 2 sheep.

bTreatment effects at P <.25.

47

H umflo 0mm ammnm 039
now musmucoo :mesm mo ammo mom mucooo mooHou HMflumuomm mmum .N ousofim

AmmIv 02.0mm... mmbhz m2;

 

 

 

 

up 3 O— 0 0 b 0 m V
1
. m
‘
‘\\‘ .0. HI
.\\\‘1§1§E§1§ MW
.’ bingo-cocoon... IA
’
’XH‘.‘ \\\ l m
K ” \ m N
‘0. "‘ l O m“
l AUX
l O m
8
I a m
l.:

is“. a; .325

8888...... now 9.25

48

N .020 so. soon. 039
new mucwucou :mEsm 00 Scam mom mucsoo chHou Howumuomm mmum .m wusmflm

AwmI. 02.0mm“. Kuhn; m:2_._.

 

 

 

Np —— Op 0 Q h 0 m V n N p
_ . . _ . .1 . . . . . . o n.
J . .nlu
..P 1 . o
“L“‘.““ao 0 s N
.\\. .2 .9 |_s.A
\ ... I.
\. 2
‘\ 00000 / l ' O
\\ oooooo O
‘\ 00000 ifl m n
“ 00000 es. N
“ 0000 i 0 II.
\\ S
“‘ 0000000 I h
‘\ ......0003‘0 x
coco-30:23....- l 0 Ir
......z... 0
9! I o.h.
6
0.0.

...... n p 0 003.0

...-.038! DOQ 3008”

49

m umwo 0mm ammnm 036
new mucwucou cwEDm mo Emuo Hmm mucsou wcoHOU Hmflumuomm wmuh .v musmflm

AwmIv 02.0mm“. mm...m< USE.

Np pp Op m m h w m Q n N p O

5/90L x smnoo ANO‘IOO

 

‘I. own .395

3:...- mOQ Qfimzw

50

hours post feeding, reaching minimum values of 2.42 x 108/9
for sheep 905 and 2.54 x 108/9 for sheep 913. Colony counts
then increased between 2 and 11 hours post feeding for both
lambs. Maximum counts obtained at 11 hours after feeding
were 9.02 x 108/9 and 7.66 x 108/9 for sheep 905 and 913,
respectively. Free colony counts for sheep fed diet 3
varied differently with time. Colony counts increased from
a minimum value of 14.04 x 108/g at 0 hours to a maximum
value of 34.72 x 108/9 at 11 hours post feeding for sheep
905. Free colony counts for sheep 913 reached a maximum
level of 12.44 x 108/9 at 0 hours after feeding, gradually
decreased to a minimum value of 10.32 x 108/9 at 8 hours

post feeding and then increased at 12 hours.

Bound Bacterial Colony Counts

Bound bacterial colony counts for sheep fed diets 1, 2
and 3 are given in Table 7. Significant diet effects
(P < .05) were observed with diet 3 consistently having
higher colony counts for each collection time. Bound colony
counts decreased between 0 and 2 hours after feeding for all
three diets. Colony counts increased between 2 and 8 hours
after feeding for diet 1, between 2 and 11 hours after
feeding for diet 2 and remained relatively stable between 2
and 11 hours after feeding for diet 3. Minimum counts were
obtained at 2 hours after feeding for diets 1 and 2, and 8
hours after feeding for diet 3. Maximum counts were

observed at 8 hours for diet 1, 11 hours for diet 2 and 0

51

 

 

 

 

TABLE 7. BOUND BACTERIAL COLONY COUNTS PER GRAM OF RUMEN
CONTENTSa
Hours After Feeding Diet 1 Diet 2 Diet 3
1 (108/9)
0 4.651 1.59 7.01: 1.48 18.00t5.04
2 3.57:1.15 5.1010.28 15.251r 3.23
s 5.78:0.90 5.25:0.14 16.61i1.84
8 7.03: 2.80 6.5013.10 15.1211.”
11 6.57:1.77 8.1312.42 154513.31
Meanb 5.52:0.72 6.4010314 16.03i1.16

 

aMean and standard error for 2 sheep.

bTreatment effects at P< .05.

52

hours after feeding for diet 3.

Variation of bound colony counts with time for
individual sheep fed diets l, 2 and 3 are shown in Figures
5, 6 and 7, respectively. For diet 1, decreases in bound
colony counts between 0 and 2 hours after feeding were
observed for both sheep. Colony counts increased from a
minimum of 2.42 x 108/9 at 2 hours after feeding to a
maximum of 6.68 x 108/9 at 5 hours after feeding and then
decreased through the next two sampling times for sheep
905. For sheep 913, colony counts increased from a minimum
of 4.72 x 108/9 at 2 hours post feeding to a maximum of 9.82
x 108/9 at 8 hours after feeding. For diet 2, bound colony
counts for sheep 905 decreased from 0 hours post feeding to
a minimum of 3.40 x 108/g at 8 hours after feeding and then
increased to a maximum of 5.70 x 108/9 at 11 hours after
feeding. Colony counts for sheep 913 decreased between 0
and 2 hours after feeding, reaching a minimum of 4.82 x
108/9 and then increased to a maximum of 10.55 x 108/9 at 11
hours after feeding. For diet 3, bound colony counts for
sheep 905 reached a maximum of 14.77 x 108/9 at 5 hours
after feeding and then decreased to a minimum of 11.84 x
108/9 at 11 hours post feeding. Bound colony counts for
sheep 913 decreased from a maximum of 23.04 x 108/9 at 0
hours after feeding to a minimum of 17.03 x 108/g at 8 hours

after feeding.

53

H uwflo com mmwsm 039
How mucwucoo :wEDm mo Emuw mom mucnou >COHOU HMflumuomm wcsom .m wusmflm

AwmIv 02.0mm“. cub“; mus—F

 

 

flu
nu
—.I
my
N
IA
03......
...-o
3.0.3.... \‘ N
\‘000000 000000 I.
\ 00.0 S
\ x
00 \ 1 s L
.... \ 08
0000 \ .. a .w
( L

 

O
p

“~L ”PO Q°°£m

0300.08.00. now 300:”

54

m umflo pom amonm 039
new mucmucov :mEsm mo Emuu mom mucsoo >20Hoo Hmwuwuomm bosom .m whomflm

AmmIv 02.0mm“. ”mm—...“: 92....

up : Op a w s 0 m v n

 

 

 

 

N r O
_ _ 0 _ 0 _ 0 — . . ..III—I 0 o
nu
l N -|
AU
... 1 s m
00000000000000000000 00000020000000.0003:- J V m
0.000000 00.000... “.““’ 1
0000000000 80*“ 9 0.0000000000800000 m n
O \\\ .1 0mm
\ I S
\\ I, s
\ I X
\\ 0.1
\\ 0
a 8
\i!\ mw
“\\\‘\.\ o 0
0‘

F
F

00.0 0.0 39.0

00000000000 DOG QfiOSW

55

m 0000 com ammnm 039

 

 

new mucmucou cmEsm mo Emuw Mom mucsou >coHOU Hmfluwuomm bosom
AwmIV 02.0mm“. EMF“? wSE.
N— pp Op m a N o m v n N p

O
O
..l
O
N
.A
O
O
n
N
II-
S
X
L
nu

8
l
O

 

‘0 9o 0005

.0000000 DOG 300...”

.5 wusmflm

Variation of RNA-Protein Ratios with Time
for Free Ruminal Bacteria

RNA-protein ratios for free rumen bacteria from sheep
fed diets l, 2 and 3 are given in Table 8. Significant
treatment effects (P < .05) were observed, with diet 3 con-
sistently having higher RNA-protein ratios for all sampling
times. For diet 1, values increased from a minimum level at
0 hours after feeding to a maximum level 4 hours after
feeding, and then decreased through 12 hours post feeding.
For diet 2, values increased from a minimum at 0 hours post
feeding to a maximum 4 hours after feeding. For diet 3,
values increased to a maximum at 6 hours after feeding and
then decreased to a minimum at 12 hours post feeding.

Variation of RNA-protein ratios with time for free
bacteria from individual sheep fed diets l, 2 and 3 are
shown in Figures 8, 9 and 10, respectively. For diet 1,
values for sheep 905, 913 and 900 (sheep 898 was off feed
and not sampled) increased between 0 and 4 hours after
feeding, reaching maximum values of .347, .297 and .325 for
sheep 905, 913 and 900, respectively. Minimum values of.
.244 and .254 were obtained at 12 hours after feeding for
sheep 913 and 900 while a minimum value of .247 was obtained
at 0 hours after feeding for sheep 905. For diet 2, RNA-
protein ratios increased from a minimum of .230 at 0 hours
after feeding to a maximum of .457 at 4 hours after feeding
for sheep 905. For sheep 898, RNA-protein ratios increased

between 0 and 2 hours after feeding, reaching a maximum of

56

TABLE 8 .

57

RNA-PROTEIN RATIOS FOR FREE RUMEN BACTERIA

 

Hours After Feeding

Diet la

Diet 2b

Diet 3b

 

0

@O‘IbN

10
12

Meanc

0.251i 0.003
0.289 i 0.004
0.323 i 0.010
0.311 1’ 0.012
0.309 t 0.013
0.291 i 0.007
0.260 1 0.008
0.290 1 0.006

0.245 1 0.022
0.314 1 0.016
0.344 i 0.040
0.268 1 0.035
0.313 1 0.024
0.269 1 0.018
0.265 i 0.019
0.288 1’ 0.011

0.3843 0.019
0.3571 0.032
0.3991 0.028
0.516i 0.140
0.471i 0.092
0.404i 0.029
0.3351 0.027
0.4091 0.025

 

aMean and standard error for 4 sheep, using calculated average

values for 1 sheep.

bMean and standard error for 4 sheep.

cTreatment effects at P.:.05.

58

H umfla numb
QmmLm Eoum maumuomm womb no“ mewumm :flmuoumldzm

AwmIv 02.0mm“. EMF”; MEI.

Np O— m o v N

 

00 com .325
“. firm QOwr-w
.0000000. mow Qmwzw

.m musmflm

NIBlOHd/VNH

59

m 0809 pom
mmwnm Eoum maumuomm mmum new moflumm aflououmu<zm

AwmIv GZEMm—u. Kuhn: m2...

No or w 0 v N

 

. I
’0 ’
4. s 0
0., Q I
0 i I Q

. l | I I s 0 000
0, .l 0
.0000000000 00003830? 0‘ \
'

t...

ooo oooem

0.0 oooem
moo oooem

moo oooem

.NIBlOt‘id/VNH.

60

00 cos 02:5
i! 0.0 82.0
....- ooo oooew

...-... moa wazm

Np

m pose cos
ammcm Eoum mflumuomm womb 00m wofiuwm :flwuoumldzm

AwmIv 62.0mm“. mmELZ mes...
Op w 0 v N

 

.oH musmfim

NIHLOHd/VNH

61

.301, then decreased to a minimum of .166 at 6 hours after
feeding, followed by an increase to a value of .300 at 8
hours and a decrease to the 12 hour sample. RNA-protein
ratios fluctuated over time for sheep 913 with a minimum of
.302 at 12 hours and a maximum of .384 at 8 hours after
feeding. For sheep 900, RNA-protein ratios increased from a
minimum of .198 at 0 hours after feeding to a maximum of
.320 at 2 hours post feeding and then decreased through 12
hours post feeding. For diet 3, RNA-protein ratios
increased from 0 hours after feeding to a maximum of .853 at
6 hours after feeding for sheep 913 and a maximum of .711 at
8 hours after feeding for sheep 898. Minimum levels of .374
at 8 hours after feeding and .258 at 12 hours post feeding
were obtained for sheep 913 and 898, respectively. RNA-
protein ratios decreased between 0 and 2 hour sampling
times, reaching a minimum of .293 and then increased to a
maximum of .506 at 8 hours after feeding for sheep 905. For
sheep 900, RNA-protein ratios fluctuated over the sampling
times with a minimum of .240 being obtained at 6 hours post,
feeding and a maximum of .420 being reached at 0 hours after

feeding.

Variation of RNA-Protein Ratios with Time for
Bound Ruminal Bacteria

RNA-protein ratios for bound rumen bacteria from sheep
fed diets 1, 2 and 3 are given in Table 9. A significant

diet effect (P < .05) was observed with diet 2 having lower

TABLE 9.

62

RNA-PROTEIN RATIOS FOR BOUND RUMEN BACTERIA

 

Hours After Feeding

Diet 1a

Diet 2b

Diet 3b

 

0

mmbN

10

12

Meanc

0.237 1 0.021
0.219 1 0.006
0.223 t 0.001
0.220 $0.007
0.219 1‘ 0.002
0.214 $0.017
0.261.10.045
0.228 10.007

0.127 t 0.011
0.151 .t 0.024
0.184 i 0.034
0.133 t 0.013
0.148 i 0.007
0.161.10.022
0.124 i0.011
0.147 10.008

0.214i0.005
0.134 10.014
023510.007
0.217 $0.015
0.21510.014
0.233 10.012
0.214 $0.006
0.216 10.005

 

aMean and standard error for 4 sheep, using calculated average

values for 1 sheep.

bMean and standard error for 4 sheep.

cTreatment effects at P‘<.05.

 

 

63

RNA-protein ratios for all sampling times. For diet 1,
values decreased between 0 and 2 hour sampling times and
between 4 and 10 hours after feeding, reaching a minimum at
10 hours and a maximum at 12 hours after feeding.
RNA-protein ratios increased from 0 hours post feeding to a
maximum at 4 hours and reached a minimum at 12 hours after
feeding for diet 2. RNA-protein ratios for diet 3 decreased
from 0 hours post feeding to a minimum at 2 hours and then
increased to a maximum at 4 hours after feeding.

Variation of RNA-protein ratios with time for bound
bacteria from individual sheep fed diets l, 2 and 3 are
shown in Figures 11, 12 and 13, respectively. For diet 1,
values for sheep 913 increased between 0 and 6 hour sampling
times, reaching a maximum of .240 at 6 hours and then
decreased to a minimum of .189 at 10 hours after feeding. A
minimum of .201 was obtained at 0, 2 and 12 hours after
feeding and a maximum of .263 at 10 hours post feeding for
sheep 905. For sheep 900, RNA-protein ratios decreased
between 0 and 6 hours, increased at 8 hours, then decreased
to a minimum of .190 at 10 hours and then increased to a
maximum of .388 at 12 hours post feeding. For diet 2, a
maximum of .132 was obtained at 0 hours after feeding and a
minimum of .095 was observed at 12 hours after feeding for
sheep 900. For sheep 905, a maximum of .268 at 4 hours and
a minimum of .113 at 6 hours were obtained. Values ranged
from .095 at 0 hours after feeding to .159 at 6 hours post

feeding for sheep 898 and for sheep 913 a maximum of .213 at

1

64

H umHD pom
mmmnw Eouh mHumuomm canom Mom onumm :Hmuoum|<zm

AwmIv OZEmmm Kuhn; NEE.

N. O. w w v N

 

000 ooo 82.0
iii 90 006:0

00000000000 D O O Q 0 w 8 w

.HH musmflm

NialoudIVNu

65

 

 

N umHo pom
moosm Eoum mHuwuomm venom 00m moHumm :Hmuoumnmzm .NH musmfim

Ham—.3 62.0mm“. mm..h.< NSF...

Np Op m o v N O

NISLOHd/VNH

 

00 ooo 000..»
1‘1 a; 00.05....
1.... mom 0025
is... nos 000:0

66

m uwHD mownH
amonm Eoum mHumuowm ccoom MOM moHumm ckuouml<zm .mH musmHm

Ham... 02.0mm“. amp“... 92....

 

a. o. 0 o v a
o
t H
mu
V
I
d
Ohm. a. ...a
000 O
4“ ‘\ m
Mn
"0
-- a
coo poem 0.

I! 0.0 002.0
III man 0095
0000000 ”CG 0003”

67

2 hours and a minimum of .125 at 12 hours after feeding were
observed. For diet 3, RNA-protein ratios of bound bacteria
decreased between 0 and 2 hours after feeding for sheep 898,
913 and 900. Minimum values of .144 and .185 at 2 hours
after feeding were obtained for sheep 913 and 900,
respectively. Minimum values of .197 at 6 hours post
feeding and .193 at 8 hours after feeding were noted for
sheep 905 and 898, respectively. Maximum values of .260 and
.240 at 10 hours after feeding were obtained for sheep 913
and 900, respectively, while maximum levels of .261 at 6
hours for sheep 898 and .225 at 4 hours post feeding for

sheep 905 were noted.

 

DISCUSSION

Numerous authors (McAllan and Smith, 1969; Cole et al.,
1976; Prigge et al., 1978) have used bacterial RNA as a
marker to determine microbial nitrogen in duodenal digesta
samples. Several assumptions are made in employing
bacterial RNA as a marker, including the theory that
bacterial RNA-protein ratios do not vary with time after
feeding. This contradicts the conclusion of other workers
(Ecker and Schaechter, 1963; Kjeldgaard and Kurland, 1963;
Rosset et al., 1966) that bacterial RNA-protein ratios are
not constant, but vary with bacterial specific growth rates
and continuous culture dilution rates.

This study was designed to determine the numbers of
free and bound bacteria and their respective RNA-protein
ratios at various times after feeding sheep corn silage,

corn silage-grain and grain diets.

General Rumen Fermentation Parameters
An increase in total volatile fatty acids was observed
after feeding for sheep fed diets 1 and 2 and similar
increases have been observed by others (Phillipson, 1942;
Gray et al., 1967). Ammonia levels increased rapidly
between 0 and 2 hours after feeding and corresponded with a

68

 

69

rapid decrease in rumen pH. The corn silage-grain diet
(diet 2) had slightly lower pH values than the corn silage
diet which would be expected as increased rumen acidity is
often observed with diets containing a high proportion of
grain (Hungate, 1966). For the high grain diet (diet 3)
there was a decrease in the acetate to propionate ratio
compared to the other two diets; however there were
decreases in total and individual volatile fatty acids over
the sampling times. It is not known why this occurred, but
it was observed that the sheep had a tendency to eat small
amounts of diet 3 throughout the day. This is reflected in
the relatively stable pH values and ammonia concentrations
over the sampling times. In addition, the ammonia values
for diet 3 were generally less than those obtained on diets
l and 2 and lower average ammonia concentrations have been
observed in the rumen of sheep which are fed continuously

(Hungate, 1966).

Rumen Bacterial Colony Counts

Free and bound bacterial colony counts were higher for
diet 3 than diets l and 2 at each sampling time.‘ The diet
effects were significant at P <.05 and P <.25 for the bound
and free bacteria, respectively and there were no signifi-
cant time effects for any of the diets. Higher bacterial
colony counts from animals on grain diets compared to
roughage diets have been observed by others (Bryant and

Robinson, 1961; Caldwell. and ‘Bryant, 1966). Bryant. and

70

Robinson (1961) obtained total colony counts ranging from
22.2 x 108/9 at one hour after feeding to 109.8 x 108/9 at 4
hours post feeding while Caldwell and Bryant (1966) obtained
47.3 x 108/9 total colony counts from a cow sampled after
feeding a grain diet. Adding together the free and bound
colony counts obtained in this study results in numbers
comparable to those reported by Caldwell and Bryant (1966),
but generally less than those observed by Bryant and
Robinson (1961). Leedle and Hespell (1980) fed a diet
similar to the high grain diet used in this study. It
consisted of 85% corn and 15% corn silage. Viable counts
obtained 3 to 4 hours after feeding were 39.4 x 108/m1
compared to 32.61 x 108/9 at 5 hours after feeding diet 3 of
this study. Colony counts obtained from sheep fed the corn
silage diet were less than numbers reported for animals on a
hay diet (Bryant and Robinson, 1961) and colony counts
observed from the sheep fed the corn silage-grain diet were
less than reported values for cows fed hay-grain diets
(Caldwell and Bryant, 1966).

Individual animal differences were observed for free
and bound colony counts from sheep 905 and 913 on each of
the three diets. The effect of individual animal
differences on total bacterial colony counts has been
reported for animals on various diets (Warner, 1962b; Bryant
and Robinson, 1968; Thorley et al., 1968; Dehority and
Grubb, 1976).

For diets l and 2 decreases in free colony counts were

71

observed between 0 and 2 hours after feeding and were
probably due to dilution of the rumen contents with feed and
saliva. Similar decreases in total colony counts after
feeding have been reported by others (Bryant and Robinson,
1961 and 1968; Warner, 1966c). For diet 3 there was only a
slight decrease in free colony counts for sheep 913 and an
increase in numbers for sheep 905 between 0 and 2 hours post
feeding. Possibly a decrease in saliva secretion on the
grain diet compared to the corn silage diets and the
provision of a readily fermentable carbohydrate source
allowed for the higher colony counts at 2 hours after
feeding on diet 3. Bound bacterial colony counts also
decreased between 0 and 2 hours post feeding for each diet
and bound colony counts for sheep 913 exhibited variations

over time which were similar to the free colony counts.

RNA-Protein Ratios for Free and Bound
Ruminal Bacteria

 

The RNA-protein ratios for free bacteria were higher on
diet 3 compared to the other two diets at every sampling
time. Diet 3 contained more grain, had a higher digestable
energy value (3.91 Mcal/kg) and contained more readily
fermentable carbohydrates than the other two diets. It was
therefore apparently able to support higher bacterial growth
rates in the rumen. Mean RNA-protein ratios for diet 3
ranged from .335 to .516. The magnitude of the bacterial

growth rates obtained with these RNA-protein ratios is not

 

 

 

72

directly ascertainable from these studies. However, at a
specific growth rate ( p = doublings per hour) of 0.40, _E_:_.
coli strain B had a RNA-protein ratio of .351 (Rosset et

al., 1966) and the RNA-protein ratio was .539 for Aerobactor

 

aerogenes when grown at 11== 0.97 (Neidhardt and Magasanik,
1960). Clostridium perfringens grown anaerobically in batch
culture had a RNA-protein ratio of .546 at u a 1.30 (Leick,
1968). Individual RNA-protein ratios for diet 3 ranged up
to .853 (sheep 913). .A similar RNA-protein ratio of .847

was reported when A, aerogenes was grown at u = 2.33 (Leick,

 

1968).

Mean RNA-protein ratios for free bacteria were similar
for diets 1 and 2. Values ranged from .251 to .323 and .245
to .344 for diets l and 2, respectively. A RNA-protein
ratio of .263 was noted when E, coli strain ML30 was grown
at u = 0.49 (Rosset et al., 1966).

Although there were no significant time effects with
any of the diets, there do appear to be similar patterns for
the RNA-protein ratios for free bacteria from the individual
sheep. For diet 1, there is less variation in individual
RNA-protein ratios over time than with diets 2 and 3. Diet
1 was the high corn silage diet and therefore did not supply
as much (if any) readily fermentable substrate to the
microorganisms. Hence rapid increases in bacterial growth
rates were unlikely. For the three sheep fed diet 1 and
sampled, similar trends were seen: a gradual increase

between the 0 and 4 hour sampling times, a plateauing effect

73

until 8 to 10 hours after feeding and then a decline to pre—
feeding values. The relatively stable RNA—protein ratios
between 4 and 10 hours post feeding corresponded with an
increase in free bacterial colony counts.

There were more fluctuations in RNA-protein ratios over
time for diet 2, which contained corn silage and grain in
about equal proportions, compared to the other two diets.
The grain supplied a readily fermentable substrate to the
bacteria which should result in increased growth, and hence
elevated RNA-protein ratios. For sheep 898 and 913 a second
increase was noted between 6 and 8 hours post feeding. This
could be caused by the free bacteria metabolizing the
degradation products of the structural carbohydrates which
have been broken down by the cellulolytic bacteria.

For sheep 905, the rapid increase in RNA-protein ratios
for free bacteria between 2 and 4 hours corresponded with a
major increase in viable free colony counts; as the RNA-
protein ratios declined, the bacterial numbers leveled off
and for sheep 913 the increase in RNA-protein ratios between
6 - 8 hours also coincided with an increase in viable free
bacterial colony counts.

RNA-protein ratios which would be considered maximal on
diet 1 or 2 were realized at 0 hours after feeding on diet
3. However, it did take longer for the diet 3 RNA-protein
ratios to reach maximum values when compared to diets l and
2. The increase of RNA-protein ratios between 2 and 8 hours

after feeding corresponded with an increase in bacterial

 

 

74

numbers for sheep 905. Colony counts remained relatively
stable over sampling times for sheep 913 and did not
parallel the sharp increase in RNA-protein ratios between 4
and 6 hours after feeding.

Significant diet effects (P‘<.05) were observed for the
RNA-protein ratios of the bound bacteria. RNA-protein
ratios were lower for diet 2 compared to diets 1 and 3 for
each sampling time. RNA-protein ratios were very similar
for diets l and 3 for bound bacteria. Mean values varied
from .214 to .261 and .184 to .235 for diets 1 and 3,
respectively. values for diet 2 varied from .124 to .184.
Bates (1981) grew several strains of rumen bacteria at
various growth rates. A linear regression calculated from
this data predicted that at a stationary phase of growth

( u = 0), the RNA-protein ratio would be .22. Aerobacter

 

aerogenes grown at stationary phase had a RNA-protein ratio

 

of .154 (Neidhardt and Magasanik, 1960). Comparing the
above results with the RNA-protein ratios obtained in this
study for the bound bacteria, it would appear that the bound
bacteria in the rumen are near or at a stationary phase of
growth. Possibly for the corn silage diets substrates were
not being liberated at a rate which would allow for rapid
growth and higher RNA-protein ratios. Higher RNA-protein
ratios were observed for the grain diet as compared to the
corn silage—grain diet, but were still generally below the
values obtained for the free bacteria. To a great extent,

the growth rate of free bacteria is controlled by the

 

)

75

dilution rate of the rumen. As the rumen dilution rate
increases, so must the growth rate of the free bacteria or
these organisms would subsequently be washed out of the
rumen. The bacteria bound to feed particles are not as
greatly affected by dilution rate. The feed particles will
remain in the rumen until they are broken down to a size
which permits them to be washed out of the rumen. Thus, the
dilution rate of the solid phase is slower than the liquid
phase of the rumen and does not require the bound organisms
to grow at as fast a rate as the free bacteria in order to
prevent being washed out.

Generally speaking, for all three diets there was less
variation over time for the RNA-protein ratios for bound
bacteria compared to the values obtained for free
bacteria. This would be expected as the bound bacteria are
being exposed to a steady supply of substrate compared to
the free bacteria which metabolize readily fermentable
compounds (if available) soon after feeding and then later
on utilize the degradation products of the structural
carbohydrates.

Smith and McAllan (1974) looked at the variation in
RNA-N/total N with time after feeding for mixed rumen
bacteria. Converting their values to RNA-protein ratios
(assuming RNA contains 14.8% nitrogen and microbial N
contains 20% nucleic acid N and 5% amino acid and peptide N)
yields the following results: .101, .118, .121 and .121 for

0, 2, 4 and 6 hours after feeding. Values obtained at 4 and

76

6 hours after feeding were significantly different from the
RNA-protein ratio obtained at the 0 hour sampling time.
Maeng and Baldwin (1976) obtained values ranging from .134
to .157 for mixed rumen bacteria sampled from 0 to 10 hours
after feeding. These values are comparable to the RNA-
protein ratios obtained for the bound bacteria in this
study, but are lower than the RNA-protein ratios of the free
bacteria.

The results of this study indicate that RNA-protein
ratios for free bacteria may vary with respect to time after
feeding, while the RNA-protein ratios for bound bacteria are
relatively constant over time. Thus, the RNA-protein ratio
for mixed rumen bacteria would vary depending on the
proportion of free and bound bacteria in the sample and the
time of sample collection in relation to feeding.

Under normal feeding practices most samples of mixed
duodenal contents will contain a large portion of bound
bacteria. As the bound bacteria have relatively constant
RNA-protein ratios over time, the RNA-protein ratio of the
mixed bacteria in the sample would also show little
variation. Using bacterial RNA as a marker for passage
studies should on the average yield relatively consistent
results, but the potential does exist for a digesta sample
to contain a high fraction of free bacteria and under these
circumstances using a bacterial RNA marker may yield

erroneous conclusions.

 

 

GENERAL CONCLUSIONS

Higher free and bound colony counts were observed on
the high grain diet as compared to the corn silage
diets.

RNA-protein ratios for free bacteria indicate that
these organisms respond physiologically to changes in
available substrate. An increase in readily
fermentable substrates was reflected by an increase in
growth rate.

The free bacteria achieve greater Specific growth rates
than the bound bacteria.

RNA-protein ratios for bacteria bound to feed particles
indicate that these bacteria grow at a relatively
constant rate which is near or at a stationary phase of
growth.

Because RNA-protein ratios for rumen bacteria are not
constant, caution should be taken in using bacterial

RNA as a marker in passage studies.

77

LIST OF REFERENCES

LIST OF REFERENCES

Allison, M. J. 1965a. Nutrition of rumen bacteria. _1:_r_1_
Physiology of Digestion in the Ruminant. R. W.
Dougherty, R. S. Allen, W. Burroughs, N. L. Jacobson
and A. D. McGilliard (Ed.). Butterworth Inc.,
Washington, D.C.

Allison, M. J. 1965b. Phenylalanine biosynthesis from
phenylacetic acid by anaerobic bacteria from the
rumen. Biochem. Biophys. Res. Comm. 18:30.

Allison, M. J. 1969. Biosynthesis of amino acids by
ruminal microorganisms. J. Anim. Sci. 29:797.

Allison, M. J. and I. M. Robinson. 1967. Tryptophan
biosynthesis from indole-3—acetic acid by anaerobic
bacteria from the rumen. Biochem. J. 102:36p.

Anonymous. 1975. GC separation of VFA C2—C5. Bulletin
749C. Supelco, Inc., Bellefonte, Pennsylvania.

Bates, D. B. 1981. Personal communication.

Bergen, W. G. 1978. Postruminal digestion and absorption
of nitrogenous components. Fed. Proc. 37:1223.

Bergen, W. G., D. B. Bates, D. E. Johnson, J. C. Waller and
J. R. Black. 1982. Intestinal protein supply.
Ruminal microbial protein synthesis and efficiency.
‘12 Protein Requirements For Cattle: Symposium. MP-
109. Division of Agriculture, Oklahoma State
University.

Blackburn, T. H. and R. E. Hungate. 1963. Succinic acid
turnover and propionate production in the bovine
rumen. Appl. Microbiol. 11:132.

Bremer, H. and P. P. Dennis. 1975. Transition period
following a nutritional shift-up in the bacterium
Escherichia coli B/r: stable RNA and protein
synthesis. J. theor. Biol. 52:365.

Bryant, M. P. 1970. Normal flora - rumen bacteria. Am.
J. Clin. Nutr. 23:1440.

78

 

 

79

Bryant, M. P. and L. A. Burkey. 1953a. Cultural methods
and some characteristics of some of the more numerous

groups of bacteria in the bovine rumen. J. Dairy
Sci. 36:205.

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

Bryant, M. P. and I. M. Robinson. 1961. An improved non-
selective culture medium for ruminal bacteria and its
use in determining diurnal variation in numbers of
bacteria in the rumen. J. Dairy Sci. 44:1446.

Bryant, M. P. and I. M. Robinson. 1968. Effects of diet,
time after feeding, and position sampled on numbers
of viable bacteria in the bovine rumen. J. Dairy
Sci. 51:1950.

Caldwell, D. R. and M. P. Bryant. 1966. Medium without
rumen fluid for nonselective enumeration and

isolation of rumen bacteria. Appl. Microbiol.
14:794.

Champney, W. S. 1977. Kinetics of ribosome synthesis
during a nutritional shift-up in Eschericha coli K-
12. Molec. gen. Genet. 152:259.

 

Chen, M. and M. J. Wolin. 1977. Influence of CH4
production by Methanobacterium ruminantium {on the
fermentation of glucose and lactate by _S_e_1enomonas
ruminantium. Appl. Environ. Microbiol. 34:756.

 

Cheng, K.-J., D. E. Akin and J. W. Costerton. 1977. Rumen
bacteria: interaction with particulate dietary
components and response to dietary variation. Fed.
Proc. 36:193.

Cheng, K.-J. and J. W. Costerton. 1980. Adherent rumen
bacteria - their role in the digestion of plant
material, urea and epitheal cells. 3;; Digestive
Physiology and Metabolism in Ruminants. Y.
Ruckebusch and P. Thivend (Ed.). .AVI Publishing
Company, Inc., Westport, Connecticut.

Chung, K.-T. and R. E. Hungate. 1976. Effect of alfalfa
fiber substrate on culture counts of rumen
bacteria. Appl. Environ. Microbiol. 32:649.

 

80

Coelho da Silva, J. F., R. C. Seeley, D. J. Thomson, D. E.
Beever and D. G. Armstrong. 1972. The effect in
sheep of physical form on the sites of digestion of a
dried lucerne diet. 2. Sites of nitrogen
digestion. Br. J. Nutr. 2843.

Cole, N. A., R. R. Johnson, F. N. Owens and J. R. Males.
1976. Influence of roughage level and corn
processing method on microbial protein synthesis by
beef steers. J. Anim. Sci. 43:497.

Dalbow, D. G. and R. Young. 1975. Synthesis time of B-
galactosidase in Escherichia coli B/r as a function
of growth rate. Biochem. J. 150:13.

Dehority, B. A. and J. A. Grubb. 1976. Basal medium for
the selective enumeration of rumen bacteria utilizing
specific energy sources. Appl. Environ. Microbiol.
32:703.

Dennis, P. P. and H. Bremer. 1947a. Differential rate of
ribosomal protein synthesis in Escherichia coli

 

 

B/r. J. Mol. Biol. 84:407.

Dennis, P. P. and H. Bremer. 1974b. Regulation of
ribonucleic acid synthesis in Escherichia coli B/r:
an analysis of a shift-up. III. Stable RNA
synthesis rate and ribosomal RNA chain growth rate
following a shift-up. J. Mol. Biol. 89:223.

Eadie, J. M., J. Hyldgaard-Jensen, S. 0. Mann, R. S. Reid
and F. G. Whitelaw. 1970. Observation on the
microbiology and biochemistry of the rumen in cattle
given different quantities of a pelleted barley
ration. Br. J. Nutr. 24:157.

Ecker, R. E. and G. Kokaisl. 1969. Synthesis of protein,
ribonucleic acid, and ribosomes by individual
bacterial cells in balanced growth. J. Bacteriol.
98:1219.

Ecker, R. E. and M. Schaechter. 1963. Ribosome content
and the rate of growth of §_a_1monella typhimurium.
Biochem. Biophys. Acta. 76:275

Forchhammer, J. and L. Lindahl. 1971. Growth rate of
polypeptide chains as a function of the cell growth
rate in a mutant of Escherichia coli 15. J. Mol.

 

Biol. 55:563.

81

Garton, G. A. 1965. The digestion and assimilation of
lipids. £1 Physiology of Digestion in the
Ruminant. R. W. Dougherty, R. S. Allen, W.
Burroughs, N. L. Jacobson and A. D. McGilliard
(Ed.). Butterworth Inc., Washington, D.C.

Gray, F. V., R. A. weller, A. F. Pilgrim and G. B. Jones.
1967. Rates of production of volatile fatty acids in
the rumen. V. Evaluation of fodders in terms of
volatile fatty acid produced in the rumen of the
sheep. Aust. J. agric. Res. 18:625.

Grubb, J. A. and B. A. Dehority. 1975. Effects of an
abrupt change in ration from all roughage to high
concentrate upon rumen microbial numbers in sheep.
Appl. Microbiol. 30:404.

Grubb, J. A. and B. A. Dehority. 1976. Variation in
colony counts of total viable anaerobic rumen
bacteria as influenced by media and cultural
methods. Appl. Environ. Microbiol. 31:262.

Hartree, E. F. 1972. Determination of protein: a
modification of the Lowry method that gives a linear
photometric response. Anal. Biochem. 48:422.

Harvey, R. J. 1970. .Regulation. of ribosomal protein
synthesis in Escherichia coli. (J. Bacteriol.
101:574.

Henning, P. A. and A. E. van der Walt. 1978. Inclusion of
xylan in a medium for the enumeration of total
culturable rumen bacteria. Appl. Environ. Microbiol.
35:1008.

Holdeman, L. V., E. P. Cato and W. E. C. Moore (Ed.).
1977. Anaerobe Laboratory Manual. Fourth Edition.
V. P. I. Anaerobe Laboratory, Virginia Polytechnic
Institute and State University, Blacksburg.

Hungate, R. E. 1950. The anaerobic mesophilic
cellulolytic bacteria. Bacteriol. Rev. 14:1.

Hungate, R. E. 1966. The Rumen and Its Microbes.
Academic Press, New York.

Hungate, R. E., M. P. Bryant and R. A. Mah. 1964. The
rumen bacteria and protozoa. Ann. Rev. Microbiol.
18:131.

82

Iannotti, E. L., D. Kafkewitz, M. J. Wolin and M. P.
Bryant. 1973. Glucose fermentation products of
Ruminococcus albus grown in continuous culture with
Vibrio succinogen_e_s_: changes caused by interspecies
transfer of H2. J. Bacteriol. 114:1231.

 

 

Isichei, C. O. 1980. The role of monensin in protein
metabolism in steers. Ph.D. Thesis. Michigan State
University, East Lansing.

Kjeldgaard, N. CL 1961. The kinetics of ribonucleic acid
and protein formation in Salmonella typhimurium
during the transition between. different states lof
balanced growth. Biochem. Biophys. Acta. 49:64.

 

 

Kjeldgaard, N. O. and C. G. Kurland. 1963. The
distribution of soluble and ribosomal RNA as a
function of growth rate. J. Mol. Biol. 6:341.

Koch, A. L. 1970. Overall controls on the biosynthesis of

ribosomes in growing bacteria. J. theor. Biol.
28:203.
Koch, A. L. 1980. The inefficiency of ribosomes

functioning in Escherichia coli growing at moderate
rates. J. Gen. Microbiol. 116:165.

 

Koch, A. L. and C. S. Deppe. 1971. In vivo assay of
protein synthesizing capacity of Escherichia coli
from slowly growing chemostat cultures. J: Mol.
Biol. 55:549.

Leedle, J. A. z. and R. B. Hespell. 1980. Differential
carbohydrate media and anaerobic replica plating
techniques in delineating carbohydrate-utilizing
subgroups in rumen bacterial populations. Appl.
Environ. Microbiol. 39:709.

Leibholz, J. and P. E. Hartmann. 1972. Nitrogen-
metabolism in sheep. I. The effect of protein and
energy intake on the flow of digesta into the
duodenum and on the digestion and absorption of
nutrients. 'Austr. J. agric. Res. 23:1059.

Leick, V3 1968. Ratios between contents of DNA, RNA and
protein in different microorganisms as a function of
maximal growth rate. Nature 217:1153.

Lewis, D. 1951. The metabolism of nitrate and nitrite in
the sheep. l. The reduction of nitrate in the rumen
of the sheep. Biochem. J. 48:175.

83

Ling, J. R. and P. J. ButteryS 1978. The simultaneous use
of ribonucleic acid, S, 2,6-diaminopime1ic acid and
2-aminoethylphosphonic acid as markers of microbial
nitrogen entering the duodenum of sheep. Br. J.
Nutr. 39:165.

Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J.
Randall. 1951. Protein measurement with the folin
phenol reagent. J. Biol. Chem. 193:265.

Maal¢e, O. and N. O. Kjeldgaard. 1966. Control of
macromolecular synthesis. A study of DNA, RNA, and
protein synthesis in bacteria. W. A. Benjamin, Inc.,
New York.

Maeng, W. J. and R. L. Baldwin. 1976a. Factors
influencing rumen microbial growth rates and
yields: effects of urea and amino acids over time.
J. Dairy Sci. 59:643.

Maeng, W. J. and R. L. Baldwin. 1976b. Factors
influencing rumen microbial growth rates and
yields: effect of amino acid additions to a purified
diet with nitrogen from urea. J. Dairy Sci. 59:648.

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

McAllan, A. B. and R. H. Smith. 1969. Nucleic acid
metabolism in the ruminant. Determination of nucleic
acids in digesta. Br. J. Nutr. 23:671.

McAllan, A. B. and R. H. Smith. 1973. Degradation of
nucleic acids in the rumen. Br. J. Nutr. 29:331.

McDonald, 1. W. 1952. The role of ammonia in ruminal
digestion of protein. Biochem. J. 51:86.

Moore, L. A. 1964. Symposium. on forage utilization:
nutritive value of forage as affected by physical
form. Part 1. General principles involved with

ruminants and effect of feeding pelleted or wafered
forage to dairy cattle. J. Anim. Sci. 23:230.

Mowbray, S. L. and D. P. Nierlich. 1975. Regulation of
RNA synthesis in Escherichia coli during a shift-up
transition. Biochem. Biophys. Acta. 395:91.

Munch-Petersen, E. and C. A. P. Boundy. 1963. Bacterial
content in samples from different sites in the rumen
of sheep and cows as determined in two culture
media. Appl. Microbiol. 11:190.

84

Neidhardt, F. C. and B. Magasanik. 1960. Studies on the
role of ribonucleic acid in the growth of bacteria.
Biochem. Biophys. Acta. 42:99.

Nierlich, D. P. 1972. Regulation of ribonucleic acid
synthesis in growing bacterial cells. 1. Control
over the total rate of RNA synthesis. J. Mol. Biol.
72:751.

Phillipson, A. T. 1942. The fluctuation of pH and organic
acids in the rumen of the sheep. J. Exp. Biol.
19:186.

Pittman, K. A., S. Lakshmaran and M. P. Bryant. 1967.
Oligopeptide uptake by Bacteroides ruminicola. J.
Bacteriol. 93:1499.

Prigge, E. C., M. L. Galyean, F. N. Owens, D. G. Wagner
and R. R. Johnson. 1978. Microbial protein syn-
thesis in steers fed processed corn rations. J.
Anim. Sci. 46:249.

Rosset, R., J. Julien and R. Monier. 1966. Ribonucleic
acid composition of bacteria as a function of growth
rate. J. Mol. Biol. 18:308.

Schaechter, M. 1973. Growth: cells and populations. E
Biochemistry of Bacterial Growth. J. Mandelstam and
K. McQuillen (Ed.). John Wiley 5 Sons, New York.

Schaechter, M., O. Maa1¢e and N. O. Kjeldgaard. 1958.
Dependency on medium and temperature of cell size and
chemical composition during balanced growth of
Salmonella typhimurium. J. gen. Microbiol. 19:592.

Shehata, A. M. E. 1958. The effect of different rations
on the number of free ruminal bacteria in sheep.
Appl. Microbiol. 6:422.

Skjold, A. C., H. Juarez and C. Hedgcoth. 1973.
Relationships among deoxyribonucleic acid,
ribonucleic acid, and specific transfer ribonucleic
acids in Escherichia coli 15T at various growth
rates. J. Bacteriol. 115:177.

 

Slyter, L. L., D. L. Kern, J. M. Weaver, R. R. Oltjen and
R. L. Wilson. 1971. Influence of starch and
nitrogen sources on ruminal microorganisms of steers
fed high fiber purified diets. J. Nutr. 101:847.

Smith, R. H. 1969. Reviews of the progress of dairy
science. Section G. General. Nitrogen metabolism
and the rumen. J. Dairy Res. 36:313.

85

Smith, C. J., R. B. Hespell and M. P. Bryant. 1980.
Ammonia assimilation and glutamate formation in the

anaerobe Selenomonas ruminantium. J. Bacteriol.
141:593.

Smith, R. H. and A. B. McAllan. 1970. Nucleic acid
metabolism in the ruminant. 2. Formation of
microbial nucleic acids in the rumen in relation to
the digestion of food nitrogen, and the fate of
dietary nucleic acids. Br. J. Nutr. 24:545.

Smith, R. H. and A. B. McAllan. 1974. Some factors
influencing the chemical composition of mixed rumen
bacteria. Br. J. Nutr. 31:27.

Smith, R. H., A. B. McAllan, D. Hewitt and P. E. Lewis.
1978. Estimation of amounts of microbial and dietary
nitrogen compounds entering the duodenum of cattle.
J. agric. Sci., Camb. 90:557.

Stanier, R. Y., E. A. Adelberg and J. L. Ingraham. 1976.
The Microbial World. Prentice-Hall, Inc., New
Jersey.

Thorley, C. M., M. E. Sharpe and M. P. Bryant. 1968.
Modification of the rumen bacterial flora by feeding
cattle ground and pelleted roughage as determined
with culture media with and without rumen fluid. J.
Dairy Sci. 51:1811.

Wallace, R. J. 1979. Effect of ammonia concentration on
the composition, hydrolytic activity and nitrogen
metabolism of the microbial flora of the rumen. J.
Appl. Bacteriol. 47:443.

Warner, A. C. I. 1962a. Enumeration of rumen micro-
organisms. J. gen. Microbiol. 28:119.

Warner, A. C. I. 1962b. Some factors influencing the

rumen microbial population. J. gen. Microbiol.
28:129.
Warner, A. C. I. 1966a. Periodic changes in the

concentrations of microorganisms in the rumen of a
sheep fed a limited ration every three hours. J.
gen. Microbiol. 45:237.

Warner, A. C. I. 1966b. Diurnal changes in the concen—
trations of microorganisms in the rumens of sheep fed
to appetite in pens or at pasture. J. gen.
Microbiol. 45:243.

 

‘1‘??— .

 

Warner,

Wolin,

Wolin,

86

A. C. I. 1966c. Diurnal changes in the concen-
trations of microorganisms in the rumens of sheep fed
limited diets once daily. J. gen. Microbiol.
45:213.

M. J: 1974. Metabolic interactions among
intestinal microorganisms. Am. J. Clin. Nutr.
27:1320.

M. J. 1981. Fermentation in the rumen and human
large intestine. Science 213:1463.

 

APPENDIX

INDIVIDUAL SHEEP DATA

 

INDIVIDUAL SHEEP DATA

TABLE A1. FREE BACTERIAL COLONY COUNTS PER GRAM OF RUMEN
CONTENTS 3 - SHEEP 905

 

 

 

 

Hours After Feeding Diet 1 Diet 2 Diet 3
(103/9)
0 3.63 1.48 6.10 11.01 14.04 12.36
2 1.21 1.15 2.42 $.45 15.00 11.66
5 3.22 1.31 7.49 $.90 21.89 $1.56
8 3.73 $.87 8.65 11.09 31.48 14.23

11 5.99 i.86 9.02 1.41 34.72 t5.33

 

TABLE A2. BOUND BACTERIAL COLONY COUNTS PER GRAM OF RUMEN
CONTENTS 3 - SHEEP 905

 

 

 

 

Hours After Feeding Diet 1 Diet 2 Diet 3
(108/9:

Too numerous

0 3.06 1.16 5.53 11.30 to count

2 2.42 1.35 5.37 1.66 12.02 11.29

5 6.68 12.06 5.39 1.37 14.77 11.65

8 4.23 11.02 3.40 1.46 13.20 11.29

11 4.80 1.18 5.70 11.43 11.84 11.29

 

3Mean and standard deviation of 4 or more roll tube cultures.

87

 

88

TABLE A3. FREE BACTERIAL COLONY COUNTS PER GRAM OF RUMEN
CONTENTS 3 - SHEEP 913

 

 

 

 

Hours After Feeding Diet 1 Diet 2 Diet 3
(103/9)
0 2.291.40 5.9611.18 12.44i.9l
2 .831.34 2.54i.13 12.201.48
5 2.171.40 2.791.41 12.1111.94
8 6.001.2.04 5.63:.44 10.32i.69
11 3.37 1 .60 7.66 11.28 11.20 i .85

 

TABLE A4. BOUND BACTERIAL COLONY COUNTS PER GRAM OF RUMEN
CONTENTS 3 - SHEEP 913

 

 

 

 

Hours After Feeding Diet 1 Diet 2 Diet 3
(108/9)
0 6.241 1.41 8.491 1.40 23.041 1.95
2 4.7211.80 4.8211.09 18.481 1.86
5 4.881 .88 5.101 .90 18.451 .60
8 9.8211.80 9.5911.22 17.0311.44

11 8.3411.72 10.5511.22 18.4612.75

 

3Mean and standard deviation of 4 or more roll tube cultures.

89

TABLE A5. RNA-PROTEIN RATIOS FOR FREE RUMEN BACTERIA

 

 

- SHEEP 905

Hours After Feeding Diet 1 Diet 2 Diet 3
0 .247 .230 .403

2 .299 .280 .293

4 .347 .457 .332

6 .339 .294 .334

8 .345 .287 .506

10 .280 .275 .351

12 .281 .281 .366

 

TABLE A6. RNA-PROTEIN RATIOS FOR BOUND RUMEN BACTERIA

 

 

- SHEEP 905

Hours After Feeding Diet 1 Diet 2 Diet 3
0 .201 .148 .202

2 .201 .134 .207

4 .221 .268 .225

6 .211 .113 .197

8 .218 .156 .201

10 .263 .205 .204

12 .201 .148 .202

 

TABLE A7. RNA-PROTEIN RATIOS FOR FREE RUMEN BACTERIA

90

 

 

- SHEEP 898

Hours After Feeding Diet 2 Diet 3
0 .248 .332

2 .301 .404

4 .274 .452

6 .166 .635

8 .300 .711

10 .247 .417

12 .263 .258

 

TABLE A8. RNA-PROTEIN RATIOS FOR BOUND RUMEN BACTERIA

 

 

- SHEEP 898

Hours After Feeding Diet 2 Diet 3
0 .095 .216

2 .158 .200

4 .152 .222

6 .159 .261

8 .150 .193

10 .133 .228

12 .128 .211

 

TABLE A9. RNA-PROTEIN RATIOS FOR FREE RUMEN BACTERIA

91

 

 

- SHEEP 913

Hours After Feeding Diet 1 Diet 2 Diet 3
0 .247 .305 .381

2 .282 .355 .419

4 .297 .344 .438

6 .279 .327 .853

8 .284 .384 .374

10 .283 .316 .480

12 .244 .302 .377

 

TABLE A10. RNA-PROTEIN RATIOS FOR BOUND RUMEN BACTERIA

 

 

- SHEEP 913

Hours After Feeding Diet 1 Diet 2 Diet 3
0 .212 .134 .211

2 .226 .213 .144

4 .226 .205 .254

6 .240 .153 .212

8 .214 .157 .256

10 .189 .191 .260

12 .195 .125 .232

 

 

92

TABLE All. RNA-PROTEIN RATIOS FOR FREE RUMEN BACTERIA

 

 

- SHEEP 900

Hours After Feeding Diet 1 Diet 2 Diet 3
0 .259 .198 .420

2 .285 .320 .310

4 .325 .301 .373

6 .316 .285 .240

8 .298 .279 .291

10 .310 .238 .369

12 .254 .212 .339

TABLE A12. RNA-PROTEIN RATIOS FOR BOUND RUMEN BACTERIA

 

 

- SHEEP 900
Hours After Feeding Diet 1 Diet 2 Diet 3
0 .297 .132 .227
2 .230 .098 .185
4 .221 .111 .238
6 .208 .107 .198
8 .225 .127 .209
10 .190 .115 .240
12 .388 .095 .212