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THE ROLE OF MONENSIN IN PROTEIN
METABOLISM IN STEERS

By

CharTes Onyeabo Isichei

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Animal Husbandry

T980

ABSTRACT

THE ROLE OF MONENSIN IN PROTEIN
METABOLISM IN STEERS

By

Charles Onyeabo Isichei

Digesta Passage Studies

 

Four abomasally cannulated steers were used in a switch-back
metabolism study to determine the effect of monensin on non-ammonia
nitrogen (NAN), dry matter (DM) and fiber passage to the abomasum.

The steers were fed high grain rations, with (HG-M) and without (HG)
monensin or high silage rations, with (CS-M) and without (CS) monensin.
Grain rations contained shelled corn, oats, alfalfa, soybean meal and
molasses. Silage rations consisted of corn silage [35% dry matter (DM)]
and soybean meal-mineral supplement. Monensin was added at 33 ppm
ration DM and all rations contained 13% crude protein (CP). Steers
were fed twice daily ad_libitum. Lignin and chromic oxide were used

as particulate and total digesta markers, and polyethylene glycol (PEG)
used to estimate liquid digesta passage.

RNA-Nztotal-N ratios of mixed rumen bacteria (isolated from
ruminally-fistulated steers receiving the same rations) and abomasal
RNA-N:total-N ratios were used to quantitate microbial contribution to
NAN flow.

From lignin passage, abomasal N and NAN passage (% of N-intake)

for rations HG-M, HG, CS-M and CS were: 96.86, 90.48, 100.91, 93.24;

Charles Onyeabo Isichei
0

94.81, 88.24, 97.23 and 89.76, respectively. Abomasal NAN, microbial N
and feed (bypass) N passage for the same respective rations were:
124.29, 74.85, 49.44; 119.25, 82.90, 36.35; 133.59, 76.12, 57.47;
133.64, 95.86 and 37.79 g/day. Feed N bypass (% NAN passage) for
rations HG-M, HG, CS-M and CS were: 39.78, 30.48, 43.02 and 28.27,
respectively. Efficiencies of microbial protein synthesis for the
above respective rations were: 17.31, 20.31, 15.13 and 21.00 9 CP per
100 9 organic matter truly digested in the rumen. Monensin decreased
microbial-N passage (P<:0.05), increased feed N bypass (P< 0.01) and
decreased efficiency of ruminal microbial cell production (P< 0.05).
The extent of ruminal degradation of 0M and fiber was not
significantly affected by monensin. Passage data derived from chromium
flow were very similar to the lignin-based estimates. In contrast,
fractional rates of ruminal PEG outflow tended to be high, thus
resulting in an overestimation of the extent of liquid digesta and

N-passage to the abomasum.

Nitrogen Balance

 

Nitrogen status of the steers used in the preceding study was
also evaluated in a balance and digestibility trial. The steers were
adapted to the same experimental rations for 18 days prior to 8-day
nitrogen balance collections.

Monensin increased nitrogen and DM digestibility of the grain
ration without significantly affecting the silage ration. Nitrogen
retention (% of N-intake) were: 37.36, 32.34, 30.90 and 27.80 for

HG-M, HG, CS-M and CS rations, respectively. These differences were,

Charles Onyeabo Isichei

however, not significant. Fiber (ADF) digestibility was higher with the

silage rations (P< 0.01), but there was no monensin effect.

Feeding Trial and Plasma Studies

A feeding trial was conducted to determine the influence of
monensin and/or elfazepam on the plasma amino acid (PAA) status and
performance of Hereford steers fed an all-silage ration. Control ration
consisted of 88% corn silage (35% DM) and 12% soybean meal-mineral sup-
plement. Monensin was fed at 33 ppm; elfazepam was added at 2 ppm
ration DM.

Overall PAA, plasma urea and glucose levels were not signifi-
cantly affected by the addition of monensin, elfazepam or the two
chemicals combined, to the rations. The trends were for slightly
reduced levels of the sulfur and branched-chain amino acids with
monensin-elfazepam combination. Essential, non-essential and total
amino acid levels were also slightly lower with the two drug combi-
nation; the other treatments were similar to the control.

In comparison with the control silage, monensin improved the
overall performance of the steers, elfazepam only stimulated intake,

while the two chemicals in combination resulted in poor performance.

In memony 06

my beflovcd patents

ii

ACKNOWLEDGMENTS

The author is deeply grateful to Dr. N. G. Bergen for his
guidance, supervision and encouragement throughout the duration of the
graduate program. His expertise and constructive advice were invaluable
both in the designing of these studies and in the preparation of the
ensuing manuscript. Sincere thanks are also due to Drs. M. T. Yokoyama,
D. R. Hawkins and R. M. Cook for their involvement in these doctoral
studies. Their scholarly comments and critical review of this disser-
tation were very much appreciated. The author also wishes to express
a deep appreciation to Dr. John Waller for his friendship and kind
indulgence.

Sincere gratitude is extended to Dr. R. H. Nelson, chairman of
the Department of Animal Husbandry for providing the necessary financial
support and research facilities that made my academic endeavors a
reality. Dr. W. T. Magee's understanding and willingness to help,
especially during the difficult periods of my graduate studies will
always be remembered. I am also deeply indebted to Dr. E. R. Miller,
who in his quiet exemplary ways was for me, a tremendous source of
inspiration and motivation.

I also wish to thank my fellow graduate students, past and
present (especially Mr. D. B. Bates and Dr. G. F. Collings), for their
intellectual stimulation and comradeship. A debt of gratitude is espe-
cially due to Ms. E. S. Rimpau, not only for her unreserved technical
assistance, but also for her generosity and thoughtfulness. I will
always cherish my association with that special lady. Special appre-
ciation is also extended to Mrs. Pat Cramer and Sherry Mileski for
their congeniality and ready assistance.

I would like to thank my beloved grandmother, Mrs. V. A. Oputa
for her unqualified love and confidence in me; my beloved brothers and
sisters, Maureen, Cyril, Valentine, Evangeline, Victor and Frederick,
for their love, patience and understanding. Immense gratitude is also
due to Ms. J. A. Okafor for her patience, support, and love. Above all,
I thank the Almighty Father for giving me such exemplary role models as
my wonderful parents, whose 'untimely' departure prevented their seeing
the successful conclusion of my turbulent quest for independence and
knowledge. But, most importantly, I thank Him for his forgiveness,
guidance and love.

For all those who, by accident or by design, did in anyway touch
my life, I hope the encounters were positive; as for me, I hope the
experiences would help to make me a better human being.

TABLE OF CONTENTS

LIST OF TABLES ..........................
LIST OF FIGURES .........................

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

Rumen Microbial Growth--Substrate Utilization .....
Overview ......................
Carbohydrate Fermentation .............
Nitrogen Metabolism ................

Quantitative Aspects of Microbial Protein Synthesis

in the Rumen .....................

Energetics of Rumen Microbial Growth--YATP .......

Digesta and Non-Ammonia-Nitrogen Passage ........
Level of Feed Intake and NAN Flow .........
Dietary Protein Source and NAN Passage .......
Forage to Concentrate Ratios and NAN Flow .....
Feed Processing and NAN Passage ..........

Influence of Microbial and Digesta Markers on

Passage Studies ...................
Fluid Markers ...................
Particulate Flow Markers ..............
Microbial Markers .................

Monensin ........................
Monensin and VFA Production ............
Feedlot Studies ..................

Monensin and Rumen Microbial Growth ..........
Effects on Microbial Population and Growth .....
Monensin and Efficiency of Microbial Growth . .

Monensin and NAN Passage ................

Monensin--Mode of Action ................

MATERIALS AND METHODS ..................
Nitrogen Metabolism Studies ..............

General Design ...................
Abomasal Cannula Design ..............

iv

Page

vii

Chapter

Rumen Microbial Studies ...............
Collection and Preparation of Samples ........
Nitrogen Balance Study ..............
Digesta Passage Studies .............
Rumen Microbial Studies .............
Further Sample Preparations ...........
Nitrogen Determination ..............
Chromium Analysis ................
Dry and Organic Matter Determination .......
Polyethylene Glycol Determination ........
Fiber Determination ...............
Nucleic Acid Analysis ..............
Statistical Analyses ...............
Feeding Trial and Blood Metabolite Studies ......
General Design ..................
Sample Preparation and Analyses ...........
Blood Samples ..................
Feed Samples ...................
Statistical Analyses ...............
Calculations .....................

IV. RESULTS ........................

Digesta Passage Studies ...............
Ruminal Dry Matter and Fiber Digestion ......
Total Nitrogen and NAN Passage to the Abomasum . .
Abomasal N Passage from Reconstituted Digesta .

Rumen Microbial Studies ...............
Microbial Contribution to Abomasal NAN Passage . .
Efficiency of Microbial Protein Synthesis
Microbial Efficiency and Ruminal Dilution Rates .

Nitrogen Balance and Digestibility Studies ......
Nitrogen Balance .................
Dry Matter and ADF Digestion ...........
Fecal Marker and ADF Recoveries .........

Feeding Trial and Plasma Metabolite Studies .....
Feeding Trial ..................

Plasma Metabolite Studies ............
V. DISCUSSION .......................

Digesta Passage Studies ...............
Digesta Flow Rates ................
Nitrogen Metabolism Studies
Ruminal Nitrogen Losses .............
NAN Passage to the Abomasum ...........
Efficiency of Rumen Microbial Protein Synthesis .

Chapter Page

Nucleic Acid Methodology .............. l20

Nitrogen Balance and Digestibility Studies ..... 123

Feeding Trial and Plasma Metabolite Studies ...... 127

Feeding Trial ................... 127

Plasma Metabolite Studies ............. 130

Monensin-Na: Mechanism of Action ......... 132

V. CONCLUSIONS ....................... l36
APPENDIX ............................. 138
LITERATURE CITED ......................... 153

vi

Table

10.

ll.

12.

LIST OF TABLES

RUMINAL OUTFLOW OF CRUDE PROTEIN AND MICROBIAL PROTEIN
SYNTHESIS--SUMMARY OF EXPERIMENTS USING VARIOUS FLOW

MARKERS ........................

COMPOSITION OF DIETS USED IN DIGESTA PASSAGE AND

NITROGEN METABOLISM STUDIES ..............

COMPOSITION OF SOYBEAN MEAL-MINERAL SUPPLEMENTS USED

IN DIGESTA PASSAGE STUDIES AND FEEDING TRIAL ......

CHEMICAL COMPOSITION OF RATIONS USED IN DIGESTA

PASSAGE STUDIES ....................

COMPOSITION OF RATIONS USED IN FEEDING TRIAL AND

PLASMA STUDIES .....................

COMPARISON OF TOTAL DIGESTA FLOW RATES ESTIMATED BY

REFERENCE TO PEG, LIGNIN AND CHROMIUM .........

DRY MATTER AND FIBER FLOW FROM THE RUMEN: ESTIMATED

FROM LIGNIN-BASED DRY MATTER FLOW ...........

DRY MATTER AND FIBER FLOW FROM THE RUMEN: ESTIMATED

FROM CHROMIUM-BASED DRY MATTER FLOW ..........

EFFECTS OF MONENSIN AND TYPE OF RATION ON THE NITROGEN
CONSTITUENTS OF ABOMASAL DIGESTA: ESTIMATED FROM

LIGNIN FLOW ......................

EFFECTS OF MONENSIN AND TYPE OF RATION ON THE NITROGEN
CONSTITUENTS OF ABOMASAL DIGESTA: ESTIMATED FROM N:

LIGNIN RATIOS .....................

EFFECTS OF MONENSIN AND TYPE OF RATION ON THE NITROGEN
CONSTITUENTS OF ABOMASAL DIGESTA: ESTIMATED FROM

CHROMIUM FLOW .....................

EFFECTS OF MONENSIN AND TYPE OF RATION ON THE NITROGEN
CONSTITUENTS OF ABOMASAL DIGESTA: ESTIMATED FROM N:

CHROMIUM RATIOS ....................

vii

49

51

52

64

7O

72

73

75

76

77

78

Table

13.

14.
15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

A.1

A.2

EFFECTS OF MONENSIN AND RATION TYPE ON NITROGEN
CONSTITUENTS OF ABOMASAL DIGESTA IN GROWING STEERS--
DETERMINED FROM RECONSTITUTED LIQUID (FROM PEG) AND

SOLID (CHROMIC OXIDE) FLOWS ..............
RATIOS OF MARKERS IN RATION AND ABOMASAL DIGESTA . . . .

COMPOSITION OF MIXED RUMEN BACTERIAL PREPARATIONS

FROM STEERS FED MONENSIN ................

MICROBIAL CONTRIBUTION TO ABOMASAL NAN FLOW IN STEERS

FED MONENSIN: DIGESTA PASSAGE STUDIES .........

EFFECTS OF MONENSIN AND TYPE OF RATION ON EFFICIENCY
OF MICROBIAL PROTEIN SYNTHESIS: ESTIMATES FROM

LIGNIN FLOW ......................

EFFECTS OF MONENSIN AND TYPE OF RATION ON EFFICIENCY
OF MICROBIAL PROTEIN SYNTHESIS: ESTIMATES FROM

CHROMIUM FLOW .....................

EFFECTS OF MONENSIN AND TYPE OF RATION ON RUMEN
DILUTION RATES AND EFFICIENCY OF MICROBIAL

PROTEIN SYNTHESIS ...................

EFFECTS OF MONENSIN AND TYPE OF RATION ON NITROGEN

BALANCE AND DIGESTIBILITY IN GROWING STEERS ......

FECAL MARKER (CHROMIUM) RECOVERY AND POST-RUMINAL

ACID DETERGENT FIBER DIGESTION .............

OVERALL DRY MATTER INTAKE, ADG AND FEED EFFICIENCY

FOR 118 DAY FEEDING TRIAL ...............

INFLUENCE OF MONENSIN AND ELFAZEPAM ON PLASMA FREE
AMINO ACID CONCENTRATIONS OF STEERS FED SILAGE

RATIONS ........................

PLASMA METABOLITE LEVELS IN STEERS FED MONENSIN

AND ELFAZEPAM .....................

INDIVIDUAL STEER APPARENT FIBER DIGESTION (FROM
LIGNIN-BASED DM FLOW) IN THE RUMEN: DIGESTA PASSAGE

STUDIES ........................

INDIVIDUAL STEER: TRUE RUMEN DRY MATTER DIGESTION

PASSAGE STUDIES-~LIGNIN BASED ESTIMATES ........

viii

Page

80
82

83

86

87

88

91

93

96

98

Table

A.3

A.4

A.13

INDIVIDUAL STEER: TRUE RUMEN DMD. PASSAGE STUDIES:
CHROMIUM-BASED ESTIMATES .................

INDIVIDUAL STEER NAN PASSAGE--CALCULATED FROM LIGNIN
OUTFLOW FROM THE RUMEN ..................

INDIVIDUAL STEER NAN PASSAGE: FROM N:LIGNIN RATIOS

INDIVIDUAL STEER NAN PASSAGE: CALCULATED FROM
CHROMIUM OUTFLOW .....................

INDIVIDUAL STEER NAN PASSAGE: FROM N:CHROMIUM
RATIOS ..........................

INDIVIDUAL STEER NAN--LIQUID OUTFLOW: FROM PARTITIONED
LIQUID (PEG) DIGESTA PASSAGE ...............

INDIVIDUAL STEER NAN--SOLID DIGESTA OUTFLOW: FROM
PARTITIONED SOLID (CHROMIUM) PASSAGE ...........

INDIVIDUAL STEER--NH3-N AND NAN PASSAGE TO THE
ABOMASUM .........................

RNA AND N CONSTITUENTS OF RUMINALLY ISOLATED
BACTERIAL PREPARATIONS ..................

MICROBIAL CONTRIBUTION TO NAN PASSAGE TO THE
ABOMASUM: INDIVIDUAL STEERS ...............

INDIVIDUAL STEER: NITROGEN BALANCE STUDIES .......

INDIVIDUAL STEER DRY MATTER DIGESTION (N2 BALANCE
CONTINUED) ........................

INDIVIDUAL STEER FECAL CHROMIUM RECOVERY--NITROGEN
BALANCE STUDIES .....................

ix

Page

140

141
142

143

144

145

146

147

148

149
150

151

Figure

2a.

2b.

LIST OF FIGURES

Flow Chart of Sampling Protocol (Digesta Passage
Studies) ........................

Scheme of Laboratory Analysis for Composited Abomasal
Samples (Digesta Passage Studies) ............

Flow Chart of Sampling Protocol and Laboratory
Analyses (Rumen Microbial Studies) ...........

ADG, DMI at Day 28, 63, 90 and 118 ...........

Time Trends for Valine, Leucine and Isoleucine .....

Page

55

57

58
99

CHAPTER I

INTRODUCTION

The ruminant animal is unique in its nutrient metabolism, due
to its symbiotic relationship with the anaerobic microorganisms that
inhabit the forepart (reticulorumen) of its digestive tract. Food
consumed by the animal is initially fermented by these rumen microbes,
in order to meet their own nutrient requirements for growth (Hungate,
1966). Microbial cells, and the unfermented feed residues are subse-
quently flushed out of the rumen to the lower gastrointestinal tract,
where they become exposed to the hydrolytic actions of the digestive
enzymes.

Of advantage to the ruminant, is the fact that these micro-
organisms can degrade B-linked polysaccharides (cellulose), which are
major constituents of most ruminant feeds. Rumen microbes can therefore
convert feed materials the animal cannot digest, into high quality,
highly digestible microbial protein. These organisms, on the other
hand, are furnished with an ecological niche conducive to their own
growth requirements, while at the same time, being given a first
opportunity at the food the animal consumes.

Since the advent of the modern feed-lot, the ruminant is
increasingly becoming a major competitor for foods more efficiently

utilized by man and other monogastrics. Feeds such as corn and soybean

are now fed to cattle in order to increase their energy and protein
intake, and thus improve their rate of growth. The idea is to shorten
the time the animal is on feed prior to slaughter. Unfortunately, such
high quality feeds can be more efficiently converted into meat protein
when they are digested in the post ruminal gastro-intestinal tract,
rather than being fermented by rumen microorganisms. Several techniques
have therefore, been developed to protect high quality plant proteins
from extensive ruminal degradation (Chalupa, 1975), and thus increase
the proportion of those feed proteins digested in the lower tract.

Over the years, the emphasis of ruminant nutrition research
has often alternated between the above objectives, methods to increase
energy intake of the animals, and manipulations aimed at maximizing
rumen microbial production. Intake stimulants (e.g. Elfazepam [Baile
_;t_;L., 1976; Baile and McLaughlin, 1979]), anabolic steroids (e.g.,
DES, Synovex) and antibiotics (e.g., Aureomycin, Monensin), are some
of the growth stimulants and feed additives widely used to manipulate
the digestive and physiological responses of the ruminant animal, in
order to improve its rate of gain and efficiency of feed conversion.
One of these feed additives, monensin, forms the subject matter of
the following studies.

Monensin is a monocarboxylic acid-polyetherin antibiotic
(Harold, 1972) produced by a strain of the actinomycete, Streptomyces

cinnamonensis. This compound was reported by several workers to reduce

 

feed intake and improve the efficiency of gains in ruminants (Potter

t a1., 1976a; Raun gt_al,, 1976; Utely, 1976). Other workers

(Richardson gt__l,, 1976) observed from jn_vitrg_studies that monensin
causes a shift in the molar ratios of volatile fatty acids in favor of
propionic acid. Reductions in rumen ammonia levels were also reported
with monensin (Dinius _t._l., 1976). These observations indicate that
monensin exerts some effects on rumen microbial metabolism and feed
degradation. It became, therefore, necessary to determine the effect
of this antibiotic on the rate of digesta passage and feed protein
degradation in the rumen.

The first study was conducted to determine the effect of
monensin on nitrogen passage to the abomasum of growing steers fed
high grain and high silage rations. Multiple markers were used to
quantitate non-ammonia-nitrogen flow, microbial protein passage and
the extent of rumen feed protein and dry matter bypass. The effects
of monensin on nitrogen retention, dry matter, fiber and nitrogen
digestion were also examined with the same rations. In another study
aimed at assessing the influence of monensin on the plasma amino acid
status of growing steers, monensin was fed separately, or in combination
with an intake stimulant (Elfazepam), in a high silage ration. In this

trial, the rate of body weight gains and efficiency of feed conversion

were also determined.

CHAPTER II

REVIEW OF LITERATURE

Rumen Microbial Growth-~Substrate Utilization

 

Overview

Rumen microorganisms rely on the ruminant not only for the
supply of the nutrients they require for their metabolism and growth,
the ruminant also provides them a habitat favorable for their active
growth and proliferation. The anaerobic nature of the rumen and the
relative constancy of its temperature (37 to 40°C) ensures theselnicro-
organisms an ecological niche that is conducive to their metabolic
needs.

Under Optimal conditions, rumen fluid is richly laden with
nutrients which are readily available to the microbes. These include
energy and nitrogen sources, metabolic intermediates and growth sub-
stances required by these organisms for the synthesis of microbial

cells.

Carbohydrate Fermentation

 

Carbohydrates (starches, soluble sugars, cellulose, hemicellu-
lose and pectins) are the primary feed components used by rumen bacteria
and protozoa to generate the energy (ATP) required for cell synthesis.
The majority of carbohydrates are converted to pyruvate via the Embden-

Meyerhoff glycolytic pathway (Hungate, 1966). Pyruvate is further

utilized to produce ATP and volatile fatty acids (VFA's), CH CO

4’ 2
and H2. The major VFA's produced (acetate, propionate, butyrate and
valerate) are absorbed via the rumen wall and utilized by the host
animal as energy substrates. Pathways for the fermentation of pyruvate
are fairly well established (Hungate, 1966; Baldwin, 1965) and the
proportion of each VFA produced depends on the type of substrate,
rumen pH and the fermentation pathways utilized by the microbial
species predominant in the rumen. Acetate is produced primarily

via phosphoroclastic reactions which involve the decarboxylation of
pyruvate. Pr0pionate is produced via the dicarboxylic acid (random-
izing) pathway, and also by the acrylate (non-randomizing or direct
reductive) pathway. The former route is usually more prominent, but
the latter becomes more important as the carbohydrate availability of
the diet increases (Baldwin, 1965). Butyrate and other higher fatty
acids are synthesized from acetyl-CoA via a reversal of B-oxidation
(Baldwin, 1965).

Even though some estimates of the theoretical ATP yield from
the above reactions are still in contention, the following stoichio-
metric relationships have been proposed (Baldwin, 1965).

2 Pyruvate —+ 2 Acetate + 2 ATP + 2 H2 + 2C02

Pyruvate + 2H2 —+~Propionate + ATP

2 Pyruvate (2 Acetate + H2) —+ Butyrate + 2ATP + 2C02

2 Pyruvate + 3H2 -+ Valerate + ATP + C02.

The presence of electron transport systems in certain rumen organisms

(especially some propionate producers) could still increase the

theoretical yield of ATP per mole of VFA produced. The currently
assigned ATP values per mole of end-product formed are: acetate, 2;
pr0pionate, 3; butyrate, 3; and methane, l. The relative energetic
efficiency for the conversion of one mole of glucose to propionate is
109% (based on bomb calorimetric determinations--Hungate, 1966); it is
62.2% and 77.9% for acetate and butyrate, respectively. This indicates
that of the three major VFA's produced, propionate fermentation results
not only in a higher ATP yield to the microorganisms, but is also

energetically more useful to the host animal.

Nitrogen Metabolism

Dietary proteins and non-protein nitrogen (NPN) are degraded by
rumen microbial proteolytic enzymes and deaminases to ammonia--nitrogen
(NH3-N),amino acids and peptides, which are differentially incorporated
into microbial protein. Ammonia is the central intermediate in the
degradation and assimilation of dietary nitrogen in the rumen, and
seems to be required by several species of rumen bacteria (Hungate,
1966; Bryant and Robinson, 1962). Many microbial species can maintain
their rates of growth with ammonia and other NPN as their sole nitrogen
source, provided they have available a readily fermentable energy
source, and other nutrient requirements such as carbon skeletons,

sulfur and other<xrfactors required for microbial protein synthesis.

Quantitative Aspects of Microbial
Protein Synthesis in the Rumen

 

 

Rumen microbial growth is dependent primarily on the
availability of substrates. These include not only adequate energy
and nitrogen supply, but also other co—factors such as sulfur, carbon
skeleton and unidentified growth substances required for optimal cell
synthesis. Any of the above requirements can limit the efficiency of
microbial cell growth and organic matter degradation in the rumen.

In recent years, considerable effort has been directed to a
quantitative estimation of the extent of microbial protein synthesis
in the rumen. The amount of microbial cells (or microbial crude pro-
tein) produced in the rumen is usually estimated in relation to some
unit of organic matter apparently digested in the rumen (DOM), or of
organic matter truly fermented (FOM). The second method is more
accurate and facilitates the calculation of ATP production, but the
former is more precise and can be applied on a wider scale, especially
when organic matter incorporation into microbial cells is not
specifically determined.

Table 1 contains a summary of rumen microbial protein estimates
obtained by several workers using different quantitation techniques.
Estimates obtained range from 6 to 34 gm of microbial crude protein
synthesized per 100 g DOM. This is equivalent to 12 to 68 g of dry
microbial cells per 100 g DOM, if a 50% crude protein content for rumen
bacteria is assumed (Smith, 1974). The wide variation in estimates
obtained is indicative of problems encountered with the use of different

markers to quantitate digesta and microbial protein flow from the rumen.

 

 

 

 

 

 

 

..Nc_ae N.NV aN_ae caxa.e.,»~= -- mm. mm_ NN_ __
38: .333 :52. 5: 3.2. 8:: :3: -- a. _: om N.

N com_o;u.z aaagm ..u=_ae a.ov aN.ae wage—N..»az -- Na mm mm c. -- NoNLu
.u.l| ANNN_V ANN NNV not: N_.N to 4.. .N.c .o m=_a N N
._ No saxmto aaagm .aae ~>ammau a Na_c~a ua..o¢ o_-e_ mN-NN_ vo_-oo_ NN.-mN o_-o_ a<o o EU

.I 183: A9. 2: :8. 583m .5 39.. cm: .38. mg m N
._. No £3.2m o.uuau uzcucaocm..aN.ae eaxape..zacum o_-o_ 0N-NN N.N-N¢N .mm-vm¢ e_-m_ <2“ o cu

AoNa_. Aux mN_. N N
gowam . ms~N__,z a_uuau =.am~u.+aN.ue eaxa_e..zacum -- No-.o oom-om_ Nno-mN_ .N-N -- o cu
.NNNPV N_o;aa.4 cvomau..actou=_ Noctuuas o mN-No Ne-me mm_-NN 0N-m_
.ANNN_V casucaz =ao:.m Lo :.amaa..amogo=oc »a_cam N_-N Nm-oo me o__-_o_ o.-q.

a N_ogaa.4 aaogm coagmaac N~_E.N o_ men on o N mmm QNN

...|chN_v Aux cc.

._. “a men: aaazm m.a>o_ aac=.+ua.c ou.e_c=a m.-a NN-OON NN-NN oo_-o_ .N-N -- oNa
AacNo_v ass: aoogm out: so.3 uu.u um.eccaa.sam N. ma .N Na __ -- aNa

.1..|haNNo_v AN. ommv .aae coonxam
._. Nu cactx o_uuau Lo saga..mp.=g uaamcoouou ON-N. oe_-mo_ omo-pqm NNm-oNN N.-m <zz
11.1».NNNNV ANN mNN. mucosa—nan”
._. Na aaocx u_uu.u ouc:...~ae eaanxcm..mm.ca omega N_-o_ NN-NN .mm-omm eoc-_N¢ op-o (xx :.=o_4
m._=g voameo»uou u_N to c +
.uo__ac ace La uaxape. =_.Lm ccou __-o oo-_e Nam-mmm eco-omm N_-N_
m__=g uuomcouuou u_N to v. .N
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I. 1 Ex 83 25:9.

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10

Also evident is the fact that both the nitrogen and energy content
of the ration do affect microbial cell yield.

Hume gt_§l, (1970) demonstrated the effects of nitrogen
insufficiency in a series of experiments in which minimal protein
diets containing urea were fed at 2-hour intervals. They fed diets
containing 3.3%, 5.9%, 11.4% and 21.0% crude protein levels to sheep
and obtained microbial protein estimates of 9.1, 10.5, 12.8 and 13.3 g
per 100 g DOM, respectively. Ruminal organic matter digestion was
similar in all four treatments. Subsequent experiments (Hume and Bird,
1970; Hume, 1970a, 1970b) showed that rumen microbial protein synthesis
can be maximized by the inclusion of sulfur, branched—chain fatty acids
and readily degradable plant proteins to the diets. These workers
reported estimates as high as 22.5 g of microbial CP/lOO g DOM. Another

33F uptake

group (Bucholtz and Bergen, 1973), from jn_vitro studies of
by washed rumen bacteria, estimated that 22.0 g of microbial protein
per 100 gm DOM were synthesized in a 4-1iter sheep rumen. Subsequent

jfl_ngg_studies in our laboratory based on the rate of 35

$04 incorpo-
ration into microbial protein in sheep fed high concentrate diets (13%
CP) at 2-hourly intervals, gave estimates of 19.3 to 25.7 g microbial
crude protein per 100 gm DOM (Isichei and Bergen, 1975--unpub1ished
data).

It is apparent from Table 1, that microbial crude protein
yields (per 100 gm DOM) are lower for high concentrate rations and

processed feeds (Prigge et_al,, 1978; Cole gt_al,, 1976; Drskov gt a1,-
1972), than for high roughage rations, or feeds with a high roughage

11

content (Coelho da Silva g__gl,, 1972a, 1972b; Ulyatt _t__l,, 1975;
Kennedy gt_gl,, 1976). This observation is related to the rate of
digesta flow. It has been shown in in_vjtrg_continuous culture studies
that the efficiency of microbial cell synthesis, or growth rate
increases with increase in turnover or dilution rate (Isaacson _t_al,,
1975). This is due to a decline in the energy expended by microbial
cells for maintenance (Stouthammer, 1976). It is not unlikely that
such situations also occur in the rumen, where it has been demonstrated
that rumen liquid turnover rates are slower for concentrates than for
forage diets (Hungate, 1966). It has also been reported from a few
jn_vivg_passage studies, that as the fractional rate of liquid outflow
from the rumen increases, the efficiency of ruminal microbial protein
synthesis (9 CP/lOO g DOM) also increases (Cole e_t_11_., 1976; KrOpp g_t_
21,, 1977a, 1977b).

Even though the amino acid composition of mixed rumen bacteria
appears to be relatively constant (Purser and Buechler, 1966), their
macromolecular composition on a dry matter basis do vary. Crude protein
content of mixed rumen bacteria vary from 32 to 57% (Smith, 1975;
Hungate, 1966). These values are lower than the 60 to 65% CP often
used. There are also some variations in the carbohydrate (polysac-
charide) content of rumen bacteria with time after feeding (Walker
and Nader, 1970) and with the starch and soluble sugar content of
the feed (McAllan and Smith, 1977). All the above variations are

bound to affect the microbial dry matter values obtained from yield

experiments. However, despite all these variable inputs, it is still

12

possible to estimate with a reasonable degree of accuracy, the
efficiency of ruminal microbial crude protein synthesis.

With a knowledge of the factors regulating rumen microbial
growth, the fermentation pathways predominant in the rumen, and the
amount of ATP generated per unit of substrate oxidized, the actual

efficiencies of microbial cell yield can be estimated.

Energetics of Rumen Microbial Growth-«YATP

It is evident from previous discussions that energy supply
forms the basis for all rumen microbial activity. Synthesis of new
cell material and maintenance of homeostasis both depend on the amount
of energy available to the organism, and with most ruminant rations,
energy availability constitutes the most common limitation to maxi-
mizing microbial output from the rumen (Owens and Isaacson, 1977).

Rumen fermentation can be likened to an open continuous flow
system (Hungate, 1966), analogous to a chemostat (Bergen and Yokoyama,
1977). This fermentation model has characteristic features that
include: the continual supply of substrates and end-products removal,
buffering capacity, low electrical potential (relative absence of
oxygen) and temperature control (Bergen and Yokoyama, 1977). Bacterial
output from the rumen is dependent on two factors:

1. Bacterial concentration or population; and

2. Growth rate (or dilution rate) (Owens and Isaacson, 1977).
Some of the nutrient and ecological factors that regulate rumen
bacterial numbers or population have been mentioned above, most

of the data having been obtained from jn_vivo and jg_vitro experiments.

13

On the other hand, the bulk of our current knowledge on the factors
that influence the growth rate and efficiency of microbial cellular
yields were obtained primarily from jn_vjtrg_studies.

Bauchop and Elsden (1960) first demonstrated from batch
culture experiments that the amount of microbial growth was directly
pr0portiona1 to the amount of ATP that could be obtained from the
degradation of an energy substrate, provided that energy was the
rate-limiting nutrient. They introduced the YATP concept, defining
it as the dry cellular yield of microbial cells (9) per mole of ATP
generated. It is clear that such a calculation is possible only when
the substrate fermentation pathways for the organism in question are
known. A knowledge of the catabolic pathways and steps at which ATP
is synthesized by the organism, makes possible the estimation of the
total amount of ATP formed for each mole of the substrate oxidized.
The absence of oxidative phosphorylation in the rumen, however, impedes
a complete oxidation of energy substrates, thus severely limiting the
amount of ATP generated. Based on Bauchop and Elsden's data (Bauchop
and Elsden, 1960), and from the work of several others (Stouthammer,
1969; Payne, 1970; Forest and Walker, 1971), it was pr0posed that
YATP was a biological constant for anaerobic microorganisms. Hence,
there must be an upper limit for anaerobic microbial cellular growth
(Hungate, 1966). A YATP value of 10.5 was generally accepted. Sub-
sequent studies (Stouthammer and Betterhausen, 1973; Hobson and Sommers,
1972, Isaacson §t_al,, 1975), however, showed that YATP was variable

for anaerobic organisms grown in continuous culture. So also is the

14

Y substrate which is defined as the molar growth microbial yield,
per mole of substrate fermented.

Reasons proposed for this variation in YATP include the
following (Stouthammer, 1976; Owens and Isaacson, 1977):

1. Differences in bacterial macromolecular cellular composition
(variable ash and polysaccharide content).

2. Transfer of metabolic intermediates between bacterial species
in mixed cultures or in the rumen, thus enhancing the efficiency
of substrate utilization and cell synthesis.

3. Specific growth rate or dilution rate influences YATP values
and consequently the molar growth yields. Increases in dilution
rate in an energy-limiting chemostat (Stouthammer, 1976;
Isaacson gt_al,, 1975) have been shown to correspondingly
increase YATP' Stouthammer and Bettenhausen (1973) determined
the YATP for A, aerogenes at various growth rates (0). By
plotting the relationship between YATP and dilution rate, they
obtained a maximum YATP value (YNTP) of 25.5, close to the
theoretical upper limit. The difference between the observed
and theoretical values represents the energy expended for
maintenance.

4. The availability of cell components would also influence cell
yields. Synthesis of required precursors and other cellular
materials involves energy expenditure. It was shown ig_yitrg
that complex media in which higher specific growth rates could

be obtained, gave higher YATP values than minimal media

(Stouthammer, 1976).

15

5. YATP is also related to the amount of available energy
utilized by the bacteria for cell maintenance (cell turnover
and maintenance of homeostasis). The amount of the energy
which is used independently of growth is called the maintenance
coefficient. Higher YATP values were obtained at higher dilu-
tion rates due to a dilution of maintenance. It has been shown
that slow-growing organisms have a much higher maintenance
coefficient. Stouthammer (1977) suggested that the low YATP
values obtained in slow-growing cultures may be due to the
uncoupling of growth, especially when bacterial cultures are
grown in minimal media or in the presence of inhibitory com-
pounds. Thus, the energy realized from substrate catabolism
may not be completely used for new cell synthesis and mainte-
nance of homeostasis. The discovery of the presence of
ATPase(s) in certain anaerobes may indicate that the excess
ATP produced under the above circumstances, would be

dissipated (Stouthammer, 1976; 1977).

Additional factors which by influencing molar growth yields
would influence the efficiency of microbial growth include, the
pathway for the degradation of the energy substrate, and the possibility
for anaerobic electron transport during fermentation. Cytochrome b has
been found in Selenomonas ruminantium, Propionibacteria and Anagrgf

vibrio lipolytica and functions in electron flow-linked (oxidative)

 

phosphorylation. Some other membrane-bound primitive electron transport

systems have been found in some other anaerobes, thus implying that

16

additional ATP could be produced in those organisms during pr0pionate
formation (Stouthammer, 1976). On the other hand, it is not impossible

that ATP-conserving schemes, such as those identified in Bacteroides

 

fragilis, a human-intestinal anaerobe (Macy gt_gl,, 1978), do occur

in rumen bacteria. In B, fragilis, the PPi-dependent 6-phosphofructo-
kinase utilizes inorganic pyrophosphate instead of ATP, for the forma-
tion of fructose-1, 6-diphosphate during glucose catabolism. Carboxy-
lation of PEP to oxaloacetate by PEP carboxykinase was also shown to
occur in the same bacteria, with the concommitant formation of ATP
(Macy gt_al,, 1978). Further studies are needed to determine whether

such in vitro pure culture findings do occur j__vivo with mixed rumen

 

bacterial populations.

Digesta and Non-Ammonia-Nitrogen Passage

 

The value to the animal of dietary protein conversion into
microbial cells, is largely dependent on the relative biological
values of the protein from both sources, although the importance
of a viable microbial population to fiber digestion and NPN utilization
cannot be overlooked. According to Smith (1969), 50 to 80% of dietary
protein is believed to be degraded in the rumen, so that the flow of
total protein should reflect microbial synthesis. Previous studies
have shown that mixed rumen bacteria have a high biological value and
digestibility (Purser, 1970; Bergen gt a1., 1968).

Generally, the rates of digesta and non-ammonia-nitrogen
passage from the rumen are both a function of the rumen dilution rate

and the rate of feed degradation. Increased food intake, feeding

17

frequency or processing of feed are some factors that have been
reported to influence the rate of digesta flow (Prigge gt_al,, 1978;
Coelho da Silva gt_al,, 1972a). Particle size and specific gravity,
however, form the basis for the selective retention of feed particles
in the rumen. Here, the extent of comminution and degradation of
roughages determines their rates of exit. With high concentrates

and more digestible feeds, the rates of microbial digestion of soluble
carbohydrates and proteins vastly exceed the rate of digesta flow or
dilution rate. This results in a greater microbial contribution to
post-ruminal digesta. The more insoluble (less degradable) proteins
such as zein or fish meal, and those high quality feed proteins pro-
tected from extensive rumen microbial attack (e.g., formaldehyde-
treated casein or soybean meal), leave the rumen at the rate of digesta
flow, thus increasing the extent of feed protein bypass (Sharma and
Ingalls, 1974; Chalupa, 1977; Bergen and Yokoyama, 1977; Williams and
Smith, 1976). Table 1 summarizes the nitrogen outflow data obtained
by several workers using different marker combinations to quantitate
total crude protein and microbial protein passage. It is difficult
to compare all the results obtained by these workers since, in most
cases, the experimental animal species, the level of intake, type of
ration, energy and protein intake differ between experiments. Also,
the validity of any one marker used over another is difficult to
establish. However, it is still possible to make some quantitative
assessments of the effects of a dietary regimen on the rate of feed
protein degradation, microbial protein output and nitrogen passage

to the lower gut.

18

Level of Feed Intake and NAN Flow

 

Few studies have been done with the primary objective of
examining the influence of the level of dry matter and protein intake
on the components of nitrogen reaching the abomasum. Using bacterial
nucleic acid -N to estimate microbial protein flow, and Cr203 to quan-
titate particulate matter flow, Coelho da Silva gt_gl, (1972a) (see
Table 1) compared the effects of high and low levels of dried rye grass
intake (19% CP) on nitrogen passage in sheep. They reported a net gain
of 6 to 18% (with low intake) and 15 to 16% (with high intake) in the
total nitrogen reaching the duodenum. Bacterial protein passage (as
a percent of total duodenal CP) was higher for the low intake than for
the high intake level (55 vs. 48%). But total bacterial protein passage
was greater for the high forage intake (156 vs. 104 gm). Microbial
nitrogen (Protozoa plus bacteria) contributed 86.8% and 76.3% to total
duodenal nitrogen, for the low and high intake levels, respectively.
NAN passage was not calculated, but the NH3-N levels averaged 5.6%
and 4.4% of total nitrogen flows for the low and high feeding levels,
respectively. If the NH3N levels are accounted for, duodenal NAN
passage showed no net ruminal N losses for the low feed intake, and
a 10% increase for the high intake, over their respective N-intakes.
Extensive ruminal nitrogen loss would have been expected with a diet
containing 19% dietary crude protein as was the case in the above
experiment. The results could probably be explained by the fact that
the rye grass was fed in ground or pelleted form, thus decreasing

digesta transit time and consequently rumen ammonia loss. Nicholson

19

and Sutton (1969) (see Table 1) fed hay plus flaked maize to sheep

at 0.9, 1.7 and 2.3 X maintenance and obtained a 3% net loss, 29% and
53% net gains, respectively, in total nitrogen flow (as % of N-intake).
The dietary nitrogen effect was confounded in this study because the
rations contained 14%, 12% and 11% CP, respectively, for the three
levels of intake, due to a progressive increase in dietary starch
content. No microbial protein values were reported.

The effect of dietary nitrogen and energy levels are more
clearly demonstrated in two studies (Leibholz, 1972; Leibholz and
Hartmann, 1972) in which sheep fitted with rumen and duodenal cannulae,
were fed barley roughages supplemented with various levels of lucerne,

casein and gluten. The diets are as follows:

1. Medium nitrogen, lucerne-~13.2% CP;

2. Low nitrogen--l.8% CP;

3. High nitrogen, lucerne plus casein-~23.8% CP;
4. Medium nitrogen, casein--12.9% CP;

5. Medium nitrogen, gluten--15.0% CP; and

6. Medium nitrogen, low energy--14.2% CP.

Diets l to 5 contained an estimated metabolizable energy (ME)
level of 1.86 mcal/kg. Diet 6 had a reduced ME level (1.34 mcal/kg),
and diet 2 was not supplemented with nitrogen. NAN passage were 64%,
339%, 31%, 66%, 60% and 50% of the dietary nitrogen intake for diets l
to 6, respectively. All the diets (except 2) showed net losses of feed
nitrogen in the rumen. Microbial nitrogen outflow were: 43%, 53%, 72%,

76% and 56% of total duodenal nitrogen for diets l, 3, 4, 5 and 6,

20

respectively. All the duodenal nitrogen in those sheep fed ration 2
was of microbial origin. Their data seem to indicate that for growing
ruminants fed sufficient energy in a 12-15% crude protein ration,

60 to 80% of the nitrogen that reaches the lower tract is probably
microbial. However, the extent of dietary nitrogen loss in the rumen
in the above studies indicate that dietary energy may have limited the
efficiency of ammonia utilization with those rations. Results with
diet 2 seem to indicate a high degree of urea-N recycling in the rumen.
Dietary nitrogen was more efficiently utilized for microbial growth and
nitrogen was probably the rate-limiting nutrient. Conclusions on the
effects of protein concentration in this study are, however, limited
because the effects of dietary protein source were not determined.

In a more recent passage study with abomasally cannulated
steers, the rations (all silage and corn-corn silage) were variously
supplemented with soybean meal (SBM) to crude protein levels of 11 to
16% (Crickenberger §t_al,, 1979). Total nitrogen passage (as a percent
of nitrogen intake) were: 79.2, 66.7, 88.4 and 67.8% for the 11.3,
12.8, 12.9 and 16.4% CP rations, respectively. NAN passage (percent
of nitrogen intake) were: 70.6, 50.9, 77.5 and 57.5% for the same
respective rations. There were considerable apparent ruminal nitrogen
losses in all four treatments. This may be due to variations in
abomasal spot sampling and poor marker (lignin) recoveries in that

experiment. Microbial protein was not estimated.

21

Dietary Protein Source and NAN Passage

The effects of dietary nitrogen source on nitrogen passage
were studied in a series of experiments with sheep fitted with rumen
and omasal cannulae (Hume, 1970b). In this study, a semi-purified
basal diet was supplemented with urea and higher VFA's (A), casein (B),
gelatin (C) and zein (D). Urea supplied 50% of dietary nitrogen in
diets B, C and D. Total crude protein averaged 14% in all rations.
Total nitrogen flow to the omasum (percent of N-intake) were 89.9,
107.9, 100.0 and 139.7% for A, B, C and 0, respectively. NAN passage
(percent of N-intake) were 82.5, 100.6, 91.8 and 135.0 for diets A, B,
C and 0, respectively. Microbial nitrogen flow (percent of total omasal
NAN) were 92.3, 91.0, 86.3 and 65.4 for the same respective dietary
treatments. Feed nitrogen bypass and total amount of crude protein
reaching the lower gut were markedly greater for zein diet than for
the other nitrogen sources. There was a net gain in omasal nitrogen
flow in the last three treatments. But some of the increase can be
accounted for by the relatively high omasal NH3-N levels (11 to 20 mg %)
obtained. Most of the NH3-N, however, would be absorbed before the
digesta reaches the abomasum (Nolan, 1975).

In another experiment, Hume (1974), compared the effects of
four protein sources (fish, lupin, peanut and soybean) on NAN and
microbial protein passage in sheep. These protein supplements were

35S label was used as the microbial

added to high concentrate rations.
marker and 51Cr.EDTA for digesta flow estimation. There were no sig-

nificant differences between treatments in the rates of digesta passage

22

to the duodenum. However, flow of NAN was greater for soybean meal
(18.2 g/day) than for lupin meal and peanut meal treatments (14.5 and
16.2 g/day, respectively). NAN flow for the fish meal treatment was
20.6 g/day. There were net gains in dietary nitrogen intake at the
duodenum with all four treatments. Abomasal nitrogen flow increased
by 22.4% over the N-intake for peanut and SBM supplements. The lupin-
meal fed sheep showed only a 5% increase in duodenal nitrogen over their
N-intake levels, while the fish meal-fed animals gained 25%. Microbial
contribution to duodenal NAN were the same (60%) for the lupin and
peanut meals, 50% for soybean and 40% for the fish meal treatment.
Dietary CP bypass was highest with the fish meal treatment (71%).
Soybean meal fed animals had 61% protein bypass, lupin meal and peanut
meal had only 35 and 33%, respectively.

In another passage study (Kr0pp gt 21,, 1977a), steers fitted
with rumen and abomasal cannulae were fed low quality roughages,
supplemented with soybean meal or soybean meal plus urea (with urea
replacing 0, 25, 50 and 75% of the soybean). All the rations were
isocaloric and isonitrogenous. Microbial protein production was
relatively constant regardless of nitrogen source. But, there were
net nitrogen losses from the rumin with all the rations. Abomasal NAN
decreased as urea partially replaced soybean meal. Feed-N bypass values
were 35.7, 29.0, 30.0 and 24% of total nitrogen intake for 0, 25, 50
and 75% urea supplemented rations, respectively.

In a second study (Kr0pp et_al,, 1977b), the steers were fed

cottonseed hulls supplemented with soybean meal or urea. Protein

23

levels were 11.1% (no urea), 8.5%, 10.8% and 12.6% for the four
treatments, and the rations were isocaloric. There were no statistical
differences in total abomasal nitrogen flow, but they reported net gains
in nitrogen reaching the abomasum with all treatments. An estimated 20%
of SBM-nitrogen escaped ruminal degradation. In both studies (Kr0pp
gt__l,, 1977a, 1977b) RNA was the microbial marker and lignin was used
to determine digesta flow.

Smith gt_al, (1978) estimated the amount of microbial and
dietary nitrogen entering the duodenum in steers fitted with rumen and
duodenal cannulae. The rations consisted of: flaked maize and hay (A);
flaked maize and straw--supplemented with decorticated groundnut meal
(8); or fishmeal (C); or heated soybean meal (0); or raw soybean meal
(E); or dried grass (F). All the rations contained 15 to 16% CP levels,
except for rations A and F which had 8.0 and 18.0%, respectively. Duo-
denal NAN passage (percent of nitrogen intake) were 71.0, 85.5, 85.7
and 76.7 for rations B, C, D and E, respectively. Treatment A resulted
in a net 5% gain in NAN output. Although there were no significant
differences among the dietary treatments, decorticated groundnut meal
tended to produce smaller amounts of microbial nitrogen output.
Microbial protein passage was the same (54 and 56% of duodenal protein)
for the groundnut meal and the fish meal, respectively. With heated
soybean meal, microbial protein contributed 61% to the total duodenal
protein. Raw soybean meal gave the highest level of microbial protein
(70%). Feed protein bypass was similar for groundnut meal, fish meal
and heated soybean meal (average 28%), and less than 18% of the raw

soy protein bypassed the rumen.

24

All the above data suggest that dietary protein source and
levels may markedly alter NAN passage and ruminal microbial protein
output. The indication is that less soluble feed proteins more
resistant to rumen degradation are more prone to enter the abomasum
undergraded (or partially degraded). Microbial protein synthesis per
unit of organic matter fermented in the rumen appears to be less
affected by the protein source. Variations in this parameter probably
reflect the extent of energy supply and rumen turnover (Theurer, 1979).

Forage to Concentrate Ratios and NAN
Flow

 

Data on the effects of forage to concentrate ratio on NAN
passage and microbial flow are rather limited. Forage rations are
believed to have a higher fluid dilution rate than concentrates
(Hungate, 1966). Addition of roughages to high concentrate ration
elevates the turnover rate of the ruminal fluid and some solids,
probably through increased saliva flow or reduced fluid space in
the rumen (Owens and Isaacson, 1977).

The preportions of bacterial and dietary protein entering
the duodenum of sheep fed a perennial ryegrass, or a short-rotation
ryegrass or white clover were assessed in a study in which 2,6-
diaminopimelic acid was used as the bacterial marker (Ulyatt _t_gl,,
1975). About 70% of all the dietary protein in the fresh forages were
degraded in the rumen. Bacterial contribution to duodenal NAN were:

43.1, 57.1 and 52.9% for the perennial rye, short-rotation ryegrass

and white clover, respectively. Increased organic matter intake

25

increased duodenal NAN passage, even though there were extensive
ruminal NH3-N losses with all treatments.

Cole §t_al, (1976) fed four levels of cottonseed hulls
(0, 7, 14, 21%) to abomasally cannulated steers on corn-based diets.
Abomasal N passage was 80 and 77% of the dietary N-intake for the
steers fed 0 and 7% cottonseed hulls, respectively. The steers that
received 14 and 21% cottonseed hulls had net abomasal nitrogen gains
of 18 and 7%, respectively. Microbial nitrogen flow was lowest with
the all-concentrate ration, increasing with increase in the roughage
content of the rations. Although an increase in the grain content of
a roughage ration is known to depress the dry matter digestibility of
the roughage component (associative effects of feeds), in contrast,
increase in the roughage content of a concentrate ration alters rumen
turnover rate, thereby increasing the rate of microbial growth (Owens

and Isaacson, 1977).

Feed Processigg and NAN Passage

 

The effect of physical and chemical alterations of forages
and grains on rumen microbial protein synthesis have both been pre-
viously discussed in this review. Since both affect rumen turnover,
they would also affect the composition of post-ruminal digesta. The
studies of Coelho da Silva gt al. (1972a) previously described, indicate
that a larger percentage of the total protein reaching the duodenum was
microbial in origin when the diets were given to sheep in chapped rather

than in pelleted form (85% vs. 69%). Pelleting of forages seems to

increase their rate of exit from the rumen, consequently increasing

26

the extent of feed protein bypass. Grinding and chopping of forages
also increase their rates of passage (Thomson, 1972). In the latter
situation, however, the rates of organic matter and feed protein
degradation in the rumen would also be increased, in contrast with
pelleted forages. Similar results were obtained with sheep fed chopped,
wafered or pelleted lucerne by the same workers (Coelho da Silva et__1,,
1972b). In that study, feed N bypass (as % of duodenal N) was 68% for
pelleted lucerne, compared to 54% for the chopped or wafered lucerne.

Current thinking is that grinding and pelleting both reduce
apparent rumen digestion (due to increased rate of digesta passage)
and metabolizable energy (ME) content of the forage, although the ME
is utilized more efficiently for growth and fat deposition in pelleted
feeds than in chopped ones (Thomson, 1972), when the organic matter in
pelleted forages is post-ruminally digestible. As a result, the net
energy (NE) of pelleted forages may be as high as that of chopped ones,
or higher, as with legumes, when grinding and pelleting cause only a
small reduction in digestibility (Thomson, 1972).

Concentrates and cereal grains are often processed by such
methods as steam-flaking, dry-rolling, grinding and acid treatment,
to enhance their utilization by the ruminant. Prigge gt Q1, (1978)
evaluated the effects of four methods of processing corn with ruminally
and abomasally cannulated steers fed 78% corn rations. The processing
methods included, dry-rolling (0R), steam-flaking (SF), propionic acid
treatment and ensiling of high moisture whole shelled corn (AHM) and

grinding and ensiling of high moisture shelled corn (GHM). The rations

27

contained an average of 11 to 12% CP. Microbial protein represented
a greater percentage of the abomasal-N for GHM than for AHM (42% vs.
35%), and was similar for DR and SF treatments (38% vs. 40.9%).
Total-N passage was greater for acid-treated whole corn than for all
the others. There were net gains in abomasal-N only with acid treat-
ment and steam flaking (24 and 14%, respectively).

The indication is that grain processing (by acid treatment and
steam flaking) increase organic matter digestion in the rumen, and
improve the efficiency of utilization over grinding and dry rolling
of grains. The above workers (Prigge et 31., 1978) showed that rumen
turnover and efficiency of microbial protein synthesis were greatly
improved by acid treatment of whole shelled high moisture corn.

Influence of Microbial and Digesta
Markers on Passage Studies

 

 

Digesta passage studies often depend on the use of techniques
or markers to monitor digesta flow and microbial contribution to post-
ruminal nitrogen. Microbial markers used include such components of
microbial cells as DAP (in bacteria) and nucleic acids. Digesta
markers may also be a normal component of the feed (e.g., lignin,
indigestible-ADF), or they may be external, relatively indigestible
material incorporated into the feed. The latter group includes soluble
markers such as polyethylene glycol (PEG) and Cr-ETDA, and solid-
particulate markers which include such compounds as chromic oxide
(Cr203), ruthenium (Ru), Lanthanium (La) and Ytterbium (Yb). The
last three are some of the series of rare-earth elements that are

recently being used in digesta passage studies.

28

Soluble and particulate markers are commonly applied
simultaneously to estimate fluid and dry matter flow, respectively.
Such an approach is often necessary because the liquid and particulate
digesta pools exhibit differential flow kinetics (Hungate, 1966), so
that markers that preferentially equilibrate with specific pools may,
in combination, provide increased accuracy in the estimation of total
flow from a reconstituted digesta (Faichney, 1975). Ideally, a marker
should meet the following criteria in order to be useful as an accurate
index of the particular component of digesta it is supposed to estimate
(Faichney, 1975):

1. It must be strictly nonabsorbable;
2. It must not affect, or be affected by, the 6.1. tract or its
microbial population;
3. It must be physically similar to, or intimately associated with
the material it is to mark; and
4. Its method of estimation in digesta samples must be specific
and sensitive, and it must not interfere with other analyses.
The most commonly used non-radioactive digesta markers such as poly-
ethylene glycol (PEG),Cr203 and lignin do not meet all the above

criteria.

Fluid Markers

Water soluble markers such as PEG, Cr-EDTA and Co-EDTA are most
often used to estimate fluid or water turnover in the rumen. However,
neither one of them fully meets those prerequisites of an ideal marker.

Hyden (1955) reported that PEG equilibrates with about 95% of rumen

29

water. But other workers (Czerkawski and Breckenridge, 1969; Malawer
and Powell, 1967) have questioned the validity of PEG as a liquid
flow marker. Even though its laboratory analysis has undergone
several modifications through the years (Malawer and Powell, 1967;
Ingham and Ling, 1978), the problems of PEG's association with dry
matter and solid digesta flow still remain. It was reported that
some PEG is absorbed from the rumen (Downes and McDonald, 1964).
Dobson et_al, (1976) found that PEG absorption was affected by the
osmotic pressure in the rumen. Others (Czerkawski and Breckenridge,
1969) demonstrated that PEG was unevenly distributed when added to
suspensions of food in aqueous buffer solutions in which PEG was
reported to associate with feed residues.

CrEDTA, on the other hand, seems to occupy a larger fluid
volume than PEG (99%), even though it is equally absorbed from the
rumen (MacRae, 1975). This marker (in the labelled form) was also
reported to associate to a variable extent with abomasal digesta.

The consequence of such aberrations in the flow of these three markers
is that liquid flow from the rumen is usually overestimated, resulting
in considerably high rumen dilution rates. Fractional rates of rumen
liquid outflow, when studied with the three markers, is highest with
PEG, and lowest with CrEDTA. CoEDTA is intermediate.

Compounding the above problems are the reliability and repro-
ducibility of Hyden's turbidimetric assay for PEG. PEG analysis, and
most of its modifications, are based on the development of an oil-in-

water emulsion of PEG when exposed to trichloroacetic acid, in the

30

presence of barium ions. Several factors such as temperature, pH,
presence of interfering ions and proteins, vibration, method of mixing
and other factors that affect colloidal solutions in general, determine
the amount and persistence of the turbidity obtained when the final
reagent is added (Ingham and Ling, 1978; Malawer and Powell, 1967).
Co-EDTA, on the other hand, can be analyzed with a fairly high degree

of accuracy.

Particulate Flow Markers

 

Lignin and chromic oxide seem to be the two particulate markers
preferentially used in a lot of passage studies. This is perhaps due
to the fact that lignin is a typical constituent of plant cell walls
and Cr203 is cheap and readily available, even though both have ques-
tionable values as digesta flow markers.

Lignin passage is usually related to digesta components that
have the slowest turnover--that is, if lignin is part of that component
(Bull 92.21:: 1979). The results of some studies indicate that lignin
is up to 10% digestible, most of the degradation occurring in the rumen
(MacRae, 1975). Another view is that lignin undergoes a partial modi-
fication in the rumen resulting in less post-ruminal recovery of feed
lignin. There is nonetheless, a tendency for rumen outflow estimates
based on lignin passage to be less than 100% of the total lignin intake,
and this results in an overestimation of protein flows. A partial
explanation of such discrepancies may rest with the lack of one
reliable and accepted laboratory technique for lignin analysis.

Acid lignin method gives lignin values often lower than those

31

obtained from the KMnO4 procedure. There is concern that the acid
detergent reagent, through its strong peptising ability, dissolves
some lignin (Van Soest, 1975). The extent to which this occurs may
vary between samples. NAN passage studies based on acid lignin/N
ratios could therefore show an inconsistent or variable ruminal
nitrogen losses (Crickenberger gt_al,, 1979).

KMnO4 lignin approach also has its problems. A comparative
evaluation of lignin content of digesta samples obtained from different
segments of the gastrointestinal tract (GIT), when analyzed by this
technique may give impossible results. Rumen and abomasal samples
have to be exposed to MKnO4 for 1% hours, while fecal samples require
up to 5 hours. Fecal samples may also have to remain longer than 30
minutes in the demineralizing solution before filtration becomes pos-
sible, and this strongly acidic solution does dissolve cellulose. The
point here is that KMnO4 technique is also non-specific, and the above
situation could overestimate the extent of lignin flow when residual
digesta components such as cellulose are dissolved during KMnO4 lignin
determination. The net effect, in passage studies, would be an under-
estimation of digesta flow rate, NAN passage and ruminal dry matter
digestion. Lignin would not become fully accepted as a digesta passage
marker until its fate in the GIT is better understood, and a more
reliable method for its laboratory estimation developed.

Chromic oxide is also regarded as a poor passage marker in some
circles because its physical properties do not correspond to those of

the solid-particulate fraction of digesta (Faichney, 1974). Various

32

alterations in the physical form of this marker reported in several
studies (e.g., impregnation in paper, baking and coarse grinding of
Cr203-flour mix) were simply aimed at simulating the particle size and
specific gravity of the particulate digesta pool, in order to obtain a
more uniform Cr203 outflow from the rumen. The independent movement
of Cr203 may not present a great problem when continuous collection
techniques are used, with duodenal re-entrant cannulae (MacRae, 1975).
Recoveries of this marker in various studies range from 56 to 100%
(MacRae, 1975; Sutton gtual., 1976). This has necessitated the use of
fecal Cr203 to obtain a corrected or adjusted chromium flow, especially
when short-term spot sampling and 24 hr. re-entrant collections are
obtained (MacRae, 1975). Depressions in dry matter flow as high as
20 to 30% due to duodenal collection procedures have been reported
with fresh forages when Cr203 was used (Ulyatt and MacRae, 1974).
With abomasal T-shaped cannulas, the use of Cr203 invites the problem
of obtaining a sample truly representative of the digesta passing the
point of cannulation. The adequacy of longer-term samplings in cor- '
recting this problem is not fully known. Rumen outflow values obtained
with Cr203 data often exceed 100% and has resulted in abomasal-N out-
flows that are greater than the amount ingested. Lignin-based data,
on the other hand, underestimates nitrogen passage (Theurer, 1979).

The current approach to passage studies is to use a dual marker
(liquid and particulate types), each of which should be closely asso-
ciated with the phase of digesta it is supposed to estimate. In this

respect, liquid-phase markers such as CoEDTA and solid-phase ones such

33

as cerium or chromium-mordanted plant cell walls may show some promise.
Rare-earth elements previously mentioned are excellent particulate and
total digesta flow markers when applied with all the metal absorbed to
feed materials. If excess metals are not washed away, these elements
may show some movement between different digesta pools. Their recov-
eries with the particulate matter of forage-fed animals were reported

to be higher than with grain fed ones (Hartnell and Satter, 1979).

Microbial Markers

 

Various methods for measuring the rate of ruminal microbial
protein output generally depend on calculations based on values for
the concentration of a particular microbial component in rumen or
post-ruminal digesta, and the ratio of this component to microbial-N.
In digesta passage studies, it is also essential to determine the
microbial contribution to nitrogen flow, if the extent of ruminal
feed protein degradation is to be assessed.

Microbial markers used have included DAP, RNA, DNA and radio-

35

isotope labelled compounds such as S (labels sulfur amino acids in

32

microbial cells), P label (for RNA's and microbial phospholipids)

15N (labels nitrogen components in microbial crude protein). All

and
the above methods depend on sampling of rumen and/or post-ruminal
digesta at specified times. Microbial markers also suffer from the
same limitations as digesta markers, since their effectiveness depends
on obtaining digesta samples that represent the true proportions of
the various components within that segment of the digestive tract.

This may lead to significant errors in the quantitative estimates

obtained.

34

It has been reported that different strains and species of
bacteria show a considerable variation in their DAP content (Purser
and Buechler, 1966). DAP-N/Total-N ratios in mixed rumen bacteria so
far reported range from 0.5 to 1.15 (Smith, 1975). Another problem
with the use of DAP is that this technique fails to account for the
contribution of protozoa to NAN passage, due to the absence of that
amino acid in protozoa. This would lead to an underestimation of the
microbial component of ruminal outflow, regardless of the extent of
protozoal passage from the rumen.

Nucleic acids, particularly RNA, is almost widely accepted
as a better index of microbial passage. DNA reflects the number of
microorganisms present, whereas RNA is associated more closely with
protein synthesis (El-Shazly and Abou Akkada, 1972). It is therefore
believed that total nucleic acid-N and the total microbial-N are pro-
portional. However, there is a greater variability in DNA-N/Microbial-
N ratios than in RNA-N/Microbial-N ratios (McAllan and Smith, 1971).
Therefore, RNA is more generally used as a microbial marker in passage
studies. The use of RNA is also based on the assumption that dietary
nucleic acids are rapidly degraded in the rumen (McAllan and Smith,
1969; Smith and McAllan, 1970), so that all the nucleic acid present
in the post-ruminal digesta should be of microbial origin. Smith gt__l.
(1978) compared microbial protein estimates based on RNA-N of duodenal

digesta in steers, some of which received doses of 32P-labelled P0

32

4.

They reported that estimates based on P-labelled RNA were on the

average 85% of those based on total RNA. They attributed these

35

differences to the presence of small amounts of dietary RNA in the
duodenum. This questions the basic assumption that all the dietary
RNA‘s are degraded in the rumen.

Another major problem with the use of RNA as the microbial
marker in passage studies lies in the laboratory methods for its
estimation. Alkali digestion of post-ruminal digesta as outlined by
McAllan and Smith (1969) is nonspecific for RNA and there is evidence
of contamination of separated ribonucleotides (in solution) by plant
lipids, pentose polymers and pigments most of which are not removed by
extraction in organic solvents prior to alkali hydrolysis. Further
purification of the RNA extracts by ion-exchange could still leave
remnants of peptides, sugars, proteins and other interfering materials
which absorb in the U.V. range of 260 nm, and also react with orcinol

reagent (Munro and Fleck, 1966).

35 15

The use of labelled markers such as S and N (urea) have
their own problems. Besides the common problem of obtaining samples
representative of the microbial population in that part of the GIT,
other concerns include, the ability to establish a steady state of
the labelled compounds within the rumen and corrections for extensive
urea, and some sulfur recycling occurring within the rumen (Nolan,
1975; Hume and Bird, 1970; Bird, 1974). If pool sizes are changing,
the appearance of labelled metabolites may show a particular metabolic
pathway but may not necessarily allow it to be quantified, especially
when recycling process is involved (Bray and Till, 1975). Other con-

siderations with all microbial markers are the effects of diurnal

variations on the composition of rumen microbes, since marker

36

concentration is usually determined in relation to the microbial cell

mass (McAllan and Smith, 1977).

Monensin
Monensin is a polyether antibiotic produced in fermentation

broths by a strain of Streptomyces cinnamonensis. It is known to

 

inhibit gram-positive microorganisms (Haney and Hoehn, 1967), and since
its discovery, has been used as an antibacterial agent effective in
controlling coccidiosis in poultry (Shumard and Callender, 1967).

This compound was discovered to cause a shift jg_yitrg_in the
molar proportions of VFA's in favor of propionate, without affecting
total VFA production (Richardson gt_al,, 1974; Richardson gt_a1,,
1976). Subsequent jg_yiyg_studies with rumen-fistulated steers
confirmed the above monensin effect. In feeding trials, it was
reported that monensin also improved the performance of beef cattle
fed different rations (Raun gt_al,, 1976; Potter gt_al,, 1976a, 1976b).

. After considerable investigations into the safety and efficacy
of this antibiotic, it was approved by the Food and Drug Administration

in 1975 for use as a feed additive in feedlot rations.

Monensin and VFA Production

 

Richardson gt_gl, (1974) conducted jn_yit§g_experiments to
characterize the effects of monensin on ruminal fermentation. They
reported that addition of 1.0 ppm of monensin increased propionate
production by 45%, without concomitant changes in total VFA levels.

This response was obtained with rumen fluid from high grain-fed

37

cattle or sheep when incubated with high grain feed as the substrate.
A similar response was also obtained by the same workers using rumen
fluid from pasture fed cattle, and high roughage feed as substrate.
The same workers, designed another series of jg_yjtrg studies
(Richardson gt_al,, 1976) to determine the optimum dose-~response level,
of monensin. Monensin was added at 0.1, 0.25, 0.5, 1.0, 5.0 and 25.0
ppm to concentrate--or high roughage—-incubation systems, containing
rumen innocula from cattle fed the same respective rations. With
concentrates, monensin reduced acetic, isovaleric and valeric acid
production at 2 1 ppm. They reported an average of 50% increase in
propionic acid production at 1.0 ppm monensin. With high roughage
substrates, acetate production was reduced at 5 ppm monensin, butyrate
at 1 and 5 ppm, while propionate levels increased at 1 and 5 ppm.
Total VFA (TVFA) production remained unchanged, indicating that equal
quantities of substrates were fermented in comparison with the controls.
A preliminary jg_yjyg_study was also conducted by the above
workers (Richardson gt_al,, 1974) in which monensin was fed at 25 to
500 mg per day for 3 weeks to rumen fistulated cattle. They reported
a 52% increase in ruminal propionic acid when 200 mg/day monensin was
fed. This increase persisted throughout the trial. In five other
jg_vjyg_dose-response studies (Richardson et_al,, 1976), ruminally-
cannulated steers were fed concentrate rations with monensin given
at 25, 50, 100, 200 and 500 mg/head/day. The results obtained were
similar to those found jn_yjtrg, but the effects on TVFA were not

consistent. Acetate decreased at dosages 2 100 mg/head/day, while

38

propionate increased , and butyrate decreased at each dosage. These
shifts in VFA patterns were shown to persist throughout the 148-day
trial. Other studies (Dinius et_al,, 1976; Perry gt_al,, 1976) have
confirmed this shift in the molar proportions of VFA towards propionic
acid in monensin-fed ruminants.
0n the basis of this redistribution of VFA's associated with

the addition of monensin to the ration, Richardson gt_al, (1976)
calculated a theoretical energy savings to the animal of 5.6% (assuming
a 10 moles per 100 moles increase in ruminal propionate). They stated
that fermentation efficiency was improved by monensin treatment as a
result of increased recovery of metabolic hydrogen in the rumen, in
the form of VFA's. Raun gt_al, (1976), however, observed that even
though propionate fermentation is energetically more efficient than
acetic and butyric acid fermentations (Hungate, 1966; Wolin, 1960),
it did not account for all the improvement in performance they
obtained in feed lot studies. They, therefore, suggested that
increased propionate may also:

1. lower heat increment (Smith, 1971);

2. spare amino acids normally used in gluconeogenesis (Reilly

and Ford, 1971; Lang et_gl,, 1967); and

3. stimulate body protein synthesis (Eskeland gt al,, 1974).

Feedlot Studies

Since the recognition of the effects of monensin on beef cattle
performance, several other studies have been conducted to determine its
optimum dose-response level and the performance of animals fed this

antibiotic, under practical feedlot conditions.

39

Raun gt 31. (1976) fed monensin at levels ranging from O to
88 ppm to steers on high energy rations and reported average daily
gain (ADG) equal to or greater than the controls (except for the 88
ppm level). Feed intake progressively declined with increase of
monensin in the rations, while the efficiencies of feed conversion
improved. The result of these studies (Raun gt_al,, 1976) indicate
that 33 ppm was the optimum level for feeding monensin to cattle.

At this level, intake declined by 13.1%, while feed efficiency (F/G)
improved by 17%.

In another study, Potter gt_gl, (1976a) fed monensin at 0
to 400 mg/head/day to beef cattle on pasture and green chop. They
reported a 17% improvement in gain and a 20% improved feed efficiency
when monensin was fed at 200 mg/head/day. Improvements in performance
were obtained at 100 to 300 mg monensin per animal per day. Further
studies in which carcass composition was examined, Potter gt_al,
(1976b) showed that monensin had minimal effects on dressing percent
and other carcass characteristics.

The results of 29 university feedlot studies (growing, finish-
ing and growing-finishing cattle performance studies) summarized by
Goodrich gt_gl, (1976) showed that monensin at 50 and 100 mg/head/day
improved ADG; the other levels (200 to 400 mg/head/day) were not
different from the controls. Dry matter intake and FIG also declined

with increasing levels of monensin. Carcass composition was not

significantly altered by feeding monensin at 200 or 300 mg/head/day.

40

The reduction in dry matter intake observed with monensin in
high energy rations prompted speculations that dietary protein intake
may need to be adjusted when this additive is fed to growing ruminants.
Several studies were therefore conducted to determine the effect of
ration protein levels and sources on the performance of cattle fed
monensin (Dartt gt_al,, 1976; Davis and Erhart, 1976; Hanson and
Klopfenstein, 1979; Gates and Embry, 1977). Dartt gt_al, (1976)
reported that withdrawal of supplemental protein from the rations
of growing steers resulted in a significant reduction in ADG (17.1%)
and a 12.9% poorer feed efficiency for those steers not given monensin.
In comparison, monensin feeding during protein withdrawal improved F/G
by 11.9% and reduced ADG by only 6.1%.

In a study with urea-supplemented rations, Davis and Erhart
(1976) obtained improvements in F/G when monensin was fed to growing
steers. In contrast, Hanson and Klopfenstein (1979) reported a reduc-
tion in the rate of gain and poorer feed efficiency with the addition
of monensin to urea-supplemented corn and corn—silage rations. Monensin
and brewers grain supplementation to the same basal rations resulted in
significant improvements in feed efficiency (Hanson and K10pfenstein,
1979). The latter studies also showed that the largest responses to
monensin occurred at lower dietary crude protein levels (10.5 vs.

12.5% CP), thus indicating that monensin may spare protein. The

above protein studies (Dartt gt_gl,, 1976; Davis and Erhart, 1976;
Hanson and Klopfenstein, 1979) all indicate that monensin is effective
with different dietary protein sources and that adjustments in protein

levels may not be necessary when monensin is fed.

41

Monensin and Rumen Microbial Growth

 

Effects on Microbial Population
and Growth

 

 

Initial characterization studies with monensin revealed that
this drug inhibits the growth of several gram-positive micro-organisms
(Haney and Hoehn, 1967). Subsequent experiments were therefore con-
ducted by various workers to determine the effect of the antibiotic
on rumen microbial population and growth (Dinius §t_gl,, 1976; Van
Nevel and Demeyer, 1977; Chen and Wolin, 1979).

Dinius gt 21, (1976) in continuous culture fermentation studies
observed small decreases in methane production with monensin in the
media. This decline was transient, however, methane production return-
ing to normal levels with prolonged monensin treatment. A similar
temporary decline was also observed jn_yiyg_by Thornton gt_al, (1976).

Other workers (Van Nevel and Demeyer, 1977) also showed in
31339 that monensin has no direct toxic effects on methanogenic rumen
bacteria. Monensin did not inhibit methane formation from H2 and CO2
by washed-cell suspensions of mixed rumen bacteria, but did inhibit
formate decomposition during carbohydrate fermentation. These workers
(Van Nevel and Demeyer, 1977) reasoned that since only about 18% of
rumen methane is derived from formate (Hungate gt_gl,, 1970), this
may explain the less dramatic inhibition of methane production found
in their studies.

In contrast, Chen and Wolin (1979) found that the sensitivity
of methanogenic bacteria to monensin varies with the specific organism.

However, none of the methanogens they examined was completely inhibited.

42

In most cases, the antibiotic appeared to cause only a delayed growth
response. The studies of Chen and Wolin (1979) were designed to
examine the effects of monensin on the growth of methanogenic and
rumen saccharolytic bacteria in a complex medium containing rumen

fluid. Bacteroides succinogenes and B, ruminicola, both important

 

 

succinate producers, were found to be less sensitive to monensin.

Selenomonas ruminantium was also resistant to the drug. 8, succinogenes

 

 

and S, ruminantium are both cellulolytics, while B, ruminicola uses

 

 

starch and other carbohydrates. Other important cellulolytics

 

(Ruminococci and B, fibrisolvens) were found to be highly sensitive

 

to monensin.

This inhibitory effect on cellulolytic rumen bacteria was
confirmed by Simpson (1978) who found that monensin strongly inhibited
cellulolytic activity in 313:9, when inoculum from animals not pre-
viously exposed to the antibiotic was used. He obtained this response
after.a 48-hour incubation, and the effects of a long-term exposure to
monensin were not examined in the study. On the other hand, Dinius gt
Q1, (1976) reported from a series of in 11539 and jg_yiyg_experiments
that there were no reductions in cellulose, hemicellulose and dry
matter digestibilities when the animals (or inocula from adapted
animals) were exposed to monensin for 21 days. Neither the numbers
of protozoa, total bacteria nor cellulolytic bacterial numbers were
affected by up to 33 ppm dietary monensin.

Chen and Wolin (1979) hypothesized from their studies, and from
others (Dinius gt_al,, 1976) that monensin acts by selecting a rumen

microbial community that produces proportionally more propionate.

43

Monensin and Efficiency of Microbial
Growth

 

Based on the results of jn_yitrg_studies conducted by
Richardson gt_al, (1976), it was demonstrated that monensin increased
propionate production without influencing either the amount of hexose
fermented, microbial cell yield and efficiency, or the production of
metabolic hydrogen (Chalupa, 1977). Other workers, however, reported
significant reductions in microbial efficiency by monensin jg XIEEZ
(Van Nevel and Demeyer, 1977). In the latter study, both the total
and net microbial growth (expressed as milligrams of N incorporated
per 100 u mole of hexose fermented) were severely inhibited by monensin.
Other workers, as previously discussed (Chen and Wolin, 1979) have
reported varying degrees of selective inhibition of different rumen
microbial species by monensin jn_yitrg.

lg_xiyg_data on the efficiency of ruminal microbial growth
are, however, scarce. Poos gt_gl, (1979), in protein passage studies,
reported significant decreases in bacterial-N flow to the abomasum
with monensin in the diet. Ruminal organic matter digestion was,
however, not determined in that study, so that microbial efficiencies
could not be accurately estimated. More jg_yiyg_studies are needed to
determine the effect of this compound on the efficiency of rumen

microbial protein synthesis.

44

Monensin and NAN Passage

In addition to the transient reductions in cellulose and
D.M. digestibilities observed when monensin is added to ruminant diets,
the results of some studies indicate that rumen proteolysis and deamina-
tion may also be inhibited. Reductions in rumen ammonia levels have
frequently been reported with monensin (Dinius gt_al,, 1976; Van Nevel
and Demeyer, 1977; Hanson and Klopfenstein, 1979), an observation that
led to earlier suggestions that the antibiotic may act in the rumen as
a deaminase inhibitor.

Van Nevel and Demeyer (1977) observed a considerable decrease
in casein degradation when monensin was added to incubations of mixed
rumen bacteria. Addition of energy substrates (cellebiose and maltose)
to the media failed to elevate the rate of casein protein breakdown.
They also reported a lowered NH3-N level and increased accumulation
of a-amino nitrogen, which indicate that deaminase activity, rather
than proteolysis may be more inhibited by monensin.

The effect of monensin on jg_yjyg_protein degradation has also
been examined in a few studies (Poos §t_al,, 1979; Lemenager gt_al,,
1979). Poos ££.91: (1979) reported 37% and 55% increases in plant-N
bypass when monensin was added to steer rations supplemented with
brewers' dried grains (806) and urea, respectively. Owens _t__1.
(1979) reported a 22% feed protein bypass when steers fed a rolled
corn-~16% CP ration were given monensin. The two studies thus indicate

that monensin reduces the extent of feed protein degradation in the

rumen .

45

Increases in the amount of NAN reaching the abomasum were also
reported in the same two studies. Monensin-fed steers showed increases
in total-N and NAN flow to the lower gut over their dietary nitrogen
intakes. Poos at al, (1979), however, reported increases in abomasal
amino acid flow only with natural protein supplements. Both the
essential and non-essential amino acids reaching the small intestines
were increased by the combination of 806 and monensin over the controls
(164 vs. 150 gm and 194 vs. 174 g, respectively). Abomasal amino acid
patterns and concentrations should reflect the level of NAN passage to
the lower gastro-intestinal tract (GIT).

Poos at_al, (1979) also reported significant decreases in
bacterial-N flow to the abomasum by the addition of monensin to steer
rations. In comparison with the control treatments, monensin decreased
bacterial-N passage by 32% for the natural protein, and 33% for the
urea supplement. Other workers (Dinius at_al,, 1976; Owens §t_al,,
1979) found no decreases in bacterial numbers or in the amount of
bacterial-N reaching the abomasum of monensin-fed steers.

It seems well-established that monensin reduces the extent of
feed protein degradation in the rumen. The effect of monensin on
rumen turnover, however, still remain to be conclusively determined.
Even though Owens gt_al, (1979) reported a 10% reduction in ruminal
DM digestion with monensin in concentrate rations, they also obtained
a reduced (24%) rumen liquid turnover rate with monensin treatment.
Another group (Lemenager gt_al,, 1978) reported a 30.8% slower rumen

liquid turnover rate and a 43.6% slower turnover of rumen solids in

46

steers fed low quality dry winter range grass, supplemented with 30%
SBM and monensin. They had, however, a 19.6% reduction in feed intake
and also a large decrease in estimated rumen liquid volume for the
monensin treatment. In a second trial in which a high concentrate
ration was limit-fed to steers (Lemenager 25.21:, 1978) monensin

still decreased rumen liquid dilution rate.

In contrast, Poos gt_al, (1979) obtained significant increases
in both the DM and liquid flow to the abomasum per day, with monensin
in both the natural protein--and urea—supplemented rations. This
indicates a faster rumen turnover, unless there were differences in

rumen volumes between treatments.

Monensin--Mode of Action

 

Despite the preponderance of information so far collected on
the effects of monensin in the metabolism of the ruminant, its mech-
anism of action still remains to be elucidated. The energetic benefits
of a shift in the molar ratios of VFA's towards propionate and, there-
fore, the increased recovery of metabolic hydrogen in propionate
coupled with subsequent improvement in the animal's amino acid
utilization, offer only partial explanations for the drug's efficacy.
The advantages of monensin's reduction of ruminal protein degradation
rate can be more fully exploited if the ruminant is given more high
quality plant proteins better utilized in the post-ruminal tract.
However, decreases in the efficiency of rumen microbial protein
synthesis and YATP (Bergen, 1979) could be a disadvantage to the
animal, depending on the nature of the diet and the amount of protein

escaping rumen degradation (Van Nevel and Demeyer, 1977).

47

Reductions in rumen ammonia levels and the overall rate of
protein degradation with monensin, suggest that rumen deaminase
activity, proteolysis and perhaps, peptide transport into bacterial
cells may be some physiological functions inhibited. And since the
inhibition of microbial growth by monensin does not affect the rate
of energy substrate catabolism (Van Nevel and Demeyer, 1977), it seems
plausible to suggest that monensin may uncouple growth from fermenta-
tion. Being an ionophore, monensin could cause an alteration in cell
membrane permeability, thus affecting the rate of substrate transport
into microbial cells.

Monensin is a strong lipophilic cation-binding ionophore
of the acid polyetherin family (Anteunis and Rodios, 1978). Other
polyether antibiotics or ionophores such as lasalocid exert similar
effects on rumen microbial fermentation as monensin (Bartley gt_a1,,

1979; Chen and Wolin, 1979).

CHAPTER III

MATERIALS AND METHODS

Nitrogen Metabolism Studies

 

General Design

Four Hereford steers fitted with abomasal cannulae and weighing
approximately 287.5 kg each, were used in a switch-back metabolism
study to determine the effects of monensin in high concentrate and
high silage rations on digesta and nitrogen passage to the lower
gastro-intestinal tract. During each period, two steers were fed
one type of ration, the other pair receiving a similar ration with
or without monensin (see Table 2). The rations were mixed and fed
twice a day ag_libitum. Feed intake and unconsumed portions were
recorded, subsamples frozen for subsequent analyses. The animals
were individually housed in 91 cm x 244 cm collection pens in an
environmentally controlled metabolism room. They had free access
to water.

Chromic oxide and feed lignin were used as particulate flow
markers, and polyethylene glycol (PEG; MW 4000) used to quantitate the
liquid digesta flow. Chromic oxide was mixed with wheat flour (1:4
ratio) and water added to form a paste which was baked at 100°C until
dry. This baked mixture was then ground coarse in a Wiley mill and

added to each ration at a level of 1% of ration dry matter (Orskov

48

TABLE 2. COMPOSITION OF DIETS USED IN DIGESTA PASSAGE AND NITROGEN
METABOLISM STUDIES

49

 

 

 

International
Ingredient reference no. 1 2 3 4
--------- % of DM ---------

Corn, aerial pt, w. ears,

ensiled, mature, well-

eared, mx 50% mn,

30% dry matter 3-08-153 -- -- 85.00 85.00
Corn, dent, yellow, grain

gr 2 US 4-02-931 58.00 58.00 -- --
Soybean, seeds, meal

solv-extd 5-04-604 4.00 4.00 -- --
Soybean meal-mineral

supplementa -- -- 13.50 13.50
Alfalfa, aerial part dehy

meal, mn 17% protein 1-00-023 20.00 20.00 -- --
Oats, grain 4-03-309 10.00 10.00 -- --
Sugarcane, molasses 4-04-696 4.00 4.00 -- --
Phosphate defluorinated,

grnd 6-01-780 0.48 0.48 -- --
Trace mineral salt 2.00 2.00 -- --
Chromic oxide-four mix 1.00 1.00 1.00 1.00
Polyethylene glycol (MW4000) 0.50 0.50 0.50 0.50
Rumensin premix -- -- -- --
Vitamin A premixc 0.01 0.01 -- --
Vitamin D premix 0.01 0.01 -- --

 

aComposition listed in Table 3 supplements l and 2 used for
diets 3 and 4, respectively.

b

C30,000 IU vitamin A per g.

d

3,000 IU vitamin 03 per g.

Diets 2 and 4 contained 30 g Rumensin per 907 kg air-dry feed.

50

.1iéfl:: 1971). PEG was mixed with the rations at 0.5% of ration dry
matter.

Total digesta flow to the abomasum and nitrogen balance were
estimated during each period. The steers were allowed an l8-day period
to adapt to each ration, followed by nitrogen balance collections for

8 days. After a two-day rest, abomasal samples were collected three

times a day (at six-hour intervals) for a period of four days.

Abomasal Cannula Design

 

A brass mold obtained from Dr. L. D. Satter of the University
of Wisconsin was used to make the cannulae. Clear plastisol (manu-
factured by Norton's Plastic and Synthetics Division, Akron, Ohio) was
placed under vacuum for at least one hour to remove air bubbles. It
was then poured into the preheated mold and allowed to solidify in
the oven at 190°C for about 20 minutes. The mold was then cooled by
immersing it in cold water for several minutes, after which the cast
was removed. Properly prepared cannulae should be pliable, transparent,
amber-colored and free of air bubbles. In a one-stage surgical proce-
dure, the cannulae were inserted through an abomasal incision, and

retained by purse—string sutures.

Rumen Microbial Studies

 

Two Hereford steers fitted with rumen fistulae were adapted for
14 days in sequential order to each of the four rations used in the
digesta passage studies (Table 2). The steers were also housed in

individual stalls in the same metabolism room. They were fed twice

TABLE 3. COMPOSITION OF SOYBEAN MEAL-MINERAL SUPPLEMENTS USED IN
DIGESTA PASSAGE STUDIES AND FEEDING TRIAL

51

 

 

Supplement No.

 

 

 

Ingredient l 2 3
---------------- % of DM ---—------------
Soybean meal (48% CP) 92.70 92.70 92.70 92.70
Defluorinated phosphate 3.00 3.00 3.00 3.00
Ground limestone 2.00 2.00 2.00 2.00
Trace mineral salt 2.00 2.00 2.00 2.00
Vitamin A premixa 0.15 0.15 0.15 0.15
Vitamin 0 premixb 0.15 0.15 0.15 0.15
Monensin (mg/454 g)C —- 125.00 -- 125.00
Elfazepam (mg/454 gm)d -— -— 8.35 8.35
Analysis of Supplement:
Crude protein (%) 49.40 49.40 49.40 49.40
Calcium (%) 2.00 2.00 2.00 2.00
Phosphorus (%) 1.20 1.20 1.20 1.20
Trace mineral salt 2.00 2.00 2.00 2.00

 

a30,000 IU vitamin A per g.

b

cAdded to provide 33 ppm monensin in total diet.

d

3,000 IU vitamin 03 per 9.

Added to provide 2 ppm elfazepam in total diet.

52

TABLE 4. CHEMICAL COMPOSITION OF RATIONSa USED IN DIGESTA PASSAGE

 

 

 

 

STUDIES
Ration no.

Ingredient 1 2 3 4

----------------- % of DM1----------------
Dry matter 93.10 93.15 35.53 35.73
Crude protein 13.10 13.25 13.26 13.20
Neutral detergent fiber 22.50 21.82 44.21 44.15
Acid detergent fiber 10.32 10.04 24.62 24.86
Permanganate lignin 3.09 3.09 3.93 3.93
Organic matter 93.31 93.04 94.38 94.39
Ash 6.70 6.97 5.62 5.62
Polyethylene glycol 0.488 0.544 0.443 0.487
Chromium (mg/g DM) 1.981 2.025 1.781 1.726

 

aActual laboratory determinations of feed samples, each from two
abomasal collection periods.

53

a day ag_libitum and had free access to water. Following each
adaptation period, rumen digesta samples were collected from each
steer at specified times and quickly transported to the laboratory

for processing.

Collection and Preparation of Samples

 

Nitrogen Balance Stugy

 

Following adaptation to each ration, total urine was collected
from each steer into large plastic carboys containing 200 m1 of 18N
sulfuric acid. The acid was added to prevent the liberation of
ammonia and other nitrogenous compounds. Carboys were emptied at
least every two days, the urine volume measured and diluted to 10
liters with water. After mixing, a 10% aliquot was stored in the
cooler. At the end of each collection period, the urine samples were
pooled, thoroughly mixed and a 500 ml subsample frozen for later
analysis.

Feces were collected in a plastic-lined steel trough placed
behind each steer. The feces were weighed, properly mixed and several
subsamples taken from different points in the mixture. Ten percent of
each collection was saved. At the end of the period, subsamples from
each steer were composited and a 10% aliquot was kept frozen for
subsequent analysis.

Several grab samples were taken from each ration as the mixed
feed was being discharged from the mixer. Samples for each steer were
kept frozen until the end of the collection period. They were then
thawed, composited and chapped in a Hobart feed grinder. Sufficient

subsamples (approximately 1 kg) were frozen to be analyzed later.

54

Digesta Passage Studies

After each nitrogen balance, abomasal samples were collected
from each of the four steers. Samples were collected three times a
day at six hour intervals for four consecutive days. The sampling
frequency was designed to give a net effect of a three-hour collection
interval, if the four days were condensed into one (see Figure 1).
Abomasal contents were agitated with air from an air-pump and
approximately 150 ml digesta were collected into a plastic beaker.
Attempts were made to keep in sequence with the wave of contraction
during abomasal emptying. Individual samples were kept frozen in
plastic bottles until the end of each period of collection. The
samples were subsequently thawed, mixed and 50 m1 aliquot from each
steer homogenized and composited. Approximately 200 ml from each
composite were freeze-dried and stored in a dessicator. Both the

dried and the wet abomasal samples were later analyzed.

Rumen Microbial Studies

 

Rumen contents were collected from the two ruminally-cannulated

steers which were fed the same rations used in the passage studies.

The contents of the rumen were mixed by inserting an arm through the
cannula prior to collection. Four hundred milliliter rumen contents
were taken at 2, 4, 6 and 8 hours after the morning feeding. The
samples were collected in vacuum bottles and quickly taken to the
laboratory for processing. Rumen liquor was obtained by squeezing

the samples through two layers of cheese cloth. The solid residues

were resuspended in 0.9% NaCl, and strained again. The liquors were

55

FOUR ABOMASALLY CANNULATED STEERS

26 days adaptation

Day 1--abomasa1 collections--12:OO AM, 6:00 AM, 12:00 PM

Day 2--abomasal collections--3:00 AM, 9:00 AM, 3:00 PM

Day 3--abomasal collections--6:OO AM, 12:00 PM, 6:00 PM

Day 4--ab0masal collections--9:00 AM, 3:00 PM, 9:00 PM

Same sampling schedule as above repeated with each
of the rations, after a 26-day adaptation

Figure 1. Flow Chart of Sampling Prot0c01*
(Digesta Passage Studies).

*Total of four collection periods. During each period, two
steers received one type of ration; the other two received the alternate
ration (with or without monensin). The rations were switched at the end
of each collection period.

56

combined and centrifuged at 150 xg for 5 minutes to remove the protozoa.
The supernatant fraction was next centrifuged at 2000 rpm (500 xg) for
10 minutes, to remove the feed residues. The supernatant was then
centrifuged at 18,000 xg for 15 minutes. The supernatant was discarded
and the residue (bacterial cells) was resuspended in 0.9% saline solu-
tion and centrifuged again. The supernatant was discarded. This
procedure was repeated three times. The resulting pellet was finally
washed in a small amount of deionized-distilled water to remove the
excess NaCl. After the final centrifugation, the bacterial pellet

was frozen and lyophilized. The protozoal fractions were subjected

to a similar series of washings and centrifugation and the pellets

also freeze-dried.

Further Sample Preparations

 

Portions of the feed samples and weighbacks (from the nitrogen
balance and abomasal collections) and fecal samples, were dried at 60°C
to a constant weight (see Figures 2a and 2b). Each was then ground
through a Wiley mill and retained for fiber and chromium analyses.

The freeze-dried abomasal, protozoal and bacterial samples were

individually finely ground with mortar and pestel.

Nitrogen Determination

 

Total nitrogen content of feed, feces, urine, abomasal and
bacterial samples was determined with the Technicon Auto Kjeldahl
System. Undried feed and fecal samples were used for the determination.

Abomasal fluid composites were separated (by high speed centrifugation)

57

Abomasal Composites (Homogenized)

100 ml 30 ml

1

Centrifuge at 45,000 xg
for 10 minutes

 

 

Oven dry at 65°C

 

 

 

I j for 48 hrs. r
T-N T-N Supernatant Residue
NHB'N NHBTN oven dry at
UM KMnO4-Lignin 65°C for 48 hrs.
OM 0M 1
RNA RNA TjN T-N
Chmmum NH3-N KMnO4-Lignin
Amino Acids PEG NH3-N
RNA 0M
Amino Acids RNA
Chromium

Amino Acids

Figure 2a. Scheme of Laboratory Analysis for Composited Abomasal
Samples (Digesta Passage Studies).

58

TWO RUMINALLY CANNULATED STEERS
14 days AAaptation
Collection of rumen contents--2, 4, 6, and 8 hrs. after feeding
squeeze through cheese cloth
Rumen Liquor

centrifuge at 150 xg for 5 min.

 

1

Residue (Protozoa) Supernatant
wash 3 x (0.9% NaCl) centrifuge at 2000 rpm
for 10 min.

centrifuge at 15,000 rpm
Qfor 10 min.

 

 

 

 

Protozoa Preparation centrifuge at 45,000 xg
for 15 min.
Residue
J (discard)
Lyophilize Bacterial Preparation

wash 3 x (0.9% NaCl)

 

T-N centrifuge at 45,000 xg
I for 15 min.
RNA j
| Bacterial Pellet
0M
Lypohilize vbT—N, RNA, 0M.

 

Figure 2b. Flow Chart of Sampling Protocol and Laboratory Analyses
(Rumen Microbial Studies).

59

into the supernatant and residue. The solids were oven dried to a
constant weight at 60°C and then finely ground. Five milliliter of
the supernatant, 2 gm dried residues and 5 gm portions of wet abomasal
composites were also analyzed for total nitrogen using the same pro-
cedure. Ammonia nitrogen was determined on the wet abomasal composites

by Conway Microdiffusion technique (1960).

Chromium Analysis

 

Dried feed, feces, and abomasal (wet, dried and residues)
composites were analyzed for chromium by nitric-perchloric acid
digestion followed by atomic emission spectrophotometry using the
I.L. 453 Atomic Absorption/Emission Spectrophotometer.

Approximately 300 mg of the dried samples (3 gm wet samples)
were digested in 30 ml of concentrated nitric acid in a 250 ml Phillips
beaker on a hot plate, until the volume was just sufficient to cover
the bottom of the beaker. The beaker was cooled to room temperature,
and the digest oxidized by adding 7 ml of 72% perchloric acid, fol-
lowed by heating again until 2 to 4 ml of the digest remained (or the
digest turned orange). The beaker was cooled and the contents diluted
to 100 ml with deionized-distilled water. The oxidized and diluted
samples were then analyzed for chromium by atomic emission spectro-
photometer at a wavelength of 425.4 nm, scale of 2.5, slit width of
80, photomultiplier voltage at 700 volts, acetylene flame, hollow-
cathode chromium lamp and nitric oxide solid burner. A standard
curve was prepared by serial dilutions of stock ammonium chromate
solution. Subjected to the same treatment as the samples were 1,

2, 5, 10, 15 and 20 ppm chromium from the solution.

6O

Dry_and Organic Matter Determination

Dry matter of the feed, feces, abomasal composites and
bacterial cells were determined by oven-drying the samples at 60°C
to constant weights. Corrections for moisture absorbed in storage
were made by second dry matter determination during other analyses.
Organic matter of the moisture—free samples was obtained by ashing

in a muffle furnace at 660°C.

Polyethylene Glycol Determination (PEG)

 

PEG in the feed, weighbacks, and abomasal supernatant were
determined by the method of Hyden (1955) as modified by Smith (1959).
Five grams of the ground feed were rehydrated in 25 gm of water,
homogenized and centrifuged to provide the supernatant used for

PEG determinations.

Fiber Determination

 

Neutral detergent fiber, acid detergent fiber and potassium
permanganate lignin contents of the dried feed, weighbacks, fecal
and abomasal composites and residues were determined by the standard
Van Soest procedures (Van Soest, 1963; Van Soest and Wine, 1967;

Robertson and Van Soest, 1977).

Nucleic Acid Analysis

 

Ribonucleic acid content of rumen bacteria, protozoa, abomasal
fluid composites, abomasal supernatant and residues were determined by
a modification of the methods of McAllan and Smith (1969) and Munro
and Fleck (1966).

61

One hundred to one hundred-fifty milligrams of dried (and
2 to 4 gm of wet) samples were homogenized under ice in 5 m1 cold
0.2 N perchloric acid (PCA) for 15 minutes. Five milliliters of
1.2 N PCA were used for the wet samples. Each was transferred into
Corex tubes in ice; the homogenizer was washed twice with 2 m1 of
cold 0.2 N PCA. The washings were added to the samples and the tubes
centrifuged at 18,000 rpm for 15 minutes. The residues were separated,
washed twice with cold 0.1 N PCA and centrifuged each time, and the
supernatants discarded. The pellets were next dispersed in 10 ml
buffered ethanol (1% potassium acetate in 95% ethanol W/V), centri-
fuged at 18,000 rpm for 15 minutes and the supernatants discarded.
The above lipid/pigment extraction procedure was repeated with
chloroformzmethanol (1:3), methanolzether (3:1) and ether, after
which the samples were dried in a vacuum-dessicator. All the above
extractions were performed at < 4°C.

The samples were next hydrolyzed in 10 ml 1.0 N KOH at
37°C for one hour. They were then cooled in ice and centrifuged
at 35,000 xg for 15 minutes. The supernatant was separated and the
residue washed twice in 2 ml 1.0 N KOH, centrifuged, and the liquid
portions combined with the above supernatant. The latter was acidified
with 9 ml of 2.0 N PCA and kept in ice (with occasional shaking) for
30 minutes. The sample was next centrifuged for 15 minutes at the
above speed. The supernatant was saved. The residue was washed in
2 ml 0.2 N PCA, centrifuged, and the supernatant therefrom combined

with the former. The solution was then adjusted to pH 7 (with

62

approximately 2 m1 of 3 N KOH), filtered into 100 m1 volumetric flasks
and the filter paper washed down with some deionized water. Five
milliliters 2 N PCA were added to the flask and the volume made up
to mark with deionized water. The acid-soluble RNA, now in 0.1 N
PCA, was analyzed by ultra-violet absorption at 260 nm. Sample DNA
content could be determined on the combined residues. Corrections for
acid-soluble peptides could be made on the RNA values by performing a
Lowry-protein analysis on the RNA solutions, and then subtracting
0.001 00 units for each mg peptide found, per m1 of the solution.

A standard curve was prepared from serial dilutions in 0.1 N
PCA (O, 12.5, 25.0, 37.5 and 50.0 ug/ml) of purified highly polymerized
yeast RNA.

Statistical Analyses

 

Analysis of variance (Snedecor and Cochran, 1967) was used to
examine the effects of treatments on the digesta passage data,
nitrogen balance and microbial growth studies. Orthogonal contrasts
(Snedecor and Cochran, 1967) were designed to compare the means of
treatment combinations of primary interest. All statistical analyses

were performed on Hewlett Packard Model 9825A Calculator.

Feeding Trial and Blood Metabolite Studies
General Design
Twenty-four Hereford steer calves weighing approximately 277 kg
each, were used in a feeding trial employing all-corn silage diets to
study the effects of monensin and elfazepam on plasma amino acid status-

blood glucose levels and performance of the animals.

63

The steers were wormed, treated for lice, vaccinated for lBR,
BVD and PI3 and then adapted to a ration containing 88% corn silage
and 12% soybean meal-mineral supplement, prior to the study. All
the steers were implanted with Synovex S.

Six steers, ranging from small to average frames, were randomly
allotted to each of the four experimental rations listed in Table 5.
The composition of the supplements are listed in Table 3. The steers
were individually fed once a day and had free access to water.

Rations were mixed immediately before feeding in a horizontal batch
mixer. Daily feed records were maintained for each steer and unconsumed
feed was removed, weighed and recorded. Feed was provided in such a
manner that the bunks were nearly clean at the next feeding period.
Several grab samples of the rations were taken as the mixed feed was
being discharged from the mixer. These were composited, comminuted,
subsampled and kept frozen for subsequent dry matter analysis. The
trial lasted for 118 days and each ration contained 13% crude protein.

The steers were individually weighed on days 0, 28, 63, 90 and
118 of the trial. Initial and final weights were taken 16 hours after
feed and water removal. During the intermediate weighing periods,
only water was removed.

Jugular blood samples were taken from each steer by veni—
puncture prior to weighing on days 28, 63, 90 and 118. Efforts were
made to keep the animals calm and restrained during blood withdrawal.

The steers were confined to individual pens (91 x 244 cm) with
completely slotted floors in an environmentally controlled metabolism

room, throughout the trial.

64

TABLE 5. COMPOSITION OF RATIONS USED IN FEEDING TRIAL AND PLASMA

 

 

 

 

STUDIES

Ingredient Percent--DM basis
Corn silage (35% DM) 88
Soybean meal-mineral supplement 1,a 2,b 3,C 4,de 12
Analysis of Supplement: .%

NX6.25 49.4
Calcium 2.0

P04 1.2

TM salt 2.0

 

aContained vitamin A and vitamin D premix.
bContained 1 plus 125 mg minensin per 454 gm.
CContained 1 plus 8.35 mg elfazepam per 454 gm.

dContained 1 plus 125 mg monensin and 8.35 mg elfazepam per
454 gm.

eSupplements 1, 2, 3 and 4 used in Rations 1, 2, 3, and 4,
respectively; ingredients listed in Table 3.

65

Sample Preparation and Analyses

Blood Samples

 

Blood samples drawn from the right jugular vein of each steer
were collected into 100 x 16 mm tubes (vacutainers) containing 2 to 3
drops of heparin. The tubes were kept under ice, deproteinized and
used for analyses of the plasma metabolites. Samples were kept frozen
until analyzed.

Plasma was prepared for amino acid analyses by the standard
procedure used in our laboratory (Bergen gt_al,, 1973). Plasma free
amino acid concentrations were determined by ion-exchange chromatography
on a Technicon Amino Acid Analyzer TSM-l as described by Bergen and
Potter (1971).

Plasma urea nitrogen levels were determined by Conway Micro-
diffusion method (1960) as outlined by Fenderson (1972).

Plasma glucose levels were determined by a modification of the
colorimetric method in which glucose is oxidized in a glucose oxidase-
peroxidase reaction (Boehringer Mannheim Corporation Cat. No. 15754,

1974).

Feed Samples

 

Dry matter of the feed and weighback were determined by drying

the samples in an oven at 65°C for 48 hours.

Statistical Analysis

 

Least square analyses (Snedecor and Cochran, 1967) were used

to examine the main effects and interactions for average daily gain

66

(ADG), average daily dry matter intake (ADDMI) and feed efficiency

(F/G).

Differences between least square means were determined

using Duncan's New Multiple Range Test.

The effects of treatment on plasma glucose, urea and amino

acid levels were examined by analysis of variance (Snedecor and

Cochran, 1967).

Calculations

 

Estimates obtained were based on the following calculations:

A. Crude protein (CP) = N x 6.25;

B. Organic matter (OM) = dry matter (DM) — ash;

C. Organic matter digested in the rumen (0M0) = OM intake - (OM

reaching the abomasum - microbial OM);

D. Non-ammonia nitrogen (NAN) = total nitrogen (TN) — ammonia-nitrogen

(NH3-N).

1:

DM flow (g/day)

= Marker1 Intake (9)
MarkerI/g Abomasal DM

 

Passage of digesta components2 (g/day): (A)

= 0M Intake x Component (g/g DMI)
DM Flow x Component (g/g Aboma§al DM)

 

TN (NAN) passage (g/day)

a. From chromium or lignin outflow

= Marker! Intake (9) , x TN(NAN)/ml Abomasal Fluid
9 Markerl/ml Abomasal F1u1d

 

 

1Chromium or lignin.

2Digesta component: OM, OM, NDF, ADF.

67

b. From N:Marker1 ratio

= NzMarkerl Ratio in Abomasal 0M x N-Intake (g)
NzMarker1 Ratio in Feed DM

c. From Reconstituted Digesta: (B) + (C)

= PEG Intake (9) . . .
(B) PEG g/ml Abomasal Fluid X N 9/m1 L‘°”1d 019°5t°

 

(C) Chromium Intake (9),

= Chromium g/g 0M Abomasal Residue x N (g/g Abomasal)

Residue

4. Proportion microbial N in abomasal N: (D)

 

= Total N/g Bacterial DM RNA-N3’“/g Abomasal DM
RNA-N“/g Bacterial 0M NAN/g Abomasal DM

5. Microbial N flow (g/day)
= (D) x NAN Passage

6. Microbial DM flow

= Microbial N Flow
N/g Bacterial 0M

 

7. True ruminal digestion5 of component2 (g/day)

= (A) -(Microbia1 DM Flow x Component/g Bacterial DM)
Component in Feed (g/day)

 

1Chromium or lignin.
2Digesta component: 0M, 0M, NDF, ADF.

3From 'adjusted RNA' per gm abomasal DM (i.e., RNA x 0.85,
see discussion: Nucleic Acid Methodology.

l’Microbial RNA assumed to contain 14.8% N (Ling and
Buttery, 1978).

sDigestion in the rumen corrected for microbial, but not for
endogenous contribution.

10.

11.

12.

13.

68

True efficiency of microbial protein synthesis (CP/lOO g OMD)

_Microbial N Flow x 6. 25
True OMD (g) x 100 9 0M

Digesta passage rate (ruminal outflow rate)
a. Liquid Digesta (liters/day)

PEG Intakeg(g)
=PEG/ml Abomasal Supernate

b. Particulate Digesta (liters/day)

Chromium Intake (9)
=Chromium7g DMTAbomasal Residue

c. Total Digesta (liters/day)

_ Marker1 Intake (g)
' Marker1 g/ml Abomasal Fluid

 

Rumen dilution rate (% per day)

= Digesta Passage Rate (liters/day)
Rumen Volumeb (liters)

 

Rumen turnover time (days)

= (Rumen Dilution Rate)-1

Post-ruminal ADF digestion (% abomasal)

_Abomasal ADF Passage (g/day) - Fecal ADF (g/day)

Abomasal ADF Passage (g/day) X 100

Fecal chromium recovery (% of intake)

_Feca1 DM (g/day) x Chromium/g Fecal DM
ChromiumTIntake (g7day)

 

 

1Chromium or lignin.

6Rumen volume separately determined. In this study, 50 liters

assumed for Hereford steers (277 kg BWT).

CHAPTER IV

RESULTS

Digesta Passage Studies

 

Total digesta outflow from the rumen was estimated from the
rates of particulate markers (lignin and chromium) appearance in
abomasal digesta. These estimates were compared with abomasal digesta
flow rates determined from the partitioning of liquid (PEG) and par-
ticulate (chromium) fractions of abomasal digesta (Faichney, 1975).
Total digesta flow rates based on the three above markers are shown
in Table 6. Estimates from the reconstituted digesta (PEG + chromium
passage) were consistently higher than the corresponding values deter-
mined from lignin and chromium flows. Lignin and chromium-estimated
digesta flow rates were 32 to 41% lower than PEG chromium flow rates.
But, laboratory values of chromium in particulate digesta (dry abomasal
residues) when reconstituted, were similar to the chromium values
obtained from the corresponding total digesta composites. Since
chromium was not detected in the liquid fraction of the abomasal
composites, it was assumed that the particulate chromium values
obtained did not, within the limits of our abomasal sampling accuracy
and sampling technique used, overestimate solid digesta flows. PEG-
estimated liquid digesta flow rates were 27 to 38% higher than total
digesta flow rates determined from abomasal lignin and chromium

passage, while total digesta flows based on the passage of the

69

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71

latter two markers were very similar within treatments (Table 6). This
implied that there could be a problem with abomasal PEG passage in the
present experiment.

However, abomasal flow of the various components of digest was
estimated from lignin, chromium, N:1ignin, Nzchromium and PEGi-chromium
passage, using the different methods of calculation previously discussed
(see section: Calculations). Here again, abomasal N recoveries esti-
mated from the reconstituted digesta were higher than the corresponding
lignin and chromium-based N recoveries. Recoveries obtained by the
former method seemed physiologically impossible for steers on the
dietary regimen used in this study (see Table 13). Based on those
observations and other reasons to be discussed later, it was decided
to calculate abomasal passage of the various components of digesta

from lignin and chromium passage rates only.

Ruminal Dry Matter and Fiber
Digestion

Abomasal passage of dry matter (0M), fiber (NDF and ADF) and

 

the extent of ruminal digestion of those feed components are shown
in Tables 7 and 8. Variations in intake and the basic differences in
fiber content of the grain and silage rations make a direct statistical
comparison of the absolute estimates untenable.

There was a trend toward a decrease in abomasal DM passage with
monensin rations, which was more evident in the silage rations. But
these differences were not significant. NDF and ADF passage to the

abomasum were similar in the high grain rations, and slightly lower

72

 

 

 

 

 

 

 

 

 

 

TABLE 7. DRY MATTER AND FIBER FLOW FROM THE RUMEN: ESTIMATED FROM
LIGNIN-BASED DRY MATTER FLOW
Treatment
High grain High silage
Without With Without With
Item monensin monensin monensin monensin SEM
Intake (g/day):
Dry matter 6468.35 6187.44 7184.21 6503.56 474.35
NDF 1455.83 1389.65 3226.75 2753.04 195.45
ADF 672.34 655.66 1785.02 1584.83 81.01
Abomasal Flow (g/day):
Dry matter 4598.19 4164.01 5668.74 4460.19 514.51
NDF 1002.10 1035.10 2276.59 1900.60 170.13
ADF a 512.16 551.26 1276.18 1140.91 82.65
Microbial DM 996.16 894.43 1148.00 909.70 78.07
Feed DM 3602.03 3269.58 4520.74 3550.49 462.67
Apparent Rumen Digestion:
Dry matter 1870.16 2023.43 1515.47 2043.37 236.23
NDF 453.72 354.55 950.16 852.45 65.92
ADF b 160.18 104.40 508.85 443.91 38.75
Rumen DMD 2866.32 2917.86 2663.47 2953.07 265.51
% Rumen Digestion
Apparent 0M0c 28.91 32.70 21.10 31.42 3.95
Apparent NDF 31.17 25.52 29.45 30.96 2.27
Apparent ADF 23.82 15.92 28.51 28.01 2,90
Rumen DMDb 44.31 47.16 37.07 45.41 3.99

 

aSignificant monensin effect (P<<0.05)

b

cSignificant monensin effect (P< 0.01)

Rumen dry matter corrected for microbial flow.

TABLE 8. DRY MATTER AND FIBER FLOW FROM THE RUMEN:
CHROMIUM-BASED DRY MATTER FLOW

73

ESTIMATED FROM

 

 

 

 

 

 

 

 

 

 

Treatment
High grain High silage
Without With Without With
Item monensin monensin monensin monensin SEM
Intake (g/day):
Dry matter 6468.35 6187.44 7184.21 6503.56 474.35
NDF 1455.83 1389.65 3226.75 2753.04 195.45
Abomasal Flow (g/day):
Dry matter 4469.88 4335.75 5584.00 4314.25 556.62
NDF a 972.88 1077.54 2364.62 1889.82 207.07
Microbial DM 980.29 926.28 1124.08 929.31 81.71
Feed DM 3489.58 3409.48 4459.93 3384.95 490.93
Apparent Rumen Digestion:
Dry matter 1998.48 1851.69 1600.21 2189.31 286.23
NDF 482.95 312.12 862.13 863.23 87.15
Rumen digestion 2978.77 2777.96 2724.28 3118.61 275.61
% Rumen Digestion:
Apparent 0M0C 30 90 29.93 22.27 33.66 4.64
Apparent EDF 33.17 22.46 26.72 31.36 3.64
Rumen DMD C 46.05 44.90 37.92 47.95 4.43

 

aSignificant monensin effect with silage ration (P<<0.05).

b

Rumen digestion corrected for microbial flow.

CMonensin effect with silage ration (P<<0.10).

74

in monensin-supplemented silage ration. These trends were also not
statistically significant. But, microbial DM passage to the abomasum
was significantly reduced by monensin (P<:0.05, Table 7). Monensin
also increased apparent DM digestibility (%) of the silage ration
(P<:0.05), without significantly affecting the high grain ration.
Apparent NDF digestibility was also slightly increased by monensin

in silage rations (Tables 7 and 8) while apparent ADF digestibility

was slightly reduced by monensin in both types of ration. These trends
were, however, not significant. True (or actual) ruminal DM digestibil-
ity (i.e., corrected for microbial DM passage) was not affected by
monensin in high grain ration, but was increased in silage ration
supplemented with monensin (P<:O.10). Mean percent DM truly digested

in the rumen (based on combined estimates determined from lignin and
chromium abomasal flows) were: 45.18, 46.03, 37.50 and 46.68 for high
grain ration, without (HG) or with (HG-M) monensin, and, high silage
ration, without (CS) or with (CS-M) monensin, respectively.

Total Nitrggen and NAN Passage
to the Abomasum

 

Passage of the nitrogen constituents to the abomasum was
estimated using lignin, chromium and reconstituted PEG and chromium
as markers. These were also compared to abomasal N passage calculated
by the marker:nutrient ratio (see section: Calculations). N passage
estimates obtained by the above methods are shown in Tables 9 to 13.
Abomasal N flow estimates from lignin, chromium and marker:nutrient

ratio methods showed some net ruminal nitrogen losses with HG and CS

TABLE 9. EFFECTS OF MONENSIN AND TYPE OF RATION ON THE NITROGEN
CONSTITUENTS OF ABOMASAL DIGESTA:

75

ESTIMATED FROM LIGNIN FLOW

 

 

 

 

 

 

 

 

 

Treatment
High grain High silage
Without With Without With
Item monensin monensin monensin monensin SEM
Intake (g/day):
Dry matter 6468.35 6187.44 7184.21 6503.56 474.35
Nitrogen 135.15 131.10 148.89 137.40 8.59
Abomasal Flow (g/day):
Total N 122.28 126.98 138.82 138.65 10.07
Ammonia-Nb 3.03 2.69 5.18 5.06 0.42
Non-NH3-N C 119.25 124.29 133.64 133.59 9.77
Microbial N 82.90 74.85 95.86 76.12 6.51
Plant N 36.35 49.44 37.79 57.47 5.08
Abomasal Flow (%):
Plant N (% NAN)°bC 30.48 39.78 28.27 43.02 2.42
NAN (% N intake) 88.24 94.81 89.76 97.23 2.72
Digesta flow ratee 42.73 42.50 60.21 61.67 3.38

 

aSignificant differences due to type of ration (P<:0.05).

bNot corrected for endogenous N secretion.

cSignificant differences due to monensin (P<:0.05).

dSignificant differences due to monensin (P<:0.01).

eLiters per day; significant differences due to ration (P<:0.01).

TABLE 10. EFFECTS OF MONENSIN AND TYPE OF RATION ON THE NITROGEN

76

 

 

 

 

 

 

 

 

 

CONSTITUENTS OF ABOMASAL DIGESTA: ESTIMATED FROM N: LIGNIN
RATIOS
Treatment
High grain High silage
Without With Without With

Item monensin monensin monensin monensin SEM
Intake (g/day):
Dry matter 6468.35 6187.44 7184.21 6503.56 474.35
Nitrogen 135.15 131.10 148.89 137.40 8.59
Abomasal Flow (g/day):
Total N a 124.04 127.46 139.38 139.66 10.01
Ammonia-N 3.03 3.17 5.74 6.07 0.42
Non-NH3-Nb 121.01 124 29 133.64 133.59 9.68
Microbial Nc 84.01 74.85 95.85 76.12 6.37
Plant N 36.99 49.44 37.79 57.47 5.11
Abomasal Flow (%):
Plant N (% NAN)°be 30.57 39.78 28.28 43.02 2.42
NAN (% N intake) 89.54 94.81 89.76 97.23 2.74

 

aSignificant ration effect (P<:0.05).

b

cSignificant monensin effect (P<:0.05).

d

Significant monensin effect (P<:0.01).

eSignificant monensin effect (P<:0.05).

Not corrected for endogenous-N secretion.

TABLE 11. EFFECTS OF MONENSIN AND TYPE OF RATION ON THE NITROGEN
CONSTITUENTS 0F ABOMASAL DIGESTA:

77

ESTIMATED FROM CHROMIUM

 

 

 

 

 

 

 

 

 

FLOW
Treatment
High grain High silage
Without With Without With

Item monensin monensin monensin monensin SEM
Intake (g/day):
Dry matter 6468.35 6187.44 7184.21 6503.56 474.35
Nitrogen 135.15 131.10 148.89 137.40 8.59
Abomasal Flow (9/day):
Total N a 120.39 131.80 135.75 141.55 10.81
Ammonia-N 2.98 2.78 5.04 5.17 0.40
Non-NH3-N 117.41 129.02 130.71 136.38 10.53
Microbial N 81.58 77.52 93.86 77.76 6.82
Plant NC 35.83 51.50 36.85 58.63 5.50
Abomasal Flows (%):
Plant N (% NAN)°bc 30.52 39.92 28.19 42.98 2.40
NAN (% N intake) d 86.87 98.41 87.79 99.26 3.12
Digesta flow ratea 42.10 44.05 58.83 62.95 3.52

 

aSignificant differences due to ration type (P1<0.05).

b

cSignificant differences due to monensin (P <0.0l).

dLiters per day.

Not corrected for endogenous N secretion.

TABLE 12. EFFECTS OF MONENSIN AND TYPE OF RATION ON THE NITROGEN
CONSTITUENTS 0F ABOMASAL DIGESTA:

CHROMIUM RATIO

78

ESTIMATED FROM N:

 

 

 

 

 

 

 

 

 

Treatment
High grain High silage
Without With Without With
Item monensin monensin monensin monensin SEM
Intake (g/day):
Dry matter 6468.35 6187.44 7184.21 6503.56 474.35
Nitrogen 135.15 131.10 148.89 137.40 8.59
Abomasal Flow (g/day):
Total N a 120.44 136.00 136.56 142.44 10.80
Ammonia-N 3.03 3.17 5.74 6.07 0.42
Non-NH -Nb 117.41 132.83 130.82 136.37 11.81
Microbial N 81.57 79.61 93.94 77.79 7.36
Plant Nc 35.84 53.22 36.88 58.58 5.99
Abomasal Flow (%):
Plant N (% NAN)°b 30.53 40.07 28.19 42.96 2.42
NAN (% N intake) C 86.87 101.32 87.86 99.25 4.01

 

aSignificant ration effect (P< 0.05).

bNot corrected for endogenous N secretion.

CSignificant effects due to monensin (P<:0.01).

79

rations. Monensin addition to both rations resulted in Virtually no
net ruminal nitrogen losses. Abomasal NH3-N recoveries were higher
when the steers received the silage rations (P<:0.05), but showed no
significant monensin effect. NAN passage (as % of N-intake) from
combined lignin and chromium passage estimates, were higher when the
rations contained monensin (P<:0.01). These, for lignin-based esti-
mates, were 88.24, 94.81, 89.76 and 97.23% for HG, HG-M, CS and CS-M
rations, respectively (P<:0.05). The same estimates from abomasal
chromium passage were 86.87, 98.41, 87.79 and 99.26% for the above
respective rations (P<:0.01).

But, microbial N passage was reduced (P<:0.05) and feed N bypass
increased upon addition of monensin to HG and CS rations. Feed N con-
tributed about 30% to abomasal NAN passage with the control rations
(HG and CS rations); with monensin added to the rations, feed N bypass
comprised 40% of abomasal NAN. Total digesta flow rates were higher
when silage rations were fed (Table 9, P<:0.01; Table 11, P< 0.05),
but monensin did not affect the rate of digesta passage. Abomasal
passage of the various nitrogen constituents of digesta calculated
by N:1ignin ratios were virtually the same as those determined from
lignin alone, while estimates from Nzchromium ratios also closely
paralleled the corresponding estimates from chromium passage.

Abomasal N Passage from
Reconstituted Digesta

 

Abomasal N passage data calculated by reconstitution of the
partitioned liquid (PEG) and solid (chromium) fractions of digesta are

shown in Table 13. The data presented in the table show marked increases

80

TABLE 13. EFFECTS OF MONENSIN AND RATION TYPE 0N NITROGEN CONSTITUENTS

0F ABOMASAL DIGESTA IN GROWING STEERS--DETERMINED FROM

RECONSTITUTED LIQUID (FROM PEG) AND SOLID (CHROMIC OXIDE)

 

 

 

 

 

FLOWS
Treatment
High grain High silage
without With without With
Item monensin monensin monensin monensin SEM
N intake (g) 135.15 131.10 148.89 137.40 8.59
Total abomasal
N (g) 153.32 161.43 162.93 162.02
Abomasal NAN (g)a 151.08 159.19 158.98 157.61 10.27
(1) Solids (g)b 61.22 60.39 84.62 79.52 4.92
(2) Liquids (g) 89.86 98.80 74.37 78.09 6.83
NH3-N flow (g)b 2.24 2.24 3.94 4.41 0.22
NAN (% N intake)a 111 79 121 43 106.78 114.71
Digesta Flow Rates
------------------ ml/day------------------
Solid f1owc 4,469.88 4,335.75 5,584.00 4,550.55 564.30
Liquid flowd 57,784.58 64,173.34 83,807.80 99,790.97 6995.28
Total f1owd 62,254.46 68,509.09 89,391.80 104,341.52

 

aNot corrected for endogenous N secretion.

bSignificant difference due to type of ration (P<:0.05).

cSignificant monensin vs. silage interaction (P<<O.10).

d

Significant monensin effect on flow rate (P<:0.05).

81

in most parameters estimated, over the corresponding estimates from
lignin and chromium passage. Total N and NAN passage to the abomasum
were significantly higher than the dietary N intakes with all the
rations fed, and there were virtually no monensin effects. Abomasal
NAN from particulate digesta (solids) and NH3-N passage were both
higher for CS and CS-M than for HG and HG-M rations (P< 0.05).
Particulate digesta flow rates were similar for the high grain
rations, and were reduced by monensin in silage rations (P<<0.lO).
Liquid and total digesta flow rates were higher for monensin supple-
mented rations (P<:0.05). Table 14 represents a direct comparison of
the marker ratios in ration and abomasal DM. Monensin increased % NAN

and lignin in abomasal 0M.

Rumen Microbial Studies

 

In order to accurately determine the effects of monensin on
feed protein and dry matter degradation in the rumen, microbial con—
tribution to abomasal N and DM flow also have to be quantitatively
estimated. Since it is rather difficult to physically separate intact
microbial cells from abomasal digesta, a microbial marker (e.g., RNA)
is usually used to estimate microbial passage. Variability in the
macromolecular composition of microbial cells with diets and time
after feeding (McAllan and Smith, 1977) make it necessary to determine
the chemical composition of the microorganisms isolated from animals
under the same experimental regimen.

Table 15 presents the total N, RNA and organic matter estimates

of mixed bacterial preparations isolated from the rumen of steers fed

TABLE 14. RATIOS OF MARKERSa IN RATION AND ABOMASAL DIGESTA

82

 

 

 

 

 

 

 

 

 

 

Treatment
High grain High silage
Without With Without With
Item monensin monensin monensin monensin SEM
% Nitrogen:
Ration be 2.10 2.10 2.10 2.10 0.10
Abomasal digesta 2.66 3.03 2.43 3.04 0.23
% Chromium:
Ration 0.21 0.20 0.18 0.17 0.01
Abomasal digesta 0.30 0.29 0.23 0.25 0.02
% Lignin:d
Ration e 3.09 3.09 3.93 3.93 0.01
Abomasal digesta 4.40 4.70 5.10 5.80 0.30
Nitrogen:Chromium:
Ration 10.33 10.48 11.79 12.29 0.63
Abomasal digesta 8.97 10.54 10.32 12.22 0.73
Nitrogen:ngnin:
Ration 0.677 0.686 0.533 0.539 0.022
Abomasal digesta 0.605 0.649 0.478 0.525 0.026

 

amg marker/g dry matter.

b

Abomasal NAN.

CSignificant monensin effect

d

Lignin determination by the

(P< 0.05).

KMnO4 procedure.

eSignificant monensin effect (P<:0.10).

83

TABLE 15. COMPOSITION OF MIXED RUMEN BACTERIAL PREPARATIONS FROM STEERS
FED MONENSINa

 

 

 

 

 

 

Treatment
High grain High silage

Without With Without With
Item monensin monensin monensin monensin SEM
Total N 83.22 83.69 83.50 83.67 2.29
RNA 87.64 87.80 87.65 87.83 3.22
RNA-N 12.97 12.99 12.97 13.00 0.46
RNA-N:TN ratio 0.156 0.155 0.156 0.155 0.14
Ash (%) 15.05 15.33 15.00 15.30
Organic matter (%) 84.95 84.67 85.00 84.70

 

aRumen samples collected at 2, 4, 6, and 8 hours after feeding,
and pooled.

bmg/g bacterial dry matter.

84

the same rations used in the passage study. Protozoa composition was
also studied, but not used in subsequent calculations since it is
believed that protozoa contribute very little to abomasal NAN (Weller
and Pilgrim, 1974).

There were no differences in total N, RNA, ash and organic
matter content between the bacterial preparations from steers fed the
four experimental rations. However, bacterial preparations isolated
two hours after feeding contained less nitrogen (0M basis) than those
obtained 4, 6 and 8 hours after the steers were fed. This response was
more evident with the HG ration and may account for the slightly lower
N content of the bacteria isolated from the steers receiving that
ration. Bacterial preparations from the steers fed monensin also
contained a slightly higher amount of ash. These differences were
not significant, however.

Microbial Contribution to Abomasal
NAN Passage

 

 

Total NzRNA-N ratios of the rumen bacterial preparations and
abomasal digesta were used to determine the microbial proportion of
abomasal NAN passage. Bacterial RNA was assumed to contain 14.8%
nitrogen (Ling and Buttery, 1978) and abomasal RNA estimates were
multiplied by 0.85 to obtain the adjusted RNA (Smith et_al,, 1978;
see discussion). Without this adjustment in digesta RNA, RNA-N values
derived were found to be exceedingly high, and in a few cases resulted
in estimates of microbial NAN passage equal to (or greater) than total
abomasal NAN passage, an unlikely occurrence with the rations used in

this experiment.

85

To assess the reliability of the nucleic acid procedure used,
RNA analyses were performed on oven-dried (previously lyophilized)
abomasal composites and compared to abomasal fluid composites. The
results obtained with the dried samples were so inflated that it was
decided to use the RNA values determined from the abomasal fluid samples
for subsequent calculations. The latter were consistently lower and
less variable between samples.

Microbial contributions to abomasal NAN passage are presented
in Table 16. Based on these calculations, monensin reduced microbial
nitrogen passage to the abomasum (P<:0.01). There were no significant
effects of ration on microbial N flow. Microbial Nzabomasal NAN ratios
averaged about 0.70 and 0.60 for the control (HG plus CS) and monensin
rations, respectively. Abomasal NAN per gram digesta DM were higher
with monensin rations (P<:0.05). Microbial-N passage to the abomasum
are presented in Tables 17 and 18. Abomasal passage of microbial N
(in g/day) determined from lignin-estimated digesta flows were lower
in monensin-fed steers (75.49 vs. 89.38; P< 0.05). The corresponding
estimates from chromium passage were 77.64 and 87.72 for ration with

and without monensin, respectively.

Efficiency of Microbial Protein
Synthesis

Ruminal organic matter digestion and efficiency of microbial
protein synthesis were determined from lignin and chromium-based

digesta passage only (see Tables 17 and 18). There were no significant

86

TABLE 16. MICROBIAL CONTRIBUTION TO ABOMASAL NAN FLOW IN STEERS FED
MONENSIN: DIGESTA PASSAGE STUDIES

 

 

 

 

 

 

Treatment
High grain High silage
without with without with

Item monensin monensin monensin monensin SEM
Bacterial RNA (mg/g DM) 87.64 87.80 87.65 87.33 3.22
Bacterial RNA-Na 13.03 12.99 12 97 13.00 0.46
Bacterial-N (mg/g 0N) 83 22 83.69 . 83.50 83.67 2.29
Bacterial TN/RNA-N 6.39 6.44 6.44 6.43 0.16
Abomasal RNA (mg/g DM) 22.64 21.81 21.27 21.31 1.73
Abomasal RNA-Na 3.35 3.23 3.19 3.15 0.26
Abomasal RNA-Nb

(adjusted) 2.85 2.74 2.21 2.68 0.22
Abomasal NANC (mg/g 0M) 26.60 30.28 24.33 30.35 2.26
Microbial-N/Ab. NANd 0.70 0.60 0.72 0.57 0.02

 

aBacterial RNA assumed to contain 14.8% N (Ling and Buttery,
1978).

bAbomasal RNA concentration x 0.85 (Smith t a1., 1978).

cSignificant monensin effect (P<:0.05).

dSignificant monensin effect (P<:0.01).

87

 

 

 

 

 

 

 

TABLE 17. EFFECTS OF MONENSIN AND TYPE OF RATION ON EFFICIENCY OF
MICROBIAL PROTEIN SYNTHESIS: ESTIMATES FROM LIGNIN FLOW
Treatment
High grain High silage
a Without With Without With

Item monensin monensin monensin monensin SEM
Organic matter intake 6033.38 5757.53 6784.31 6139.76 448.33
Abomasal OM flow 4218.72 3768.58 4863.78 3750.21 469.01
Microbial 0M flowb 846.73 757.59 975.80 770.52 66.28
Feed 0M flowC 3371.99 3010.99 3887.98 2979.69 422.40
Rumen OMD 2661.39 2746.54 2896.33 3160.88 235.07
Rumen 0M0 (%) 43.99 47.93 43.56 51.71 4.66
Microbial Efficiengy:
MicrobialdNAN

(g/day) 82.90 74.86 95.86 76.12 6.51
Microbial CP

synthesis

(g/100 g 0M0) 20.31 17.31 21.00 15.13 1.94
Microbial CP

synthesisde

(g CP/lOO g OMD) 20.31 17.31 21.00 15.13 1.94

 

ag/day.
b

Significant monensin effect (P<<0.05).

CSignificant monensin vs. silage (P<=O.lO).

dSignificant monensin effect (P‘<0.05).

eEstimated from N:1ignin ratios.

TABLE 18. EFFECTS OF MONENSIN AND TYPE OF RATION 0N EFFICIENCY OF
MICROBIAL PROTEIN SYNTHESIS:

88

ESTIMATES FROM CHROMIUM FLOW

 

 

 

 

 

 

 

 

Treatment
High grain High silage
a Without With Without With

Item monensin monensin monensin monensin SEM
Organic matter intake 6033.38 5757.53 6784.31 6139.76 448.33
Abomasal 0M flow 4101.53 3919.37 4788.76 3623.68 469.51
Microbial 0M flowb 833.25 784.56 955.47 787.12 69.34
Feed OM flow 3268.28 3134.82 3833.29 2836.56 439.12
Rumen OMD 2765.10 2622.71 2951.02 3303.20 244.52
Rumen 0M0 (%) 45.83 45.55 43.50 53.80 4.23
Microbial Efficiency:
Microbial NAN

(g/day) 81.58 77.52 93.86 77.76 6.82
Microbial CP

synthesis

(g/100 g OMD) 19.38 18.75 20.25 15.00 2.19
Microbial CP

synthesisCd

(9/100 9 OMD) 19.38 19.25 20.25 15.00 2.31

ag/day.
b

Significant monensin effect (P<:0.10).

cSignificant differences, monensin vs. silage ration (P<:0.10).

dEstimated from Nzchromium ratios.

89

differences due to monensin or the type of ration in the extent of
organic matter degradation in the rumen. The trends were, however,
for a slight increase with monensin-feeding. About 50% of the organic
matter fed with monensin was digested in the rumen compared to 44% for
rations without monensin. These differences were, however, not
statistically significant.

Based on lignin flow, true efficiency of ruminal microbial
protein (N x 6.25) synthesis were higher without monensin than with
monensin in the rations (P<:0.05). Microbial efficiencies estimated
from abomasal chromium passage showed the same trend, but reached a
level of significance at a 0.10, with silage rations only. Microbial
crude protein synthesized per 100 gm of organic matter actually degraded
in the rumen were approximately 20.7 g for rations without monensin and
16.20 gm for rations supplemented with monensin. Combination of silage
and monensin consistently produced the lowest microbial efficiencies
(15 gm CP per 100 g 0M0).

Microbial Efficiency and Ruminal
Dilution Rates

 

 

Rumen dilution rates and turnover times were calculated from
lignin and chromium flows, and also from PEG flows. Since rumen liquid
pool size was not determined with each experimental animal for each
ration fed, a 50-1iter rumen liquid volume was assumed for all the
steers in each treatment. There were variations in animal size and
level of feed intake, and this could have affected the dilution rates

obtained. The relationship between microbial efficiencies and rumen

90

dilution rates are shown in Table 19. There were no significant
differences in dilution rates and rumen turnover time which can be
attributed to monensin in the rations. However, rumen turnover time
(in days) was shorter and fractional digesta outflow rates (% per hour)
higher, with silage rations than with high grain rations (P<:0.01).
Trends toward increases in rumen dilution rates, did not seem to
increase the efficiency of microbial protein synthesis when monensin
was fed.

In contrast, fractional liquid outflow rates derived from PEG
were much higher, and consequently rumen turnover times, much faster
than those estimated from either lignin or chromium outflows. Based
on analytical reasons to be discussed later, it was decided that PEG
outflow was too fast and would not be useful in this study as an index
of digesta flow. Microbial efficiencies were therefore not determined
from PEG data.

Also, the variations in animal size and level of intake,
coupled with an across-the-board assumption of a 50-liter rumen volume
made the establishment of a direct relationship between microbial
efficiency and rumen fractional dilution rate difficult. A direct

linear trend was more evident with monensin treatment than without.

TABLE 19. EFFECTS OF MONENSIN AND TYPE OF RATION ON RUMEN DILUTION

91

 

 

 

 

 

 

 

 

 

RATESa AND EFFICIENCY OF MICROBIAL PROTEIN SYNTHESIS
Treatment
High grain High silage
Without With Without With

Item monensin monensin monensin momensin SEM
From Lignin Flow:
Microbial CP

synthesis

(9 CP/lOO g 0M0) 20.31 17.31 21.00 15.13 1.94
Dilution rate

(%/hour)b 3.56 3.54 5.02 5.14 0.28
Rumen tugnover time

(days) C 1.19 1.22 0.84 0.81 0.09
From Chromium Flow:
Microbial CP

synthesis

(9/100 9 OMD) 19.38 18.75 20.25 15 00 2.19
Dilution rate (%/hr)b 3.51 3.67 4.91 5.25 0.29
Turnover time (days)b 1.20 1.18 0.86 0.80 0.08
From PEG Flow:
Liquid gilution

(PEG) 4.82 5.35 6.99 8.32 0.58
Turnoveg time

(day) C 0.90 0.80 0.60 0.52 0.07

 

aFifty liter rumen volume assumed.

bSignificant differences due to ration type (P<:0.01).

CReciprocal of the dilution rates.

92

Nitrggen Balance and Digestibility Studies

 

Nitrogen Balance

 

Table 20 summarizes the data obtained from nitrogen balance
studies. Variations in nitrogen intake between treatments may account
for the absence of significant differences between the mean estimates
of some parameters measured. There were, however, significant monensin
effects on fecal nitrogen excretion (P<:0.025). Fecal N (gm/day) were
44.69, 37.01, 54.65 and 48.30 for HG, HG-M, CS and CS-M rations,
respectively. Although monensin had no significant effect on urinary
nitrogen excretion, more nitrogen (in gms) were excreted in the urine
by steers on silage in comparison with steers on grain rations (44.44
and 44.00 9 vs. 59.40 and 52.36 9). Daily nitrogen retentions were
similar between rations HG, CS and CS-M (43 to 44 gm); monensin in high
grain rations resulted in a slightly higher amount of nitrogen retained
(48.32 g for ration 2), but this was not statistically significant.
Apparent nitrogen digestion (%) followed a similar trend (66.07, 65.46,
66.60% for rations HG, CS and CS-M, respectively) with a significantly
higher percent digestion for monensin-high grain rations (P<:0.01).
More nitrogen (as % of N-intake) was also retained by steers given
monensin in high grain rations (37.36%), in comparison with the other
three rations (32.34, 27.80 and 30.90 for rations HG, CS and CS-M,
respectively). Even though the above differences were not statistically
significant, the trends were for higher percent of dietary nitrogen

retention with monensin in the silage and grain rations.

93

TABLE 20. EFFECTS OF MONENSIN AND TYPE OF RATION 0N NITROGEN BALANCE
AND DIGESTIBILITY IN GROWING STEERS

 

 

 

 

 

 

 

 

 

 

Treatment
High grain High silage
Without With Without With
Item monensin monensin monensin monensin SEM
Nitrogen Balance (g/day):
N intake 131.73 129.32 158.21 144.40 9.33
Fecal Na 44.69 37.01 54.65 48.30 3.24
Urinary N 44.44 44.00 59.40 52.36 7.27
N retained b 42.61 48.32 44.16 43.75 4.73
% N digestion 66.07 71.38 65.46 66.60 1.45
% N retention 32.34 37.36 27.80 30.90 3.12
DM Digestion (g/day):
0M intake c 6438.97 6217.25 6812.18 6493.89 388.09
DM excreted 1586.90 1141.86 2022.24 2007.17 126.54
DM digestion 4852.07 5075.39 4789.94 4486.73 298.86
% DM digestiond 75.36 81.63 70.31 69.08 1.18
ADF Digestion (g/day):
ADF intake 670.58 631.74 1680.36 1565.65 62.46
Fecal ADF 359.25 338.43 667.30 636.00 33.74
ADF digestion 311.33 293.32 1013.06 929.65 43.10
% ADF digestione 46.43 46.40 60.29 59.38 3.32
°5ignificant monensin effect (P< 0.025).
bSignificant monensin effect in grain rations (P< 0.01).

cSignificant monensin effect in grain rations (P<:0.025);

overall (P < 0.05).

dSignificant ration effect (P<:0.01); monensin effect in grain

rations (P<:0.05).

eSignificant ration effect (P<:0.01).

94

Dry Matter and ADF Digestion

Dry matter and ADF digestibility data are also presented in
Table 20. Fecal DM excretion were similar for the high silage rations
(2022.24 vs. 2007.17 g/day), but monensin decreased the amount of daily
fecal dry matter output with high grain rations (1586.90 vs. 1141.86 gm;
P<:0.01). Consequently, the trend was for a greater amount feed DM
apparently digested in the total tract with monensin in the high grain
rations (5075.39 9), in comparison with the other three treatments
(4852.07, 4789.94 and 4486.73 gm for HG, CS and CS-M rations, respec-
tively). Apparent dry matter digestion (as % of 0M intake) were higher
on concentrate rations than on silage (75.36 and 81.63 vs. 70.31, 69.08;
P<:0.01) and monensin increased DM digestion with steers on HG-M ration
(75.36 vs. 81.63%; P<:0.05).

Because of the differences in ADF content between the grain
and silage rations which resulted in significantly different dietary
‘ADF intakes between the two rations, a direct comparison of the extent
of ADF digestion could not be made. It is evident from fecal ADF
excretion, and ADF digestion data obtained, that monensin had no effect
on both parameters. There were, however, greater proportions of dietary
ADF digested in the animals on silage than in those receiving concen-
trate rations. ADF digestion (as % of intakes) were 46.43 and 46.40
for HG and HG-M rations compared to 60.29 and 59.38 for CS and CS-M

rations, respectively (P<:0.01).

95

Fecal Marker and ADF Recoveries

 

The proportions of feed chromium intake and abomasal ADF
recovered in the feces are shown in Table 21. Chromium recovery was
higher for the silage than for the grain rations. There were also
smaller variations between individual recoveries when the steers
received silage. Fecal chromium recovered (as % of intakes) were
74.53, 70.42, 83.31 and 92.31 for HG, HG-M, CS and CS-M, respectively.

In contrast, fecal recoveries of abomasal ADF were generally
lower on silage than on grain rations. Percent ADF recoveries were
also low in comparison with the values obtained for chromium. This
implies either a high degree of post-ruminal ADF digestion or problems
with fecal ADF analysis. ADF recoveries (% of abomasal) were 69.60,
63.72, 55.80 and 56.49 for HG, HG-M, CS and CS-M rations, respectively.
This means that 30.31, 36.28, 44.20 and 43.51% of ruminal ADF output
were digested in the lower gastro-intestinal tract for HG, HG-M, CS

and CS-M rations, respectively.

Feeding Trial and Plasma Metabolite Studies

Feeding Trial

 

As previously reported, the steers were fed basal silage
rations consisting of 88% corn silage (35% DM) and 12% soybean meal-
mineral supplement during the feeding trial and plasma studies. The
composition of the rations are listed in Tables 3 and 5. Control
ration consisted of the basal corn silage; monensin ration contained
33 ppm monensin; elfazepam contained 2 ppm elfazepam, and monensin-

elfazepam contained 33 ppm monensin and 2 ppm elfazepam.

96

TABLE 21. FECAL MARKER (CHROMIUM)a RECOVERY AND POST-RUMINAL ACID
DETERGENT FIBER DIGESTION

 

 

 

 

 

 

 

Treatment
High grain High silage
Without With Without With
Item monensin monensin monensin monensin SEM
Chromgc oxide recovery
(%) 74.33 70.42 83.31 92.31 3.41
18.29 110.45 :2.19 11.91
ADF Recoveny:
Abomasal ADFC 515.51 531.16 1195.90 1125.91 60.42
Fecal ADF 359.25 338.43 667.30 636.00 33.74
Fecal ADF recovery
(%)d 69.69 63.72 55.80 56.49 3.57
17.41 i10.60 $5.45 :2.85
Post-ruminal ADF
digestion (%) 30.31 36.28 44.20 43.51 3.57
17.41 :10.60 :5.45 :2.85

 

aFrom nitrogen balance trial.

bPercent of Cr203 intake.

cAdjusted for ADF intake at abomasal collection.

dPercent of abomasal ADF flow.

97

The overall performance results are presented in Table 22.
Initial and final weights were similar across treatments, but monensin-
fed steers had the highest average final weight. Dry matter intake
(DMI) varied between treatments, but these were not statistically
different from control. In comparison with control, DMI was 7.62 kg
per day, highest for the elfazepam, while monensin treatment resulted
in slightly less dry matter consumed (6.85 kg). Monensin-elfazepam
combination resulted in a significantly reduced intake, when compared
with elfazepam alone (6.60 kg; p.:o,05), Monensin-fed steers had the
highest average daily gains (ADG), whilethe combination of both drugs
resulted in the lowest rate of gains. ADG (in kg) were: 0.98, 1.08,
0.99, and 0.86 kg for the control, monensin, elfazepam and monensin-
elfazepam supplemented rations, respectively. Monensin treatment also
produced the best efficiency of feed conversion, while elfazepam alone
produced the poorest. F/G ratios were: 7.20, 6.34, 7.74 and 7.69 for
the control monensin, elfazepam and monensin-elfazepam rations, respec-
tively. These differences in ADG and feed efficiency were, however,
not statistically significant.

To further evaluate the performance of the steers, A00 and DMI
for the different treatment groups were compared through the monthly
weighing periods. The trends are graphically illustrated in Figure 3.
Some differences in DMI were observed during the first and third months
of the experiment. During these periods, monensin-fed steers consumed
slightly less feed than the control steers, but gained more. Elfazepam

treatment enhanced feed intake most effectively during the first three

98

TABLE 22. OVERALL DRY MATTER INTAKE, ADG AND FEED EFFICIENCY FOR 118
DAY FEEDING TRIAL

 

 

 

 

Treatment
Monensin and
Control Monensin Elfazepam elfazepam
Initial wt. (kg) 280 278 278 282
Final wt. (kg) 400 409.5 392 385
A06° (kg) 0.98 1.08 0.99 0.86
0N1°° (kg/day) 7.02 6.85 7.62 6.60
DM/kg gain 7.20 6.34 7.74 7.69

 

aLeast square means.

bElfazepam different from monensin-elfazepam (P< 0.05).

months, but produced gains better than controls only during the first
two months. Gains in the last half of the experiment were lower.
Steers on the two-drug combination showed similar, but much lower
intake patterns as the control and monensin groups. However, these
animals gained slightly less than the control groups during the first

half of the trial and subsequent gains were poor.

99

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100

Plasma Metabolite Studies

The effects of monensin and elfazepam on the overall plasma
free amino acid profiles are presented in Table 23. Several of the
amino acids most commonly involved in amino-group transfer were similar
across treatments, but the trends were for slightly higher levels with
monensin in the rations. Proline concentration was also highest with
monensin treatment (8.00 0 moles per d1), elfazepam treatment and the
two-drug combination also resulted in proline levels slightly higher
than controls (7.20 and 7.10 vs. 6.70 0 moles per d1). Glycine levels
were highest in elfazepam fed steers; the other treatment groups showed
levels similar to control. Alanine levels were highest in the control
steers and similar among the three other treatments. Monensin and
elfazepam treatments resulted in methionine concentrations similar to
control, but slightly less for the two-drug combination; but cystine
levels were virtually the same across treatments. Levels of the
branched-chain amino acids were similar between rations, with monensin-
elfazepam combination producing slightly lower values for valine and
isoleucine. Despite the above trends in amino acid concentrations,
none of the observed treatment differences were significant (P<:0.05).

A summary of the plasma amino acid levels is presented in
Table 24. Plasma urea-nitrogen and glucose levels are also reported
in the same table. There were no significant differences in either
the total essential amino acid (TEAA), non-essential amino acids (NEAA),
or total amino acid (TAA) levels between the treatments. The trends

were for slightly lower levels in the three parameters, when the

101

TABLE 23. INFLUENCE OF MONENSIN AND ELFAZEPAM ON PLASMA FREE AMINO ACID
CONCENTRATIONS 0F STEERS FED SILAGE RATIONSa

 

 

 

 

 

 

 

Treatment
Monensin and
Control Monensin Elfazepam elfazepam SEM
-------------------- u moles/d1 --------------------
Aspartate + Asp-

NH2 3.20 3.50 3.10 3.20 0.20
Threonine 6.00 6.40 6.30 6.00 0.50
Serine 5.90 6.50 5.90 5.80 0.40
Glutamate + Glu-

NHZ 26.30 26.90 26.60 25.80 1.20
Proline 6.70 8.00 7.20 7.10 0.50
Glycine 24.00 24.30 26.00 24.30 1.40
Alanine 25.10 24.30 23.70 22.80 1.40
Valine 21.50 21.10 21.70 20.50 0.90
Methionine 2.20 2.10 2.10 1.80 0.10
1/2 Cystine 1.60 1.60 1.54 1.60 0.10
Isoleucine 10.30 10.10 10.00 9.20 0.50
Leucine 11.40 11.70 11.50 11.50 0.50
Tyrosine 4.66 4.58 4.50 4.26 0.20
Phenylalanine 4.47 4.65 4.61 4.41 0.20
Lysine 9.90 9.70 9.60 8.30 0.60
Histidine 6.00 5.90 6.40 6.30 0.30
Arginine _jfl1gfl1 9.70 9.60 8.60 0,50
Total amino acids 178.90 180.90 180.30 170.70 6.20

 

aAll treatment comparisons N.S. at P<:0.05.

102

Table 24. PLASMA METABOLITE LEVELS IN STEERS FED MONENSIN AND ELFAZEPAMa

 

 

Treatment

 

Monensin and
Control Monensin Elfazepam elfazepam SEM

 

------------- u moles/d1 plasma -------------

Essential amino
acid 82.10 81.30 81.80 76.60 3.00

Non-essential
amino acid 96.80 99.50 98.50 94.10 3.80

Total amino
acid 178.90 180.90 180.30 170.70 6.20

---------------- mg/dl plasma ----------------
Urea-nitrogen 12.10 13.50 12.20 13.60 0.46
Glucose 98.80 95.70 93.50 93.40 3.80

 

aAll comparisons N.S. at P<<0.05.

103

two drugs were combined. These treatment comparisons were also not
significant (P< 0.05).

With plasma urea levels, elfazepam group had levels similar
to control (12.20 vs. 12.10 mg/dl), while the monensin and monensin-
elfazepam groups gave similar, but slightly higher levels (13.50 vs.
13.60 mg/dl). These differences were also not significant (P<:0.05).

Plasma glucose levels were relatively high in all treatments.
Levels were highest in the control steers and similar among the three
treatment groups. Glucose levels (in mg per d1 plasma) were: 98.80,
95.70, 93.50 and 93.40 for the controls, monensin, elfazepam and
monensin-elfazepam treatments, respectively.

Figure 4 represents a graphic illustration of the changes in
the levels of plasma valine, leucine and isoleucine with time through
the monthly sampling periods. There was an observable decline, from
days 28 to 63, in plasma valine and isoleucine of those animals on the
two monensin-supplemented rations. Steers on elfazepam alone, showed
the same patterns in the time trends for the three amino acids as the

controls.

104

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CHAPTER V

DISCUSSION

Digesta Passage Studies

 

The digesta passage data indicate that, with the grain
ration, monensin did not significantly affect the amount of dry matter
apparently (and truly) degraded in the rumen. With the silage ration
monensin increased the extent of ruminal degradation of the dry matter
consumed by the steers. Based on lignin passage to the abomasum,

% DMI truly digested in the rumen was similar for the grain rations

(HG, 44.31 vs. HG-M, 47.16%), and increased with monensin added to the
silage (37.07 vs. 45.41%). True DMD estimates from chromium passage
followed the same pattern. These were: 46.05, 44.90, 37.92 and 47.95%
for H0, HG-M, CS and CS-M rations, respectively. Apparent ruminal fiber
(ADF and NDF) digestibility also followed the above trends. The trends
were for lower apparent ADF digestion with monesin rations.

Both the apparent and true rumen 0M and fiber digestion appear
to be somewhat low in this study. Such values have, however, been
reported for concentrate rations (Orskov et_a1,, 1971; Nicholson and
Sutton, 1969; Kennedy et_al,, 1976), and for rations containing high
amounts of roughages (Cole et_al,, 1976, Kropp et_al,, 1977a, 1977b).
These workers (Cole et_al,, 1976) progressively increased the roughage
content of corn rations from 0 to 21% by adding cottonseed hulls, and

obtained an almost linear decrease in rumen DM digestion, ranging from

105

106

58.9 to 43.4%. Some other workers (Drennan at al,, 1970) have even
obtained negative net ruminal 0M digestion using duodenal spot sampling
techniques, with chromic oxide (corrected for 100% chromium recoveries)
as digesta marker, while at the same time reporting plausible rumen
digestibility estimates when lignin was used. Kr0pp et_al, (1977a)
reported a 34% apparent ruminal 0M digestion of roughage rations
supplemented with soybean meal.

Despite the relatively low ruminal dry matter and fiber
digestion obtained in the present study, a comparative assessment
of abomasal passage of the various components of digesta determined
from both lignin and chromium flows through the abomasum, indicate a
close agreement between the two digesta markers. It is not unlikely
that variations between and within the experimental animals in the
rates of digesta flow, coupled with the spot-sampling method used to
obtain abomasal digesta samples may have been responsible for the low
rumen digestibility estimates obtained.

Fluctuations in abomasal digesta flow rates were commonly
observed throughout the experiment. Despite the attempts to simulate
a three hourly abomasal collection interval (extended over a four-day
period), there were periods during the trial when digesta flows were
so minimal that sufficient digesta samples could not be obtained to
make up the digesta composite. This situation was also more prevalent
with specific animals during the trial. Abomasal samples taken during
periods of reduced digesta passage would disproportionately contribute
to a composite sample, while samples obtained during periods of elevated

digesta flow, would be underrepresented in the composite. However,

107

attempts were made during the trial to keep digesta sampling in
sequence with the waves of omaso-abomasal contractions.

Other problems which may contribute to low ruminal digestibility
estimates may arise from the laboratory analytical procedures employed.
KMnO4-lignin technique was used to determine the lignin content of
abomasal digesta, and this method, rather than the acid-lignin method
is more likely to result in an overestimation of digesta lignin, due
to the fact that some of the reagents utilized can also degrade the
expected residual fiber component (cellulose) after the delignification
process. (For details, see Literature Review: Influence of Markers
on Digesta Passage Studies.) In contrast, several investigators have
suggested that chromic oxide flows independently of both the liquid
and solid portions of digesta, with a tendency toward a faster ruminal
outflow rate than both digesta pools (Faichney, 1975; MacRae, 1975;
Drennan et.al,, 1970). This would also result in high post-ruminal
chromium recoveries and consequently, decreased rumen digestion
estimates.

In spite of all the above marker problems, it was evident from
the results obtained in the present study, that monensin did not affect
rumen DMD of the grain ration. This is contrary to results reported by
other workers (Owens et_al,, 1979). In another study (Lemenager et_al,.
1978), no significant differences in rumen DM digestion were reported
in steers fed high energy rations with and without monensin, although
estimates of non-microbial-ruminal dry matter were not reported. Owens

e§_a1, (1979) reported a 10% decrease in rumen 0M digestion for steers

108

fed monensin and high energy rations. Feed DM intake was kept constant
during the trial, and that may explain the relative reduction in rumen
DM digestion. In contrast, the animals were fed ad libitum throughout
the present study and consumed 4 to 9.5% less feed 0M when monensin
was included in the rations. Reductions in feed intake could increase
rumen 0M retention time, thus increasing ruminal digestion (Owens and
Isaacson, 1977). However, the more probable explanation was that in
this experiment, the animals were adapted to each ration for 28 days
prior to abomasal digesta collection. By that time, feed intake on
monensin rations had gradually increased again, after the initial

12 to 18% reduction observed when the animals were first placed on
monensin-supplemented rations. Other workers (Dinius et_al,, 1976;
Van Nevel and Demeyer, 1977; Chen and Wolin, 1979) have reported that
monensin does not inhibit microbial fermentation of energy substrates
when incubated with cultures from animals previously adapted to monen-
sin. But the significant increase in ruminal 0M digestion of the
monensin-silage rations found in this study, may also be related to

a longer rumen solid retention time of roughages (Bergen and Yokoyama,
1977), together with a slower rumen solid turnover reported to occur

when monensin is added to rations (Lemenager 23.21:’ 1978).

Digesta Flow Rates

 

Total digesta flows were quite variable between steers for each
ration fed, but there were no significant interaction of monensin with
ration. Monensin did not affect total digesta flow rates, even though

ruminal digesta outflows were higher for silage than for the high grain

109

rations (42.42 to 43.28 vs. 59.52 to 62.31 liters per day--mean of
combined lignin and chromium abomasal flows). Total digesta passage
estimates from ruminal chromium outflows closely agree with those
determined from lignin passage (Table 6). Mean digesta flow rates
obtained for high grain rations (HG, 42.42 vs. HG-M, 43.28) were 23

to 30% lower than digesta flow rates reported by other workers (Poos
_tLal,, 1979) for a concentrate ration with or without monensin. A
direct comparison is, however, not realistic because Poos et_al, (1979)
maintained the animals on a constant level of feed intake, using a
continuous feeding system, and fed supplemental protein sources which
were slowly degraded in the rumen (Brewers dried grains). Increased
feeding frequency may therefore have increased the rates of digesta
passage from the rumen in that study. However, those workers (Poos
et_al,, 1979) also observed no monensin effect on the rates of digesta
passage; and that agrees with the result obtained in this study. Some
other investigators (Lemenager —E.El:’ 1978; Owens et_al,, 1979)
reported that monensin significantly depressed ruminal fluid turnover
rates in steers fed high roughage and concentrate rations. Lemenager
_t_al, (1979) also found that feed intake, and rumen liquid volume
(even though unusually high with the forage rations) were reduced by
monensin. They, however, attributed the excessive rumen liquid volume
to incomplete rumen PEG equilibration. With concentrate rations,
Lemenager 93.21: (1979) related the reduced rumen liquid outflow rates

by monensin, to other factors besides the observed depressions in feed

intake. It is, however, interesting that rumen liquid volumes for

110

monensin-fed steers were 9 to 28% higher than in the control animals
in that experiment. But those workers did not indicate any possible
relationship between those results to the slower rumen fluid turnover
rates they obtained when monensin was fed.

Liquid digesta passage estimated from abomasal PEG flows are
presented in Table 6. These estimates are 30 to 40% higher than total
digesta flows obtained from abomasal lignin and chromium passage.
Hume et_al, (1970) reported a 10% faster mean ruminal PEG dilution
rate relative to lignin. Their data also showed wide variations
between individual estimates of digesta flows determined from ruminal
PEG disappearance, with some estimates 20 to 60% higher than their
corresponding lignin-based estimates. Harrison et_al, (1975) showed
that rumen dilution rate could be increased by intraruminal infusion
of artificial saliva. They also reported further increases by addition
of PEG to the saliva.

Such results question the validity of PEG as a fluid marker
in digesta passage studies. Excessively high PEG flows may, however,
indicate an underestimation of PEG in rumen or abomasal digesta.

This is a likely possibility, because it has been reported that
several digesta components such as proteins and by-products of enzyme
degradation in the GI tract do interfere in laboratory analysis of
PEG in digesta fluids (Smith, 1959; Ingham and Ling, 1978).

In the present study, abomasal passage of the various com-
ponents of digesta was estimated using chromium, lignin and PEG as

digesta markers. Estimates of nitrogen passage from lignin and

111

chromium flows were very similar (Tables 9 to 12), and generally agree
with results obtained by other workers from studies in which similar
levels of dietary crude protein (12 to 13%) were fed to growing
ruminants (Orskov et_al,, 1971; Leibholz and Hartmann, 1972; Hume

at al,, 1970). In contrast, nitrogen passage data estimated from

PEG flows were much higher, and showed excessive net abomasal gains
relative to N intake (Table 13). Such ruminal nitrogen outputs are
expected and have been reported with diets containing low levels of
nitrogen (Hume et_al,, 1970; Orskov et_al,, 1971). The suspicion,
therefore, was that ruminal PEG outflow was excessively high in this
study and resulted in an overestimation of the amounts of various
components of digesta leaving the rumen. Since PEG estimates obtained
by analysis of PEG in the experimental rations used were in close agree-
ment with the calculated amounts added (Table 4), it was assumed that
abomasal PEG recoveries were relatively low. PEG analysis of abomasal
digesta samples may have been hindered by the presence of the inter-
fering materials discussed above. It was therefore decided to use
only lignin and chromium flow data for the calculation of abomasal

passage of the various components of digesta.

Nitrogen Metabolism Studies

 

Ruminal Nitrogen Losses

 

Results of the present studies indicate that net ruminal
losses of dietary nitrogen occurred to a varying extent with each
ration fed (Tables 9 to 12). Based on the combined estimates from

lignin and chromium flows, 8.0 to 8.9% of feed nitrogen consumed

112

was lost during rumen digestion in the animals consuming rations not
supplemented with monensin. Steers on monensin-supplemented rations
showed only a :1.80% net change in abomasal nitrogen flow over their
dietary nitrogen intakes. Net feed nitrogen losses in the rumen were
also slightly higher for concentrates than for silage rations.

Losses of dietary nitrogen during ruminal digestion have
generally been reported for rations containing more than 10 to 11%
crude protein, even though these rations may contain adequate amounts
of digestible energy to meet the energy needs of the rumen microorga-
nisms (Hume et_a1,, 1970; Leibholz and Hartmann, 1972). With such
rations, post—ruminal N and NAN flows are often lower than the amounts
of feed nitrogen consumed by the experimental animals.

The extent of ruminal nitrogen loss is controlled by factors
which regulate the efficiency of rumen microbial protein synthesis.
The relationship between rumen nitrogen utilization and dietary protein,
energy and co-factors, have already been reviewed. Other equally
important considerations include the degradability (or solubility)
of the feed protein source and its retention time in the rumen (Bergen
and Yokoyama, 1977). Orskov et__l, (1971) reported a 7 to 10% dietary
nitrogen loss when sheep were fed maize and barley rations containing
up to 15% crude protein. As the soy protein of the ration was in-
creased, the extent of ruminal nitrogen loss was reported to also
increase, especially with rations containing high levels of crude
protein (Orskov at al,, 1971; Hume et_al,, 1970). Soybean meal and

similar protein sources such as peanut meal are extensively degraded

113

in the rumen (Mathers et_al,, 1976; Sniffen, 1978). In contrast, less
degradable proteins such as fish meal or zein are less prone to exten-
sive ruminal nitrogen losses (Orskov at 41., 1974; Mathers _tual.,
1976; Hume, 1974). It is also unlikely that ruminal nitrogen losses
from corn silage rations would be as high as losses from corn-soy
rations. Fermented feeds such as corn silage have been shown to
contain 40 to 50% water soluble nitrogen (Bergen et__l,, 1974;
Wilkinson et_a1,, 1976), which are not readily degraded to ammonia
in the rumen (Bergen et_al,, 1974). This is consistent with results
obtained from this study. Overall, slightly less ruminal nitrogen
losses were found to occur on corn silage than on the high grain
rations.

With rations containing crude protein levels higher than
10 to 11% rumen microbes would produce ammonia in excess of their
needs, and it has been shown that the rate of rumen ammonia release
is proportional to the dietary protein content, rather than to microbial
requirements for nitrogen (Laughren and Young, 1979). Extensive ruminal
nitrogen losses with high quality feed proteins such as soybean may
limit the amount of total protein intake that becomes available to
the animal, thus limiting the efficiency of feed protein utilization
and consequently, the performance of the animal.

This may be of significance only in very high producing (dairy)

cows since ordinarily, the beef animal would obtain sufficient nitrogen

from such a ration to maintain its optimal rate of growth.

114

It is evident from this study that monensin reduced the extent
of dietary nitrogen loss in the rumen (Tables 9 to 12). This agrees
with the results obtained in other nitrogen passage studies (Poos e3.
al,, 1979; Hanson and Klopfenstein, 1979). Monensin has been reported
to cause a decrease in the rates of rumen microbial proteolysis and
deamination of feed protein (Van Nevel and Demeyer, 1977). Rumen
ammonia levels were reported to decrease with addition of monensin
to ruminant rations (Dinius et_al,, 1976; Thornton e£_al,, 1976;

Poos e;_al,, 1979). Rumen ammonia levels were not determined in

this experiment, but abomasal ammonia levels were slightly lower

in monensin-fed steers. However, these may not accurately reflect

the relative ruminal ammonia concentrations, since ruminal and omasal
ammonia absorption rates may vary (Nolan, 1975). Reductions in dietary
nitrogen losses in the rumen by monensin would enhance the efficiency
of feed protein utilization in the ruminant given rations containing
high quality and more degradable preformed plant proteins, especially

when dietary nitrogen levels are low.

NAN Passage to the Abomasum

 

Abomasal NAN passage data are presented in Tables 9 to 13.
As previously noted, estimates of NAN passage calculated from abomasal
lignin and chromium flows (Tables 9 vs. 11), were in close agreement,
and those calculated from N:marker ratios were also similar (Tables
10 vs. 11). Based on the combined average of the four, NAN passage
(as % of N intake) were: 87.88, 97.34, 88.79 and 98.24 for H0, HG-M,

CS and CS-M, respectively. These estimates for the control grain and

115

silage rations (HG and CS) are higher than those obtained by
Crickenberger et_a1, (1979) for steers fed similar rations although
the high grain rations used in their study contained about 42% corn
silage 0M (HG, 87.88 vs. 50.90%; CS, 88.79 vs. 77.50%). Crickenberger
et_al, (1979) also encountered digesta sampling problems and varying
abomasal lignin recoveries.

In contrast, others have reported abomasal NAN recoveries
within the range of estimates obtained in the present study, using
rations containing similar crude protein concentrations (Hume, l970d;
Nicholson and Sutton, 1969; Orskov et_al,, 1971; Prigge _t_al,, 1978;
Kropp et_al,, 1976b). Hume (19700) fed urea, casein, gelatin and zein
as alternative proteins to sheep on rations containing about 14% CP.

He recovered 82.5, 100.6, 91.8 and 135.2% of the dietary N from the
four respective rations as abomasal NAN. Orskov et_al, (1971)
recovered even higher amounts with barley-soybean rations. In steer
nitrogen passage studies, Kropp et_al, (1977b) recovered 99.7 to 136.0%
of the feed N as abomasal NAN using roughage rations supplemented with
soybean meal or urea (8.55 to 12.76% CP).

The increased recovery of NAN with monensin-supplemented ration
found in the present study, is consistent with previous findings that
monensin reduces the rate of protein degradation in the rumen. Poos
at al, (1979) also reported increases in abomasal NAN passage in steers
fed concentrate rations containing monensin. Daily abomasal NAN passage
(as gm crude protein) from this study were: 742.3, 797.6, 826.3 and
843.6 for H0, HG-M, CS and CS-M rations, respectively. These amounts

116

of total daily protein are sufficient to meet the maintenance and
growth protein needs of steer calves, estimated to gain between

0.9 to 1.3 kg per day, based on NRC estimates. The extra protein
passage obtained in monensin-fed steers may be realized in terms of
additional growth, only if the net efficiencies of N utilization are
generally low, due to high maintenance costs (e.g., stresses due to
confinement). Otherwise, the expected optimal growth rates would be
51.1 kg per day, and the NAN passage estimates obtained from all the
rations would seem adequate for optimal growth.

Based on the combined estimates from lignin and chromium flow
data, feed N contributed 30.53 and 28.23% to abomasal NAN passage in
the steers fed the control rations (H0 or CS). The corresponding
values for monensin rations were 39.89 and 43.00% (HG-M or CS-M).
Therefore, 26.82 and 25.07% of feed N passed the rumen undegraded
in steers receiving H0 and CS, respectively. Feed N bypass values
for HG-M and CS-M were 38.83 and 42.24%, respectively. These estimates
are comparable to those reported from digesta passage studies in which
grain and roughage rations, supplemented with soybean meal (Kropp et_
al,, 1977a, 1977b; Cole at al,, 1976) or rations containing monensin
(Poos et_al,, 1979) were fed to growing steers. Soy and ground-corn
proteins have been estimated to be 60 to 90% degraded in the rumen
(Mathers et_al,, 1976; Orskov et_al,, 1971) while ruminal degradation
of corn silage was estimated to be lower--50 to 60% (Orskov and Mehrez-
1977). These estimates do not differ between the two ration-types
used in this study, but monensin increased feed protein bypass by

12 and 17% with the high grain and silage rations, respectively.

117

In contrast to this increase in feed N passage observed,
monensin reduced the amount of microbial nitrogen leaving the rumen.
Microbial N as a percent of abomasal NAN were: 69.48, 60.1, 71.77
and 57.70 for the combined mean estimates from steers fed HG, HG-M,

CS and CS-M, respectively. This observation agrees with the results
reported by others from ig_yiyg_nitrogen passage studies (Poos e§__l,,
1979). Similar findings have also been reported from jg_yit:9_studies
with monensin (Bartley et_al,, 1979; Chen and Wolin, 1979). In the
present study, monensin decreased microbial N passage by 9.48 and
14.07% for the grain and all silage rations, respectively. This
reduction in microbial N flow should be offset by the increases in
feed N bypass observed in this experiment, if the nutritional quality
of the bypassed feed N is similar to that of microbial N. The nutri-
tional quality of soybean meal is only slightly lower than that of
microbial protein (Bergen et_al,, 1978). However, some corn protein
and water soluble nitrogen would also be expected to pass the rumen
undegraded, or partially degraded. The nutritional value to the
animal of the amino acids and aromatic nitrogen constituents of

water soluble nitrogen fraction of silages has not been clearly
determined.

But the added advantage of monensin in this experiment is
that these nitrogen passage estimates were obtained, despite reductions
in nitrogen and 0M intake observed when the steers received monensin
rations. The net gains of monensin supplementation in this study
should therefore be realized primarily in terms of reduced feed

costs and enhanced efficiency of feed N utilization.

118

Efficiency of Rumen Microbial
Protein Synthesis

 

 

As indicated earlier, there was no significant monensin effect
on the amount of organic matter digested in the rumen. However, since
there were differences in daily microbial nitrogen passage to the
abomasum, lower efficiencies of ruminal microbial protein production
were obtained with monensin supplemented rations.

Estimates of percent apparent rumen organic matter digestion
in the present study are similar to those reported in other studies
for steers fed roughage (Kennedy, 1980; Thomson et_a1,, 1972), and
concentrate rations (Kropp et_al,, 1977a, 1977b), but lower than values
obtained from studies in which sheep were given concentrate or semi-
purified diets (Orskov e__a1,, 1971; Hume, l970a). Percent organic
matter actually digested in the rumen was slightly higher for rations
containing monensin, although these estimates were not statistically
different (avg. 50 vs. 44%). As was previously explained for ruminal
DM digestion estimates, the steers were allowed a 28 day adaptation to
the rations, before digesta samples were collected. The results
obtained from the present study do not contradict the conclusion
reached by other investigators that monensin decreases the rate of
rumen microbial protein breakdown, without inhibiting microbial fer-
mentation rate (Dinius _t_al,, 1976; Van Nevel and Demeyer, 1977).
Since monensin also decreased ruminal microbial protein output in

this study, the net effect was a decrease in the efficiency of

microbial synthesis.

119

Microbial crude protein synthesized per 100 gm of organic
matter actually digested in the rumen, were lower for rations con-
taining monensin than for the controls (Tables 17 and 18). The mean
combined estimates from the four quantitation methods used were 16.76 g
for monensin-supplemented rations and 20.24 g for the controls. True
microbial efficiencies were, however, only slightly reduced by monensin
in the grain ration (19.85 vs. 18.44 g per 100 g OMB), and more drasti-
cally reduced in monensin-silage ration (20.63 vs. 15.07 g/100 g OMD).
Perhaps rumen ammonia levels were less than Optimum in the steers fed
the CS-M rations. Since a major proportion of silage nitrogen consists
of the ruminally less degradable water soluble nitrogen (Bergen et_al,,
1974), this, in addition to the reduced rate of feed protein degradation
by monensin, may have created suboptimal rumen ammonia levels which
limited microbial growth.

Van Nevel and Demeyer (1977) had previously reported that
monensin may inhibit ruminal protein deamination, rather than pro-
teolysis. They further suggested that peptide uptake by bacteria
may be another step in bacterial protein metabolism that is also
inhibited by monensin. Bartely et_al, (1979) observed decreases
in the rate of urea hydrolysis by monensin jg_yjt§g, Such findings,
together with previous reports of reduced rumen ammonia levels by this
antibiotic (Dinius e;_al,, 1976; Thorton et_al,, 1976), suggest that
the generation of ammonia in the rumen may be one step at which
monensin interferes with microbial growth. Since the results

presented here and those from other studies (Van Nevel and Demeyer,

120

1977) indicate that monensin does not impede microbial fermentation
of organic matter, ammonia supply may be limiting the growth of many
rumen bacteria to such an extent that energetic uncoupling occurs.
This would inhibit bacterial growth since many rumen bacteria use
ammonia as their preferred nitrogen source, even in the presence of
amino acids (Bryant and Robinson, 1962).

Energetic uncoupling occurs with most bacteria when their
potential to produce energy from substrates exceeds their ability to
utilize that energy for biosynthetic purposes (Hespell and Bryant,
1979). As a result of such an ammonia insufficiency as proposed above,
microbial cell yield would decrease without a concomitant decrease in
fermentation rate (Hespell and Bryant, 1979). Alternatively (or in
addition to the above), monensin may also affect the rate of amino acid
and peptide (as well as other nutrient) uptake by rumen microbes and
cause a growth rate depression.

Other investigators have offered various suggestions as to the
mode of action of this antibiotic. Chen and Wolin (1979) concluded
from their jg_yjt§g studies that monensin may act by selectively
inhibiting, to a variable extent, the growth of certain bacteria
species. They further hypothesized that monensin may act in the
rumen by selecting for a microbial population that produces more

propionate.

Nucleic Acid Methodology

 

The use of bacterial RNA-Nztotal N ratios to estimate the
proportion of microbial N passage from the rumen, hinges on the

assumption that this ratio is similar to that of the microorganism

121

passing through the abomasum. The validity of that assumption depends
on the similarity of this ratio between rumen protozoa and bacteria
and also on the contribution of protozoa to post-ruminal digesta flow.
Contrary to the findings of Smith et_al, (1978), RNA estimates of
protozoal preparations isolated from rumen contents obtained in this
study were much lower than the corresponding estimates from bacterial
preparations. A similar trend was indicated in the data presented

by Ling and Buttery (1978), if it is assumed that total N content of
the bacteria and protozoa they isolated in that study were similar.
The use of bacterial RNA-Nztotal N ratio alone to estimate microbial

N passage to the abomasum in this study would therefore underestimate
microbial passage to a varying extent, depending on the rate of pro-
tozoal N exit from the rumen of each experimental animal with each of
the rations fed.

The assumption that all the nucleic acid passing out of the
rumen is microbial in origin has also been disputed by other inves-
tigators (Ling and Buttery, 1978; Smith e§_al,, 1978). These workers
compared duodenal microbial-N'passage data estimated by the RNA approach

35 32F labels incorporated

with the same estimates obtained using S or
into microbial cells and observed that RNA-based estimates were con-
sistently higher. Smith et_al, (1978) found that RNA-based estimates
of duodenal microbial N flow were consistently about 15% higher than
those determined by the radioisotope incorporation method and hence
corrected duodenal RNA values by multiplying by 0.85 (adjusted RNA).

Smith e§_a1, (1978) reasoned that this correction approach would equate

122

35s

RNA-based microbial N flow with the more quantitatively accurate
incorporation method (Ling and Buttery, 1978). They (Smith et_al,,
1978) also concluded that some dietary RNA may have escaped degradation
in the rumen. Based on the above considerations and also on the fact
that RNA estimates from abomasal digesta in the present study appeared
exceedingly high and, in a few cases, resulted in calculated microbial
NAN passage equal to (or greater than) total abomasal NAN passage, the
adjusted RNA values were used to determine microbial contribution to
abomasal NAN flow. Estimates of RNA in bacterial preparations in this
study gave RNA-Nztotal N ratios within the range of those obtained by
Smith et_al, (1976; 1978) from animals fed similar rations (0.156 vs.
0.12 to 0.19), even though the extensive ion—exchange procedure was
eliminated from our analysis.

Wide variations in RNA values between and within digesta
samples obtained from individual steers were observed. Such problems
were also encountered by others (Ling and Buttery, 1978; Smith e3_al,-
1978). RNA concentrations determined from oven-dried abomasal digesta
samples were 20 to 30% higher than the corresponding values from
undried abomasal digesta samples. The reason for such a discrepancy
is not clear. It is possible that more of the lipid/pigment and other
materials which absorb light at 260 nm were not removed to the same
extent from both samples during the initial extraction with organic
solvents. RNA values from undried abomasal digesta were also less
variable within animals and were therefore used for subsequent

calculations.

123

Nitrogen Balance and Digestibility
Studies

 

-The nitrogen balance trials were conducted to examine the
effects of monensin on overall dry matter digestibility and efficiency
of nitrogen utilization. Monensin reduced the amount of nitrogen
excreted in the feces without increasing urinary nitrogen excretion.
Nitrogen digestion as a percent of N-intake was higher for HG—M than
for H0 rations (71.38 vs. 66.07), and similar for CS and CS-M rations
(65.46 vs. 66.60, respectively). This increase in N digestion by
monensin in the grain ration also resulted in an increase in the
percent of N-intake retained, although these were not significant.
Poos et_al, (1979) reported increases in apparent N digestibility
and retention in lambs fed monensin in concentrate rations in one
trial, although these authors observed the opposite effect in another
balance study. In the present study, the trend was for an increase in
the amounts of nitrogen retained with HG-M and CS-M rations, which may
be attributed primarily to increases in total N digestion. There is
no ready explanation for these results. Monensin-fed steers consumed
less amount of feed DM and protein since the rations were offered ad_
libitum. It may be that the feed protein that bypassed ruminal
degradation with monensin rations was high in nutritional quality.
This protein, together with microbial protein, may have been more
digestible in the gastrointestines. As indicated above, the slightly
higher (though not statistically different) percent N digestibility
and retention with CS-M ration (in comparison with control silage)

may simply be due to a lower N intake by the steers when fed the

124

CS-M ration (see Table 20). The data were not adjusted to equal
intakes. However, the above speculation may be valid for HG-M versus
HG rations, although N retention for the grain rations were also not
statistically different.

The estimates of DM digestion followed the same pattern as that
observed in N digestibility. Monensin increased DM digestibility of
the high grain rations (75.36 vs. 81.63%), but did not affect corn
silage dry matter digestion. There was also no monensin influence
on ADF digestibility. ADF digestibility values were: 46.43, 46.40,
60.29 and 59.38 for HG, HG-M, CS and CS-M rations, respectively. The
higher amounts of ADF digested in the silage rations may indicate a
longer rumen retention time of forage particulate matter (size factor),
and perhaps a more extensive silage fiber digestion in the lower G.I.
tract. The latter observation appears to be a credible explanation,
in view of the percent fecal ADF recoveries obtained (Table 21).
Fecal recoveries of abomasal ADF were about 10% higher for CS and
CS-M rations, than for H0 and HG-M rations. This implies that about
10% more ADF was digested in the lower G.I. tract of the steers when
they received the silage rations.

Nitrogen retention and 0M digestibility estimates obtained in
the present study are about 5% higher than those reported by Poos et_
a1, (1979) for lambs fed monensin and concentrate rations, but are
similar to values obtained by Crickenberger et_al, (1979) for growing
steers receiving similar grain and silage rations (without monensin).

Poos e§_al, (1979), however, reported reductions in DM and ADF

125

digestibility in lambs fed monensin and concentrate rations.

The latter investigators also observed no monensin effect on both
parameters, when the lambs were fed for a longer period. This latter
observation may explain the absence of a significant monensin effect
on 0M and fiber digestion in this study since, as previously reported,
the animals were allowed a relatively long period to adapt to each of
the rations. Dinius et_al, (1976) also reported that monensin did not
affect DM and fiber digestion with steers adapted to monensin rations
for 21 days.

As was previously stated, microbial protein passage to the
abomasum was reduced in the steers that received monensin (especially
in the silage rations). Yet those steers had a slightly greater amount
of nitrogen retained. A possible explanation is that the increased
abomasal NAN passage with HG-M and CS-M must have compensated for
reduced microbial-N passage in steers given those rations. Since
microbial protein is high in nutritional quality, the protein that
bypassed ruminal degradation with monensin rations must have had a
quality comparable to that of microbial protein. Perhaps more soy-
bean, which has a slightly lower nutritional quality than microbial
protein (Bergen et_al,, 1978) escaped ruminal degradation in monensin
rations. On the other hand, maybe the bypassed protein and the
microbial protein blended better in terms of the total amino acid
profile.

The amino acid profile of bypass protein and the relative

proportion of that protein that is unavailable to the animal (bound

126

protein) should both be considered when assessing the nutritional
value of that protein to the animal. In this regard, soy protein
contains only about 2.5% bound protein, and the amino acid profile
of its total and insoluble protein (75% of the total) are quite
similar (Sniffen and Hoover, 1978). The animal would therefore
benefit from an increase in soy protein bypass.

The amount of nitrogen retained by the animal is an approximate
indicator of the efficiency with which a dietary nitrogen source is
utilized. It is possible to estimate from the amounts of nitrogen
retained (NR), the expected daily weight gains (DWG) of the steers
for each of the rations fed during the nitrogen metabolism studies.
This was calculated from the following equation, which converts
nitrogen retention into muscle protein accretion (muscle protein
was assumed to contain 80% water, although this should be lower in

the yearling steers):
DWG (kg) = NR (kg) x 6.25 x 5.

Based on such calculations, the expected daily weight gains
on the HG, HG-M, CS and CS-M rations were 1.33, 1.51, 1.38 and 1.37 kg,
respectively. The values are slightly higher than the previous esti-
mates from abomasal crude protein passage (see discussion: NAN
Passage). The NAN passage data are closer to the actual daily weight
gains obtained during the study. The steers gained about 0.97 kg per
day on HG and HG-M rations combined. The effect of monensin on ADG

during the periods the steers received the grain rations cannot be

127

determined because the animals were not weighed at the end of period 1,
when the grain rations were switched. But ADG on the CS and CS-M
rations were 0.87 and 0.92 kg, respectively, and these also fall within
the range of daily gains estimated from the abomasal crude protein flows
(and NRC tables). In addition to the fact that nitrogen retention tends
to overestimate weight gains, such deviations from the expected growth
rates may also relate to the stresses of confinement to which the steers
were subjected during the entire study. The slightly better performance
observed with the high grain rations may reflect a relatively higher
efficiency of corn nitrogen and energy utilization for rumen microbial

synthesis.

Feeding Trial and Plasma Metabolite Studies

 

Feeding Trial

 

The indication from the results presented in Table 22 is that
the combination of monensin and elfazepam resulted in the highest
reduction in feed intake. In comparison with controls, monensin-
elfazepam treatment reduced intake by 8.0%, while monensin alone
resulted in a 3% reduction in feed consumption. In contrast, elfazepam
alone increased feed intake by 8.5%. Despite this stimulation of
intake, elfazepam did not significantly affect ADG (0.99 vs. 0.98 kg
for the control). Monensin improved ADG by 10%, while monensin-
elfazepam depressed ADG by 12%. Monensin treatment also resulted
in the best efficiency of feed conversion (12.5% improvement over
the control treatment), while elfazepam and the two-drug combination

each produced F/G ratios that were 8% poorer than the controls.

128

Elfazepam has long been recognized as a potent feed intake
stimulant when given to various animals (Baile et_al,, 1976; Wise
and Dawson, 1974; Dinius and Baile, 1977). Dinius and Baile (1977)
fed elfazepam at increasing dosage levels to steers on a ground hay
ration and reported 26.8, 7.3 and 16.4% improvement in ADG, feed intake
and feed efficiency, respectively. They found no differences in
response between the different levels (up to 2 ppm) of elfazepam
fed. The only major similarity between their findings and the results
obtained in the present study is that elfazepam stimulated intake in
this study. However, differences in both performance results may be
due to differences in the type of rations used in the two studies.

Prior et_al, (1978) did not even ellicit an intake response
in steers fed elfazepam and corn silage rations. In the latter study,
the steers were fed to a constant weight, and elfazepam reduced ADG
by 9%. Some other workers (Gonzalez et_al,, 1977; Wangsness et_al,,
1977).have, however, obtained increases in intake with elfazepam and
roughage rations, along with increased 0M and protein digestibility.

It seems that positive responses to elfazepam were often
obtained with dry roughage and concentrate rations (Gonzalez e§_al,.
1977; Farlin and Baile, 1977; Dinius and Baile, 1977). With fermented
feeds such as corn silage rations, elfazepam may stimulate intake, but
has not improved performance (Prior e§_al, 1978). Prior egnal. (1978)
speCulated that the lack of growth response they observed with corn
silage may be related to the ration moisture content and perhaps to

the stability of elfazepam in fermented feeds.

129

The results obtained in the present study for monensin-elfazepam
treatment combination also contradicts those reported by Farlin and
Baile (1977). These workers obtained improvements in ADG and F/G
ratios in heifers fed concentrate rations with monensin and elfazepam
included in the same diets. Dinius and Baile (1977) also reported that
elfazepam tended to alleviate the depression in feed intake induced by
monensin in steers given both chemicals. In contrast, the results here
indicate that the two drugs when combined further inhibited feed intake
(Table 22). It is interesting to note that the former investigators
(Dinius and Baile, 1977) also reported that one of the steers became
hyp0phagic when placed on monensin-elfazepam rations, and therefore
had to be removed from the experiment. Baile and McLaughlin (1979)
nonetheless concluded that elfazepam may still be helpful, in some
rations, in overcoming the initial reduction in feed intake caused
by monensin, but that it may not be effective with all rations.

The results obtained in this study with monensin treatment
are consistent with the performance data reported by several other
investigators (Potter et_al,, 1976a; Raun et_al,, 1976; Hanson and
Klopfenstein, 1979). Monensin appears to increase the efficiency
of protein and feed ME utilization. This would result in decreased
feed costs and increased net returns to the ruminant livestock pro-
ducer. On the other hand, elfazepam may be beneficial in situations
in which feed consumption by the ruminant is restricted or regulated
by factors other than energy balance. This may apply to high roughage

rations and fermented feeds such as corn silage, where intake may be

130

limited by factors such as bulk (rumen fill) and ruminal solid digesta

flow rates.

Plasma Metabolite Studies

As previously stated, there were no significant effects of
treatments on overall plasma amino acid (PAA) profiles, blood glucose
and blood urea levels (P<:0.05). The overall patterns were for slightly
elevated PAA levels in steers fed the two monensin rations. These
trends were more apparent for glutamate, aspartate, threonine and
serine, the typical amino acids involved in transamination and glu-
coneogenesis. Although there were no ration differences in the overall
levels of the branched-chain amino acids (Table 24), it is evident from
Figure 4 that monensin caused some depression in the plasma levels of
valine and isoleucine during the first half of the experiment. Plasma
urea-nitrogen (PUN) was also slightly elevated with both monensin-
supplemented rations, in comparison to the responses obtained with
elfazepam and control rations. As previously reported, plasma glucose
levels seemed relatively high with all treatments, but were highest in
the control steers.

The relative usefulness of PAA profile as an indicator of the
nutritional status of the ruminant has been extensively reviewed by
some investigators (Bergman and Heitmann, 1978; Bergen, 1979b). Plasma
or tissue free amino acids represent only a small pr0portion of total
body amino acids and may not accurately reflect the magnitude of AA
fluxes between the different tissue pools. Changes in PAA patterns

in the ruminant also do not usually reflect the dietary AA profile,

131

since microbial protein generally constitutes a major proportion of
post-ruminal nitrogen flow. The exception to this are proteins that
are relatively more resistant to ruminal degradation, but digestible
in the lower tract. Large excesses or deficiencies of AA in proteins
that escape ruminal degradation will be reflected to some extent, in
the PAA profile (Bergen et_al,, 1973). Also, PAA levels of the
branched-chain amino acids can often indicate the animal's nutritional
state. Under starvation or protein deprivation, there is an elevation
in plasma levels of those amino acids, and this indicates muscle
protein catabolism (Leibholz, 1970).

In contrast, intravenous administration of glucose, propionate
or other VFA's results in an insulin-mediated depression of PAA levels
(Call et_a1,, 1972), with subsequent increase in nitrogen retention
(Eskeland et.al,, 1974). Glucose and propionate were shown to produce
greater decline in PAA levels, with the branched-chain AA's most rapidly
declining (Potter et_al,, 1968). The above response was interpreted to
mean an increase or induction of tissue protein synthesis (Eskeland et_
al,, 1974). In the ruminant, 50 to 60% of blood glucose may be syn-
thesized from pr0pionate (Young, 1977). Since dietary carbohydrates
are usually fermented in the rumen to VFA's, often less than 10% of
the animal's glucose needs is obtained from glucose absorption in the
gastro intestines. This implies that gluconeogenesis is of tremendous
importance to the ruminant. It also means that other glucogenic sub-
strates have to provide up to 40% of the animal's glucose needs, and

these are usually the glucogenic amino acids, which could be more

132

efficiently utilized for tissue synthesis. Since it has been
definitely shown that monensin increases rumen propionate levels,
while simultaneously decreasing the rate of feed protein degradation
in the rumen, it is apparent why the efficiency of nitrogen utilization
would be improved in ruminants fed monensin-supplemented good quality
plant proteins.

The trends in PAA, PUN and plasma glucose found in the present
study are consistent with those reported by Pendlum _§_a1, (1980).
The decline in plasma levels of valine and isoleucine in the steers
receiving monensin rations during the first half of this study, may
indicate an increase in the rate of protein synthesis and growth.
Since no adjustments in the dietary crude protein level were made
as the trial progressed, the subsequent rise in PAA and PUN levels
may be due to protein reaching the lower gut in excess of the animal's

protein needs, during the latter phase of growth.

Monensin-Na: Mechanism of Action

 

Monensin belongs to the class of carboxylic ion0phores that
catalyzes the electro-neutral exchange of cations for protons. It
is similar in structure and function to nigericin, but mediates the
exchange of Na+ for H+ across cell membranes (Harold, 1972). These
compounds have strong affinity for specific monovalent metal cations,
which they capture by ligand-binding to several oxygen atoms within
their framework (Pressman, 1973). The terminal carboxylic acid
must be deprotonated in order to form the complex (Pressman, 1973).

Consequently, an ionophore such as monensin forms a charged-neutral

133

isostoichiometric complex (zwitterion) with Na+. Monensic acid would
most likely adopt a similar conformation when in solution abundant
with monovalent cations, such as rumen fluid. Monensic acid has a
pka of 6.65 (Haney and Hoehn, 1968), so that it would be easily
deprotonated in rumen fluid.

It has been demonstrated in many facultative and anaerobic
bacteria (e.g., E, 9911, S, faecalis) that, as in mitochondria and
aerobic microorganisms, nutrient uptake depends on the maintenance
of a proton-motive force (comprising of a pH and an electro-chemical
gradient) between the cell and the surrounding media. According to
the chemi-osmotic hypothesis, protons produced as a result of substrate
oxidation are extruded from the cell, thereby generating a pH gradient
(interior alkaline) and an electro-chemical gradient, to which nutrient
transport is coupled (Mitchell, 1972). Addition of monensin to the
media may initially reverse the flow of protons, thereby collapsing
the pH gradient (Harold, 1972). Since the ion0phore facilitates
antiport exchange of Na+ for H+, the electrochemical gradient would
remain intact. This reduction in total proton-motive force may perhaps
reduce the rate of uptake of nutrients required for microbial synthesis.

Rumen fluid usually has a high concentration of Na+ and KT,
both of which are obligate growth requirements for rumen bacteria,
although K+ requirement is less stringent than that of Na+ (Caldwell
and Anderson, 1974). Both cations are required for many cellular
functions which include nutrient transport and activation of many

enzymes (e.g., ATPases; Harold, 1977). Rumen bacteria would therefore

134

tend to have a high intracellular concentration of those cations under
normal feed lot conditions. Addition of monensin to ruminant rations
may initially result in the rapid extrusion of those intracellular
cations coupled to an influx of protons into rumen microbial cells,
thereby inhibiting nutrient uptake. It has been shown that amino acid
and P04 transport in S, faecalis, and in many other bacteria is inhib-
ited by monensin, indicating that the pH gradient may play a greater
role in the transport of those nutrients (Harold, 1972).

Other investigators (Estrada-0 and Calderon, 1970) reported
that monensin inhibited the energy-dependent PO4 uptake, and its
incorporation into ADP in mitochondrial preparations, a situation
they described as analogous to the inhibition of mitochondrial oxi-
dation of TCA intermediates under conditions of cellular hyperosmo-
larity. Estrada-0 and Calderon (1970) further reported that the above
inhibition was reversed by the addition of succinate, B—hydroxybutyrate,
glutamate + malate, or by increasing the P04 concentration of the media.
The preferential accumulation and oxidation of succinate and B-
hydroxybutyrate thus, provided the energy required for the uptake
of P04's. It is not unlikely that the initial inhibition of energy
substrate degradation and the increased production of pr0pionate, both
of which occur when ruminants are initially exposed to monensin, are
based on similar physiological responses. In such a situation,
bacterial species that can metabolize those metabolic intermediates
(e.g., succinate and propionate producers with membrane-bound cyto-

chromes) would have a selective advantage for growth in the rumen.

135

Adaptation to monensin by rumen microbial species probably
occurs as a result of the cyclical conductance of ions, since that
seems to be the basic mode of action of most carboxylic ionophores
(Pressman, 1973). A gradient of monovalent cations has to exist for
the ionophore to functionally exchange cations for protons. This
exchange, which is facilitated by the ionophore moving in an alter-
nating fashion to either surface of the lipid membrane, continues
until equilibrium is established (Mitchell, 1976). In this situation,
rumen bacteria may probably have sufficient intracellular cations (Na+
and KT) for adequate growth. The specific mechanism by which monensin
alters or influences nutrient metabolism in rumen microbial cells can-
not be conclusively determined until adequate jg_yj§[9_and jg_yjyg.
studies with rumen microorganisms are conducted. However, it seems
likely that alterations in microbial membrane permeability caused by
monensin, may also result in tremendous expenditure of ATP in order

to maintain the transmembrane potential necessary for nutrient uptake.

CHAPTER V

CONCLUSIONS

Based on the data obtained in these studies, the following

conclusions are made:

1.

Monensin increased ruminal dry matter digestion of the corn
silage rations, but did not significantly affect DM digestion
of the high grain rations in the rumen.

Apparent ADF and NDF digestion in the rumen were not affected
by monensin.

Monensin increased NAN (% of N-intake) passage to the abomasum
in silage and high grain rations.

Monensin increased the proportion of dietary nitrogen that
escaped degradation in the rumen, but did not affect abomasal
digesta flow rates.

Ruminal organic matter digestion was increased by monensin in
steers fed silage, but not in steers that received grain
rations.

Monensin reduced the amount of microbial N leaving the rumen.
Efficiency of ruminal microbial protein synthesis was reduced
by monensin in the rations.

Abomasal appearance of the various components of digesta based
on lignin and chromium passage were similar between the two

digesta markers.

136

10.

11.

12.

137

PEG tended to overestimate liquid digesta passage to the
abomasum.

Monensin did not influence nitrogen retention or fiber
digestion in the steers, but increased apparent N digestibility
of grain rations.

From the results of the feeding trial, it is concluded that
plasma amino acid, plasma urea and plasma glucose levels of
growing steers were not affected by addition of monensin,
elfazepam and the two chemicals combined, to all-silage
rations.

Monensin improved the overall performance of steers fed
all-silage rations; elfazepam only stimulated silage intake,
while the combination of elfazepam and monensin in the silage

resulted in poor performance.

APPENDIX

138

TABLE A.1 INDIVIDUAL STEER APPARENT FIBER DIGESTION (FROM LIGNIN-BASED
DM FLOW) IN THE RUMEN: DIGESTA PASSAGE STUDIES

 

 

Feed Abomasal App.rumen Feed Abomasal App. rumen
a NDF-intake NDF NDF ADF-i ntake ADF ADFD
Steer (9m) (9m) (9m) (9m) (9m) (9m)

 

992-1b 1439.02 895.79 543.24 663.87 537.16 126.61
528-1 1343.09 980.67 362.42 619.61 525.42 94.19
994-2 1617.01 1174.59 442.42 745.98 640.40 105.57
799-2 1271.22 1035.22 236.00 586.46 511.16 75.30

992-2 1430.86 1009.71 421.15 691.18 549.12 142.06
528-2 1239.52 920.88 318.64 599.02 504.36 94.66
994-1 1808.06 1155.57 652.49 824.11 566.24 257.87
799-1 1216.33 870.07 346.26 573.31 444.82 128.49

992-3 2722.73 1905.69 817.04 1607.69 1152.51 455.18
528-3 2683.76 1728.30 955.46 1584.76 974.25 610.52
994-4 2976.64 1968.87 1007.79 1766.75 1186.44 580.31
799-4 2422.78 1702.21 720.57 1431.73 1058.26 373.47

992-4 2877.01 1990.52 886.49 1595.78 1161.05 434.73
528-4 2735.74 1940.80 794.95 1545.04 1157.89 387.14
994-3 4000.27 3113.43 886.84 2105.41 1682.09 423.32
799-3 3500.24 2358.93 1141.31 1842.23 1295.87 546.36

 

aSteer notations represent rations: HG, HG-M, CS and CS-M,
respectively, for Tables A.1 through A.10.

bNot used in calculations; steer developed abomasal lacerations.

139

 

 

 

TABLE A.2 INDIVIDUAL STEER: TRUE RUMEN DRY MATTER DIGESTION. PASSAGE
STUDIES--LIGNIN BASED ESTIMATES
Abom. Microb. Microb. Feed DM Rumen
DM flow 9 Microb. NAN flow DM flow flow DMD
Steer (gm) N/g DM (g/day) (g/day) (9) (g/day)
992-1a 5160.05 .08322 72.20 867.58 4292.47 2103.18
528-1 3674.30 .08322 69.85 839.34 . 2834.96 3134.32
994-2 5058.54 .08369 83.88 1002.27 4056.27 3130.45
799-2 4227.12 .08369 66.70 796.99 3430.13 2219.73
992-2 4382.44 .08369 85.54 1022.11 3360.33 3021.72
528-2 2987.94 .08369 63.30 756.37 2231.58 3299.53
994-1 5772.09 .08322 106.29 1277.22 4494.87 3537.40
799-1 4348.18 .08322 72.56 871.91 3476.27 1927.24
992-3 4501.98 .08350 89.73 1074.61 3427.37 2995.68
528-3 4745.47 .08350 95.95 1149.10 3596.37 2735.07
994-4 4575.54 .08367 81.72 976.69 3598.85 3423.19
799-4 3656.74 .08367 68.38 817.26 2839.48 2875.99
992-4 5101.29 .08367 67.85 810.924 4290.37 2674.08
528-4 4507.19 .08367 86.51 1033.94 3473.25 2839.02
994-3 7621.22 .08350 111.60 1336.53 6284.69 2239.22
799-3 5805.88 .08350 I86.15 1031.74 4774.14 2684.28

 

aNot used in calculations; steer developed abomasal lacerations.

140

 

 

 

TABLE A.3 INDIVIDUAL STEER: TRUE RUMEN DMD. PASSAGE STUDIES:
CHROMIUM-BASED ESTIMATES
Microb. Microb. Feed DM Rumen
DM flow 9 Microb. NAN flow DM flow flow DMD

Steer (9m) N/g DM (g/day) (g/day) (9) (glday)
992-1a 4975.00 .08322 69.62 836.58 4138.42 2257.23
528-1 3515.00 .08322 70.00 841.14 2673.86 3295.42
994-2 5788.00 .08369 94.98 1134.90 4653.10 2533.62
799-2 4175.00 .08369 65.86 786.95 3388.05 2261.81
992-2 4252.00 .08369 83.00 991.76 3260.24 3121.81
528-2 3128.00 .08369 66.24 791.49 2336.51 3194.69
994-1 5586.00 .08322 102.85 1235.88 4350.12 3682.15
799-1 4308.63 .08322 71.89 863.86 3444.77 1958.74
992-3 4444.00 .08350 88.26 1057.01 3386.99 3036.06
528-3 4325.00 .08350 87.43 1047.07 3277.93 3053.51
994-4 4631.00 .08367 82.55 986.61 3644.39 3377.65
799-4 3767.00 .08367 70.38 841.16 2925.84 2789.63
992-4 4161.00 .08367 67.92 811.76 3349.24 3615.21
528-4 4698.00 .08367 90.17 1077.69 3620.31 2691.96
994-3 7654.00 .08350 112.04 1341.80 6312.20 2211.71
799-3 5913.00 .08350 87.71 1050.42 4862.58 2595.84

 

aNot used in calculations; steer developed abomasal lacerations.

141

 

 

 

TABLE A.4 INDIVIDUAL STEER NAN PASSAGE--CALCULATED FROM LIGNIN OUTFLOW
FROM THE RUMEN

Lignin mg Lignin mg g NAN

intake Lignin/m1 outflow NAN/m1 flow
Steer DMI (gm) (g/day) ab. fluid liters ab. fluid day
992-1a 6395.65 197.63 2.0840 94.830 2.2394 212.36
528-1 5969.28 176.02 4.4879 39.220 2.800 109.82
994-2 7186.72 222.07 4.4300 50.129 2.920 146.38
799-2 5649.86 174.58 3.890 44.880 2.549 114.40
992-2 6382.05 197.21 4.4910 43.911 2.9923 131.40
528-2 5531.11 170.91 5.5027 31.060 3.380 104.98
994-1 8032.21 248.20 4.9622 50.017 3.010 150.55
799-1 5403.51 166.97 4.2860 38.957 2.500 97.39
992-3 6423.05 249.86 4.0515 61.670 2.0464 126.20
528-3 6331.44 246.29 3.8670 63.691 2.180 138.85
994-4 7022.04 273.16 4.1432 65.929 2.3610 155.66
799-4 5715.47 222.33 4.0067 55.490 2.2819 126.62
992-4 6964.45 276.49 4.401 62.824 1.960 123.14
528-4 6312.27 250.60 4.0143 62.427 2.0652 128.92
994-3 8523.91 338.40 5.11932 66.102 2.3579 155.86
799-3 7458.42 296.10 5.9976 49.370 2.3021 113.66

 

aNot used in calculations; steer developed abomasal lacerations.

142

TABLE A.5 INDIVIDUAL STEER NAN PASSAGE: FROM N:LIGNIN RATIOS

 

 

 

Abom. Feed NAN

lignin lignin Abom. Feed passage
Steer (g/g DM) (g/g DM) N/lignin N/lignin (9/day)
992-1a .0383 .0309 1.07490 .68350 212.46
528-1 .0502 .0309 .62390 .68366 115.07
994-2 .0439 .0309 .65923 .68350 146.41
799-2 .0413 .0309 .65521 .6822 114.38
992-2 .0450 .0309 .66629 .68867 131 40
528-2 .0572 .0309 .61425 .68867 104.98
994-1 .0430 .0309 .60658 .67327 150.55
799-1 .0384 .0309 .58333 .67259 97.40
992.3 .0555 .0389 .50504 .58393 126.19
528-3_ .0519 .0389 .56377 .57979 138.85
994-4 .0597 .0389 .56985 .59229 155.66
799-4 .0608 .0389 .56952 .59596 126.63
992-4 .0542 .0397 .4454 .47786 123.16
528-4 .0556 .0397 .51446 .49159 128.92
994-3 .0444 .0397 .46058 .48363 155.86
799-3 .0510 .0397 .3838 .48363 113.64

 

aNot used in calculations; steer developed abomasal lacerations.

143

 

 

 

 

 

 

 

TABLE A.6 INDIVIDUAL STEER NAN PASSAGE: CALCULATED FROM CHROMIUM
OUTFLOW
Chromium mg Chromium mg g NAN
intake chr./ml outflow NAN/m1 per

Steer feed DM (g/day) ab. fluid (liters) ab. fluid day
Collection 1:
992-1a 1.8042 11.54 0.1262 91.44 2.2394 204.76
528-1 1.8042 10.77 0.2740 39.31 2.800 110.06
994-2 1.9406 13.95 0.2457 56.76 2.920 165.75
799-2 1.9406 10.964 0.2474 44.32 2.549 112.97
Collection 2:
992-2 2.1095 13.463 0.3160 42.60 2.9923 127.59
528-2 2.1095 11.67 0.359 32.50 3.380 109.85
994-1 2.1572 17.33 0.358 48.40 3.010 145.68
799-1 2.1572 11.66 0.302 38.60 2.50 96.494
Collection 3:
992-3 1.7188 11.04 0.18134 60.66 2.0464 124.13
528-3 1.7188 10.88 0.18754 58.040 2.180 126.53
994-4 1.7020 11.95 0.1791 66.731 2.3610 157.55
799-4 1.7020 9.73 0.17022 57.121 2.2819 130.34
Collection 4:
992-4 1.7490 12.18 0.19360 62.885 1.960 123.26
528-4 1.7490 11.04 0.16967 65.068 2.0652 134.38
994-3 1.8429 15.71 0.2367 66.366 2.3579 156.48
799-3 1.8429 13.75 0.2735 50.263 2.3021 115.71

 

aNot used in calculations;

steer developed abomasal lacerations.

144

 

 

 

 

 

 

 

TABLE A.7 INDIVIDUAL STEER NAN PASSAGE: FROM N:CHROMIUM RATIOS
Abom. Feed Feed NAN

N2 9 Chr./g nitrogen chromium Abom. N: Feed N: passage
Steer (g/g DM) abom. DM (g/g DM) (g/g DM) chromium Chromium (g/day)
Collection 1:
992-1a .04117 .0023195 .02112 .0018042 17.750 11.706 204.85
528-1 .03132 .0030643 .021125 .0018042 10.221 11.709 110.07
994-2 .02894 .0022373 .02112 .0019406 12.935 10.8626 180.76
799-2 .02706 .0026264 .02108 .0019406 10.3031 10.8626 112.97
Collection 2:
992-2 .02998 .0031600 .02128 .0021095 9.4873 10.088 127.72
528-2 .035135 .0037312 .02128 .0021095 9.4170 10.088 109.87
994-1 .026083 .0031026 .020804 .0021572 8.4068 9.644 145.66
799-1 .022400 .0027062 .020783 .0021572 8.2773 9.634 96.49
Collection 3:
992-3 .02803 .0024841 .022715 .0017188 11.284 13.216 124.57
528-3 .02926 .0025173 .022554 .0017188 11.6236 13.123 126.48
994-4 .03402 .0025805 .023040 .0017020 13.1707 13.537 157.41
799-4 .03463 .0025830 .023183 .0017020 13.407 13.621 130.42
Collection 4:
992-4 .02414 .0023853 .018971 .001749 10.1203 10.847 123.27
528-4 .028604 .002350 .019516 .001749 12.172 11.1584 134.38
994-3 .02045 .0020526 .019200 .0018429 9.963 10.4184 156.51
799-3 .019576 .0023254 .019200 .0018429 8.41834 10.4184 115.71

 

aNot used in calculations; steer developed

abomasal lacerations.

145

TABLE A.8 INDIVIDUAL NAN--LIQUID OUTFLOW:
(PEG) DIGESTA PASSAGE

FROM PARTITIONED LIQUID

 

 

 

 

PEG gm PEG/m1 PEG NAN/day
(gm) abomasal outflow NAN/m1 supernate
Steer intake supernate m1 supernate (gm)
992-1a 32.60 0.3136 103,954.10 1.19975 124.72
528-1 30.41 0.6882 44,187.70 1.60560 70.95
994-2 37.54 0.5459 68,767.20 1.68990 116.21
799-2 28.13 0.3870 72,687.34 1.03881 75.51
992-2 36.17 0.5351 67,594.80 1.66340 112.44
528-2 31.35 0.6580 47,644.00 1.91070 91.03
994-1 34.535 0.6285 59,721.60 1.72780 103.19
799-1 24.50 0.3528 69,444.44 1.37430 95.44
992-3 33.81 0.3696 91.477.27 0.86670 79.28
528-3 33.15 0.4513 74,008.42 0.91560 67.76
994-4 37.342 0.3936 94,872.97 0.89412 84.83
799-4 30.513 0.2818 108,278.92 0.7707 83.45
992-4 29.82 0.2439 122,267.32 0.65712 80.34
528-4 27.61 0.3744 73,744.66 0.86404 63.72
994-3 30.83 0.3744 82,318.40 0.9675 79.64
799-3 26.98 0.3086 87,427.10 0.8097 79.79
aNot used in calculations; steer developed abomasal lacerations.

146

 

 

 

 

TABLE A.9 INDIVIDUAL STEER NAN--SOLID DIGESTA OUTFLOW: FROM

PARTITIONED SOLID (CHROMIUM) PASSAGE

Chromium mg Chromium Residue g NAN

intake per gm. abom. outflow NAN/gm solid
Steer (g/day) residue (gm) residue flow/day
992-1a 11 54 2.3195 4,975.00 .02007 99.85
528—1 10.77 3.0643 3,515.00 .01638 57.57
994-2 13.95 2.2373 5,788.00 .01273 73.684
799-2 10.964 2.6264 4,175.00 .012604 52.62
992-2 13.463 3.1666 4,252.00 .01448 61.56
528-2 11.67 3.7312 3,128.00 .01717 53.70
994-1 17.33 3.1026 5,586.00 .01364 76.19
799-1 11.66 2.7062 4,308.63 .01158 49.89
992—3 11.04 2.4841 4,444.30 .01764 78.40
528-3 10.88 2.5173 4,325.00 .01849 79.97
994-4 11.95 2.5805 4,630.90 .018404 85.23
799-4 9.73 2.5830 3,767.00 .02070 77.98
992-4 12.18 2.3853 5,106.30 .01423 72.66
528-4 11.04 2.3500 4,698.00 .01750 82.21
994-3 15.71 2.0526 7,653.70 .01310 100.26
799-3 13.75 2.3254 5,913.00 .01350 79.83
aNot used in calculations; steer developed abomasal lacerations.

147

 

 

 

TABLE A.10 INDIVIDUAL STEER--NH3-N AND NAN PASSAGE TO THE ABOMASUM
mg NAN/ml mg NH3/m1 mg TN/ml % DM mg NH3-N/ mg TN/g
abomasal abomasal abomasal abomasal abomasal abomasal

Steer fluid fluid fluid fluid 0M 0M

992-1a 2.2394 0.056015 2.29542 5.44 1.0297 42.20
528-1 2.800 0.07163 2.87163 8.94 0.80123 32.12
994-2 2.920 0.06157 2.98157 10.09 0.61021 29.60
799-2 2.549 0.062115 2.611115 9.42 0.6594 27.72
992-2 2.9923 0.06490 3.0572 9.98 0.6503 30.63
528-2 3.380 0.06491 3.4449 9.62 0.6747 35.81
994—1 3.010 0.07446 3.08446 11.54 0.64523 26.73
799—1 2.5000 0.06548 2.5655 11.16 0.58674 22.99
992-3 2.0464 0.08396 2.13036 7.30 1.15014 29.18
528-3 2.180 0.10024 2.28024 7.45 1.3455 30.61
994-4 2.3610 0.09574 2.45674 6.94 1.3795 35.40
799-4 2.2819 0.07948 2.3614 6.59 1.20607 35.83
992-4 1.960 0.06272 2.02272 8.12 0.7724 24.91
528-4 2.0652 0.08955 2.15475 7.22 1.2403 29.84
994-3 2.3579 0.07840 2.4363 11.53 0.67996 21.13
799-3 2.3021 0.08008 2.3822 11.76 0.6810 20.26

 

aNot used in calculations; steer developed abomasal lacerations.

148

TABLE A.11 RNA AND N CONSTITUENTS 0F RUMINALLY ISOLATED BACTERIAL

 

 

 

 

 

 

 

PREPARATIONS
mg RNA/g mg i Bacteria
sample RNA-N/g mg N/g Bacteria __ N (mg) per

DM bact. DM bacteria TNzRNA-N x g DM
High Grain without Monensin:
A2 Bact. 72.20 10.867 76.19 7.0113
A4 Bact. -- -- -- --
A5 Bact. 94.22 13.945 87.18 6.252 6'44 83°22
A8 Bact. 96.49 14.281 86.29 6.0423
High Grain with Monensin:
B2 Bact. 86.54 12.808 85.94 6.7098
B4 Bact. 86.68 12.829 83.22 6.4869 6.44 83.69
B6 Bact. 91.17 13.49 84.82 6.2876
B8 Bact. 86.82 12.85 80.76 6.2848
High Silage without Monensin:
C2 Bact. 82.40 12.20 75.23 6.1701
C4 Bact. 87.65 12.97 85.79 6.6211 6.43 83.50
C6 Bact. 94.04 13.92 90.06 6.470
C8 Bact. 86.52 12.81 82.92 6.4802
High Silage with Monensin:
02 Bact. 85.21 12.61 82.94 6.58
04 Bact. 87.98 13.02 79.90 6.14 6.44 83.67
06 Bact. 90.23 13.35 86.10 6.45
08 Bact. 87.89 13.01 85.74 6.59

 

149

TABLE A.12 MICROBIAL CONTRIBUTION TO NAN PASSAGE TO THE ABOMASUM:
INDIVIDUAL STEERS

 

 

 

mg NAN/g mg RNA/g Ab. RNA-Nb Y T-N Microbial-N:
abomasal abomasal adjusted bacteria: 9 abomasal-
Steer DM DM (mg) RNA-N NAN
992-1a 41.17 17.19 2.1624 6.435 0.340
528-1 31.32 24.59 3.0934 6.435 0.636
994-2 28.94 20.46 2.5739 6.44 0.573
799-2 27.06 16.55 2.0820 6.44 0.583
992-2 29.983 24.10 3.0318 6.44 0.651
528-2 35.14 26.14 3.2884 6.44 0.603
994-1 26.08 22.73 2.8594 6.435 0.706
799-1 22.40 20.61 2.5928 6.435 0.745
992-3 28.03 24.64 3.0997 6.432 0.711
528-3 29.26 25.01 3.1463 6.432 0.691
994-4 34.02 22.06 2.7752 6.438 0.525
799-4 34.63 23.07 2.9022 6.438 0.540
992-4 24.14 16.41 2.0644 6.438 0.551
528-4 28.604 23.68 2.9789 6.438 0.671
994-3 20.45 18.09 2.2757 6.432 0.716
.799-3 19.58 18.33 2.3059 6.432 0.758

 

aNot used in calculations; steer deve10ped abomasal lacerations.

bmg RNA x 14.8% x 0.85, see discussions; nucleic acid
methodology.

150

TABLE A.13 INDIVIDUAL STEER: NITROGEN BALANCE STUDIES

 

 

 

N-
DMI N-intake Fecal -N Urin-N retained % N- % N-
Steer (kg) (gm) (gm) (gm) (gm) digested retained
992-1 6.36 132.18 45.62 35.00 51.57 65.49 39.01
528-1 5.93 123.37 47.75 30.00 45.62 61.30 36.98
994-2 7.24 150.61 49.78 40.87 59.96 66.95 39.81

799-2 5.89 122.57 32.21 43.18 47.18 73.72 38.50

992-2 6.29 130.77 35.91 46.80 48.06 72.54 36.75
528-2 5.49 113.33 30.12 45.13 38.09 73.42 33.61
994-1 7.998 166.35 47.96 79.76 38.63 71.17 23.22
799-1 5.47 105.02 37.42 33.00 34.60 64.37 32.95
992-3 6.563 150.63 57.60 63.97 29.06 61.76 19.29
528-3 6.57 149.89 50.84 59.29 39.76 66.08 26.52
994-4 7.35 169.81 58.42 72.47 38.91 65.59 22.91
799-4 6.01 138.93 45.77 53.33 39.84 67.06 28.68
992-4 6.55 139.88 45.55 51.33 43.00 67.44 30.74
528-4 6.08 128.97 43.45 32.29 53.23 66.31 41.28
994-3 7.54 177.44 59.04 57.57 60.84 66.73 34.29
799-3 6.57 154.87 51.12 56.77 46.96 66.99 30.33

 

151

TABLE A.14 INDIVIDUAL STEER DRY MATTER DIGESTION (N2 BALANCE CONTINUED)

 

 

 

DMI/day DM feces DMD
Steer (kg) (kg) (kg) % Digested
992-1 6.35483 .56645 4.78838 75.35
528-1 5.931174 .75540 4.17577 70.40
994-2 7.2407 .51755 5.72315 79.04
799-2 5.8929 .95401 4.93891 83.81
992-2 6.28679 .98856 5.29823 84.28
528-2 5.4486 .107303 4.34125 79.68
994-1 7.99774 .79481 6.20293 77.56
799-1 5.47214 .23094 4.2412 77.51
992-3 6.56291 .97798 4.5849 69.86
528-3 6.5673 .89153 4.6758 71.20
994-4 7.34457 .43266 4.91191 66.88
799-4 6.00919 .84735 4.16184 69.26
992-4 6.547305 .96195 4.58536 70.03
528-4 6.07451 .78671 4.2878 70.59
994-3 7.54415 .30614 5.2301 69.43
799-3 6.574368 .91332 4.66105 70.90

 

152

TABLE A.15 INDIVIDUAL STEER FECAL CHROMIUM RECOVERY--NITROGEN BALANCE

 

 

 

STUDIES
Fecal Chromium Chromium
mg Cr/g DM excretion intake % Recovery
Steer DM feces per day per day (g/day) chromium
992-1 5.166 1566.45 8.0923 11.089 72.98
528-1 5.074 1755.40 8.9070 10.350 86.06
994-2 6.619 1517.55 10.0450 12.264 81.91
799-2 6.990 954.01 6.6690 9.08 73.45
992-2 6.698 988.56 6.6214 11.66 56.79
528-2 6.346 1107.30 7.0270 10.11 69.51
994-1 5.483 1794.81 9.8410 14.84 66.31
799-1 5.327 1230.94 6.5572 9.01 72.78
992-3 4.450 1977.98 8.8020 10.54 83.51
528-3 4.5186 1891.53 8.5471 10.65 80.25
994-4 4.823 2311.03 11.1460 11.79 94.54
799-4 5.125 1754.99 8.9943 9.65 93.21
992-4 4.801 1961.95 9.4193 10.33 91.12
528-4 4.845 1786.71 8.657 9.58 90.37
994-3 4.598 2306.14 10.604 12.614 84.07
799-3 4.905 1913.32 9.385 10.99 85.40

 

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