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This is to certify that the

thesis entitled

A Study on the Effect of the Combination of
Monensin and Isoacids on Rumen Fermentation
In Vitro

presented by

Patrice Kone
has been accepted towards fulfillment

of the requirements for

M.Sc. degree in Animal Science

L "Mr/é a

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A STUDY ON THE EFFECT OF
THE COMBINATION OF MONENSIN AND
ISOACIDS ON RUMEN FERMENTATION LN VITRO.

BY

Patrice Kone

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1987

 

//‘._,‘/;‘ y (VI/I"

ABSTRACT

A STUDY ON THE EFFECT OF THE COMBINATION OF MONENSIN
AND ISOACIDS ON RUMEN FERMENTATION fl VITRO.

BY

Patrice Kone

A semi-continuous technique was adapted to investigate the
interaction of isoacids and Monensin on rumen fermentation.
The culture was established using inoculum from a cow fed
timothy hay; The media contained timothy hay, urea and
ammonium sulfate (1.0, 0.02 and 0.003 g/100 ml, respectively),
minerals and vitamins. The culture was for 12 days and was
allowed to stabilize 4 days before adding treatment level of
isoacids and Monensin. Comparisons were made for the last 2
days of each trial. Isoacids (equal proportions of isobutyric,
2-m-butyric, isovaleric and valeric acids) at 15 mg/100m1
increased‘acetate (6.11 vs 5.60 meq/lOOml) and total volatile
fatty acid production (8.97 vs. 8.13 meq/100m1); Monensin at
150 ug/100m1 reduced acetate (3.99 vs. 6.08 meg/100ml) and VFA
(6v84 vs. 8.54 meg/100ml) but increased propionate production
(2.28 vs. 1,73 meg/100ml). The combination of isoacids and
Monensin increased acetate in relation to Monensin Ui24 vs.
4.00) but did not eliminate the effect of the ionophore on
propionate. 'Total gas production, hydrogen and ammonia-

nitrogen levels were not influenced by isoacids and Monensin.

DEDICATION

To my parents, who instilled in me the belief that
education was the greatest gift they could bestow in me,
I thank them for both moral and financial support; to my
wife Rachel who has been a source of constant strength during

the course of this study.

iii

 

ACKNOWLEDGEMENTS

To acknowledge all the people that have assisted me with my
career to date is impossible; I would like to especially
recognize a few, however.

I would like to express my deepest gratitude to the
following people: to Drsm Dave Hawkins and Robert Cook who
function very much like co-advisors on my graduate committee;
to Dr. Dave Hawkins for his advice and encouragement throughout
the program; to Dr. Robert Cook, whose guidance, and high
interest made the completion of this investigation possible. I
especially appreciate his help and suggestions in the writing
of this thesis.

I also want to acknowledge the other members of my graduate
committee, Dr.‘William Magee, for his high interest to my
academic progress, and Dr.2Kim Wilson for graciously consenting
to serve on my committee.

The advice and instruction of Dr.1Paulo Machado was
absolutely indispensable throughout all phases of this project,
particularly his assistance with operation of the computer.
Statistical advice from Angelica Machado was an absolute
essential and gratefully received. I also sincerely appreciate
the suggestions of Dr. M. Yokoyama in the interpretation of the
results.

Finally, I would like to thank members of my family,
friends, teachers, colleagues, students that have assisted with

my career to date.

iv

 

ABSTRACT................

TITLE...................

DEDICATION..............

ACKNOWLEDGEMENTS........

TABLE CONTENTS..........

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

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

LIST OF ABBREVIATIONS...

INTRODUCTION............

TABLE

OF

CONTENTS

. ........... o oooooo .00.
ooooo-oooooolllocooooo-
o0.000000ouooooooocoooocoooo
oooooooooooooooooooooo oooooo
0.0.0.0000 ooooo ooo ..... on...

o. ooooooooooooooooo o ........... o.
oooooooooooooooooooooooooooo
one... ooooooo o. ooooooooo

.000 0.0.0000... ........ coo-o

LITERATURE REVIEW... .......................... . ........ ...

Monensin.............

to...

0

Animal Response to Monensin ........ . ......... .......

Effect on Rumen Fermentation ...........

Effect on Rumen Microbes.....

Isoacids ....... .................

Isoacids and Monensin .....................

Long-Term Culture Experiments...

MATERIALS AND METHODS .............. . ....... .

A. MATERIALS ........................................ ...

Description of the incubation vessel.

Establishment of culture......... ..................

Transfer of culture ....... ...........

Preparation of isoacids and Monensin

solutions.... ..... ... ..........

Sampling culture media. ......... .....

Measurements of total gas production.

0

...-u ooooooooo
ooo ooooooooooo
ocean-00.00.000.090.

uoooooooooo

Page

10
11
11
14
16

16

17
l7

l8

B. ANALYTICALMETHODSOO............OOOOOOOOOOOOOO ..... 0
Gas composition............... ....... ......... .....
Ammonia Nitrogen Determination.....................

Volatile Fatty Acids...............................

C. EXPERIMENTAL DESIGN.................................
Validation of the system. ................. ... ......
Effect of Chemicals.... ..... ... ............... .....

RESULTS AND DISCUSSION....................................

A. Validation of the system............................
Source of inoculum .............. ...................
Substrate...... .................. . .................
Protein level......................................
Ethanol and Methanol...............................

B. Influence of Chemicals on Rumen Fermentation ........
Isoacids...........................................
Monensin...........................................

Combination of isoacids and Monensin..... ..........

SMARYAND CONCLUSIONSOOOOOOOOOO000............OOOOOOO...

LIST OF REFERENCES............ ............... .. ...........

19

19

21

21

24

25

25

25

26

28

3O

35

35

4O

45

54

56

LIST OF TABLES

Table Page
1. Composition of 1 liter of medium... ..... ............ 12

2. Composition of medium stock solutions............... 13

3. Composition of vitamin solution..................... 14

4. Standard stock solution............................. 20

5. Number of experiments to validate the system and

test the effect of isoacids and Monensin............ 21

6. Nutrient Content of the source of inoculum.......... 22
7. Nutrient Content of the substrate............ ....... 23
8. Effect of the inoculum on rumen VFA concentration

and total gas production in vitro using grass

hay as substrateOOOOOOOOO.........OOOOOOOOOOOOOOOOO. 25
9. Effect of substrate on rumen VFA concentration and

total gas production in vitro using inoculum
from a cow fed grass hay............................ 27

10. Effect of ammonia nitrogen level and isoacid
concentration on rumen VFA and total gas production
in vitro using inoculum from a cow fed grass hay.... 28

11. Effect of isoacids on rumen VFA concentration
in vitro using inoculum from a cow fed a high
roughage dietOOOOOOO.....0.0.0.000.........OOOOOOOOO 34

12. Effect of isoacids on rumen gas production and
ammonia nitrogen concentration in vitro using
inoculum from a cow fed a high roughage diet ..... 38

 

13. Effect of Monensin on Rumen VFA concentration
in vitro using inoculum from a cow fed a high
roughage dietOO0.00.0.0000000000000000000000.00.. 39

14. Effect of Monensin on rumen gas production and
ammonia nitrogen concentration in vitro using
inoculum from a cow fed a high roughage dietu.n 49

 

15. Effect of isoacids and Monensin on rumen VFA
concentration and gas production in vitro using
inoculum from a cow fed a high roughage dietu.n 44

 

16. Effect of isoacids and Monensin on rumen gas
production, ammonia nitrogen concentration and pH
using inoculum from a cow fed a high rouhage diet... 45

vii

LIST OF FIGURES

Figure Page

1. Anaerobic digester and gas measuring apparatus.”.u 15

2. Daily rumen VFA concentration in vitro using
grass hay as a substrate .................. .......... 32

3. Daily rumen VFA concentration in vitro using
grass hay asa substrate ........ 33

4. Effect of isoacids on average daily rumen propionate
concentration in vitro using grass hay as a
substrate.... .......... ...... ....... ................ 37

5. Effect of Monensin on average daily rumen acetate
concentration in vitro using grass hay as a
substrate........................................... 40

6. Effect of Monensin on average daily propionate
concentration in vitro using grass hay as a
substrate........................................... 41

 

7. Proposed mechanism of action of the effect of the
combination of isoacids and Monensin on rumen
fermentation ......... . .............................. 47

8. Effect of the combination isoacids and Monensin on
Daily rumen acetate concentration in vitro using
grass hay as a substrate ...................... ...... 50

9. Effect of the combination isoacids and Monensin on
daily propionate concentration in vitro using
grass hay as substrate .............................. 51

10. Effect of the combination isoacids and Monensin on

daily rumen methane concentration in vitro using
grass hay as a substrate ...... . ..................... 52

viii

C2

C3

C2C3

C4

C4-C5

CS

CH4

C02

GH

GP

H2

IVA

ISO

MON

2MB

N-NH3

VAL

LIST OF ABBREVIATIONS
Acetate
Propionate
Ratio of C2 to C3
Butyrate
Four and five-carbon branched-chain fatty acids
Corn silage
Methane
Carbon Dioxide
Grass hay
Gas Production
Hydrogen
Isovalerate
Equimolar weight mixture of isobutyric acid,
isovaleric acid, 2—methyl butyric acid and valeric
acid.
Monensin
2-Methyl Butyrate
Ammonia nitrogen

Valerate

ix

INTRODUCTION

 

The efficiency of nutrient utilization by ruminants is
influenced by the balance of rumen fermentation end-products,
which is dependent upon the types of microorganisms present in
the rumen. Consequently, effective manipulation of microbial
species in the rumen and their activities can result in
improved animal performance.

Monensin has been used extensively in diets for feedlot
cattle since 1975. IRecently, isoacids have been commercialized
for dairy cattle and may improve growth of feedlot cattle.
However, little is known about the effects of the combination
of the two compounds in the rumen.

In yitgg rumen fermentations have shown that Monensin
decreases the acetatezpropionate ratio (Smith, 1971; Richardson
gt al., 1976; Chalupa, 1977). These results have been

attributed to a toxic effect of the antibiotic upon Ruminococci

 

(Brulla and Bryant, 1980). Isoacids (isobutyric, isovaleric, 2-
methylbutyric, and valeric acids), however when added to in
vitro systems, have increased acetate production due to an

enhanced growth of Ruminococci (Allison and Bryant, 1958;

 

Gorosito _e_t_ 31., 1985).

The mode of action of the ionophore is believed to be
through an interruption in Na+/K+ transport at the membrane
level (Romatowski, 1979). Isoacids have their effect at the
membrane level too. They are carbon skeletons for the
synthesis of branched chain aminoacids that are incorporated

into membrane proteins (Allison and Bryant, 1958).

The objective of this work was to study the effects of
adding Isoacids to an in'vitro rumen fermentation system

containing Monens in.

 

LITTERATURE REVIEW

 

MONENSIN.

Monensin, a carboxylic polyether ionophore, is a

biologically active compound produced by Streptomyces

 

Cinnamonensis (Haney and Hoehn, 1967). Initial use of Monensin

 

as an anticoccidial feed additives was launched by the poultry
industry; However during the past 15 years chemical agents
including Monensin have been identified as having the potential
to affect rumen metabolism” Thus, because of its potent effect
on ruminal ecology and animal production, Monensin has been
approved since 1975 by the Food and Drug Administration as a

feed additive for cattle.

Animal response to monensin

 

Numerous studies conducted on animal response to
monensin, have shown that when it is incorporated at a
recommended level into high concentrate diets for finishing
cattle, body weight gain is unchanged, even though feed intake
is generally depressed. Therefore, the efficiency of feed
conversion is improved (Owen, 1980; Bergen, 1984). The
influence of Monensin on feed intake is well documented.
Furthermore, studies have shown that in high grain diets,
supplementation with Monensin depressed feed intake by 5 to 6%
(Anonymous, 1975; Owens, 1980).

When animals were fed roughage, Monensin did not depress
intake and body weight gain improved (Potter and Richardson

1975). Therefore, efficiency of feed conversion is improved.

Cattle receiving Monensin under pasture conditions have
responded with a 17% increase in rate of gain (Anonymous 1975).
Monensin has been shown to increase the efficiency of feed
utilization (kg of feed per kg of live weight gain) in both
sheep and cattle fed a variety of rations (Potter gt gt 1976).
Carcass data analysis indicated that the main effect of the
Monensin is to increase the efficiency of dietary energy

retention in the carcass.

Effect g2 Rumen Fermentation.

 

The effect of Monensin on rumen fermentation is well
known. Monensin has consistently increased the molar proportion
of propionate with a concomitant decline in the molar
proportion of acetate and butyrate (Chalupa, 1980; Bergen and
Bate, 1984). Van Maanen gt gl,, (1978) found using isotope
dilution techniques that higher production of propionate occurs
at the expense of acetate. The shift in VFA is often
associated with a decrease in methane production without
accumulation of gaseous hydrogen (Van Nevel and Demeyer, 1979;
Chalupa, 1980).

The concept that propionate is more efficiently utilized
than acetate is based on two hypotheses. Hungate (1966)
estimated the efficiency of conversion of hexose to propionate
to be higher than the efficiency of conversion of hexose to
acetate or butyrate. Therefore, it was proposed that
propionate is more efficiently utilized than acetate and
butyrate. The second theory proposed by Smith (1971) was more

controversial. Smith proposed that propionate is more

efficiently used than acetate by the tissue.

Allen and Harrison (1979) reported a decrease in branched
chain fatty acids when Monensin was added at 22 ppm to the diet
of sheep fed dried grass/maize.

t2 xtttg studies have indicated a decrease in microbial

methane production with monensin (Barley gt gt., 1979;
Chalupa gt gl., 1980). Similar responses have been obtained in
viva“ The inhibition of methane production by methane
producers is only partial. Studies have shown a decrease of 4%
to 31%. Van Nevel and Demeyer (1977) reported a decrease in
the metabolism of formate to carbon dioxide and hydrogen when
Monensin was fed and proposed that this effect of Monensin
could account for the decrease in methane production. Monensin
has no effect on carbon dioxide production at low level, but
high levels of the chemical significantly depresses carbon
dioxide (Bartley gt gt” 1979; Chalupa gt gt, 1980). Chen and
Wolin (1979) reported a selection against hydrogen-producing
rumen bacteria and a selection in favor of succinate-forming
bacteria. They proposed that the decrease in hydrogen and
carbon dioxide production could also account for the decrease
in methane production when Monensin is fed.

Other ruminal effects due to Monensin were: a lower
ruminal lactate production in stressed animals (Denis gt gt”
1980), an increase in ruminal forage fill (Ellis and Delaney,
1982), a decrease in ruminal rate of passage (Lemenger gt gt
1978), and an increase in dry matter digestibility and ammonia

nitrogen retention (Rust gt gt., 1978). Schelling (1983) has

 

 

defined seven probable system modes of action of Monensin:
modification of acid production, modification of feed intake,
change in gas production, modification in digestibility,
changes in protein utilization, modification in rumen fill and
rate of passage, and perhaps other ruminal modes of action.

The effect of Monensin on volatile fatty acids is accepted
as one of the main modes of action. However this probably does
not account for all the effects of Monensin.

Reilly and Ford (1971) suggested that Monensin spares amino
acids normally used for gluconeogenesis. Eskeland gt gt;
(1974) proposed a possible stimulation of protein synthesis
when Monensin is fed and Smith (1979) suggested that the

increase in propionate may also lower heat increment.

Effect 93 rumen microbes

 

Numerous $2 vivo and it vitro studies were conducted to
determined how Monensin controlled microbial activity. Chen
and Wolin (1979) and Denis gt gt. (1981) found that

Ruminoccoccus albus, Ruminoccocus flavefaciens and Butyrivibrio

 

 

 

fibrisolvens are very sensitive to Monensin. These species are

 

very important in the production of acetate, butyrate, carbon

dioxide and hydrogen. Selenomonas ruminantium which

 

decarboxylate succinate to propionate were resistant to

Monensin. Bacteriodes, Selenomonads and Succinivibrio, all

 

 

succinate producers, were not inhibited by Monensin. Lactate

producing species such as Lactobacillus vitulinus and

 

Lactobacillus ruminus were inhibited by Monensin, whereas at

 

the same level, lactate fermentors (Megasphaera elsdenii and

 

Selenomonas ruminantium) were not sensitive to Monensin.

 

Therefore it was proposed that Monensin could be effective in
preventing lactic acidosis. These findings have been confirmed
by’Nagaraja.gtflgt.,(1981).

Attempts have been made to explain the changes brought
about by Monensin on the predominant rumen microbes (Chen and
‘Wolin, 1979; Anderson gt g;., 1981). The sensitivity pattern
suggested that the antibiotic acted by selecting for the rumen
microbes that produce proportionally more propionate.

Consequently, species such as Selonomonas ruminantium and

 

Bacteriodes ruminicola were selected. Ruminoccocci and

 

 

Butyrivibrio that are major producers of acetate, butyrate,

 

hydrogen and carbon dioxide were inhibited.

ISOACIDS
Branched-chain carbon skeletons referred to as isobutyric

acid, 2-methylbutyric acid and isovaleric acid are required for
growth by a wide variety of cellulolytic anaerobic
microorganisms (Hungate, 1966; Cook, 1985). Valerie acid has
been shown to improve cellulose degradation it gtttg_(Cummins
and Papas, 1984; Amos and Little, 1971). The ruminal source of
branched-chain fatty acids are degraded feed protein and
endogenous branched-chain aminoacids (Pittman and Bryant 1964).

Addition of isoacids to 12;!iEEQ fermentation systems has
increased digestibility of soybean stover (Soofi gt gt., 1982;

Cummins and Papas, 1984).

 

In the rumen, bacteria use branched chain fatty acids to
synthesize aminoacids, but they also use isoacids for the
biosynthesis of long chain fatty acids (Allison gt gl., 1961
Dehority gt gt, 1967).

IE XiEEQ studies by Felix (1976) and by Cummins and Papas
(1984), have shown that the addition of isoacids increased
microbial growth and dry matter digestion. By the same token,
Gorosito and Russel (1984) have reported that the addition of
these acids not only increases cell wall digestion of intact
forage but also increases ammonia nitrogen utilization by the
rumen microbes.

lg ztgg studies reported that isoacids have constantly
increased nitrogen retention and have lowered urinary nitrogen
loss when steers were fed isolated soy protein (Oltjen gt gt”
1970). Felix and Cook, (1980) and Cook, (1985) pointed out
that isoacids plus urea improved milk production. 'These
authors reported that isoacids increased growth rate in young
animals but not in older animals and that the supplementation
of Isoacids and urea to high producing cows fed corn silage as
the sole roughage had a positive effect on milk production,
persistancy of lactation, body weight, feed intake and nitrogen
balance.

The requirement for branched chain fatty acids by the
rumen microbes is well known. .Allison and Bryant (1963) and
Yokoyama and Johnson (1984) reported a requirement of branched
chain fatty acids for growth of several rumen cellulolytic

bacterial species including Ruminococcus albus and Bacteriodes

 

9

succinogenes. Isoacids are also required for growth of

 

methanogenic bacteria, Treponema and Megasphaera elsdenii.

 

Towns and Cook (1984) reported an alteration of growth
hormone and insulin and an increase in milk production in
lactating cows fed high concentrate diets. Fieo gt gt. (1984)
also found a higher growth hormone concentration.

Towns and Cook (1984) found that during an eight-hour
sampling period, isoacid treated cows had higher growth
hormone but lower blood glucose than control cows.

Bines and Hart (1984) and Istasse and Orskov (1984)
reported that the decrease in propionate production in the
rumen resulted in lower stimulus for insulin secretion.

Brondani (1986) found that the decrease in insulin found
for the first time by Towns and Cook (1984) when cows were fed
high concentrate diet plus isoacids was due to a decreased

I

propionate production in the rumen.

MONENSIN AND ISOACIDS

Monensin, when used in lactating cow diets, has caused a
decrease in milk production and milk fat percentage. This
response may be due to a high level of propionate in relation
to acetate and butyrate, which stimulates the release of
insulin. Insulin is responsible for partitioning nutrients
away from milk production to fat deposition. The addition of
isoacids in the diets of cows receiving Monensin could increase
the acetate to propionate ratio, because isoacids would
increase acetate production. The problem of combining the two

chemicals is that isoacids is a growth factor for cellulolytic

 

10

bacteria and Monensin is toxic to this class of microorganisms.
Thus, our intent was to investigate the effect of the two
compounds on the rumen microorganism tg vitro using a long-term

culture technique.

LONG TERM CULTURE EXPERIMENTS

Numerous tg ytttg continuous culture experiments have
attempted to use substrate concentrations characteristic of the
rumen and to substitute artificially for the supply and removal
functions of the rumen (Hungate gt gt., 1942; Gray gt gt, 1962;
Short, 1978). Survival of protozoa in number and kinds
comparable to the rumen is the easiest means to ascertain
whether the rumen population is maintained. Rates of
production of fermentation acids and gases are criteria for
comparing tg gtttg activity with that in the rumen.(Hungate,
1966).

The objectives of the present study were first to
duplicate the rumen conditions sufficiently well to support the
fermentation rate characteristic of the rumen, secondly to
investigate through a quantitative measurement of the
production of VFA and gasses under completely controlled
conditions, and finally to determine the effects of the

combination of Isoacids and Monensin on rumen microbes.

MATERIALS AND METHODS

 

A. MATERIALS

 

A series of tg gtttg experiments were conducted to
investigate the effect of Monensin and isoacids upon a mixed
population of rumen microorganisms. The experimental technique
consisted of replacing one half the volume (50 ml) of a 24-hour
culture by a new medium of equal volume. There were ten
trials lasting from eight to twelve days, with some variations
in the cow diet, the media composition and the concentration of
chemicals.'

A mature non-pregnant and non-lactating rumen-canulated
Holstein cow of approximately 550 kg body weight served as a
donor. Throughout the entire experimental period, the cowwas
fed four different diets, one of each separated by a ten-day
period of adaptation. The cow was fed three hours prior to
taking the rumen samples. Samples from different parts of the
rumen were strained through two layers of surgical gauze into
1-liter glass bottles kept at 40 degrees Celsius and rapidly
brought to the laboratory. A total of 1 liter of rumen fluid
was added to the same amount of medium. The medium composition

is shown in Tables 1, 2 and 3.

11

 

12

Table 1. COMPOSITION OF 1 LITER OF MEDIUM

 

Dry nutrients:
Grass hay
Urea
Ammonium Sulfate
Mineral solution 1
Mineral solution 2
Micromineral solution
Sodium Bicarbonate solution 6.32%
Sodium Sulfide solution
Vitamins

Distilled water

20.00

.40

.06

80

8O

20

110

685

 

Adapted from Phillips,ILS., and JuM Tadman.(1980)
(unpublished data)

gr
gr
gr
ml
ml
ml
ml
ml
ml

ml

13
Table 2. COMPOSITION OF MEDIUM STOCK SOLUTIONS

 

Mineral solution 1 g/liter

K2HPO4.3(H20) 12.5

Mineral solution 2

KHZPO4 12.5
MgSO4.7(H20) 3.0
NaCl 12.0
CaC12.2(H20) 1.6

Micromineral.solution

Disodium Dihydroxide. E.D.T.A 5.000
FeSO4.7 (H20) 2.000
H3303 0.030
CoCL2.6 (H20) 0.020
ZnSO4.7 (H20) 0.010
MnC12.4 (H20) 0.003
Na2MoO4.2 (H20) 0.003
NiC12.6 (H20) 0.002
CuC12.2 (H20) 0.001

Sodium Bicarbonate 6.33%
NaHC03 63.300
Sodium Sulfide 2.5%

Na28.9H20 25.000

 

Adapted from Phillips and Tadman 1980 -(unpublished data)

14

Table 3. COMPOSITION OF VITAMIN SOLUTION

 

Composition g/liter
'EEEQQIQZEEESEEISLIE. "”23""
Riboflavin 2.0
Thiamine Hydrochloride 2.0
Nicotinamide 2.0
Calcium Panthotenate 2.0
Lipoic Acid 1.0
Para-Aminobenzoic Acid .1
Folic Acid .05
Biotin .05
Coenzyme B12 .05

 

DESCRIPTION _O_F THE INCUBATION VESSEL

 

A 250 m1 erlenmeyer flask, with a liquid port on one side
for culture sampling and a gas sampling port on the other
side, was fitted with a graduate cylinder for gas measurements
(Figure 1). .A 12 cm flexible tube was placed at the extremity
of the liquid port in order to allow for easy transfer. A
pinch clamp was placed at the extremity of each tube to avoid

any oxygen entry into the flask (Figure 1).

15

639.09? 9.7382». moo mco tobmoma 059.005. é 0.59m

 

 

 

 

 

 

 

 

 

 

 

 

..ooncoto .. n
l_:._ omN:
‘ tom
2%:
ton moo
mmctam tow ..A r omctxm
meow mmU. . I” _CL 00
Ml-
3050.6
x23 .5 on:
r
[lit

 

 

 

 

16

ESTABLISHMENT OF CULTURE

 

On the first day of culture establishment, 1 liter of
:medium was prepared and the same amount of rumen fluid was
collected from a cow 3 hours after feeding. Inoculum and
medium were mixed under C02. A 100 ml aliquot of this mixture
was then dispensed anaerobically into the incubation flasks
flushed with ammonia nitrogen. The fermentation flasks were
then placed in a Grant water bath shaking at a rate of 45/min

and the temperature of the water was maintained at (39°C)t

TRANSFER OF CULTURE

The procedure was adapted from Short (1980) and modified as
follows: after 24 hrs of incubation, one half (50 m1) of the
incubated culture was replaced by 50 ml of a new medium in the
following way; The digestors were individually removed from
the water bath. Fifty ml of the old medium was withdrawn while
the fermentor was being flushed with nitrogen. Then, 50 ml
from a fresh medium were collected from a round flask gassed
with 100% C02 and transferred via a 60 ml syringe into the

flask containing the remaining 50 ml of the old culture.

lBench Scale Equipment Co., Dayton, Ohio.

17

PREPARATION 9_F_ ISOACIDS AND MONENSIN SOLUTIONS

Isoacids (isobutyric, 2-methylbutyric, isovaleric, and
valeric acids) were obtained from Eastman Chem. Co. The four
isoacids were mixed at equimolar concentration, then
neutralized with sodium hydroxide and diluted in double-
distilled water to final concentrations of 10, 15, 20, and 40
mg/dl in the fermentors.

Monensin, obtained from Sigma Company, was primarily
dissolved in 10 ml of methanol and then diluted with double-
distilled water. The final medium concentrations were 100,

150, 200, and 400 ug/dl.

SAMPLING THE CULTURE MEDIA

Fifty milliliters of a 24-hour culture were collected
daily from each digestor and divided as follows: 25 ml was
used to measure the pH. The remaining 25 ml was centrifuged at
1800 x 9. Two ml of the supernatant was treated with sulfuric
acid (1N) and sodium tungstate (10%), and was then saved for
ammonia nitrogen determination. One ml was treated with 200 111
of formic acid (88%) and immediately analyzed for volatile

fatty acids.

18

MEASUREMENT 3 TOTAL GAS PRODUCTION

The system used to measure the rate of gas production was
devised by Phillips and Tadman (1973), and is based on the tg
ttttg technique of using gas production rates for obtaining an
estimate of microbial net growth (El-Shalzy and Hungate,
1965). The gas produced in the fermentation vessel caused the
barrier solution to move down in the manometer, and is
monitored as a function of time. The barrier solution was
double distilled water containing 20% NaCl. A vacuum pump was
used to bring the solution to the 90 ml mark. Readings were

taken during the next three hours.

B - ANALYTICAL PROCEDURE

Gas composition

 

After 24 hrs, 0.5 m1 gas samples were collected and allowed
to remain in the Pressure-Lok A2 gas syringes3 until injection
into the gas chromatograph. .A Hewlett-Packard 5750 Gas
Chromatograph4 equipped with a thermal conductivity detector
was used for the analysis. Analysis was performed at the

following operating conditions:

3 Precision Sampling Corp., Baton Rouge Louisiana.
2 Hewlett-Packard. Route 41, Avondale, Pensylvania.
g Supelco, Inc. Bellefonte, Pensylvania.

19

1. Column: Pyrex, 3m long x 2 mm i.d. packed
with carbosieve, 100/120 mesh.5

2. Detector temperature : 250°C

3. Injection port temperature : 150°C

4. Column temperature : 125°C

5. Carrier gas (Argon) flow rate : 20 ml/min

6. Bridge current : 100 milliamperes

A model 7227A Strip Chart Recorder and a Model 3370B Integrator
were used for recording.

Ratio between gases were calculated, based upon peak
length of individual gases detected by the gas chromatograph

and recorded on the Strip Chart Recorder.

AMMONIA NITROGEN

 

.Ammonia.nitrogen from rumen.and fermentor samples
were analyzed using the indophenol reaction (Chaney and
Menbach, 1961) adapted from the determination of urea after
hydrolysis with urease. Samples were prepared according to the

method of KMlasek (1959) and Okuda gt a1. (1963).

VOLATILE FATTY ACIDS

 

Prior to the injection, 1 ml of incubation medium was
acidified using formic acid. Injection volume was 1
microliter; The standard was obtained by a 1/10 dilution of the

stock solution (Table 4).

20
TABLE 4. STANDARD STOCK SOLUTION

 

VFA (umoles/ml)
ACETATE . . . . . . . . . . . . 57.430
PROPIONATE . . . . . . . . . . . 19.490
ISOBUTYRATE . . . . . . . . . . . 0.518
BUTYRATE . . . . . . . . . . . . 14.470
2-METHYLBUTYRATE . . . . . . . . . 1.420
ISOVALERATE . . . . . . . . . . . 1.864
VALERATE . . . . . . . . . . . . 2.924

 

One ml of the supernatant from the fermented sample was
mixed with 200 microliter of 88% formic acid inside an
automatic sampler vials (Hewlett-Packard C04. The samples
were then loaded onto an automatic injector.

Volatile fatty acids (VFA) were determined on a Hewlett
Packard Gas-Liquid Chromatograph equipped with flame ionization
detector. Analysis was performed at the following operating
conditions:

1. Column: Pyrex, 6 ft long x 2 mm i.d. packed
with 3% carbowax. 20M + 5% HCOOH on 60/80
carbopack B 1-1825 (Supelco. Inc)

2. Detector temperature : 200°C

3. Injection port temperature : 200°C

4. Column (Oven) temperature: program 116°C to 180°C
8°C/min

5. Carrier gas (Nitrogen) flow rate : 20 ml/min

6. Hydrogen pressure: 30 psi

21

7. Air pressure: 40 psi

An integrator-recorder, Model 3380A, was used for recording.

C. EXPERIMENTAL DESIGN.

 

A series of ten experiments was conducted, first to
validate the tg xtttg system and then to investigate the
interaction of isoacids and monensin on rumen microbes. See
summary Table 5.

TABLE 5 Number of experiments to validate the system and test
the effect of isoacids and Monensin.

 

 

 

 

; 1Inoc. 2Subs. 3Prot. 4Mon 5Iso 6M+I
VALIDATION ; 2 3 2 - - -
INTERACTION; - - - 1 1 1
1

inoculum 2Eubstrate, 3protein level.
monensin, isoacids, monensin + isoacids.

Validation gt the System.

 

 

To reach the first objective, seven experiments were
conducted over a period of eight days each, with a variation in
the Inoculum, the Substrate and the Urea level. In addition,

the effect of methanol and time were also evaluated.

 

22

In order to evaluate the Inoculum effect, two experiments
were conducted: diets were changed after each experiment and a
ten-day adaptation period was observed. The basal diets were
corn silage, and grass hay. The proximal analysis of the
ration is shown in table 6. The difference between rations
could not be tested with complete validity because cows were

not replicated within ration.

Table 6. Nutrient Content of the Source of Inoculum.

DM CP NDF NEM NEG NEL
grass hay 100 6.8 66.8 1.23 1.15 .50
corn silage 100 8.7 42.8 1.60 1.58 .99

In the second phase, three experiments were conducted.
The objectives were to investigate the effects of the substrate
on the fermentation pattern. Straw, grass hay, and corn silage
were used (Table 7). Data were analyzed by AOV, using a
completely randomized design. Differences between treatment

means were tested using orthogonal contrasts.

23

TABLE 7. Nutrient Content of the Substrate.

 

DM CP NDF NEM NEG NEL
grass hay 100 6.8 66.8 1.23 1.15 .50
corn silage 100 8.7 42.8 1.60 1.58 .99
straw 100 4.3 73.2 0.90 0.20 1.02

 

In the third phase, two experiments were conducted where
the Inoculum and the substrate used were the same in both
experiments. To assess the effects of low and high
concentrations of nitrogen, 0.2 g of urea were added to a liter
of medium for the first experiment ‘vs.(L6 g of urea for the
second experiment. These two levels were chosen based on
previous dose response study on the effect of different levels
of urea on ammonia concentration in the culture media.

Because Monensin would not dissolve in aqueous solution, it was
therefore necessary to find an adequate solvent that would not
affect the fermentation pattern. Thus, for this purpose,
ethanol and methanol were tested at 10% and 1% of the medium
volume. During these experiments, the effect of time was
investigated as well. Consequently, daily measurements of VFA,
gas composition, and ammonia nitrogen content were obtained
throughout the 8-day period and comparisons of all the data
were made. Protein level data were analyzed using a 2x2

factorial.

24

Effect gt Chemicals.

 

To reach the second objective, three trials were
conducted in order to observe the artificial rumen
fermentation as affected by isoacids and Monensin. The first
two experiments had either isoacids or Monensin as treatments
whereas the third received a combination of the two chemicals.
The incubation time was twelve days using triplicate
fermentors. Isoacids and Monensin were added to the flasks
after the fourth day of incubation. The final concentrations
of isoacids in the fermentors were 10, 15, and 20 mg/dl of
medium in trial one. In trial two, Monensin concentrations
were 100, 150, and 200 ug/dl whereas in trial three, levels
were 150 ug/dl for Monensin and 10 and 15 mg/dl for isoacids.

All statistical analysis were carried out using the
Statistical Analysis System (SAS) (SAS-MSU,1982). Data were
analyzed by analysis of variance using split-plot for
repeated measurements. Differences between treatment means

were tested by Tukey's test for all experiments.

RESULTS AND DISCUSSION
VALIDATION OF THE SYSTEM
In order to set up a semi-continuous culture technique
that will approach the rumen conditions, a series of seven
experiments were carried out. Source of inoculum, substrate,
protein level and other factors affecting microbial activity in

the rumen were investigated.

Source gt inoculum

Two experiments were conducted to investigate the effect
of the type of ration on the long term culture technique over a
period of eight days. Ground hay was used as substrate in
both trials. The results are summarized in Table 8.

Table 8. Effect of the inoculum on Rumen VFA concentration and
total gas production tg vitro using grass hay as

 

 

 

 

 

 

 

 

 

substrate.
day 1 day 4 DAY 8

Variable csl 6H2 cs GH cs GH
mmoles /d1

C2 8.03 6.50 6.63 6.65 6.64 6.50

C3 2.77 1.64 1.88 1.86 1.87 1.84

C4 1.59 1.23 0.79 0.78 0.78 0.79

ISO 0.26 0.21 0.15 0.13 0.14 0.13

TOT 12.60 9.62 9.45 9.42 9.43 9.26
ml/hr

GAS 28 20 18 18 17 17

 

1rumen fluid from a cow fed corn silage.
rumen fluid from a cow fed grass hay.

25

26

VFA are expressed in mmoles/d1 of the culture volume, and
gas production in ml/hr. On the first day of incubation
acetate, propionate, butyrate and total gas production were
affected by the source of inoculum, however on the fourth day
of incubation and thereafter there was no difference on rate of
gas production nor on VFA production between the two types of
ration.

One of the main factors influencing the rumen fermentation
is the variability in the components of the feed (Hungate
1955). The rumen microbial population depends on the
continuous supply of the digestible feeds included in the
ration. In this study, since the substrate used was the same
in both experiments, an adaptation of the microbial population

to thesubstrate may have occurred over time.

Substrate

 

After determining that source of inoculum had no effect on
the system the effect of substrate on the fermentation was
studied. The results are presented in Table 9.

VFA and gas productions were higher when corn silage was
fed compared to grass hay, and lower than for grass hay when
straw was used as substrate.

Of the digestible feeds, carbohydrates are the most
important quantitatively because of their superiority as a
source of energy under anaerobic conditions (Hungate,1955).
Soluble carbohydrates, starch, and insoluble carbohydrates are

the common types present in forage plants. Protein, in

27
addition to nitrogen and carbohydrates, is also required for

fermentation and growth. Protein may influence rumen
fermentation, not only directly as a source of nitrogen for

assimilation, but also as a source of isoacids.

Table 9. Effect of substrate on rumen VFA concentration and
total gas production tg vitro using inoculum from
a cow fed grass hay.

 

 

 

 

 

 

 

 

 

Substrate
Variable Corn silage Grass hay Straw SEM
mmoles /dl
VFA 11.22a 9.98b 7.75C .51
Acetate 6.37a 5.93b 5.45c .13
Propionate 2.86a 1.76b 1.77b .18
Butyrate .87a .87a .97a .02
molar %
Isoacids 1.65a 1.47b .83c .12
ml/hr
Gas 41.44a 24.77b 16(77c 3.63

 

a,b,c Means in a row with different superscript differ p(<.05).

In the present study the difference in VFA production was
due to a difference in energy and/or protein content of the
different substrates used. ‘When straw was used as substrate,
isoacid concentrations remained very low in the culture.
Therefore, they may have been a limiting factor for growth of

cellulolytic bacteria.

Protein Level

 

28

Two experiments were conducted to investigate the effect

of protein level on the semi-continuous culture system and to

adjust theiammonia nitrogen level in the medium.

two components of protein degradation ammonia nitrogen and

isoacids on rumen fermentation,

was studied (Table 10).

The effect of

order to keep energy constant, the same substrate (Grass hay)

was used in both trials.

 

 

 

 

 

 

 

 

 

Table 10. iEffect ofiammonia nitrogen level and Isoacid

concentration on rumen VFA and Total gas production

E Etro using inoculum from a cow fed grass hay.
.Ammonia Nitrogen Level (mg/d1)
5.57 0.24
Variable 1130- 2mm ISO- ISO+ SEM
mmoles/d1
VFA 8.114a 8.918b 7.955a 8.107a .10
Acetate 5.607a 6.118b 5.510a 5.408a .02
Propionate 1.637a 1.679a 1.630b 1.635b .02
Butyrate 0.609a 0.667b 0.602a 0.604a .03
Isoacids 0.261a 0.454b 0.213a 0.460b .1
ml/hr

Total gas 27.72a 28.49a 26.33a 27.40a 2.

 

1isoacids not added.
2isoacids at 10 mg/100ml of final incubation media.
a,b,c Means in a row with different superscript differ (p<.05)

 

29

There were no differences in total gas production and VFA
production wheniammonia concentrations in the media were low
(0.24 mg/dl) or high.(5.57 mg/dl) and when isoacids were not
added to the media. The addition of isoacids to the culture
containing low ammonia nitrogen resulted in no change in VFA
and gas production. IHowever, when urea was added in larger
amount to the medium, the addition of isoacids significantly
increased acetate and total VFA concentrations.

Several studies related to protein metabolism have been
conducted. Bergen (1979) reported that the ruminal fermentation
is a coupled process between carbohydrate degradation and
microbial cell synthesis. .Ammonia-N, isoacids and other
factors such as carbon skeletons and sulfur are required for
this process (Brondani, 1986). A major concern in ruminant
nutrition is to define the nutrients required by rumen
microorganisms for maximum fermentation of feedstuffs,
particularly for low protein, high fiber plant material Cook
(1985). Cook and Felix (1976) found that isoacids were
limiting factors for growth of rumen microorganisms when
animals were fed high levels of urea as source of supplemental
nitrogen. Urea is hydrolyzed in the rumen to ammonia and
carbon dioxide (Hungate and Gall, 1955). Many types of
bacteria contribute to this process (Muhrer and Caroll, 1964).
Since little energy is released, the splitting of urea to
ammonia would.be of value to the microorganisms mainly for
growth.

In our study no difference was observed between low and

high ammonia concentration (Table 10), although VFA

30

production was slightly lower when ammonia concentration was
low. The low concentration of isoacids may have limited the
microbial growth in the culture containing sufficient amount of
nitrogen. The addition of isoacids to the culture containing
5.57 mg/dl NeNH3 increased total VFA and gas production
whereas isoacids had no effect when added to the culture
containing 0.24 mg/dl of N-NH3. .kmmonia nitrogen may have been
a limiting factor for growth in this case. Previous
experiments conducted using grain as substrate failed to show a
significant increase in VFA production when isoacids were added
to the system. The probable reason for the no effect of
isoacids on VFA may be that the high level of protein present
in the Substrate provide sufficient branched chain fatty acids
for the bacteria. Therefore isoacids were not limiting

in the fermentation process.

Ethanol and Methanol

In order to find an adequate solvent that would dissolve
monensin, the effect of ethanol and methanol was assessed.

tg gtttg and tg gttg studies related to ethanol metabolism
in the rumen have shown that Ethanol is formed in pure cultures
of a number of species of rumen bacteria, but does not occur in
the normal rumen at a significant concentration. Emery EE.El
(1959) reported that Ethanol added to the rumen exerts little
effect on oxygen consumption and methane production. IEthanol
was not rapidly metabolized in the rumen, nor was it rapidly
attacked tg ytttg (Leroy, 1958; Emery 1959). Ethanol slowly

disappeared, probably by absorption and passage to the omasum.

 

31

In our study Ethanol at 1% caused a drastic decline in all
parameters after 48 hrs of incubation. The explanation of the
decrease in VFA and GAS production when Ethanol was added to
the system is uncertain. Ethanol may have been toxic for
certain rumen microbes. 'Therefore, we did not use ethanol as a
solvent for Monensin even though it is widely used in pure
culture as a solvent.

Czerkawski and Breckenridge (1972) investigated the
metabolism of the primary alcohols, methanol through butanol,
by rumen microorganisms 12:21529- They reported that methanol
is initially oxidized to formic acid with the resulting
hydrogen used by other microorganisms for methane production.
The rate of oxidation of these alcohols are relatively slow in
comparison to the rate of hexose fermentation. When methanol
was added to the fermentation vessel Czerkawski and
Breckenridge (1972) found no change in the amount of acetic,
propionic or butyric acids produced, but there was an increase
in methane production.

In our study, addition of methanol at 1% of incubation
medium decreased VFA, C02 and Total gas production and
increased methane production. However, at 0.1% there was no
change in total VFA nor C02 concentration and only a slight
increase in methane production was observed. In order to
eliminate any possible error due to addition of methanol,
during the treatment period the control flasks received 051% of

methanol.

32

The length of the long term culture technique was then
extended over a period of twelve days in order to observe the
effect of time on the system. There was a decrease of the
variables measured, from day 1 to day 3, followed by a
relatively a stable condition from day four to day twelve..a
summary of these results is presented in Figures 2 and 3.

Based on these results, the fourth day of incubation was
chosen as the first day of the treatment.‘The treatment

injected through the gas port of the incubations flasks.

33

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35
INFLUENCE OF CHEMICALS ON RUMEN FERMENTATION.

The effects of different concentrations of Isoacids are

summarized in Tables 11 and 12.

TABLE 11. Effect of isoacids on rumen VFA concentration tg

vitro using inoculum from a cow fed high roughage diet.

 

Isoacid concentration (mg/d1)

 

 

 

 

Variable CTRL 10 15 20 SEM
mmoles/d1
VFA 7.8780 8.728a 8.939a 8.413b .08
Acetate 5.480b 6.072a 6.170a 5.613b .07
Propionate 1.552b 1.698a 1.638a 1.520b .02
Butyrate .609a .586a .667a .659a .03
Isoacids .284d .406c .498b .597a .01
Isobutyrate .034d .060c .076b .089a .001
2-Methylbutyrate .066d .108c .134b .150a .003
isovalerate .049d .103c .129b .133a .003
Valerate .127c .143c .169b .220a .006

 

a,b,c Means in a row with different superscript differ (p<.05)

Isoacids at 10 and 15 mg/dl increased acetate concentration

but had no effect on acetate concentration at 20 mg/dl.

Isoacids at 10 and 15 mg/dl of the culture media,

increased total VFA, but there was a trend toward a decrease at

20 mg/dl compare to the previous levels.

As expected, there

36
was an increase in branched-chain fatty acid concentrations in
respect to the amounts added in the media.

Isoacids at 10 mg/dl and 15 mg/dl increased propionate
concentration. 1However, a trend toward a decline of propionate
production back to the control level was observed at 20 mg/dl.
Figure 4.

Previous studies conducted by Felix and Cook (1980),
reported that isoacids increased growth rate in young animals
but not in older animals and that the supplementation of
Isoacids and urea to high producing cows fed corn silage as the
sole roughage had a positive effect on milk production,
persistancy of lactation, body weight, feed intake and nitrogen
balance. Towns and Cook, (1984) found that during an eight-hour
sampling period, isoacid-treated cows had higher growth hormone
but lower glucose and lower insulin. Brondani (1986) found that
during an eight hour sampling period, a low concentration of
insulin observed in isoacid-treated cows was associated with a
decrease in propionate production. Brondani therefore pointed
out that the decrease in insulin found for the first time by
Towns and Cook, (1984) when cows where fed a high concentrate
diet plus isoacids was due to a decreased propionate production
in the rumen.

The present dose response study of the effect of isoacids
on mixed rumen microbes was the first to demonstrated that
isoacids have different effects at different concentrations.

At low concentrations of branched chain fatty acids, the
supplementation of isoacids increase all VFA. Perhaps by

serving as a growth factor for cellulolytic bacteria.

 

37

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38
However, when the concentration is optimal, further
supplementation of isoacids tend to shift the carbon flux
toward more acetate, less propionate, more microbial growth and
a trend toward more butyrate (Table 11). These results clearly
indicate that it is likely that the decrease in propionate
found by Brondani, (1986) was due to the high ruminal
concentration of isoacids brought about by the diet and the
supplementation of isoacids.

The mechanism by which isoacids induces these changes in
bacteria is unknown. The fact that propionate concentration
decreased suggests that propionate producers are the primary
targets and that metabolic adaptations occur in these species.
The fact that growth yield increased suggests that a shift in
bacterial electron flow occurs such that the production of end
products is couple with optimal response in net microbial
growth.

In the present study it was also noted that high
concentrations of isoacids tend to decrease VFA and gas
production. This suggests that supplementation of isoacids
far above normal concentrations may affect the efficiency of

fermentation.

 

39

TABLE 12. Effect of Isoacids on rumen gas production and
ammonia nitrogen concentration tg vitro using
inoculum from a cow fed high roughage diet.

 

Isoacid concentration (mg/d1)

 

 

 

 

 

 

 

 

Variable Control 10 15 20 SEM
ml/hr

Total gas 27.72a 27.71a 28.49a 27.55a 0.4
percent

CH4 35.15a 36.15a 34.38a 33.08a 1.99

C02 49.02a 54.94a 49.03a 47.37a 3.15

H2 3.83a 4.00a 3.67a 3.83a 0.2
mg/dl

NNH3 5.57b 6.10ab 6.34a 6.11ab 0.05

 

a,b Means in a row with different superscript differ p(<.05).

In contrast to VFA production, total gas production as
well as CH4, C02 and H2 were not affected by isoacids. An tg
v_i_t_r_o study conducted by K.A Cummins (1984) using ammonium salt
of isoacids at 1% of dry matter in a diet composed of
cottonseed meal or alfalfa haylage and soybean meal (16% CP),
showed an increased microbial nitrogen incorporation, increased
dry matter digestion and microbial growth. But, when isoacids
were added above 1% of the dry matter, digestion was decreased.

In our study, we did not observe any significant change in
ammonia nitrogen concentration in the medium. However, in the
presence of very low concentrations of ammonia nitrogen,
isoacids did not increase acetate nor total VFA. This

suggests that the increase in VFA production was associated

40

with an increase in ammonia nitrogen.

The effect of different concentrations of Monensin are

summarized in Tables 13 and 14.

 

 

 

 

 

 

Table 13 Effect of monensin on rumen VFA concentration
Q vita using inoculum from a cow fed high
roughage diet.
Monensin Level (ug /100ml
Variable Ctrl. 100 150 200 SEM
mmoles / d1
VFA 8.543a 7.221b 6.847b 6.887b 0.14
Acetate 6.026c 4.286b 3.743a 3.601a 0.11
Propionate 1.736c 2.272b 2.281b 2.420a 0.03
Butyrate 0.523a 0.467b 0.429ab 0.359a 0.02
Isoacids 0.184 0.162 0.162 0.195 0.01
Isobutyrate 0.027a 0.028a 0.027a 0.027a 0.01
2M-butyrate 0.042a 0.038a 0.036a 0.054a 0.01
Isovalerate 0.035a 0.028a 0.030a 0.047a 0.01
Valerate 0.080a 0.072a 0.069a 0.067a 0.01
AC/PR 3.509a 1.907b 1.755bc 1.614c 0.07

 

a,b,c Means in row with different superscript differ p<.05)

In contrast to isoacids, Monensin drastically decreased

acetate concentration at all levels tested.

Propionate

concentration was significantly increased at all levels tested

whereas butyrate concentration dramatically decreased at all

levels tested.

Figures 5 and 6.

A summary of these results is presented in

 

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43

Table 14. Effect of Monensin on rumen gas production and
ammonia nitrogen concentration tg vitro
using inoculum from a cow fed high roughage diet.

 

 

Monensin level (ug/100ml)

 

Variable Control 100 150 200 SEM

 

 

 

 

 

 

 

 

ml/hr
Total gas 25.88a 22.93b 20.72c 20.16c 0.68
%
CH4 36.33a 27.07b 26.77b 26.00b 0.91
C02 49.83a 48.33a 42.50a 47.50a 3.2
H2 3.5b 3.5b 3.5b 4.0a 0
mg/dl

N-NH3 3.37a 4.25a 4.27a 4.37a 0:76
PH 6.80c 6.82c 6.85b 6.89a 0.07

 

a,b,c Means in a row with different superscript differ p(<.05).
The addition of Monensin decreased total gas production at
all levels, and a drastic decrease in methane production.
Neither C02 nor H2 were significantly affected. The results
from the Monensin experiment agreed with similar studies
conducted by Short (1976) and Chalupa (1977). Monensin
constantly decreased acetate and butyrate production, and
increased propionate production. This shift in rumen
fermentation by Monensin is due to a selective action of the
antibiotic on the rumen microbes by interfering with the

passage of ions across cell membranes (Bergen, 1984).

 

44

Isoacid concentrations did decrease in the
presence of Monensin, but not at a significant level.
Matsumoto gt gt,, (1984) reported a decrease in NH3 and total
amino acids, a decrease in protozoa number and a decrease in
isoacid concentration whereas leucine, isoleucine and valine
concentrations were higher when 40ppm monensin was added to the
diet of steers fed concentrate and rye grass silage. Monensin,
by increasing ammonia nitrogen utilization, also increased

isoacid utilization.

45

The effect of the combination of isoacids and monensin

are presented in tables 15 and 16.

Table 15. Effect of Isoacids and Monensin on rumen VFA
concentration and gas production tg vitro
using inoculum from a cow fed a high roughage diet.

 

 

 

 

 

 

 

 

Treatment
Variable Ctrl. 1150-15 2MON—15 3M+I~1o 4M+I—15 SEM
mmoles/d1
VFA 8.835ab 9.268a 6.8500 8.530ab 8.350b .25
oz 6.047a 6.335a 3.743c 5.245b 4.986b .11
c3 2.033ab 1.821b 2.282a 2.267a 2.320a .11
C4 .579a .594a .405a .620a .589a .01
ISO .252b .647a .162b .398ab .435ab .09
IB .034b .143a .027c .065b .076b .003
2MB .050b .128a .036c .092b - .107ab .01
IV .052b .153a .030c .094b .115b .01
VA .127b .263a .069c .149b .161b .01
ml/hr
Gas 25.55ab 26.50a 20.72c 24.50ab 23.72b .79

 

1Isoacids at 15mg/100ml of final incubation media.

Monensin at 150ug/100ml of final incubation media.

Monensin at 150ug/100ml and isoacids at 10mg/100ml.

Monensin at 150ug/100ml and isoacids at 15mg/100ml.

a,b,c,d Means in row with different superscript differ (p<.05)

46

Table 16. Effect of isoacids and monensin on rumen gas
production, ammonia nitrogen concentration and pH
tg vitro using inoculum from a cow a fed high
roughage diet.

 

 

 

 

 

 

 

 

 

 

 

Treatment
Variable Ctrl. 1130-15 2MON-15 3M+I-1o 4M+I-15 SEM
ml/hr
gas 25.55ab 26.50a 20.72c 24.50ab 23.72b .79
%
CH4 35.15a 34.38a 26.90b 29.40ab 28.35b 1.06
C02 49.37a 49.03a 32.65b 48.03a 49.20a 2.01
mg/dl
N-NHB 4.90a 4.40a 4.37a 4.68a 5.13a .19
ph 6.79a 6.78a 6.87b 6.79a 6.79a .01

 

1Isoacids at 15mg/100m1 of incubation media.
Monensin at 150ug/100m1 of incubation media.
4Monensin at 150ug/100ml and isoacids at 10mg/100ml.
Monensin at 150ug/100ml and isoacids at 15mg/100m1.
a,b,c, Means in row with different superscript differ (p<.05)
The addition of Monensin caused a decrease in acetate and
propionate after 24 hrs of incubation, but after 48 hrs,
propionate was higher than control values whereas acetate
remained lower than the controls. (Figures 8 and 9). The
addition of isoacids at 10 mg/100ml to flasks containing
monensin increased acetate and total VFA concentration
compared to monensin alone, and increased butyrate
concentration above the control. However, the addition of

isoacids to flasks containing Monensin did not alter propionate

methane and total gas production.

47

The possible mode of action of the combination of isoacids
and Monensin is shown in figure 7. Bryant (1964), reported the
distribution of predominent rumen bacteria cultured from rumen

contents of cattle fed differents diets. Bacteriodes

 

succinogenes, Butyrivibrio fibrisolvens, Bacteriodes ruminicola

 

 

 

and Ruminococcus albus were the most predominant bacteria when

 

cows were fed wheat straw. They represented respectively 20,
19, 12 and 8% of total isolates. According to Wolin and Miller
(1983), Monensin affects the rumen fermentation by selecting
for organisms that participate in the production of relatively
more propionate and against those that contribute to the
production of relatively more acetate, butyrate and precursors

of methane. ZRuminococci and Butyrivibrio are inhibited by very

 

 

low concentrations of monensin. These species are important
producers of acetate, butyrate and the substrate for

methanogens, H2 and C02. Selenomonads are very insensitive,

 

whereas Bacteroides, although sensitive, rapidly become

 

resistant to the antibiotic. Both organisms are important in
the production of propionate. The addition of isoacids to the
culture containing Monensin would cause an outgrowth of

Bacteroides resulting in more acetate, succinate, and formate.

 

Succinate would be decarboxylated to propionate by selenomonads

 

and formate would be used as an energy source by methanogens.
The end result would be an increase in acetate, propionate and
probably more substrate degradation as indicated by higher VFA
and total gas production observed in the last experiment

(Table 15).

48

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50

The addition of isoacids to a culture containing Monensin

will serve as a factor for growth not only for Bacteriodes

 

succinogenes but also for Ruminoccoccus flavefaciens and

methanogenic bacteria, which require also isoacids. Bacteriodes

 

succinogenes in concert with Ruminoccoccus flavefaciens and
Methanogenic bacteria will produce more acetate and methane.
This was confirmed in our experiment by an increase in acetate
and a trend toward an increase in methane production initially
depressed by Monensin Figures 8 & 10. Based on these results
we proposed that the combination of isoacids and Monensin may
be of practical application in cattle rations. The addition of
Monensin alone to the diet of growing steers increase the
efficiency of growth by increasing propionate production.
Because of the importance of propionate in the metabolism of
glucose and the regulation of insulin secretion in ruminants,
the increase in ruminal propionate production will promote
growth. Isoacids increase acetate, total VFA and microbial
synthesis. Since the addition of isoacids to cultures
containing Monensin does not alter the effect of Monensin on
propionate production the excess of acetate and microbial
protein may further improve the efficiency of growth in these

animals.

 

51

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SUMMARY AND CONCLUSIONS.

A semi-continuous culture technique was set up to
investigate the interaction of isoacids and Monensin on rumen
fermentation in yitrg. Initial studies validated the system.
Inoculum, Substrate and protein level were evaluated. Three
experiments were then conducted. The first two tested different
levels of Isoacids and MOnensin seperately. The third
investigated the interaction of the two chemicals. 'The dose
level study and the interaction of the two chemicals led to the
following conclusions:

-Isoacids at 1% of DM increased acetate and propionate
production and consequently total VFA production. At 1.5% of DM
isoacids increased acetate and total VFA production and did not
change propionate production. At 2% Isoacids decreased
propionate production, but total VFA was not significantly
affected.

-In contrast to isoacids the effect/response of Monensin
drastically decreased acetate, butyrate, methane and total VFA
production at all levels tested, and dramatically increased
propionate production at all levels tested.

-Isoacids when added to a system containing Monensin
restored acetate, methane and total VFA production to the
control level and increased propionate production above the
control.

-Isoacids when added to a culture containing Monensin may

favor an outgrowth of Bacteriodes succinogenes and perhaps

 

Ruminoccoccus flavefaciens and methanogens that require

 

54

55

isoacids as growth factor. The addition of isoacids and

Monensin may have practical application in cattle rations.

 

 

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