Influence of amino acids and branch chain organic acids on ruminant fiber fermentation
by Connie Kay Clark
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Animal Science
Montana State University
© Copyright by Connie Kay Clark (1986)
Abstract:
Amino acids and branched chain organic acids were examined for possible positive effects on fiber
fermentation in the rumen. Nine L-isomer amino acids and three branch chain organic acids were added
as single amendments to in vitro buffer to evaluate influence of these additions on fermentation kinetics
of native winter range forage collected via esophageal fistula. Amino acids provided 50 mg nitrogen
per 100 ml buffer and branch chain organic acids were added at a .01% level. Dry matter (DM) and
neutral detergent fiber (NDF) disappearance at 48 h (41.2% and 53.1%) were similar (P>.05) for buffer
amendments and the control, although treatments differed significantly (P<.05). In comparison to the
control, methionine, arginine, and cysteine increased (PC.05) both DM and NDF fermentation rate, and
histidine and leucine influenced only DM fermentation rate. The correlation between DM fermentation
rate and extent was positive and significant (P<.01). Lag time calculated for DM (29.1 h) and NDF
(23.3 h) was similar for all treatments.
Methionine was further evaluated in an in situ trial. Three isonitrogenous isocaloric (.32 kg crude
protein and 1.23 kg TDN) supplements, DL-methionine-urea, soybean meal (SBM), and urea, were
formulated which contained: 4, 0, 63, 33; 0, 81, 0, 19; and 0, 0, 67, 33% nitrogen from DL-methionine,
SBM, urea and beet pulp. Cannulated cows receiving the supplements were maintained on a mixture of
75% mature grass hay and 25% barley straw. DL-methionine-urea supplementation increased (P<.05)
DM fermentation rate in comparison to the other supplements. Estimated rumen digestibility (48%)
was improved (PC.01) and particulate passage rate (4.2 %/h) was slower (PC.05) with
DL-methionine-urea and urea supplementation as compared to SBM. Neutral detergent fiber
fermentation followed similar trends; however, no significant differences could be detected. Lag times
(1.5 h) were calculated for NDF only.
A heifer growth trial was conducted utilizing the three supplements evaluated in the in situ trial and a
SBM-urea supplement. Heifers were fed the same low quality roughage as described for the in situ
trial. No differences were detected for average daily gain, intake, or feed efficiency. 
INFLUENCE OF AMINO ACIDS AND BRANCH CHAIN ORGANIC ACIDS
ON RUMINANT FIBER FERMENTATION
by
Connie Kay Clark
A thesis submitted in partial fulfillment 
of the requirements for the degree
of
Master of Science 
in
Animal Science
MONTANA STATE UNIVERSITY 
Bozeman, Montana
May, I 986
ii
MAIN L ie.
/1/3-72
CsV V
APPROVAL
of a thesis submitted by
Connie Kay Clark
This thesis has been read by each member of the thesis committee 
and has been found to be satisfactory regarding content, English 
usage, format, citations, bibliographic style, and consistency, and is 
ready for submission to the College of Graduate Studies.
■ s jn/Sb___ //7 ,/ /
Date f Chairperson, Graduate Committee
Approval for the Major Department
■
Date
0
Head, Major Department
Approved for the College of Graduate Studies
Date Graduate Dean
iii
STATEMENT OF PERMISSION TO COPY
In presenting this thesis in partial fulfillment of the 
requirements for a master's degree at Montana State University, I 
agree that the Library shall make it available to borrowers under the 
rules of the Library. Brief quotations from this thesis are allowable 
without special permission, provided that accurate acknowledgement of 
source is made.
Permission for extensive quotation from or reproduction of this
thesis may be granted by my major professor, or in his absence, by the
>
Director of Libraries when, in the opinion of either, the proposed use 
of the material is for scholarly purposes. Any copying or use of the 
material in this thesis for financial gain shall not be allowed 
without my written permission.
Signature.
VACKNOWLEDGMENTS
I wish to express sincere appreciation to the following people:
Mark Petersen for his guidance, assistance and support in all 
areas of my graduate program;
Kris Havstad, Walt Newman, Rodney Kott and Ray Ansotegui for 
serving as members of my graduate committee;
Nancy Roth and Meg LaVigne for assistance with laboratory 
analysis;
Jim Fadal and Mike McInerny for their knowledge and help with the 
statistical analysis and;
Jeanne Blee for her guidance with typing the thesis.
I would like to extend a special thanks to my husband, Mike, and 
our family for their understanding and support. I dedicate this 
thesis to my late grandpa, Lawrence H. Swenson, from whom, I developed 
an interest in agriculture.
vi
TABLE OF CONTENTS
LIST OF TABLES ..............................................vii
LIST OF F I G U R E S ........................................ .....
ABSTRACT............................................. - • ix
INTRODUCTION................................... ..... . I
LITERATURE REVIEW ..............................  • %
Bacterial Nitrogen and Branch Chain Organic
Acid Requirements . . . . . . . .  5
Bacterial Growth Rate . . . . . . .  7
Fiber Fermentation . . . . . . . .  10
EXPERIMENTAL PROCEDURES . . .    • 16
R E S U L T S ................................... ....  . . 25
In Vitro Trial • • • • • • • • • ■  25
In Situ Trial 27
Heifer Growth Trial . . . . . . . .  29
D I S C U S S I O N .......................................   30
CONCLUSION AND RECOMMENDATIONS .........................  #
REFERENCES CITED ..................................  %7
APPENDIX .    . 55
vii
LIST OF TABLES
Table
I Composition of the supplements evaluated in the in situ 
trial and the heifer growth trial ................
2 Influence of in vitro buffer amendments on dry matter
(DM) disappearance and fermentation rate of native 
winter range forage esophageal extrusa . . . .  •
3 Influence of in vitro buffer amendments on neutral
detergent fiber (NDF) disappearance and fermentation 
rate of native winter range forage esophageal extrusa .
4 Influence of in vitro buffer amendments on dry matter
(DM) and neutral detergent fiber (NDF) fermentation lag 
time of native winter range forage esophageal extrusa .
5 In situ dry matter fermentation rate, particulate
passage rate and rumen digestibility for forage as 
influenced by supplement treatments ................
6 In situ neutral detergent fiber fermentation rate,
particulate passage rate, rumen digestibility and
fermentation lag time for forage as influenced by 
supplement treatments ................  . • •
7 Rumen liquid parameters as influenced by supplement
treatments during the in situ trial ................
8 Rumen ammonia levels as influenced by supplement
treatments in the in situ t r i a l ................ ...
9 Average daily gain, feed intake, and feed efficiency of
heifer calves as influenced by supplements . .
Page
56
57
58
59
60
61 . 
62
63
64
viii
LIST OF FIGURES
Figure
1 In vitro dry matter digestibility of native winter
range forage . • . . . . •
2 In vitro dry matter digestibility of native winter,
range forage »
3 In vitro dry matter digestibility of native winter
range forage . . . . . . .
4 In vitro neutral detergent fiber digestibiliy of native
winter range forage . . . • . •
5 In vitro neutral detergent fiber digestibiliy of native 
winter range forage . . . . . . .
6 In vitro neutral detergent fiber digestibiliy of native 
wipter range forage . . . . . . .
Page
31
32
33
35
36
37
ix
ABSTRACT
Amino acids and branched chain organic acids were examined for 
possible positive effects on fiber fermentation in the rumen. Nine 
L~isomer amino acids and three branch chain organic acids were added 
as single amendments to in vitro buffer to evaluate influence of these 
additions on fermentation kinetics of native winter range forage 
collected via esophageal fistula. Amino acids provided 50 mg nitrogen 
per 100 ml buffer and branch chain organic acids were added at a .01% 
level. Dry matter (DM) and neutral detergent fiber (NDF) disappearance 
at 48 h (41.2% and 53.1%) were similar (P>.05) for buffer amendments 
and the control, although treatments differed significantly (P<.05). 
In comparison to the control, methionine, arginine, and cysteine 
increased (P<.05) both DM and NDF fermentation rate, and histidine and 
leucine influenced only DM fermentation rate. The correlation between 
DM fermentation rate and extent was positive and significant (P<.01). 
Lag time calculated for DM (29.1 h) and NDF (23.3 h) was similar for 
all treatments.
Methionine was further evaluated in an in situ trial. Three 
isonitrogenous isocaloric (.32 kg crude protein and 1.23 kg TDN) 
supplements, DL-methionine-urea, soybean meal (SBM), and urea, were 
formulated which contained: 4, 0, 63, 33; 0, 81, 0, 19; and 0, 0, 67,
33% nitrogen from DL-methionine, SBM, urea and beet pulp. Cannulated 
cows receiving the supplements were maintained on a mixture of 75% 
mature grass hay and 25% barley straw. DL-methionine-urea
supplementation increased (P<.05) DM fermentation rate in comparison 
to the other supplements. Estimated rumen digestibility (48%) was 
improved (PC.01) and particulate passage rate (4.2 %/h) was slower 
(PC.05) with DL-methionine-urea and urea supplementation as compared 
to SBM. Neutral detergent fiber fermentation followed similar 
trends; however, no significant differences could be detected. Lag 
times (1.5 h) were calculated for NDF only.
A heifer growth trial was conducted utilizing the three 
supplements evaluated in the in situ trial and a SBM-urea supplement. 
Heifers were fed the same low quality roughage as described for the in 
situ trial. No differences were detected for average daily gain, 
intake, or feed efficiency.
IChapter I 
INTRODUCTION
Agriculture is challenged to feed an ever-increasing population 
with declining available resources. The ruminant provides man with a 
vital link to an abundant store of organic energy, forage, which 
supplies a source of nutrients for the ruminant that cannot be used by 
man. Fiber is a resource found in many areas unsuitable for 
cultivation or human residence. Equipped with a unique fermentation 
vat, ruminants are capable of utilizing fiber and complement man's 
need for energy and high quality protein without direct competition 
with man for food or land.
Forage harvesting by the ruminant animal may be the sole means of 
utilizing the rangelands in Montana. Although range plants can 
provide an adequate diet for the ruminant, the nutritional status of 
range plants vary substantially depending on the season of the year. 
Fiber digestibility may range from sixty-seven percent in June down to 
forty-nine percent or lower in December. Winter range supplementation 
with a protein source has been a common management practice to help 
maintain animals on winter range. A supplement program capable of 
increasing fiber digestibility and improving diet utilization by the 
animal could benefit the producer. In viewing the price-cost squeeze 
experienced by animal industries, a decrease in supplemental feed cost 
as well as better diet utilization by animals, may improve profits for 
producers.
IIn order to approach optimum rumen fermentation of winter range 
forage, minimum ruminal conditions are required. The rumen provides a 
suitable environment for anaerobic microorganisms to ferment fiber the 
animal consumed, and subsequently produce volatile fatty acids which 
are used by the animal as energy. The microbial population of the 
rumen may have specific nutritional requirements to obtain an optimum 
level of growth and reproduction which could enhance fiber 
fermentation. The importance of meeting nutritional needs of microbes 
may be magnified for ruminants on winter range as the nutrient supply 
for rumen microorganisms fermenting low quality forage could put 
limiting restraints on microbial, growth and reproduction.
Cellulolytic bacteria are necessary for fiber digestion, but 
other bacteria must not be ignored because many of the microorganisms 
work in a synergistic fashion to accomplish fermentation. Providing 
rumen microorganisms with readily accessible amino acids or branch 
chain organic acids may enhance turnover of the microbial population. 
Although cellulolytic bacteria utilize ammonia as a major source of 
nitrogen, small amounts of amino acids or branch chain organic acids 
added to rumen bacteria are known to stimulate growth. It has. been 
demonstrated that amino acids and branch chain organic acids improved 
microbial growth rate, but it is not known how this may influence 
fermentation.
This study had three objectives:
1) evaluate influence of amino acids and branch chain 
organ!cacids on fermentation of winter range forage,
2) incorporate an amino acid or branch chainorganic acid 
into a supplement for cows consuming a low quality
2
3roughage and:evaluate influence of the supplement on 
fermentation parameters, and
.3) evaluate influence of the supplement o!performance of 
growing heifers.
The study consisted of three phases. The first phase investigated the
influence of single amino acid or branch chain organic acid addition
■ ' !
to in vitro buffer solution on fermentation of native winter range 
forage, and served as a screening procedure for the following phases. 
An in situ trial was completed for phase two, in which a Di­
me thio nine-urea supplement was compared to soybean meal and urea 
supplements. The final phase evaluated feedlot performance of heifers 
receiving a low quality forage supplemented with one of four 
supplements; DL-methionine-urea, urea, soybean meal, or soybean meal-
urea.
4Chapter 2 
LITERATURE REVIEW
The ruminant is unique in its ability to harvest highly fibrous 
plants and produce nutritious food and products for man. To 
accomplish fiber utilization, the ruminant animal and rumen microbe 
populations live in a symbiotic relationship. With ingestion of feed, 
the ruminant provides a nutrient source for the microorganisms to grow 
and reproduce. The ruminant also contributes a suitable microbial 
environment with optimum temperature, pH, and mixing of rumen 
contents.
The rumen microorganisms provide energy substrate, protein and 
vitamins for the ruminant animal. An important function of the 
microbes is the production of cellulase and hemicellulase (Smith et 
al., 1973; Dehority, 1968), enzymes which enzymatically degrade 
fibrous digests. Cellulose and hemicellulose supply an abundant 
source of carbon and energy in the ruminant’s diet and without the 
fermentation action of the rumen bacteria these nutrients would be 
unavailable to the animal. The microbe population utilizes the carbon 
and energy of cellulose to produce volatile fatty acids such as 
acetic, propionic and butyric acid which the animal absorbs into the 
blood and uses for energy.
The microbial biomass of the rumen is composed of a multitude of 
differing microbe species capable of many biochemical functions 
(Hungate, 1966). Rumen bacteria appear to be more essential than
5protozoa because rumen fermentation can proceed normally in defaunated 
animals (Eadie, 1962). Bacteria can be roughly divided into three 
groups according to their activity: cellulolytic bacteria, amylolytic 
bacteria and proteolytic bacteria, however many of the activities of 
individual species overlap with other species and it is difficult to 
designate bacteria into specific groups. Important cellulolytic 
species include Ruminocooous albus. Ruminococcus flavefaciehs. 
Bacteroldes succinoeens. and Butvrivibrio fibrisolvens (Wolin, 1979).
To understand the process of cellulose fermentation, all species 
of rumen microbes need to be considered as fermentation occurs through 
the interaction of many species. Only a few of the predominant 
species produce cellulase for degradation. The hydrolysis products 
released from cellulose degradation are utilized as fermentation 
substrates by noncellulolytic as well as cellulolytic microorganisms. 
This reduces end product inhibition which improves the efficiency of 
enzymatic substrate degradation.
Bacterial Nitrogen and Branch Chain Organic Acid Requirements
Bryant (1973) reviewed nutrient requirements of cellulolytic 
bacteria. Three of the major cellulolytic species, Bacteroides 
succinogens. Rumlnoooccus albus. and Ruminoooccus flavefaciens, 
required ammonia as the essential nitrogen source during growth 
regardless of the amount of amino acid and peptide nitrogen in the 
medium (Bryant and Robinson, 1962). Some strains are incapable of 
utilizing organic nitrogen source. These bacteria possibly lack the 
mechanisms to transport most amino acids or peptides into the cell.
6Studies with Butvrivibrio fibrisolvens. a cellulolytic species, have 
indicated amino acids are essential for growth in some strains (Bryant 
and Robinson, 1962; Gill and King, 1958).
Branch chain organic acids, isovaleric, isobutyric and 2- 
methylbutyric, are required by major cellulolytic species (Bryant and 
Robinson, I 962). Specific requirements for branched chain organic 
acids of cellulolytic bacteria has been studied (Dehority et al., 
1967; Allison et al., 1962a). Bacteroides suooinogens require a 
combination of either isobutyric acid or 2~ me thy I butyric acid with 
valeric acid (straight chain fatty acid). Ruminococcus albus 
exhibited a requirement for isobutyrate or 2-methylbutyerate to 
achieve maximum growth. The Ruminoooocus albus bacteria incorporated 
the branched chain organic holds into branched chain 14- and l6-car,bon 
fatty acids or 14- and 16-carbon aldehydes (Allison et al., I 962b). 
Ruminococcus flavefaoiens can utilize isobutyric, isovaleric, or 2- 
methylbutyric acid to maximize growth. Isovaleric acid was found to 
serve as a carbon skeleton for leucine synthesis bv R. flavefaciens 
and isobutyric acid served the same function for valine synthesis 
(Allison et al., 1962a). Ruminococcus flavefaciens have limited 
ability to incorporate exogenous branched chain amino acids. Allison 
(1969) later reported that in addition to synthesis of valine, 
leucine, long chain branched fatty acids and aldehydes, the bacteria 
utilize shorter branched chain organic acids to synthesize isoleucine.
There is evidence of nutritional interdependence among rumen 
bacteria. Miura, et al. (1980) reported growth stimulation of 
Bacteroides amvlophilus by starch resulted in successive growth of
7amino acid-dependent Meaasphaera elsdenii and branch chain fatty acid- 
dependent Ruminococcus albus in a media lacking both amino acids and 
branch chain fatty acids, indicating interdependence among the three 
bacteria species. Allison (1978) has also reported branch chain 
organic acid production by MsaaaRhaera elsdenii. Further 
investigation by Muira, et al. (1983) demonstrated mixed rumen 
bacteria interact nutritionally in the same manner as revealed in pure 
culture mixture. The microbial population changes in relation to 
alterations of amino acid and branch chain fatty acid concentrations. 
Preformed amino acids or branch chain fatty acids are not essential 
for growth of branch chain fatty acid-dependent bacteria (Slyter and 
Weaver, 1971); however lag in the appearance of cellulose digestion 
may reflect time required for producing branch chain fatty acids via 
nutritional interdependence.
Bacterial Growth Rate
Currently in the literature, several publications report 
responses in rumen bacterial growth rate with the addition of amino 
acids, however; not much is available on the influence of amino acid 
additions on fermentation rate in the rumen. There are several 
factors in the complex ecosystem of the rumen that can have an effect 
on growth rate of microorganisms. Rates of hydrolysis of polymeric 
carbon and nitrogen sources, concentrations of permeable 
macronutrients used by microorganisms, concentrations of potentially 
growth-limiting micronutrients, inhibitory agents, and pH may be 
influential factors on growth rate (Wolin, 1979). The effect of
8supplying readily available amino acids to rumen microorganisms may 
have an impact on microbial growth rate. An estimation that 60 to 80$ 
of microbial nitrogen was derived from ammonia and 40 to 60$ from 
amino acid- and peptide-nitrogen has been reported (Al-Rabbat et al., 
1971; Nolan and Leng, 1972). An optimum ratio, 75$ nonprotein 
nitrogen to 25$ amino acid nitrogen, for rumen microbial growth has 
been indicated (Maeng et al., 1976).
A study conducted by Maeng and Baldwin (1976a) revealed that the 
addition of small amounts of amino acids to a diet containing urea as 
the nitrogen source improved rumen microbial yield. The greatest 
impact upon microbial yields with amino acid additions occured 
immediately after feeding and became less evident as time passed. The 
microbial nitrogen, protein and cell yield per mol ATP and per mol VFA 
was increased (P<.01) by amino acid containing treatments. The 
projected microbial nitrogen, protein, and cell yields per day and per 
kg of diet dry matter also was significantly improved with the 
addition of amino acids. A second study completed by Maeng and 
Baldwin (1976b) indicated a stimulatory microbial response with 
partial substitution of amino acids for urea. Amino acids replaced 
25$ of urea-nitrogen in a growth media. Growth rate of rumen bacteria 
was twofold and mean doubling time was reduced by half.
The relative degree to which ATP or other energy-rich compounds 
produced from catabolic activities are utilized by anabolic activities 
of the cell is referred to as energetic uncoupling. To obtain maximum 
microbial cell yields per unit of nutrient input, the rate of ATP 
production from fermentation reactions must equal the usage rate by
9biosynthetic reactions at all times or in other words, the energetic 
uncoupling must be minimized. Hespell and Bryant (1979) suggest the 
stimulated bacterial growth response observed with amino acid 
additions is due to a decrease in the degree of energetic uncoupling 
with the rumen microbial population as a whole.
Studies have indicated the addition of branched chain organic 
acids have a positive influence on bacterial growth in the rumen. 
This is especially true of cellulolytics which have been shown to have 
nutrional requirements for branched chain organic acids. Russell and 
Sniffen (1984) reported the addition of low concentrations of 
isovaleric and 2-methylbutyric acid improved synthesis efficiency of 
rumen bacterial protein from carbohydrates in vitro. Bacterial growth 
measured by cell protein synthesis was increased 11.2% and 16.4% with 
the addition of isovaleric and 2-methylbutyric acid. A combination of 
isovaleric, iso butyric, 2-methylbutyric, and valeric acids produced a 
18.7% increase in protein synthesis when added to in vitro inoculum. 
Branched chain organic acids have also increased bacterial growth- 
measured in the rumen of sheep consuming a low quality forage 
supplemented with urea, isovalerate, isobutyerate, and 2- 
methylbutyerate (Hemsley and Moir, 1963).
Branched chain organic acids have been shown to effect digestion 
of forage. Isovaleric and isobutyric acid were added to an in vitro 
system fermenting purified cellulose with urea serving as the nitrogen 
source. Digestibility values at 30 hours revealed a substantial 
increase for the branched chain fatty acid treatment in comparison to 
the control (Bentley et al., 1955). Soofi et al. (1982) more recently
10
?
reported similar results. The addition of branched chain volatile 
acids increased (P<.05) in vitro dry matter digestibility at 48 hours, 
48.1% digested verses 45.3% digested in the control. Gorosito et al. 
(1985) also studied in vitro digestion of forage as influenced by 
additions of isovaleric, isobutyric, 2-methy!butyric and valeric acid. 
All acids except valeric had positive effects on fermentation as 
indicated by increased cell wall digestibility at 24 hours and 
improved ammonia utilization. The greatest improvement for cell wall 
digestion was noted for the grass hay although alfalfa hay cell wall 
digestibility was also increased.
Hemsley and Moir (1963) reported an increase in intake and 
passage rate when isobutyric, isovaleric and 2-methylbutyric acid were 
added to a urea supplement provided to sheep consuming a low quality 
forage. Felix et al. (1980) fed isoacids with urea to lactating cows 
and indicated no influence on nitrogen or dry matter digestibility; 
however loss of urinary nitrogen was decreased and percent of absorbed 
nitrogen retained was higher indicating an improvement in utilization 
of nitrogen.
Fiber Fermentation
Forage digestion in the rumen involves complex relationships and 
interactions among the animal, microbial populations in the rumen, and 
plant components. Understanding ruminal digestion is simplified when 
the process is divided into four components: digestion rate, 
digestion lag, extent of digestibility, and passage rate (Mertens, 
1977).
r>
11
The first component to be addressed is digestion rate. Rate of 
digestion has been suggested as being important in assessing forage 
quality and voluntary intake (Crampton, I 957; Thorton and Minson, 
1972). The theory is that enhancing rate of digestion would increase 
disappearance of fiber from the rumen; therefore, freeing space for 
additional intake. Mertens and Ely (1979) reported simulation results 
in which values for coastal bermuda grass suggested that a 1.0% 
increase in digestion rate resulted in a .67% increase in maximum 
digestible dry matter intake. Digestion rate can have significant 
effects on dry matter digestibility (Mertens, I 977). The effect of 
rate is magnified in grasses with high levels of cell wall content.
Several factors have been suggested which may have an affect on 
digestion rate. Limited growth and reproduction of microorganisms in 
the rumen may influence rate because microorganisms produce enzymes 
necessary for fiber degradation; however, few factors that affect 
microbial population have been studied for their impact on rate of 
digestion. Plant composition has an impact on rate of digestion. The 
morphological types of leaf tissues vary in rate of digestion with the 
mesophyll and phloem tissue being rapidly digested, while bundle 
sheaths and epidermis tissue are slowly degraded and the vascular 
bundles and sclerynchema are almost entirely indigestible (Akin et 
al., 1974; Akin and Amos, 1975). In addition, individual animals may 
affect digestion rate through mastication and rumination. It has been 
reported (Dehoirity and Johnson, 1961) that reducing particle size can 
increase cell wall digestibility in vitro. The animal also influences 
rurneq pH through buffering capacity. Rumen pH affects the ratio of
12
species in the rumen microbe population (Russell et al., I 97 9), and 
inhibits cellulase activity when pH falls below 6.0 (Stewart, 1977).
Fiber digestion lag time is a period at the initial onset of 
digestion in which very little digestion occurs. The effect of lag 
time increases exponentially with increasing levels of fiber in the 
diet, indicating its importance as a variable in digestion (Mertens, 
1977). There have been several explanations to account for the 
phenomenon of lag time. Brazle and Harbers (1977) completed work that 
indicated penetration of the epidermal layer may provide an initial 
barrier to digestion. It may also be possible that physical or 
chemical inhibitors must be removed or that swelling of fibers is 
required before enzymes can contact and react with fiber particles. 
Attachment of bacteria to fiber is necessary for some fiber digestion 
(Akin, 1979). These suggestions all imply that plant and bacteria 
interactions are responsible for lag time.
Mertens and Ely (1982) reviewed other explanations for lag time. 
Period of lag may be due to delay in digestion until bacterial numbers 
and concentration of fiber-digesting enzymes reach levels that are no 
longer limiting to digestion. A preference for starch or other 
readily fermentable carbohydrates may also be a factor involved in lag 
time. The addition of starch to forage has shown to increase fiber 
digestion lag time (Mertens and Loften, 1980).
The third component of fiber digestion, extent of digestibility, 
has been shown to have a great impact on digestibility of forage. In 
a simulation (Mertens and Ely, 1979), an increase in the indigestible 
fraction resulted in a decrease in digestibility and an increase in
13
neutral detergent fiber content of the rumen and intestines. The dry- 
matter intake was also affected and showed a decrease. Values for 
coastal bermuda grass indicated that a 1.0% decrease in 
indigestibility resulted in a 1.0% increase in maximum digestible dry 
matter intake.
Lignin is generally accepted as an anti-quality factor which 
inhibits utilization of fibrous carbohydrates in forage. Smith et al. 
(1972) noted correlations of .78 or higher between lignin and the 72 
hour cell wall indigestibility used to predict potential 
digestibility. This relates to work reported by Akin and Amos (1975) 
in which the indigestible portion of plant material contains high 
levels of lignin. Waldo and Smith (1979) suggest cellulose is 
composed of two fractions; potentially digestible and indigestible. 
The potentially digestible fraction disappears from the rumen by both 
passage and digestion whereas the indigestible disappears only with 
passage. Lignin concentration influences extent of digestibility but 
increasing lignin does not slow rate of digestion.
Passage rate is the fourth component of fiber digestion. When 
long forage is consumed by an animal, particle size must be reduced 
before the forage can escape the rumen (Pearce, 1967): Particle
reduction in the rumen may be considered as a rate limiting step in 
the process of forage escape from the rumen (Smith et al., 1983). 
Prehension, rumination (Welch, 1982), and microbial weakening of 
fiber (Akin and Amos, 1975) are factors involved in particle 
reduction. Contribution of microbial action to particle reduction has 
been questioned. Murphy and Nicoletti (1984) evaluated the particle
14
size of coarse ground hay after incubation in vitro for 48 hours and 
indicated minor contribution of microbial action to particle 
reduction. Conflicting results were reported by Pearce and Moir 
(1964) in which sheep fitted with devices to inhibit rumination were 
able to consume a diet of known high rumination potential, and it was 
concluded that microbial breakdown was sufficient in the rumen for 
passage of digesta. In a model presented by Mertens (1977) ruminal 
digestion of grass decreased exponentially as passage rate increased. 
With a passage rate of 1.0% per hour grass dry matter digestibility 
was 67.2%, and when rate was increased to 5.0% disappearance per hour 
the dry matter digestibility value dropped to 58.4%. Studies have 
shown that grinding a diet to decrease particle size decreases 
retention time in the rumen (Moore, I 964, Alwash and Thomas, 1971). 
Retention time has also been'effected by feed intake (Crovum and 
Williams, I 979). As intake increases retention time in the rumen 
decreases. It is evident that passage rate can be influenced by 
physical form of the diet, rumination, and to a lesser degree, 
microbial action, and passage of feed through the rumen can have an 
effect on digestion.
The relationship of liquid passage and fermentation is receiving 
more attention. Fermentation responses to change in fluid dilution 
rate are complex. Bull et al. (1979) suggest that increased liquid 
turnover may benefit the animal in terms of microbial growth 
efficiency, improvement in utilization of nonprotein nitrogen and 
reduction in fermentation losses.
15
Adams and Kartchner (1984) reported that increased intake in 
steers consuming a hay diet increased the fluid dilution rate. Fluid 
dilution rate was 4.3 %/h for steers consuming 1.40% body weight, and 
7.2 %/h for those consuming 2.4% body weight. Estell and Galyean 
( 1985) indicate similar results with compiled data collected from 
seven beef steer trials.
Diet composition is another factor that may influence liquid 
dilution rate. Owens' and Isaacson (1977) related elevation of percent 
roughage in the diet to increased intake resulting in higher rate of 
liquid passage. Other studies revealed a decrease in liquid dilution 
rate when the level of concentrates were elevated in the diet (Cole et 
al., 1976; Goetsch and Galyean, 1982).
Liquid dilution rate has also been related to ammonia levels and 
osmolarity in the rumen (Prigge et. al., 1984). Estell and Galyean 
(1985) showed a positive relationship (P<.01) existed between ammonia 
concentration and fluid dilution rate in a trial where beef steers 
were grazing blue grama rangeland. Effecting fluid dilution rate with 
hypertonic solutions infused in the rumen was studied by Rogers et al. 
(1979). Holstein steers fed a high roughage diet were infused with 
sodium chloride. Fluid dilution rates increased (P<.05) from 5.85 %/h 
with no infusion to 7.11 %/h with infusion of sodium chloride. The 
increased dilution rate was not accompanied by changes in rumen 
volume. Increased transruminal water flux accounted for the higher 
level of liquid required for increased flow rate.
16
EXPERIMENTAL PROCEDURE
In Vitro Trials
Nine amino acids, arginine, cysteine, histidine, leucine, lysine, 
methionine, tryptophan, phenylalanine, and isoleucine, and three 
branched chain organic acids, isovaleric, isobutyric, and 2- 
methy!butyric, were evaluated to determine their influence on in vitro 
fermentation of winter range forage esophageal extrusa. Fermentation 
rate, digestibility and lag times were calculated for both dry matter 
(DM) and neutral detergent fiber (NDF) content of fermented samples. 
Two replications of triplicate samples incubated with either single 
amino acid or organic acid additions were conducted.
The winter range forage was a composite of esophageal collections 
from cows grazing winter pastures on the Red Bluff Range Research 
Ranch located 56 km west of Bozeman, Montana on the northwest slope of 
the Madison Range. Grasses composed 65 to 75% of the vegetation, with 
forbs and woody species making up the remaining 25 to 35%. The forage 
samples were freeze dried, ground through a I mm screen and weighed 
(.25 gram) into 50 ml in vitro fermentation tubes. McDougall’s buffer 
solution ('McDougall, 1948) was modified to contain 50 mg nitrogen per 
100 ml buffer. Amino acid was added to the buffer to provide 25% of 
buffer nitrogen with 75% supplied by urea. All amino acids were L- 
isomers. Branch chain organic acids were added to buffer at 0.1%, and 
100% of buffer nitrogen was contributed by urea. Urea supplied all the
Chapter 3
17
buffer nitrogen in the buffer solution for the control. Following pH 
adjustment to 6.9» 20 ml of buffer were added to the forage sample. 
Rumen ihnoculum was obtained from two fistulated crossbred beef cows 
fed mature grass hay and barley straw ad libitum. Fluid from donor 
cows was composited and strained through sixteen layers of cheesecloth 
to remove large digesta particles. The fluid was continuously 
agitated and maintained at 39° C while 5 ml were innoculated into each 
in vitro tube. Fermentation tubes were flushed with COg» capped, and 
incubated at 39° C. At 2, 4, 6, 8, 10, 12, 18, 24, 36, and 48 hours 
postinoculation, fermentation was ceased with the addition of .5 ml 556 
mercuric chloride in two blank, triplicate treatment and control 
tubes. After incubation, tubes were centrifuged at 2000 rpm for 15 
min, decanted, dried for 48 hours'at 60° C, and weighed to determine 
DM disappearance. The concentration of NDF was determined for each 
residue (Van Soest and Robertson, 1980),
Fermentation rates for DM and NDF were determined by nonlinear 
regression techniques (SAS, 1978). Variable b in the equation, dig. = 
ae*3^ , estimated the rate of change of DM or NDF for the entire 
incubation period. Lag times for DM and NDF were calculated using the 
first order kinetic equation (Mertens, 1977):
log A0 = K(t-tL) , '
A 2.303
where A0 = concentration at zero hour, A = concentration at time t, K= 
rate of disappearance, and tL = lag time.
Data were subjected to analysis of variance for randomized block 
design, whereas the replicated in vitro runs were designated as
18
blocks. When a significant f-value was indicated, means were tested 
by Least Significant Difference multiple comparison (Snededcor and 
Cochran, 1980).
In Situ Trial
Methionine additions to in vitro buffers increased (P<.05) DM and 
NDF fermentation rate in comparison to the control and exhibited 
higher values for DM and NDF digestibility although not significant. 
Based on the in vitro results methionine was selected for further 
evaluation. Feed grade methionine is an amino acid commercially 
available, which could be incorporated into supplements at competitive 
costs.
Three supplements designated as, DL-methionine-urea, SBM and 
urea, (table I) were formulated to provide 1.23 kg total digestible 
nutrients and .32 kg crude protein equivalence daily, and were 
evaluated for their influence on forage DM and NDF rumen fermentation. 
The nitrogen sources for the supplements were DL-methionine, SBM, 
urea, and beet pulp. DL-methionine provided 4%, urea 63%, and beet 
pulp 33% of the crude protein in the DL-methionine-urea supplement. 
Soybean meal contributed 81% of the protein content in the soybean 
meal supplement and beet pulp supplied 19%. Fifty-three percent of 
the protein in the urea supplement was derived from urea, 12% from 
ammonium sulfate, and 31% from beet pulp. Ammonium, sulfate was added 
to the urea supplement to provide 7.9 grams sulfur, an equivalent 
amount of sulfur as, contributed by methionine in the DL-methionine-
urea supplement
19
Six mature crossbred beef cows fitted with rumen cannulas were 
utilized in this trial. Each treatment replication (two cows) were 
confined in one pen and maintained on a forage mix containing. 75% 
mature grass hay and 25% barley straw, chopped by a tub grinder thru a 
5 cm screen, and fed ad libitum. The forage was 7.0% crude protein 
and 62.96% NDF on an as fed basis. Cows consumed approximately 13.33 
kg DM of the chopped forage mix daily. The supplements were fed daily 
at 0800 hour when cows were haltered and tethered to assure each cow 
consumed all of the daily supplement treatment. The amount of . 
supplement fed per head per day was as follows: 1.95 kg DL-
methionine-urea supplements, 1.97 kg urea supplement, and 1.85 kg SBM 
supplement. Each supplement supplied .32 kg protein and 1.23 kg total 
digestible nutrients. The experimental periods allowed for 10 days of 
adaptation to supplements followed by a five day sample collection 
period.
Bags were made of nylon monofilament material with a pore size of 
44 microns. Each nylon bag was double stitched around the bag 
perimeter and the outer dimensions were 15.2x17.8 cm. The sample 
fermented in the nylon bags was identical to the chopped forage fed to 
the cows. The forage was ground by a Wiley mill through a 2 mm screen 
and three gram samples were placed in each bag. This allowed for a 
ratio of 7 mg of sample per cm^ of nylon material (Uden and Van
i
Soest, 1984). Bags were attached to snap swivels on a hoop of tygon 
tubing filled with a chrome ball chain for suspension Rumen digesta 
collected from cannulated cows fed the diet previously described was 
prepared as a mordant according to Uden et al. (1980) and contained
20
3.2756 chromium on the fiber. The mordanted fiber served as a 
particulate passage marker in the rumen. Cobalt EDTA was prepared as 
a liquid passage marker in the rumen (Uden et al., 1980).
At zero hour on day one of the collection periods, each 
cannulated cow was dosed with 100 g of chromium mordanted fiber and 15 
g cobalt EDTA. The bags were suspended in the rumen and 15 minutes 
later rumen contents were sampled. All rumen samples were grab samples 
(500 ml) from approximately the same location in the cranial sac of 
the rumen. Collection times were 4, 8, 12, 18, 24, 36, 72, and 96 
hours. For each time period, two sample filled bags and rumen extrusa 
were collected from every cow, and one blank bag was collected from 
one cow on each treatment.
Rumen samples were immediately treated with I ml 556 mercuric 
chloride to cease any further microbial activity, centrifuged at 2000 
rpm for 15 minutes, the fluid was decanted, and both fluid and fiber 
samples were frozen at -4° C for later analysis. The nylon bags were 
washed with cold water until the rinse water was clear when squeezed 
out of the bag. The bags were dried in a forced air oven at 60° C for 
48 hours.
Dry matter and NDF (Van Soest and Robertson, 1980) content was 
determined for each bag. Rumen digesta samples were prepared 
(Williams, David and Iismaa, 1962) and the chromium concentration was 
determined with atomic absorption spectrophotometry. Rumen fluid 
samples were analyzed for cobalt and rumen ammonia concentration. A 
portion of each fluid sample was centrifuged at 10,000 rpm for 10 
minutes and the supernatant was evaluated for cobalt concentration
21
with an atomic absorption spectrophotometer (Gaylean and McCollum, 
1985). Rumen ammonia levels (mg/100 ml rumen fluid) were determined 
by a macro Kjeldahl procedure (AOAC, I960).
Dry matter and NDF disappearance values were subjected to 
nonlinear regression (SAS, 1985) to estimate rate of fermentation for 
each parameter. The equation used for the nonlinear regression 
analysis was; Y = aeE-k (t-tlag)] + where as Y = digestibility, a = 
potential digestibility, -k = rate of fermentation, t = time, tlag = 
lag period, and u = undigestible fraction. Dry matter data did not 
indicate a period of fermentation lag, therefore the tlag variable was 
not included in the DM equation for fermentation parameters estimates. 
Cobalt and chromium concentrations were transformed into natural 
logarithms and linear regression techniques (Snedecor and Cochran, 
1982) were applied to determine the rate of passage from the rumen of 
the liquid and particulate fractions.
Dry matter and NDF rumen digestibilities of the forage sample 
were calculated with the following equation:
Rumen Digestibility = 100 - [a (k1/(k1+k2)) + b ], 
a = potential digestibility, k1 = particulate rate of escape from the 
rumen, kg = fermentation rate, and b = undegradable portion. The 
potential digestibility value used for calculation was the percent 
digestible at 72 hours.
\
The liquid dilution rate constant was utilized to predict three 
liquid parameters of the rumen, fluid volume, fluid outflow, and fluid 
turnover time as influenced by the supplements. Fluid volume was 
estimated using the equation, volume = (ml/a)(b), whereas a =
22
concentration of cobalt at zero hour (intercept of the linear 
regression line) and b = amount of cobalt EDTA given at time zero 
hour. The equation, outflow = liquid volume/ escape rate, provided a 
measure of fluid outflow from the rumen. The third parameter, fluid 
turnover time, equals the inverse of the rate constant.
The experimental design of this trial was a 3x3 replicated La tin- 
square; whereas, over the course of the three periods all cows 
received each treatment. All estimates were subjected to analysis of 
variance for replicated Latin-squares (Snedecor and Cochran, I 980). 
When a significant f-value was indicated then mean differences were 
determined by the Newman-Keuls means test.
Heifer Growth Trial
Four isonitrogenous isocaloric supplements (table 1) incorporated 
into roughage diets fed to weanling heifer calves were evaluated by 
performance measurements. Three supplements, DL-methionine-urea, SBM 
and urea, were formulated similar to those described in the in situ 
trial. In the fourth supplement, soybean meal-urea, urea contributed 
47%, soybean meal 28%, and beet pulp 25% to the protein content. The 
supplements and a chopped roughage mixture consisting of 75% mature 
grass hay and 25% barley straw, were used to formulate rations to meet 
Nutritional Research Council requirements (NRC, 1976) for heifers 
gaining .3 kg per day.
The feeding trial was conducted at the Montana Agriculture 
Experiment Station Feedlot, Bozeman. Fifty-six weanling heifer calves 
with an average initial weight of 231.5 kg were used in the feeding
I
I
23
trial. Breeds of the heifers included Angus, Hereford, Angus-Hereford 
crossbred, and Tarantaise crossbred. Initial weights were an average 
of two consecutive day weights. The heifers were randomly assigned to 
groups within breed and body weight stratification. Four pens were
I
allotted to each treatment group; two of the pens held four heifers 
while the other two pens contained three. The pens had fence line 
bunks protected by partial roof cover, and were bedded with straw. 
Automatic waterers provided water and trace mineral salt blocks were 
available in the bunks for each pen.
The heifers were fed once daily in the morning., Diets were mixed 
individually by treatment in a feed wagon equipped with a scale to 
weigh the forage. For those diets containing urea, the urea was 
dissolved in 11.748 liters of water and sprayed over the ration as it 
was mixing in the feed wagon. This procedure allowed for a more even 
distribution of the urea and reduction in palatability problems. 
Water without urea was sprayed on the SBM diet in the same fashion. 
Feed remaining in the bunks was removed and weighed every other day to 
estimate DM intake. When heifers were consuming all of their diets 
the forage level in the ration was increased, however the amount of 
supplement remained constant throughout the entire feeding period. 
The supplements were fed to provide the following levels per head per 
day: 1.95 kg for the DL-methionine-urea treatment, 1.97 kg for the 
urea treatment, 1.68 kg for the SBM treatment, and 1.85 kg for the 
SBM-urea treatment.
The duration of the feeding trial was ninety days. During the 
trial the heifers were weighed every fourteen days before the morning
24
feeding to monitor animal performance. Water was withheld for twelve 
hours prior to weighing time. A final weight was an average of 
weights collected on the last two days of the trial. Intake, daily 
gain, and feed efficiency, were calculated as indications of 
performance. Individual animals were used as the experimental unit for 
average daily gain while pen was the experimental unit for intake and 
feed efficiency. Data was analyzed by analysis of variance for 
complex randomized block designs utilizing y = treatment, 
pen(treatment) as the model (SAS, 1985).
25
Chapter 4 
RESULTS
In Vitro Trial
Extent of in vitro fermentation at 48 hours (table 2) for all 
amino acid, arginine, cysteine, histidine, leucine, lysine, 
methionine, tryptophan, phenylalanine, and isoleucine, and branch 
chain organic acid, isobutyric, isovaleric, and 2-methyl butyric, 
treatments in comparison to forage alone was similar (P>.05). The 
control, containing no amendments, had 41.2% disappearance of DM at 48 
hours. Other dry matter disappearance values ranged 36.9% (isobutyric 
acid) to 47.4% (methionine). The addition of arginine (47.1%) or 
methionine (47.4%) improved (P<.05) disappearance when compared to 
isobutyric acid, lysine or phenylalanine (36.9» 37.1, and 39.9%, 
respectively). It is interesting to note that the control had the 
third lowest disappearance extent (41.5%) which was nearly six 
percentage units lower than either arginine or methionine addition.
Dry matter fermentation rate (table 2) was increased (P<.05) by 
over 30% with addition of arginine, methionine, histidine, leucine, or 
cysteine (3.6, 3.6, 3.5, 3.5, and 3.4 %/h, respectively) in comparison 
to forage alone (2.7 %/h) or lysine (2.6 %/h) addition. Tryptophan, 
isoleucine, phenylalanine, isobutyric, leucine and 2-methyl butyric 
(3.1, 3.3, 2.8, 2.9, 3.0, and 3.1 %/h, respectively) were similar 
(PX05) to forage alone. Linear regression analysis indicated a
26
positive (P<.01) correlation (R^ _ .73) between extent and rate of dry 
matter forage.
Neutral detergent fiber 48 hours disappearance (table 3) was 
similar (P>.05) for all amendments in comparison to the control with 
no amendments. The addition of histidine, arginine, leucine, 
methionine, isoleucine, isovaleric and 2-methy!butyric acid (57.8, 
56.9, 54.5, 56.2, and 56.6%, respectively) improved NDF disappearance
in comparison to phenylalanine (47.9%). Tryptophan, phenylalanine, 
lysine, isobutyric acid, and forage alone consistently ranked 
numerically among those treatments with the least DM and NDF 
fermentation extent. Arginine, histidine, methionine, and isoleucine 
appeared in the four treatments which ranked highest for both DM and 
NDF digestion extent. NDF analysis of the winter range forage 
indicated the sample contained 67.9% NDF which may account for similar 
trends noted between DM and NDF data.
The rate of NDF fermentation (table 3) for the control was 
numerically the slowest (2.5 %/h) of all treatments tested, which was 
slower (P<.05) than cysteine, methionine, and leucine (3.8, 3.8, and 
3.4 %/h). These amendments improved NDF fermentation rate by 52, 52, 
and 36%. Linear regression analysis showed no significant correlation 
between NDF fermentation extent and rate.
Lag times calculated for DM and NDF fermentation appear in table 
4. Statistical analysis revealed no significant differences between 
treatments for DM or NDF lag times. The estimated lag time for in 
vitro digestion of the DM ranged from 28.0 hours (control) to 32.1
27
hours (isobutyric acid). Neutral detergent fiber lag time values were 
between 20.2 hours (lysine) and 25.2 hours (methionine).
In Situ Trial
Dry matter fermentation rates (table 5) were affected in a 
similar fashion. Fermentation rate was 9«54 %/h when cows received 
DL-methionine-urea supplement which is an improvement (P<.05) compared 
to 7.28 %/h for SBM and 7«74 %/h for urea supplementation. The 
values indicate that the DL-methionine-urea supplement increased 
fermentation rate 29 and 31% in comparison to rates determined when 
cows received the SBM or urea supplements.
Supplements also had an influence (P<.05) on particulate passage 
rate from the rumen (table 5). Digesta particulate passage rate when 
animals received the SBM supplement (4.92 %/h) was approximately 
sixteen percent more rapid than passage rate for urea (4.24%) and DL- 
methionine-urea (4.20%) supplementation.
The estimated DM rumen digestibility (table 5) of the forage was 
influenced by the supplements. The calculated rumen digestibility was 
48.49% for the DL-methionine-urea treatment and 47.55% for the urea 
treatment which was higher! (PC.10) than than the digestibility value,
42.40%, for the SBM treatment. In comparison to SBM, DL-methionine-
i
urea and urea supplements increased rumen digestibility of DM 14.4%I'
and 12.1% respectively. ?
The results for NDF fermentation rate and rumen digestibility 
(table 6) followed similar trends as the DM fermentation measurements 
however no differences (PC.05) could be detected. Rumen NDF
28
digestibility was increased by DL-methionine-urea treatment (39.21/6) 
in comparison to urea (35.28%) and SBM (36.53%) treatments. Neutral 
detergent fiber fermentation rate was improved 21 to 28% when cows 
were supplemented with DL-methionine-urea (5.13 %/h). Fermentation 
rate was 4.01 %/h when cows received SBM supplement, and 4.24 %/h when 
urea was supplemented.
Two of four rumen liquid parameters were influenced by 
supplements (table 7). Rumen fluid dilution rate was increased 
(PC.OI) with DL-methionine-urea (10.62 %/h) and urea (10.53 %/h) 
supplements when compared to SBM supplement (9.77 %/h). Supplements 
had a similar effect on fluid turnover time. During the trial when 
cows were supplemented with SBM the fluid turnover time, 10.25 h, 
increased (P<.01) in comparison to DL-methionine-urea (9.50 h) and 
urea (9.56 h) supplementation.
Fluid volume was similar between all treatments; 40.91 liters for 
DL-methionine-urea, 41.75 liters for SBM and 41.86 liters for urea. 
Outflow of fluid from the rumen was also not affected by the different 
supplements. Fluid outflow values reported are 3.89, 4.28, and 4.0 
liters per hour for Dl-methionine-urea, SBM and urea supplements, 
respectively.
Rumen ammonia levels (table 8) are reported for each, collection 
hour and an average for the entire trial. Ammonia levels are not 
significantly different for treatments. Over the entire trial, the 
rumen ammonia levels were 9.53, 8.26 and 9.22 mg/100 ml rumen fluid 
respectively for the following treatments, DL-methionine-urea, SBM 
and urea. Ammonia levels at the 4 hour collection was higher (P<.05)
29
with DL-methionine-urea and urea supplements (26.38 and 25.72 mg/100 
ml) compared to SBM (11.01 mg/100 ml).
Heifer Growth Trial
Average daily gain (table 9) for heifer calves consuming a low 
quality forage was similar for all supplements, DL-methionine-urea, 
SBM, urea, and SBM-urea. The average daily gains for the supplemental 
regimes were .60 kg (DL-methionine urea), .64 kg (SBM) .62 kg (urea) 
and .60 kg (SBM-urea). The feed intake (table 9) is reported as an 
average intake per head per day and indicates no difference between 
treatments. Feed intake over the entire feeding trial was 6.83, 
6.74, 6.74, and 7.29 kg/(h d” )^ for DL-methionine urea, SBM, urea, and 
SBM-urea supplementation. The third measurement calculated, feed 
efficiency (table 9), was also not influenced by the treatments. Feed 
efficiency (kg DM ingested /kg gain) was 11.44, 10.71» 11.75, and 
11.26 for DL-methionine , SBM, urea, and SBM-urea supplemented 
heifers, respectively.
30
Chapter 5
DISCUSSION
In Vitro Trial
The association of various fermentation factors such as rate, 
digestibility extent of potentially digestible fraction, lag time and 
rumen passage rate all effect digestibility in the animal. An 
understanding of the dynamics of these factors in relation to winter 
range forage utilization may prove beneficial. The in vitro technique 
provided results for use as a screening technique; however, there are 
limitations in evaluating digestion extent and the impact of digesta 
passage rate. In situ methods are more adapted for evaluating 
digestion extent and rumen passage rate simultaneously.
Results from the in vitro trial revealed trends that provide a 
strong basis for future research. Mertens (1977) indicated rate can 
have significant impact on DM digestibility especially in grasses with 
high cell wall contents. This study presented significant DM rate 
differences and a positive correlation between rate and extent of 
digestion.
Lag times determined in this study were longer than 20 hours. 
The duration of lag time was much longer than anticipated when 
compared to previous work with other forages (Mertens and Loften,
. i
1980; Simpson, 1984) where lag time was reported to be four to five 
hours. Graphs (figures 1-3) of the Indigestible DM portion plotted 
against time reveal three phases of fermentation: a rapidly
%
 D
M
 R
em
ai
ni
ng
100
BOn
60 -
CONTROL
AR GINJ N E
CYSTEINE
HISTIDINE
LEUCINE
LYSINE
I I i I r i i i i i I I r~
2 4 6 8 10 12 18 24
• I i i I r- - -
36 4
Hour
Figure I. In vitro dry matter digestibility of native winter range forage
CO
%
 D
M
 R
em
ai
ni
ng
100
60-
60-
CONTROL
METHIONINE
TRYPTOPHAN
PHENYLALANINE
ISOLEUCINE
LUrx>
X--.
40 L
2 4 6 8 10 12
• I I I I I I I I I I i' I 1
18 24 36
Hour
Figure 2. In vitro dry matter digestibility of native winter range forage
%
 D
M
 R
em
ai
ni
ng
100
40
U)
LU
2 4 6 8 10 12 "24 ' ' ' ' 36
I 1 1
48
Hour
Figure 3. In vitro dry matter digestibility of native winter range forage
34
degradable phase in which the cell. solubles are released, a slow phase 
indicating lag time for the non-soluble fraction, and a fermentation 
phase in which the rate increases with breakdown of the less 
degradable fraction. Graphs (figures 4-6) of NDF fermentation reveal 
similar phases; however, the second phase is more pronounced 
indicating a more definite lag period for NDF in comparison to DM. 
Mertens (1977) reported cellulose fermentation rates of 8-16 %/h for 
readily digested cell wall parts and 2-3 %/h for the slowly digested 
fractions indicating rate is not constant during the fermentation 
process. The value of 2-4 %/h for slowly digested fractions is in 
agreement with the in vitro NDF rates which had a 2.5-3.4 %/h range.
The lag times reported where calculated assuming 100% potential 
digestibility of the forage, which would increase the lag time. A 
more realistic lag time could be obtained if potential digestibility 
of the forage was known. Thus amount digested for a given incubation 
period could be compared directly to the potentially digestible 
fraction. This supports the need for determining potential DM and 
NDF digestibility for the winter range forage esophageal extrusa 
samples in order to improve lag time estimates.
The effect of lag time on fermentation increases at an 
exponential rate according to the model outlined by Mertens (1977). 
This model indicates that lag time may be an extremely important 
variable in fermentation. The elements responsible for lag time are 
not well documented, ■ There are several possible causes for lag time: 
physical characteristics of the forage, bacterial characteristics,
%
 N
D
F 
R
em
ai
ni
ng
100
60-|
CONTROL
ARGmjNE
CYSTEINE
HISTIDINE
LEUCINE
LYSINE
40 - * I I I I I I I I i I I I I I I i I i----1---1---1---r
2 4 6 8 10 12 18 24 36
UU
V l
Hour
Figure 4. In vitro neutral detergent fiber digestibility of native winter range forage
%
 N
D
F 
R
em
ai
ni
ng
100
CONTROL
METHIONINE
TRYPTOPHAN
PHENYLALANINE
ISOLEUCINE
UU
n I I I I I i i i i i r i —
362 4 6 8 10 12
Hour
Figure 5. In vitro neutral detergent fiber digestibility of native winter range forage
%
 N
D
F 
R
em
ai
ni
ng
100
LU
-J
6 8 10 12
Hour
Figure 6. In vitro neutral detergent fiber digestibility of native winter range forage
38
polarity of bacteria and forage particle or chemical reactions that 
may be involved.
In Situ Trial
Results of the in situ trial agree with those of the in vitro 
trial. Methionine had a positive influence on fermentation rate both 
in vitro and in situ. Salter et al. (1979) determined methionine to 
be a limiting amino acid for bacteria on diets low in protein. 
Providing methionine to rumen bacteria may remove this limitation, 
allowing for better bacterial growth and rate of fermentation. When 
comparing results from supplementation, DL-methionine-urea increased 
DM fermentation, particulate passage rate increased with SBM 
supplementation, and both DL-methionine-urea and urea improved rumen 
digestibility and had similar particulate passage rate. This implies 
that passage rate has a more influential effect on rumen 
digestibility than fermentation rate and .may help to explain why no 
digestibility differences were found in the in vitro trial when 
fermentation rate was significantly influenced. Hoover et al. (1982) 
reported DM and acid detergent fiber digestibilities increased with 
increasing retention of solids in a continuous culture device 
fermenting a semipurified diet with zein as the major protein source.
The in situ NDF results followed similar trends as DM but no 
significant differences were detected as in the in vitro trial where 
methionine significantly increased NDF fermentation rate. This may be 
due to laboratory technique involved in determining the NDF content of 
the sample in the nylon bags. Neutral detergent fiber was determined
39
by refluxing the bag and sample in NDF solution. Individual bags were 
rinsed manually with hot water. A more uniform method for rinsing 
the soap out of the sample and nylon material could reduce 
experimental error and provide more accurate NDF values.
Dry matter fermentation in situ exhibited no lag period and 
lag times were not included in the rate equation. The lag period for 
NDF was approximately one hour for each of the treatments. A lag 
period is not evident in DM fermentation because while lag is 
occurring in the NDF fraction, .the readily soluble portion is being 
released. These results are quite different from lag times 
calculated for in vitro fermentation, and as mentioned previously the 
in vitro lag times were based on 100% potential digestibility of the 
forage sample. The in situ results are more in agreement with other 
reported lag times and strongly supports the need for determining the 
potential digestibility of forage samples in order to estimate 
realistic lag times. Varga and Hoover (1983) calculated a .90 hour 
lag period for orchard grass in situ.
From the results it appears that particulate and liquid passage 
rates were influenced independmntly. The passage of the particulate 
fraction was 17% slower (P<.05) for both DL-methionine-urea and urea 
supplementatin compared to SBM. Particulate passage rate has been 
shown to be influmnced by intake, as intake increases so does the 
pavticulate passage rate (Owens arid Isaacwon, 1977). It is feasible 
that intake may be driven by metabolic eneroy needs of an animal.
Maintenance energy expenditures were found to be highly 
correlated to body protein mass maintenance (Ferrel et al., 1979).
40
Energy expenditure for daily protein comprised 89% of the basal energy 
expenditure. In a later study, Ferrel and Jenkins (1985) concluded 
after several different approaches that metabolism of visceral organs 
constitute a major proportion of total animal energy, expenditures. 
Results observed by the same scientists indicated weight of the organs 
can vary in response to nutritional treatments. Animals on a higher 
plane of nutrition had heavier weights for the stomachs, small 
intestines, large intestines, livers and kidneys. Animals in a 
positive nitrogen balance may require more energy to maintain current 
protein mass compared to an animal in a negative nitrogen balance. 
The higher maintenance energy requirement possibly could influence 
intake in that the animal consumes more to meet its needs.
Bypass protein can place an animal in a state of positive 
nitrogen balance. Cows on winter range receiving a blood meal protein 
supplement had higher (P<.05) intake, 1.46 % of body weight, than cows 
not receiving the supplement, 1.17 % of body weight, (unpublished 
data, Dunn, 1984; Miner, 1984). Cows on the blood meal supplement 
could be in a higher positive nitrogen balance than those receiving no 
supplement, and this may support the ideathat maintenance energy 
requirement to meet protein body mass maintenance may influence 
intake.
When cows on the in situ trial consumed the urea containing 
supplements, they could be under, nitrogen balance stress compared to 
the period when cows consumed the SBM. Supplementation with SBM did 
increase passage rate compared to supplementation with DL-methionine- 
urea and urea. In consideration of the previous discussion, the
41
influence of supplement treatments on increased passage rate could be 
a result, of intake driven by energy maintenance requirement.
ONR methionine-urea and urea supplementation increased fluid 
dilution rate 8.7% (P<.01) when compared to SBM. The increase in rate 
may be explained by the concentration of ammonia in the rumen 
contributed by urea in the DL-methionine-urea and urea supplements. 
Ammonia levels in the rumen have been noted as an influential factor 
for fluid dilution rate (Prigge et al., 1984; Estell and Galyean, 
1985). Rogers et al. (1979) was able to affect fluid dilution rate by 
infusing a hypertonic solution into the rumen.
It may be possible that the ammonium ions affect the osmolarity 
in the rumen which changes the transruminal water flux' providing more 
metabolic water. The liquid passage increases to dilute ammonia 
levels so osmolarity is returned to normal. Faster passage rates 
noted for the urea containing diets could also explain why ammonia 
levels were the same for all three supplements.
Rumen fluid volume was approximately 41.0 liters and was not 
influenced by treatments. These values are similar to rumen volume 
(55 I) reported by Rogers et al. (1979), but lower than volume values 
(130 I) reported by Adams and Kartchner (1984). All compared values 
are for animals consuming a high roughage diet. It is not uncommon to 
observe similar rumen volume when turnover is altered (Harrrison et 
al., 1975) The rumen turnover time differences correspond to liquid 
dilution rate, as dilution rate increases the turnover time decreases.
The rumen ammonia levels were different (P<.05) only at the four 
hour collection period. Supplementation with DL-methionine-urea and
42
urea significantly !increased ammonia level in comparison to soybean 
meal. The four hour collection was the only collection closely 
following supplementation as all other morning collections occured 
prior to supplementation. The significant difference found indicates 
the DL-methibnine-urea and urea supplements influenced ammonia levels 
in the rumen at four hours post-supplementation. This increase would 
reflect a more rapid ammonia release from urea contained in the 
supplements in comparison SBM. Eight hours after supplementation no 
differences were observed.
Rumen ammonia levels for optimum microbial growth have been 
reported to be between 5 and 9.0 mg / 100 ml rumen fluid (Hume et al., 
1970; Satter and Slyter, 1974; Kang-Meznarich and Broderick, 1981; 
Pritchard and Males, 1982). Mehrez et al. (1977) indicated a much 
higher level of 23.2 mg / 100 ml rumen fluid necessary to maximize 
microbial growth. The ammonia levels for the entire in situ trial 
were adequate for microbial yield in comparison to the first values 
listed above.
Ammonia levels do vary among collection periods and no consistant 
pattern is evident. The cows were fed the forage ad libitum and 
ingestion of the feed could occur at any time. Wohlt et al. (1976) 
demonstrated time after feeding influenced ammonia levels. If cows 
were consuming the forage at various uncontrolled times in relation to 
collection periods, time after feeding could account for variation in
ammonia levels for the trial.
43
Growth Trial
Average daily gain, feed intake and feed efficiency Were not 
influenced by the supplements, DL-methionine-urea, SBM, urea or SBM- 
urea fed to heifer calves consuming a low quality forage. The forage 
intake was not limited for heifers on the trial and they consumed 
approximately 7.0 kg / (h d ” )^, which was 2.6 kg / (h d“ )^ more than 
had been the anticipated. The high intake values could possibly be 
attributed to the forage being chopped. Alwash and Thomas (1971) 
reported that grinding a diet decreases retention time in the rumen 
and an increase in intake occurs..
Even though the crude protein value was only 7.0%, the heifers 
consumed enough roughage to almost meet their protein requirements. To 
gain .3 k/day, the NRC (1976) requirement for protein is .42 kg /day 
and the heifers were receiving approximately .67 kg/day. The 
excessive protein provided to the heifers made it difficult to detect 
any differences in performance measurements due to supplemental 
protein source. Feeding the supplements while limiting intake may 
have produced a response due to supplement treatment.
In summary, methionine had an influential effect on fermentation 
rate of fiber in vitro and in situ. The in situ data indicated the 
importance of passage rate for determining digestibility of forage. 
Passage rate had a larger impact on digestibility than fermentation 
rate. The particulate passage rate and liquid dilution rate appeared 
to be independently affected. Providing amino acids to rumen bacteria 
have shown to affect fermentation rate of DM and NDF; however, passage 
rate appeared to have a greater impact on digestibility.
44
Chapter 6
CONCLUSIONS AND RECOMMENDATIONS
In vitro and in situ results of this study indicate that 
methionine influenced fermentation rate of low quality roughage. In 
both systems, the addition of methionine enhanced fermentation rate by 
at least 30%. Methionine has been identified as a limiting amino acid 
for rumen bacteria fermenting low protein diets. Providing readily 
available methionine to rumen bacteria may satisfy the methionine 
requirement; therefore, increasing bacterial growth rate. The 
fermentation rate response to methionine may be a result of increased 
microbial growth rate.
Particulate and liquid passage rates measured during the in situ 
trial were inversely affected by treatments. DL-methionine-urea and 
urea supplementation increased fluid passage rate and decreased 
particulate passage rate compared to SBM. The increase in fluid 
passage rate for urea containing supplements may be attributed to 
a change in rumen osmolarity due to higher ammonia concentration 
following morning supplementation. The decrease in particulate 
passage rate is not easily explained. Protein supplementation may 
have changed body protein mass. As protein mass increased, 
maintenance energy requirements also increased. This difference may 
have caused a metabolic stimulation of intake which in turn increased 
particle passage rate; however, further research is required to 
determine if this is occurring.
45
Calculated rumen digestibility was lower for the DL-methionine- 
urea and urea supplementation in comparison to SBM. Even though DL- 
methionine-urea supplementation increased fermentation rate, the 
slower particulate passage rate appeared to have a greater impact on 
calculated digestibility. Supplements containing urea affected 
passage rates and digestibility in a similar manner indicating that 
urea may have an independent impact on rumen activity. Comparing 
supplemental methionine to a natural protein source such as soybean 
meal may be difficult in the presence of urea.
The heifer growth trial revealed no differences in daily gain, 
intake or feed efficiency due to supplements. The method of feeding 
chopped roughage ad libitum with a constant level of supplement 
allowed heifers to consume 112# of the protein necessary for a .3 kg 
daily gain. Differences between supplements may have been detected 
had the heifers been limited in protein intake.
The following is a list of recommendations that may be considered 
if research is continued in this area.
1) Bacterial growth rate should be measured to determine if 
the increase in fermentation rate with the addition of 
methionine is due to an increase in bacterial growth rate.
2) The in vitro technique could be used to determine an 
optimum level of methionine for enhancing fermentation of 
low quality roughages.
3) The method of rinsing nylon bags especially during the 
determination of neutral detergent fiber could be improved 
to help decrease experimental error and to obtain more
accurate values. Rinsing the nylon bags needs to be more 
consistent.
4) Intake values for animals used during the in situ trial 
would have been useful to determine if intake was 
influencing the particulate passage rate.
5) Ammonia concentration in the rumen should be measured 
more regularly rather than at bag collection time. This 
would allow a better understanding of differences in ammonia 
concentration due to supplemental treatments.
6) Methionine supplementation without urea may have 
different responses that would enhance the utilization of 
low quality roughages.
7) Feeding a long roughage rather than one which has been 
chopped would possibly show more of a benefit from 
increased fermentation rate in the rumen.
8) If a second heifer growth trial were to be conducted the 
level of protein should be limited to determine if the 
supplements influences weight gain and feed efficiency.
Results from this study do indicate that the addition of methionine 
can increase fermentation rate. Further evaluation is needed to 
better understand the impact of methionine supplementation on rumen 
dynamics and animal performance on low quality roughages. It may 
become economically feasible to incorporate methionine into protein 
supplements for improved animal production.
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48
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55
APPENDIX
56
Table I. Composition of the supplements3 evaluated in the in 
situ trial and the heifer growth trial
Ingredients
%
IFN*3 DL-methionine- 
urea
SBM0 Urea SBM-urea
Beet Pulp 4-00-669 93.22 66.68 92.72 85.37
DL-Methionine 1.14 — —
Urea 5-05-070 3.68 . 3.40 2.85
Soybean Meal 5-04-612 ——1 31.00 — 9.82
Molasses 4-00-669 1.94 2.30 1.93 1.94
Vitamin A & Dc* .02 .02 .02 .02
Ammonium Sulfate 6-09-339 — • 1.93
Crude Protein, 
as fed basis
10.67 9.93 10.10 9.93
aThe SBM-urea supplement was used in the heifer growth trial only. 
^International Feed Number 
cSoybean Meal
^Vitamin A & D premix contained 44,000,000 ID/kg vitamin A and 
8,800,00 lU/kg vitamin D.
57
Table 2. Influence of in vitro buffer amendments on dry matter
(DM) disappearance and fermentation rate of native
winter range forage esophageal extrusa
Amendment DM disappearance3 Fermentation rate*30
% %/hour
None 41.2def 2.7d®
Arginine 47. Id 3.6d
Cysteine 44.8de 3.4fShl
Histidine 46.3d® 3.5hl.
Leucine 44.6de 3.4Shi
Lysine 37.If 2-6d
Methionine 47.4d 3.6hl
Tryptophan 43.2def 3i1defghi
Phenylalanine 39.9ef 2.8def
Isoleucine 45.8de 3.3®fghl
Isobutyric acid 36.95 2.9defg
Isovaleric acid 43.3def 3.OdeCSh
2-Methylbutyric acid 44.3d® ^o-Jdefghi
aAverage for both runs and standard error ±2.1. 
kAverage for both runs and standard erhor ±0.2.
cDig = ae^x, whereas b = rate of digestion and are the values listed 
above.
defghij4eans within columns with different superscripts are different 
(P<.05).
58
Table 3. Influence of in vitro buffer amendments on neutral detergent
fiber (NDF) disappearance and fermentation rate of native
winter range forage esophageal extrusa
Amendment NDF disappearance3 
%
Fermentation rate*3 
%/hour
Forage only
Arginine
Cysteine
Histidine
Leucine
Lysine
Methionine
Tryptophan
Phenylalanine
Isoleucine
Isobutyric acid 
Isovaleric acid 
2-Methy!butyric acid
53. Icde 2.5°
56.90d 3.Ocde
53.2cde 3.9f .
57.8° 2.9cde
54.5cd 3.4d®
51.5cde 2.8°d
55.8cd 3.8d®
51.Ida 3.Ocde
47.9® 2.7°
56.6cd ' 2.9cde
50.9de 2.9°d°
56.2cd 2.7°
56.6cd 2.9cd
aAverage for both runs and standard error ± 2.2.
^Average for both runs and standard error ±0.2.
c<^ e^Means within columns with different superscripts are different 
(P<.05).
59
Table 4. Influence of in vitro buffer amendments on dry matter (DM)
and neutral detergent fiber (NDF) fermentation lag time of
native winter range forage esophageal extrusa
Amendment DM lag timea NDF lag timeb
hours hours
Forage only 28.Oc 21.9°
Arginine 30.3° 24.6°
Cysteine 29.9° 25.o°
Histidine 30.2C 23.5°
Leucine 29.9° 25.1°
Lysine 30.2° 20.2°
Methionine 29.9C 25.2°
Tryptophan 29.8° 25.Ic
Phenylalanine 29.5° 24.3°
Isoleucine 29.4° 22.5° .
Isobutyric acid 32.1° 23.4°
Isovaleric acid 29.1° 20.3°
2-Methylbutyric acid 28.8° 22.4°
aAverage for both runs and standard e r r o r 2.1.
^Average for both runs and standard error ±2.3.
cMeans within columns with different superscripts are different 
(P<.05).
60
Table 5. In situ dry matter fermentation rate, particulate passage 
rate and rumen digestibility for fOragea as influenced by 
supplement treatments
Supplements
Measurements DL-methionine-
urea
Soybean Meal Urea SEb
Fermentation rate, %/h 9.53° 7.28d 7.74d .071
Particulate passage . 
rate, %/h 4.20° 4.92d 4.24° .026
Rumen Digestibility, %
(calculated) 48.49° 42.40: 47.55° 2.37
aA low quality forage consisting of 75% mature grass hay and 25% 
barley straw.
b±. Standard error of the mean.
c^Means within the same row with different superscripts are different 
(P<.05).
e^Means within the same row with different superscripts are different 
(PC.10).
61
Table 6. In situ neutral detergent fiber fermentation rate, 
particulate passage rate, rumen ■ digestibility and 
fermentation lag time for foragea as influenced by 
supplement treatments
Supplements
Measurements DL-methionine-
urea
Soybean Meal Urea SEb
Fermentation rate, %/h 5.13 4.01 4.23 .05
Particulate passage 
rate, %/h 4.20° 4.92d 4.24° .03
Rumen Digestibility, % 
(calculated) 39-21 36.53 35.28 3.42
Fermentation lag time, h 1.09 1.05 1.77 .41
aA low quality forage consisting of 75% mature grass hay and 25%
barley straw.
k± Standard error of the mean.
cc^ Means within the same row with different superscripts are different 
(P<.05).
62
Table 7. Rumen liquid parameters as influenced by supplement 
treatments during the in situ trial
Supplements
Rumen measurements DL-methionine-
urea
Soybean Meal Urea SEa
Fluid volume, liters 40.91 41.75 41.86 .41
Fluid dilution rate, %/h 10.62b 9.77° 10.53b .02
Fluid outflow, liters/h 3.89 4.28 4.00 .23
Fluid turnover time, h 9.50b 10.25° 9.56b .18
a±  Standard error of the mean.
^0Means within the same row with, different supercripts are different 
(P<.01).
63
Table 8. Rumen ammonia Ievelsa as influenced by supplement treatments 
in the in situ trial
Supplements
Collection hours DL-methionine-
urea
Soybean Meal Urea SEb
4 26.38° 11.Old 25.72° 5.72
8 8.30 4.93 9.32 4.18
12 5.01 4.25 3.73 .68
18 4.90 5.25 4.16 1.39
24 23.53 9.18 8.15 6.25
36 3.81 5.34 4.51 1.61
48 8.72 9.84 9.04 1.91
72 8.58 10.46 9.44 1.03
96 6.24 7.97 5.35 2.07
Entire trial 9.53 8.26 9.22 3.96
aAmmonia levels are reported as mg/100 ml rumen fluid.
^Standard error of the mean.
0^Means within the same row with different superscripts are different 
(P<.05).
Table 9. Average daily gain, feed intake, and feed efficiency of 
heifer calves as influenced by supplements
64
SuDDlements
Measurements DL-methionine-
urea
SBMa Urea SBM-urea SEb
Average Daily Gain, 
kg/(head day-1)
.60 .64 . 6 2 .60 . 0 2
Feed Intake,
kg/(head day-1)
6.83 6.74 6.74 7.29
C
OCM
Feed Efficiency, 
kg dm/kg gain
11.44 10.71 11.75 11.26 .55
aSoybean Meal
HtStandard error of the mean
I
MONTANA STATE UNIVERSITY LIBRARIES
stks N378.C5471 RL
Influence of amino acids and branch chai
3 1762 00512047 O
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NTfS Cl^rV , Connie Kay
rsU71 Influence of amino a— '
cop.? cids and branch chain...
DATE ISSUED TO
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