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EFFECT OF A SILAGE MICROBIAL
IN OCULANT ON ANIMAL PERFORMANCE
AND SILAGE DIGESTIBILITY

By

Susan Lowe Fish

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1991

ABSTRACT

EFFECT OF A SILAGE MICROBIAL
INOCULANT 0N ANIMAL PERFORMANCE
AND SILAGE DIGESTIBILITY
By

Susan Lowe Fish

Alfalfa forage was ensiled in two concrete stave silos. One silo served as a
control (C), while the other was inoculated (I) with M plantarum.
Silages in combination with slowly degradable (SD) and rapidly degradable (RD)
protein sources were fed to lactating Holsteins and beef heifers. Silage digestion
was evaluated by a feeding trial with Holstein steers and in; m dry matter
digestibility (IVDMD). Digestion of fresh alfalfa leaves by rumen cellulolytic
species alone or in combination with L; W ,i_n_ yi_t_rg were viewed with
scanning electron microscopy.

Lactic acid bacteria (LAB) counts were greater in I than C by d 3. Lactic acid
was greater (p<.05) and ammonia-N was lower (p<.01) in I than C during feedout.
Fat corrected milk, protein and fat was greater (p<.05) for cows fed ISD than CSD.
Steers fed CRD had the greatest feed efficiency, and lowest (p<.10) average daily

gain. No differences were observed in digestibility.

ii

To my mother and father, Alice and Jim, whose
encouragement, love and support throughout

the years has made all things possible.

111

ACKNOWLEDGEMENTS

I would like to thank Dr. Melvin T. Yokoyama for serving as my major
professor. Thanks to Drs. Herbert Bucholtz, Bob Hausinger, Steven Rust and Stan
Flegler for serving on my graduate committee and their guidance throughout my
program. My gratitude is extended to Dr. Steve Rust, Dr. Melvin Yokoyama and
the Department of Animal Science for the financial support of my program.

Special thanks to Stan Flegler and all of the people at the Center for Electron
Optics for their excellent instruction and assistance in electron microscopy and to
Elaine Fink at the Beef Research Farm for laboratory assistance. Special thanks
to Jim Leisman for his help on statistical analyses. To Drs. Roy Emery, Bill
Thomas and Steve Rust, thank you for all your help on research projects and the
writing of abstracts. To fellow graduate students, especially Bob Patton and Alan
Grant, I cannot begin to thank you enough for your constructive criticism and loyal
friendship. For the people who have helped me and I have neglected to mention,

as well as those who have helped me put this manuscript together....Thank You!

iv

TABLE OF CONTENTS

Page
LIST OF TABLES .............................................................................................. viii
LIST OF FIGURES ................................................................................................ x
1.0 INTRODUCTION ................................................................................. 1
2.0 REVIEW OF LITERATURE ............................................................... 3
2.1 Fermentation of Alfalfa Forage ............................................... 3
2.1.1 The Fermentation Process ..................................... 3
2.1.2 Efi'ect of Lactic Acid Producing Bacteria
During Fermentation . ........................................... 4
2.1.3 Plant Proteolysis ...................................................... 7
2.1.4 Substrate Utilization During Silage
Fermentation ......................................................... 10
2.1.5 Aerobic Stability of Silage ..................................... 14
2.1.6 Silage Inoculants .................................................... 16
2.2 Rumen Cellulotytic Bacteria and Their Role In Fiber
Digestion ................................................................................... 18
2.2.1 Rumen Microbial Fermentation and Digestion....18
2.2.2 Plant Cell Wall Constituents ................................. 18
2.2.3 Rumen Cellulolytic Species .................................... 21
2.2.4 Cellular Attachment and Digestion of Plant

Material ................................................................... 25

TABLE OF CONTENTS (CONT)

Base

3.0 FERMENTATION CHARACTERISTICS AND NUTRITIVE VALUE
OF ALFALFA FORAGE EN SILED WITH AND WITHOUT

ADDITION OF A BACTERIAL IN OCULANT ....................................... 27
3.1 Introduction ............................................................................. 27
3.2 Materials and Methods .......................................................... 29
3.2.1 Silo Filling and Sampling .................................... 29
3.2.2 Lactic Acid Bacteria Enumeration ...................... 30
3.2.3 Aerobic Stability .................................................... 30
3.2.4 Preparation of Forage Samples ............................ 31
3.2.5 Lactation Trial ....................................................... 32
3.2.6 Growth Trial. ......................................................... 34
3.3 Statistical Analysis ................................................................. 36
3.4. Results and Discussion. .......................................................... 37
3.4.1 Silage Composition ................................................ 37
3.4.2 Silage Aerobic Stability ......................................... 46
3.4.3 Lactation Trial ....................................................... 49
3.4.4 Growth Trial ........................................................... 54
3.5. Conclusion ........................................................... . .................................. 5 6
4.0 EFFECT OF A BACTERIAL SILAGE INOCULANT ON FIBER
DIGESTION AND RUMEN CELLULOLYTIC SPECIES ........................ 57
4.1 Introduction .................................................................................... 57
4.2 Materials and Methods ................................................................. 59
4.2.1 Digestion Trial ............................................................. 59
4.2.2 Electron Microscopy .................................................... 61

vi

TABLE OF CONTENTS (CON’TJ

Page
4.2.3 Growth Enhancement ......................................................... 63
4.2.4 Q Xit_rg Trial ....................................................................... 63
4.3 Statistical Analysis ..................................................................................... 65
4.4 Results and Discussion ............................................................................. 66
4.4.1 Digestion Trial ..................................................................... 66
4.4.2 Electron Microscopy ............................................................. 69
4.4.3 Growth Enhancement .......................................................... 75
4.4.4 In £1132 Trial. ....................................................................... 79
4.5. Conclusion ........................................................................................................ 82 i
5.0 BIBLIOGRAPHY .............................................................................................. 83

vii

Tale

10

11

12

13

14

15

LET OF TABLES

Page
Classification of Lactic Acid Bacteria Important
in Silage ........................................................................................................ 6
Anaerobic Pathways of Sugar Metabolism by Lactic
Acid Bacteria ................................................................................................. 8
Biological Reactions Associated with Clostridial
Fermentation ................................................................................................ 11
Fermentation of Organic Acids as Substrates by Lactic
Acid Bacteria. ............................................................................................... 13
Diet Ingredients Fed to Holstein Cows During
Lactation Trial ........................... ' ................................................................... 33
Diet Ingredients Fed to Beef Heifers During Growth
Trial ............................................................................................................... 35
Composition of Forage Material Placed Into The Silo .............................. 38
Lactobacilli Numbers in Silage Material Post-Ensiling ............................ 40
Mean Temperatures at the Various Elevations Within
Each Silo ....................................................................................................... 42
Efi'ect of Elevation and Treatment on Silo
Temperatures ................................................................................................ 42
Fermentation Characteristics of Alfalfa Silage in
Buried Bags .................................................................................................. 44
Characteristics of Fermentation During Ensiling for
Inoculated (I) and Control (C) Silage ......................................................... 45
Chemical Indices of Fermented Forage During Feedout .......................... 47
Silage Characteristics Throughout Aerobic Exposure
of 14 Days ..................................................................................................... 50

Response of Holstein Cows Fed Alfalfa Silage With and
Without the Addition of a Microbial Inoculant ......................................... 52

viii

16

17

18

19

20

21

23

24

25
26
27

28

29

LET OF TABLES (991321;)

Ease
Performance of Crossbred Heifers Fed Alfalfa Silage With
and Without the Addition of a Microbial Inoculant. ........................ 55
Characteristics of Alfalfa Silage Fed to Holstein
Steers During the Digestibility Trial ................................................ 60
Performance of Holstein Steers Fed Alfalfa Silage
With and Without the Addition of a Microbial Inoculant ............... 67
Digestibility of Alfalfa Silage With and Without the
Addition of a Microbial Inoculant ...................................................... 68
I; m Digestibility of Dry Matter (IVDMD) of Control
and Inoculated Silage .......................................................................... 80

Composition of Alfalfa Forage Entering Silos Before Treatment

Composition of Samples Bored from Ports 1.5 m from Bottom of
Each Silo.

Composition of Silage During Feedout.

Medium Used to Grow Rumen Cellulolytic Bacteria to Mid-
Exponential Phase.

Mineral Mixes Used in Rumen Cellulolytic and Digestion Media.
Lactobodlli (LBS) Media. '

Medium Used in Digestion of Alfalfa Leaf With Individual and
Co—cultures.

Volatile Fatty Add Mixture Used for Digestibility Medium.
GCS-RF Medium.

ix

10

11

12

13

14

LET QF FIQQES

 

Page
Transformation of N Constituents During Ensiling ............................... 9
Average pH of the Control and Inoculated Silages During
First 45 Days Post-ensiling ....................................................................... 39
Average Temperature of the Control and Inoculated Silages
During First 45 Days Post-ensiling .......................................................... 41
Silage Temperature and Dry Matter Recovery for Control and
Inoculated Silage During Aerobic Exposure ............................................. 48
Calculations for Digestibility Using Accumulation of
Chromium Concentrations in Feces .......................................................... 62
Digested Alfalfa Leaf After 24 h Digestion in Rumen
Fluid. ........................................................................................................... 70
Undigested Alfalfa Leaf With Cut Surface of Transverse
Leaf Section Exposed. ................................................................................ 70
3mm gym in Monoculture, Attached To a Degraded
Alfalfa Leaf. ................................................................................................ 71
.R_. albus Attached to an Alfalfa Leaf Stomata While in Co-
Culture with L; plantarum ........................................................................ 72
B_. albg in Co-Culture With L plantarum Attached To An
Alfalfa Leaf. ................................................................................................ 72
B, W' m Co-Culture WithL 1m Attached
To An Alfalfa Leaf... -- .......................... 74
B, W in Monoculture, Attached To An
Alfalfa Leaf. ................................................................................................ 74
Growth of Mum albus 7 With and Without
Silage Extract ............................................................................................. 76
Growth of W flgvgfgg’ens FD-l With and
Without Silage Extract .............................................................................. 77

X

TFFI N’T

mm Essa

15 Growth of W M 8-85 With and Without
Silage Extract ............................................................................................. 78

16 km Digestibility of Dry Matter (IVDMD) of C and I
Silages ......................................................................................................... 81

xi

1.0 INTRODUCTION

The ensiling of forage crops, such as alfalfa has increased in popularity over the
past few decades. The ability to harvest a high quality feedstufi‘ without nutrient
losses assodated ,with poor drying conditions and the ability to store this material
for long periods of time, makes ensiling economical. Successful fermentation of
forages, however, relies on the quality of the ensiled material and fermentation
rate (McCullough, 1978). Forage material must contain a sufident amount of
plant sugars to be used as substrate by epiphytic lactic add bacteria (LAB), as well
as sumdent numbers of these bacteria to convert sugars into lactic add (Weinberg,
et al., 1988). The rate and efidency of add production by epiphytic LAB are
important factors in efident silage maidng. A low pH inhibits other microbial
activity thereby, restricting the breakdown of plant proteins into a highly soluble
form, which is ineffidently utilized by the cow (Chamberlain, et al., 1986). Growth
of clostridia resulting from plant protein degradation can cause high ammonia and
butyric add concentrations, as well as support a poor preservation and lower dry
matter intakes.

In order to improve fermentation, a suitable LAB inoculant should be added to
forage material at the time of ensilement. An inoculation containing sumdent
homofermentative LAB would ensure rapid and effident utilization of soluble

carbohydrates and a faster decline in pH. A more rapid fermentation could

increase dry matter recovery and may preserve plant proteins, produdng a higher
quality silage.

Several studies using microbial inoculants have reported variable results.
Many of these (Ohyama et al., 1975; Carpintero et al., 1979; Lindgren et al., 1983;
Rooke et al., 1985) have reported advantages with the addition of a microbial
inoculant while others ( Throne, 1981; Ely et al., 1982; Buchanan-Smith and Yao,
1981; and Moon et al., 1981) have had negative efi'ects with inoculation. The
conditions under which these inoculants are effective have not been defined. Crop
characteristics such as dry matter (DM) content, water soluble carbohydrate (W CS)
content, buffering capadty and initial pH all affect the ensiling process and thus
could afl‘ect inoculation (Pitt and Leibensperger, 1987).

The concept of inoculating forages has been widely accepted as common practice
throughout many parts of the world. Although several studies have indicated
changes in fermentation which occur with inoculation, little attention has been
directed toward the efi‘ect of spedfic inoculants on the nutritional quality of silage.

The objectives of the following studies were designed to measure fermentation
efi‘ects as well as the nutritional value of inoculated alfalfa silage through livestock

production trials, in vitro laboratory experiments and electron microscopy.

2.0 REVIEW OF LITERATURE

2.1 Fermentation of Alfalfa Forage

2.1.1 The Fermentation Process

Silage fermentation consists of biochemical changes which occur in fresh plant
material during ensilement. Activity is initiated during wilting when epiphytic
bacteria use soluble plant sugars as a substrate and multiply. Once ensiled, plant
enzymes use glucose, fructose, sucrose and fructans along with trapped oxygen to
produce water, carbon dioxide and energy. The energy which is produced cannot
escape the forage matter, thus is liberated as heat, increasing the temperature of
the plant material. These reactions continue as long as oxygen and sugars are
available, and can continue through feedout. Such an event could result in large
amounts of nutrients broken down into carbon dioxide and water, leading to a
considerable amount of DM loss (Woolford, 1984). Control of this activity is a
question of chop length, rate of ensiling, silo design, sealing and general
management (McDonald, 1979).

The majority of organisms found on growing crops are aerobes. The number of
LAB is generally low (Stirling, 1953; Keddie, 1959; Stirling and Whittenbury,
1963). However, counts usually rise significantly by the time the herbage reaches
the silo. The microbial increase is due to inoculation of forage by farm machinery

(Gibson, et al., 1961; Henderson, et al., 1972), and liberated sap made available as

3

4

substrate during the chopping and laceration of fodder (Greenhill, 1964).

Multiplication of these microbes can continue until sugar substrates are
depleted. Forage spedes, DM content, substrate availability and buffering capadty
are all factors which afi‘ect fermentation. In addition, the number and spedes of
anaerobic bacteria can play a major role in the quality of fermentation (Carr, et al.,
1984; Ely, et al., 1982; Ely, et al., 1981; Kung, Jr., et al., 1984; McDonald and
Henderson, 1962; Moon, et al., 1981). The greater the number of homofermen-
tative LAB, the more lactic add is produced and the quicker the drop in pH (Muck,
1989). A decrease in the pH of the ensiled mass needs to be low enough to inhibit
undesirable microbial activity and endogenous plant catabolic processes (Shockey,
1988). It is also important that the pH declines at a rapid rate to prevent
proteolysis, thus preserving the maximum amount of nitrogen (N) as protein N
(McDonald, 1981).

2.1.2 Efl‘ect of Lactic Add Produdng Bacteria During Fermentation

The prindple of microbial inoculation was first adopted in 1909 by Bouillant
and Crolbds, when they applied lactic add inoculants to beet pulp to improve
fermentation (Watson and Nash, 1960). Later, in 1930, Ruschmann and Koch and
in 1934, Rushmann and Meyer (Fenton, 1987) documented that the rate of
addification during silage fermentation is dependent on epiphytic bacteria found
on fodder plants.

There are numerous microorganisms found on growing plants (Woolford, 1984),
with the number tending to increase with plant maturity and advancement of the
season (Kroulick, et al., 1955). The majority of these are Gram negative aerobes,
which will not thrive in the anaerobic environment of the silo. Thus their

enzymatic processes contribute little to silage preservation. However, the Gram
positive lactic add produdng bacteria, are facultative anaerobes, which enables
them to utilize soluble sugars to carry out metabolic functions aerobically on the
plant or anaerobically in the silo. The number of LAB on growing alfalfa is
generally low, usually less than 100 cfu/g and reduced further during wilting
(Keddie, 1959; Stirling and Whittenbury, 1963). However, counts of lactobadlli
usually rise significantly by the time they reach the silo. This is partly due to
inoculation of microorganisms from farm machinery (Henderson, et al., 1972;
Gibson, et al., 1961).

Until 1978, there was little known about the composition of microflora during
silage fermentation. However, Beck (1978) studied the qualitative changes in LAB
during the fermentation of grass and red clover with high and low DM contents.
He reported that fermentation in wilted and unwilted silage was initiated by
homofermentative LAB being 5% of total lactobale present by day 4. However,
after 142 d of fermentation, 75% of all lactobadlli in the silage with the low DM
and 98% of the lactobadlli in the silage with high DM were heterofermentative.
Beck suggested that bacteriologic shift could be due to a greater acetate tolerance
in heterofermentative bacteria. Table 1 shows the bacterial spedes commonly
found in silage (McDonald, 1981). The dominant organisms in silage according to
Langston and Bouma (1960) are L. plum, M and M sp.

Gibson, et al., (1958) reported that L, planta_ru_m_ and L. addophilus were the
dominant homofermentative bacteria in fermentation. While others (Langston, et
al., 1962; Moon, 1981, and Moon, et al., 1981) revealed evidence that streptococd
and leuconostocs initiate fermentation and are superceded by spedes of Lactobadlli

and Pediococd.

TABLE 1. Classification of Lactic Acid Bacteria Important in Silage

 

(A) Heterofermentgtive
999mg

Leuconostoc mesenteroides
Leuconostoc dextranicum
Leuconostoc cremoris

M

Lactobadllus brevis
Lactobadllus fermentum
Lactobadllus buchneri
Lactobadllus viridesceno

(B) Hgmgfermentgtivg
rocug

Streptococcus faecalis
Streptococcus faedum
Pediococcus addilactid
Pediococcus cerevisiae
Pediococcus pentosaceus

.324

Lactobadnus plantarum

Lactobadllus curvatus

Lactobadllus casei

Lactohacillus coryniformis subsp. coryniformis

 

McDonald, P. 1981

Table 2 illustrates the products of an anaerobic sugar fermentation by LAB
described by Whittenbury and coworkers (1967). Glucose and fructose are the most
common soluble sugars utilized by LAB, however LAB can also ferment pentoses,
xylose and arabinose, which are formed from the degradation of hemicellulose

(Dewar, et al, 1963) and amino adds (Rodwell, 1953).
2.1.3 Plant Proteolysis

The deamination of protein in silage is another process resulting from plant
enzyme activity. The breakdown of fresh plant material can be caused by plant
proteases (Bergen, et al., 1974; Ohshima and McDonald, 1978), however, most
proteolytic activity is a result of aerobic conditions inside the silo.

Figure 1 illustrates post-harvest nitrogen metabolism in ensiled plant material
from hay and cereal crops (Bergen, 1974). Fresh forage material contains 70-90%
ofthe total nitrogen in the form ofprotein while the remaining 10-30% is non-
protein nitrogen consisting of free amino adds, amides and small concentrations of
urides, amines, nucleotides, chlorophyll, low molecular weight peptides and amino
adds bound in non-protein form (Hegarty and Peterson, 1973). It is not uncommon
for 50-60% of the true protein nitrogen to be broken down into simpler non-protein
nitrogenous compounds in preserved forage (Whittenbury, 1967).

Amino adds resulting from proteolysis can be metabolized into ammonia
(deamination), amines (decarboxylation) and unidentified nitrogenous compounds
(Bergen, et al., 1974; Ohshima and McDonald, 1978). A good quality silage is
characterized by low concentrations of ammonia-N, amines and other compounds
produced from the break down of amino adds (Bergen, 1984). If aerobic conditions

remain in the silo it creates an environment which allows yeast and mold to

TABLE 2. Anaerobic Pathways of sugar Metabolism by Lactic Add Bacteria

 

W

1 glucose- -> 2 Lactic add

 

1 fructose-mm» 2 Lactic add

1 pentose-----> 1 Lactic add + 1 Acetic add

W
1 glucose-mm» 1 Lactic add + 1 Ethanol + 1 Carbon dioxide

3 fructose-----> 1 Lactic add + 2 Mannitol + 1 Acetic add

1 Pentose-----> 1 Lactic add + 1 Acetic add

 

Whittenbury, et al., 1967

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multiply and increase the silage temperature (Bergen, 1984). Clostridial
fermentation is assodated with ammonia, butyric add and a higher pH than that
found with lactic add bacteria. This results in an unstable and often unpalatable
silage. Butyric add produced by sacchrolytic organisms which metabolize lactate
and sugars, (Table 3) often serves as an indicator of clostridial activity. The result
of this type of fermentation occurs at a high DM or a low pH (Whittenbury, et al.,
1967). Woolford (1984) suggested that clostridial activity is suppressed at a dry
matter above 31% and/or a pH below 4.5. Under ideal conditions, sufident
numbers of lactic add produdng bacteria occurring naturally, would produce a
drop in pH during day 2-5 of ensilement. Bergen and coworkers (1974) suggested
that DM of forage material at the time of ensilement is the most dedsive factor
infiuendng the amount of protein degradation which will occur during
fermentation. The lower the DM, the larger the amount of plant protein escaping
proteolysis. Thus, DM at the time of ensiling and rate at which the pH falls

during fermentation are factors one must consider during silage preservation.
2.1.4 Substrate Utilization During Silage Fermentation

The major water soluble carbohydrates (WSC) found in forage material are
glucose, fructose, sucrose and fructosans. The most available sugars for microbial
substrate are glucose and fructose, due to the continual hydrolysis of sucrose and
fructosans to glucose and fructose monomers (Whittenbury, et al., 1967).

The WSC content as well as the fi'uctose/glucose ratio of green fodder plants
varies depending on spedes, weather conditions, stage of growth, time of day,
wilting conditions and fertilizer application (Woolford et. al., 1982).

Soluble carbohydrates present in forage material after aerobic metabolism are

11

TABLE 3. Biological Reactions Associated with Clostridial Fermentation

 

Organic Adds
2 Lactic add > 1 Butyric add + 2002 + 2H2

 

Amino Adds
(A) Coupled oxidation-reduction reactions
1 Alanine + 2 Glydne--->3 Acetic add + 3NH3 + 1002
(B) De-amination
3 Alanine-«~--> 2 Propionic add + 1 Acetic add + 3NH3 + 1CO2
1 Valine-----> 1 lsobutyric add + 1 NH3 + 1 CO2
1 Leudne-«m-o 1 Isovaleric add + 1 NH3 + 1 CO,U
(C) Decarboxylation
Histidine-«---> Histamine
Lysine»-—----> Cadaverine
Arginine-----> Ornithine-»--->Putresdne
Tryptophan----> Tryptamine
Tyrosine-«mm> Tyramine

 

12

fermented by a variety of microorganisms, however, under ideal conditions LAB
ferment sugars and produce an intolerable addic environment for other
microorganisms (Whittenbury, et al., 1967). Lactic add bacteria utilize soluble
sugars through two fermentable pathways to produce lactate (Table 2,
Whittenbury, et al., 1967), as previously described. Homofermentative LAB are
the most desirable for they are more emdent in produdng lactate than
heterofermentative LAB (produdng 2 moles of lactic add versus one mole), and
more efident in conservation of DM (McDonald, et al., 1973). One cannot predict
a final ratio of fermentation products, for it is possible to have 100% variation
occur in the amount of lactic add produced under two similar drcumstances.

In addition to phosphate, several organic adds also are commonly found in
fresh herbage and silage. These adds include malate, citrate, and glycerate
(McDonald, 1979). Organic adds in combination with their salts comprise a
bufl'ering system in plants (Playne and McDonald, 1966). Legumes contain higher
amounts of add (0.6 to 0.8% of DM) than grasses (0.2 to 0.6% of DM), as well as
higher protein and more cations which contribute to a much greater bufi‘ering
system.

Considerable interest has been given to those organic adds in silage which
bufi'er within the pH range of 4-6. Early stages of fermentation are characterized
by the dissimilation of organic adds by LAB (Edwards and McDonald, 1978). The
main products of citrate and malate fermentation by LAB are shown in Table 4
(Whittenbury, et al.,1967). Products from these reactions include formation of
organic salts (lactate, acetate), neutral products (ethanol, acetone and 2,3
butanediol) and alkaline released cations (Whittenbury, et al., 1967). Other
substrates which can be fermented by LAB include amino adds (Rodwell, 1953).

13

TABLE 4. Fermentation of Organic Adds as Substrates by Lactic Add Bacteria

 

A. 1 Citric add------> 2 Acetic add + 1 formic add + 1 carbon dioxide
or

2 Citric add-----> 2 Acetic add + 1 acetone (or 2,3 butanediol) + 4 carbon
dioxide.

or
2 Citric add-~«-> 3 Acetic add + 1 lactic add + 3 carbon dioxide
B. 1 Malic add-mm—> 1 Acetone (or 2,3 butanediol) + 4 carbon dioxide

or
2 Malic add----> 1 Acetone (or 2,3 butanediol) + 4 carbon dioxide

01'

1 Malic add -> 1 Acetic add (or ethanol) + 1 formic add + 1 carbon

dioxide

 

 

Whittenbury et al., 1967

11+

Brady (1966) demonstrated that 14.11am and m can deaminate serine,

arginine, glutamine and aspargine.
2.1.5 Aerobic Stability of Silage

The most important factor in achieving high quality silage is rapid occurrence
of anaerobiosis in the silo. Other factors influendng aerobic deterioration include
quantity of substrate, DM of the ensiled crop, botanic origin and ambient
temperature (Woolford, 1990).

Aerobic deterioration of silage ultimately results in complete mineralization of
easily oxidized nutrients which are broken down into CO, and 11,0, generating
heat and resulting in DM losses (Woolford, 1984). Studies have shown that DM
lossesoveraperiodof5-l5 dayscanbeasgreatas32%. Oncetheprocessof
aerobic deterioration commences, it is practically impossible to stop (Honig and
Woolford, 197 9).

Aningressofairassmallas 100tol50mgO,/ngMisadequatetomake
silage highly susceptible to aerobic deterioration (Woolford, et al. 1979). Upon
exposure to oxygen, conditions become favorable for proliferation of aerobic
bacteria, yeasts and fungi (Moon et al., 1980 and Woolford et al., 1982). In most
silages, yeasts have the ability to increase in numbers from <102 to 1013 cfu/g DM
by day 3 ofaerobic exposure (Beck 1963, as cited by Woolford, 1990).

Yeasts involved in aerobic deterioration have been classified as add-utilizers
comprised dmmfisdsmmsflmdsandficbisse and sugar-
utilizers which are m sp. (Gross and Beck, 1970, as cited by Woolford,
1990; Moon and Ely 197 9; Johnsson and Pahlow, 1984). A high population of
yeasts does not necessarily mean a silage will deteriorate. Instead, quantity of

15

lactate-utilizing yeasts deddes whether a silage will deteriorate or not upon
aerobic exposure (Johnnson and Pahlow, 1984).

Thermophilic filamentous fungi are also found in deteriorating silage, however
theirgrowthisgenerallyslowerandthus havelittle afi'ectonsilageasafeed.

Woolford and Cook (1978) treawd silage material with antibiotics that had
antibacterial and antifungal properties. Their studies revealed the involvement of
proteolytic bacteria from the genus Badllus. Bacteria appear to initiate
deterioration in maize silages, followed by yeasts (Woolford et al., 1978).
Deterioration in cereal crops and grass silages on the other hand, begins with
yeasts (Woolford et al., 1979). However, Woolford (1984) concluded that this
inconsistency concerning the identity of microbial groups responsible for the onset
ofaerobic deterioration lies in the properties ofensiled material, spedfically DM
content rather than botanic origin.

Primary substrates ofaerobic deterioration have been described as nitrogen
free extracts which included water soluble carbohydrates and organic adds (Honig
and Woolford, 1979). Woolford (1990) suggests that the organisms involved in
aerobic deterioration will use a wide range of substrates which include those found
in the original crop and others which are produced by fermentation. Regardless of
the substrate utilized, deterioration in forage crops is always accompanied by a loss
of residual sugars and the evolution of ammonia and carbon dioxide. The latter
canbedirectlyequatedteDMlossanditsmeasurementcanbeusedtomonitor
the progress of deterioration (Woolford, 1990). Fermentation adds (such as acetic
and lactic adds), amino adds and proteins are all used as substrates (Woolford,
1984). The pH increases with add depletion and tends to be greatest at the silage

surface where exposure to oxygen is greatest (Woolford, 1978).

16

Aerobic deterioration occurs in all silages to some varying degree, except for
those undergoing an extensive secondary fermentation. This deterioration
depredates conservation efidency, causes nutritional losses and can even pose a
potential health hazard to livestock. Such management practices as rapid silo
filling, spedal cutting equipment for forage removal, rescaling between feed-outs
and use of an effective inoculant at the proper application rate can minimize

aerobic deterioration.
2.1.6 Silage Inoculants

At the present time, there are several silage inoculants on the market. They
have been reported to influence the rate and extent of silage fermentation. Typical
ingredients found in inoculant may include enzymes, bacteria, molds,
micronutrients for microorganisms or a mixtures of all these to influence forage
respiration and fermentation rate (Parker, 1979). Bolsen (1978) has described
silage inoculants as "those products that supply lactic add produdng
microorganisms and enzymes and/or microorganisms that increase the availability
of carbohydrates and other nutrients to lactic add produdng microorganisms".

Commerdally available inoculants not only vary in ingredients but in type of
preparation (dried,.liquid, freeze—dried) and packaging (bottles, vacuum packs and
paper sacks). .

Whittenbury (as cited by Beck, 1978) described the requirements of a quality

silage microorganism as follows:

1. It must be fast growing and able to compete with and
dominate other microorganisms present in silage.

17

2. It must be homofermentative.
3. It must be add tolerant down to a silage pH of 4.0.

4. It must possess the ability to ferment glucose, fructose,
sucrose, and preferably fructosans and pentosans.

5. It should have no action on organic adds.
And in 1975, McCullough described the following as requirements of a cost

efi'ective quality inoculant:

1. The cost of the additive must be less than the silage lost
without the additive.

2. Addition of the additive must result in a more efident
fermentation than occurs naturally.

3. The additive should produce a silage with a greater digestibility
energy and/or protein than untreated silage.

Several workers have shown varying results from inoculation, including
advantageous results (Rooke et al., 1985; Ohyama et al., 1975 and Owens, 1977)
and non-significant results (Ely et al., 1982; Moon et al., 1981 and Buchanan-

Smith and Yao, 1981).

2.2 Rumen Cellulolytic Bacteria and Their Role in Fiber Digestion
2.2.1 Rumen Microbial Fermentation and Digestion

The rumen is an ideal fermentation site. It makes up one-seventh of the total
mass of a ruminant’s body weight (Russell and Hespell, 1981). The rumen remains
at a constant temperature of 39°C and is well buffered by salivary secretions. The
microflora inhabiting the rumen is dense containing approximately 1010 to 1011
bacterial and 10‘ protozoal cells per milliliter of rumen contents. There is an
extensive diversity and synergism in the ecosystem which contains more than 200
spedes of bacteria and over 20 spedes of protozoa (Bryant and Robinson, 1962).

During ruminal fermentation, feedstuffs are broken down and fermented into
short chain fatty adds through microbial metabolism and are used as the
ruminant’s energy source, while the animal relies heavily on the microbial mass as
a protein source. Methane, heat, and ammonia are formed as well, representing a
loss of energy and nitrogen to the animal. The balance of fermentation products
determines the efidency of nutrient utilization in ruminants. In turn, this balance

is ultimately controlled by the various microorganisms found in the rumen.
2.2.2 Plant Cell Wall Constituent

In ruminants the plant cell wall is extensively degraded and utilized as an
energy source by the rumen microflora. Plant cell walls are indigestible by animal

enzymes, however, gastrointestinal microflora partially degrade cell wall material.

18

19

The cell wall of plants is made up of an organic matrix of cellulose, hemicellulose,
lignin and other small fractions of pectins, gums mucilages, cutin, tannin, bound

cell wall protein and cell wall minerals.

Cellulose

First recognized by Payen in 1939 (Whistler and Smart, 1953), cellulose is the
most abundant carbohydrate in the world. Its recycling is dependent on microbial
activity which produces carbon dioxide during degradation. An enormous amount
of energy lies in these cellulosic carbohydrates, making them an excellent food
source for herbivores. Cellulose is the largest component of plant cell walls, thus
serving as a primary structural element. Linked at the C-1 and C-4 position
through glycosidic linkages, individual anhydrous glucose molecules make up the
linear polymer in a beta configuration. Glucan chains consist of 100 to 10,000 or
more units of glucose (Ott and Tennent, 1954), and are held together by tight
hydrogen bonds (Albersheim, 197 5) between the hydroxyl group of a sugar on one
chain and an oxygen atom of another. Chains are also held together by

VanderWaals forces.
Hemicellulose

Hemicellulose is the second largest constituent found in plant material
(Phillips, 1940). First named in 1891 by Schultz (Whistler and Richards, 1970),
hemicellulose has been defined as the polysaccharide in plant tissues other than
cellulose which is extracted with alkali and hydrolyzed in add (Collings, 1979).

Hemicellulose is a complex mixture of polysaccharides which constitute much of

the cell wall matrix (Bailey and Gaillard, 1965). It is a polybeta 1-4 D-

20

xylanopyranose based on a backbone'of xylose residues, with branches of arabinose,

glucose and/or galactopyranosides (Akin and Barton, 1983).
Lignin

Lignin is a polymer of phenylpropanoid units intimately assodated with
structural carbohydrates (Himmelsbach and Barton, 1980), and plays a major role
in redudng microbial attack on cell walls ( Akin and Barton, 1983). Phenolic adds
such as p-coumaric add and ferulic add which are precursors of lignin can bind to
structural carbohydrates which inhibits carbohydrate degradation (Hartley et al.,
1974).

Other Constituents

Pectin is comprised of chains of galacturonic add, galactans and arabinans
(Aspinall, 1973). Pectins are not pure polysaccharides, but mixed and branched,
forming complex polysaccharide structures. It is found in intracellular spaces in
the cell wall and is assodated with cellulose in other cell layers (Esau, 1965).
Hemicellulose, pectin and lignin play an important role as matrix substances for
the cell wall.

Cowling (197 6) demonstrated that crystallinity and lignification are the most
important factors in determining the susceptibility of cellulose to enzyme
degradation. It has been shown that spedfic enzymes which attack glucan bonds
in cellulose chains are incapable of attacking an intact plant fiber (Albersheim,
1975). Thus accessibility of cellulose to microbial enzymes and chemical magenta
depends on the arrangement of cellulose within the cell wall ( Collings, 1979).

Although some plant material is accessible and easily digested, the degradation

21

of fiber material in the rumen is a result of complex microbial processes (Cheng et
al., 1980). These processes include the digestion of plant cell walls, to yield
microbial cell growth and fatty adds end products. As with any ecological system,
the microorganism should be attracted to its nutrient substrate. It has been
demonstrated that plant material undergoing colonization and digestion by rumen
microorganisms includes the adherence of bacteria, protozoa and fungi, however,
bacteria are responsible for the majority of the digestion which takes place in the
rumen (Hungate, 1966). Akin and Barton (1983) found through the use of the
scanning electron microscope (SEM) that plant cell wall digestion did not occur
unless rumen bacteria were closely assodated with or completely adhered to the
cell walls.

2.2.3 Rumen Cellulolytic Spades

Based on relative numbers in the rumen of domestic ruminants and their
ability to attack various forms of cellulose in pure cultures, the major rumen
cellulolytic bacteria are W W (Sijpestein, 1951),
Walling (Hungate, 1957). adherents: fibrobacter masses
(Hungate, 1950). These are the three major spedes which obtain their energy for
growth solely through cellulose fermentation (Bryant, 197 3). B, M will
digest cellulose to a lesser extent (Bryant, 1973; Hungate, 1966). Each of these
spedes except B, W are capable of utilizing hemicellulose-type
components from forage (Dehority and Scott, 1967). B, W is the most
active cellulolytic, bacterium digesting the more resistent cellulose such as cotton

fibers and mature bay to a greater extent than Mm which are active,

22

but show much more variation between strains in ability to degrade more resistant
cellulose (Bryant, 1973).

Minato and coworkers (1966), noted that both Ruminm and B,
W adhere to fiber during digestion, however, B, sugg’nogeneg was firmly
attached to the cell wall. 'A few other cellulolytic spedes of the genus Qlostridium
(Hungate, 1957; Shane et al., 1969) and Egbagterium ELM (Bryant et al.,
1958; Van Gylswyck and Hofi'man, 1971) have been found in the rumen
occasionally.

The largest numbers of cellulolytic bacteria are found when the ruminant is fed
a high roughage diet, however in ruminants fed cellulose as the total feed source,
cellulolytic bacteria only comprise 25% of the total rumen microbial population
(Slyter et al., 1971). Many non-cellulolytic bacteria found in the rumen are
responsible for the degradation of pectins and xylans. Numerous synergistic
interactions between cellulolytics and noncellulolytic spedes occur and has been
shown to enhance cellulose degradation (Dehority and Scott, 1967).

Rumen cellulolytics produce cellulose enzymes which hydrolyze insoluble
cellulose into soluble cellulodextrins or sugars, some of which they can absorb and
ferment to obtain energy for growth (Schaefer and King, 1965; Sheth and
Alexander, 1969).

End products of cellulose degradation include acetate, propionate, butyrate, CO,
methane, and microbial cells. This includes interacting populations of 1) rumen
cellulolytic bacteria, 2) carbohydrate fermenting spedes which can use products
hydrolyzed from cellulose, 3) spedes which will degrade sucdnate, formats and any
lactate produced from microbes in 2 and 4) methanogenic bacteria which will

reduce CO, using H, or formats as an electron donor (Hungate, 1950).

23

All rumen cellulolytic bacteria require one or more B-vitamins for growth.
Biotin is the most common vitamin required by the cellulolytics. However, some
strains ofgflbJualsorequire pyridoxine. Afew strains ofggaljmgmay require
folic add, riboflavin or thiamine (Bryant, 1973). The vitamins required by B,
W strains are similar to those required by Edible (Bryant and Robinson,
1961; Gill and King, 1958; Scott and Dehority, 1965), with some strains requiring
pyridoxine and cobalamine which in some cases can be replaced by methionine
(Scott and Dehority, 1965). L W requires biotin, using this as its
primary B-vitamin. P-aminobenzoic add has been shown to stimulate the growth
in some strains of B, m (Bryant and Robinson, 1961; Scott and Dehority, 1956).
B, W has a requirement for Na+ and a great demand for Ca“ (Bryant et
al., 1959). The other cellulolytics have a lower demand for K‘, Na“, and Ca“.
Ferrous iron and Zn2+ has been found to stimulate microbial activities even further
(Matturi, 1972).

All of the rumen cellulolytics have a mquirement for sulfur. W
utilizes cysteine or sulfide, but not sulfate (Bryant et al, 1959). The W
grow well in media containing sulfide or sulfate (Bryant, 1973).

The main nitrogen source for cellulolytic bacteria is ammonia (Bryant and
Robinson, 1961; Bryant et al., 1959, Dehority, 1963). The ammonia is a product of
non-cellulolytic bacteria metabolism. This is just another example of co-existence
and cooperation between rumen spedes. Cellulolytic bacteria lack the ability to
use organic nitrogen sources for growth and though not established, it appears that
they probably lack the mechanism for transporting amino adds or peptides into the
cell (Pittmann et. al, 1967). Although W bacteria cannot use amino

adds if present, B, W will utilize the amide nitrogen from glutamine

24

and asparagine for growth and function (Bryant and Robinson, 1961).

Many strains of rumen cellulolytic bacteria require a carbon source beyond that
of the energy source. The source commonly used by these bacteria is CO, or
bicarbonate. B, W and & gm require large amounts of 00,,
which is fixed into pyruvate during glycolysis (Caldwell et al., 1969). Without 00,,
these bacteria are unable to obtain energy in the form of carbon, for growth
(Bryant, 1973). They also use CO, for biosynthetic purposes (Allison, 1969; Allison
1970). B, 31113; does not require large amounts of CO, for growth, but requires
small amounts for optimal growth and for biosynthetic processes (Bryant, 197 3).

Short chain fatty adds, better known as volatile fatty adds are essential for
growth of the three major rumen cellulolytics at 0.5-0.3mM in batch cultures
(Dehority and Scott, 1967). Carbon skeletons from these fatty adds are not
degraded, but incorporated into certain cellular constituents (Bryant, 1973 ).
W W is the only cellulolytic that requires the straight chain
valeric add, which can be replaced by longer chain adds (Wegner and Foster,
1963). The cellulolytic bacteria utilize the various branched chain fatty adds, such
as C“ and Cu from isobutyric, Cu5 and C1, fiom isovaleric, and anteisa Cus and Or,
from 2-methyl-butyrate (Allison, et al., 1962; Wegner and Foster, 1963). These
branched chain fatty adds are also precursors for fatty aldehydes in these bacteria.
One or more of the above fatty acids are used for the biosynthesis of amino adds:
valine, leudne, and isoleudne respectively (Allison et al., 1962; Robinson and
Allison, 1969; Allison, 1970) via reductive carboxylation reactions (Bryant and

Robinson, 1961; Allison, 1969).

25

2.2.4 Cellular Attachment and Digestion of Plant Material

There are many factors which influence the rate and extent of forage cell wall
digestion. Feeds containing fractions of cellulose and hemicellulose are relatively
insoluble in the rumen and are degraded slowly (Dehority, 197 3; Van Soest, 197 3).
Degradation is highly influenced by structural factors. Such factors would include
the close assodation of lignin with cellulosic materials, acting as a barrier against
bacterial cellulases (Russell and Hespell, 1981). Crystallinity also efi'ects digestion
(Bryant and Robinson, 1962). Russell and coworker (1981), showed that high
crystalline fibers were readily degraded by cellulases from certain cellulolytic
bacteria while fiber digestion was much slower for other cellulolytic spedes. Those
who have made extensive observations (Akin and Amos, 1975; Akin et al., 1974) of
mixed cultures of rumen bacteria have observed that many rumen bacteria appear
to adhere to plant cell walls by means of thin fibrous capsules. In many of these
observations, it has been noted that the bacteria digest plant cell wall material and
infiltrate the resultant cavities.

Cheng and coworkers (1977) found that bacteria in the rumen of cows fed corn
silage versus other forage based diets had the least bacterial slime formation, but
every bacterial cell showed some extracellular structure. Although some plant
material is accessible and easily digested, the process is. long and sequential (Akin
and Amos, 197 5). Digestion begins with penetration through the stomata (Baker
and Harris 1947) and colonization on fiber macerations produced from mastication.
Dinsdale et al., (1978) in an my study demonstrated that mixed populations of
rumen bacteria released 12 to 36% of the dry matter of damaged cells in legume
leaves. These organic nutrients are used to support enormous proliferations of

bacteria in intracellular space and at the leaf surface. Subsequently, plant cell

26

walls are ruptured by certain spedes of bacteria who digest cellulose in grasses
and cellulose and pectins in legumes (Dinsdale et al., 1978). Plant protoplasm
which remains to be digested supports a further proliferation of bacteria until
bacterial microcolonies fill plant cell wall compartments, while refractory cells

remain uncolonized (Akin and Amos, 1975).

3.0 FERMENTATION CHARACTERISTICS AND NUTRITIVE VALUE
OF ALFALFA FORAGE EN SILED WITH AND WITHOUT ADDITION
OF A BACTERIAL INOCULANT

3.1 Introduction

Preservation of forage crops as silage has increased in popularity over the past
years due to excellent conservation of nutrients and the ability to obtain a higher
quality roughage. The success of ensiling forage relies on the presence of adequate
numbers of microorganisms, soluble sugars for use as substrates and an anaerobic
environment. Fulfillment of these conditions will allow a lactic add fermentation to
predominate (Whittenbury, et al., 1967). Kroulik, et al., (1955) reported that there
was a considerable variation in the numbers of bacteria found on green plants and cut
forages. Bacterial populations varied with the type of plant, anatomical location,
season, weather conditions and plant maturity.

Bacteria responsible for a rapid fermentation and production of a quality forage are
predominately lactic acid producers (Kempton and Clement, 1959; Langston and
Bouma, 1960). The addition of m sp. to fresh forages has been
recommended for control of silage fermentation (Lesens and Shultz, 1968; McDonald,
et al., 1964). Previous efforts (Bolsen, 1978; Thomas, 1978) to utilize microbial
additions to silage have varied from no response to increased DM and protein
recovery.

As milk production increases, the requirement for total N for the lactating cow

27

28

increases. The intake of ruminally degradable N often exceeds the amount which is
converted into microbial protein. Consequently, protein nitrogen supply to the small
intestine may be limiting. Efiidency of N utilization is improved as more rumen
undegradable protein is fed (Waldo and Glenn, 1984). _ Titgemeyer, et al., (1989)
evaluated amino add disappearance floor the small intestine with four dietary protein
supplements. In their study, each protein supplement was inadequate in at least one
of the essential amino adds, thus suggesting that amino add requirements of
ruminants should be supplied by a combination of protein supplements.

The objectives of this study were to examine the ensiling characteristics of alfalfa
forage treated with or without the addition of a bacterial inoculant and to evaluate the
response of lactating Holsteins and crossbred beef heifers fed the silage in combination

with a slow or rapidly degradable rumen protein source.

3.2 Materials and Methods
3.2.1 Silo Filling and Sampling

Two hundred and sixty tons of 1/10th bloom first cutting alfalfa forage was
wilted to 45% dry matter (DM), chopped to .6 cm length and ensiled in two top
unloading upright concrete stave silos (4.3 x 18.3 M). One silo served as a control
silo, containing uninoculated forage material (C), while the other was inoculated (I)
with a commerdal inoculant (Ecosyl, CIL Inc., Ontario, Canada N6A 4L6). The
inoculant contained a strain of WW and was applied in liquid
form at the blower to provide 2.5 x 10‘ colony forming units cfu/g of chopped
forage. Each silo was filled in an alternate load sequence. Incoming loads of
forage were sampled for DM determination and composited based on whether they
were harvested in the AM or PM of each filling day. Samples were frozen (-10 °C)
for later laboratory analyses. Thermocouples positioned at the center and outer
perimeter of the silos. Two were placed at four elevations (1.5, 5.3, 9.1 and 12.9 m)
in each silo. Temperature changes were monitored over a 45 d post ensilement
period. Three nylon bags were buried near the thermocouples at each of the four
elevations in each silo. Upon retrieval, bags were emptied and the contents were
frozen for later laboratory analyses. Differences in DM weights in each bag before
and after ensiling were used to estimate DM recovery. Samples of fermented
silage were taken with a Pennsylvania State Forage Sampler ( Nasco, Fort.
Athnson, WI 53538) from ports in a door 1.5 m from the bottom of the silo on d 0,
1, 2, 3, 5, 7, 10, 13 and 45 post ensiling for LAB enumeration and chemical

analyses. During feedout, samples of silage were taken twice weekly from each

29

30

silo. Dry matter was determined, and samples were composited and frozen (-10 0C)

for later laboratory analyses.
3.2.2 Lactic Add Bacteria Enumeration

One hundred g of forage material were diluted with 900 ml of sterilized,
distilled water, placed in a Waring blender (Waring Products Inc., New York, NY),
and agitated for 30 s. The homogenate was strained through 2 layers of
cheesecloth. Serial dilutions (1:10 ml) were prepared using a 0.1% peptone (Difco,
Detroit, MI) medium. Microbial enumeration was determined on LBS (BBL,
Cockeysville, MD) 8881’ Plates inoculated with .2 ml of appropriate dilutions, using
a micropipetter. Plates were incubated aerobically for 45 hrs and colony forming

units were counted presumptively as lactic add produdng bacteria.
3.2.3 Aerobic Stability

Aerobic stability of inoculated and uninoculated forage was studied eight
months post-ensiling to determine the quality of the silage upon exposure to air.
Approximately 1.3 kg of alfalfa silage from each silo was placed into each of 16
styrofoam containers (1600 cm’) and stored at room temperature (23 °C).
Temperature was monitored on a daily basis for 14 d. Duplicate containers were
emptied and subsamples obtained for both treatments on d 0, 1, 3, 5, 7, 10, and 14
ofairexposure. One hundredgofsilage were collectedbymixingthe entire
contents of each container and tahng random subsamples. These samples were
fiozen (-10 °C) for future laboratory analyses.

31

Temperature, pH, DM, total N, lactic 'add, ammonia N, soluble carbohydrate and

VFA’s served as indices of silage stability.

3.2.4 Preparation of Forage Samples

Fresh and fermented samples were removed from the freezer and minced
through a Hobart macerator. Approximately 100 g of material were placed in a
convection oven (60 °C) for 48 hrs, to determine DM (AOAC, 1984). Dried samples
were ground through a Cyclotec sample mill (Tecator Inc., Hemdon, VA), for
further analyses. Dried plant material was ashed in a mufile furnace (600 °C)
overnight to determine ash content (AOAC, 1984). Gross energy was determined
on the wet minced samples using an Automatic Adiabatic Bomb Calorimeter (Parr
Instrument Co., Moline, IL).

Neutral detergent fiber (NDF) and add detergent fiber (ADF) was determined
according to the procedures of Goering and Van Soest (1970).

A 10% homogenate was prepared by mixing 20 g of fresh or fermented forage
material with 180 g of distilled water and blended in a Sorvall Omnimixer (Ivan
Sorvall Inc., Newton, CT). The homogenate was strained through two layers of
cheesecloth, and allowed to stand for 15 min. before pH determinations were made.

Total N concentrations of fresh and fermented plant material was determined
by semi-micro Kjeldahl digestion followed by colorimetric N analysis (AOAC, 1984)
using a Technicon Autoanalyzer II (Technicon, Terryton, NY). The difference
between total N and N content after protein predpitation with 50% sulfosalicylilic
add (SSA), 1 part SSA to 10 parts of 10% homogenate, and centrifuged at 15,000 x

g for 20 min., was used to represent soluble N. Add detergent insoluble nitrogen

3 2
was determined by Kjeldahl nitrogen analysis on the ADF residue. Ammonia-N
concentration was determined on 10% homogenates using the Technicon
Autoanalyzer II.

Lactic add concentration was determined using appropriate aliquots of water
soluble extract according to the procedure of Barker and Summerson (1941).

Soluble carbohydrate analysis (Dubois et al., 1956) was performed on the 10%
plant homogenates.

Volatile fatty add concentrations in fresh and fermented plant tissues were
determined by gas chromatography. Twenty ml of 10% homogenate was diluted
with 4 ml of 25% metaphosphoric add and centrifuged at 15,000 x g for 20 min.
Two ul of supernatant were injected into a Hewlett-Packard Gas Chromatograph
(5840A, Hewlett-Packard, Farmington Hills, MI 48024) with flame ionization
detector equipped with a 1.8 m x .2 mm stainless steel column (Supelco MR56559)
packed with 10% SP—1200 and 1% H,PO, on chromosorb WAW (80/100-Supelco
Inc., Bellefonte, PA).

3.2.5 Lactation Trial

Thirty-two Holstein cows were blocked according to calving date and parity. At
initiation of the trial, cows averaged 59 d post-partum. Cows were fed a 40:60
alfalfa silagezconcentrate total mixed ration ad libitum, along with five pounds of
alfalfahayperday. Attheendofthe21dpreliminaryperiod,cowsbegana56d
experimental period and were fed a ration comprised of 50% alfalfa silage and 50%
concentrateinsufidentquantitiestoallowa 10%refusal. A2x2factorial

arrangement of treatments was utilized to difi'erentiate difi'erences in milk

33

TABLE 5. Diet Ingredients Fed to Holstein Cows During Lactation Trial

 

Rumen Degr_adability

 

Ingredients Slow (SD) Rapid (RD)
% DM Basis
Alfalfa silage 50.00 50.00
High moisture corn 41.03 41.80
Soybean meal 2.05 8.20
Corn gluten meal 3.77 0.00
Blood and meat meal 2.05 0.00
Mono-dicaldum phosphate 0.00 0.41

Trace Mineral Salt 0.33 0.35

 

enzymatic processes contribute little to silage preservation. However, the Gram
positive lactic add produdng bacteria, are facultative anaerobes, which enables
them to utilize soluble sugars to carry out metabolic functions aerobically on the
plant or anaerobically in the silo. The number of LAB on growing alfalfa is
generally low, usually less than 100 cfu/g and reduced further during wilting
(Keddie, 1959; Stirling and Whittenbury, 1963). However, counts of lactobale
usually rise significantly by the time they reach the silo. This is partly due to
inoculation of microorganisms from farm machinery (Henderson, et al., 1972;
Gibson, et al., 1961).

Until 197 8, there was little known about the composition of microflora during
silage fermentation. However, Beck (197 8) studied the qualitative changes in LAB
during the fermentation of grass and red clover with high and low DM contents.
He reported that fermentation in wilted and unwilted silage was initiated by
homofermentative LAB being 5% of total lactobadlli present by day 4. However,
after 142 d of fermentation, 75% of all lactobadlli in the silage with the low DM
and 98% of the lactobadlli in the silage with high DM were heterofermentative.
Beck suggested that bacteriologic shift could be due to a greater acetate tolerance
in heterofermentative bacteria. Table 1 shows the bacterial spedes commonly
found in silage (McDonald, 1981). The dominant organisms in silage according to
Langston and Bouma (1960) are L. planta;u_m, L. bgvigr and W sp.

Gibson, et al., (1958) reported that L. plum and L. ag'dgphilus were the
dominant homofermentative bacteria in fermentation. While others (Langston, et
al., 1962; Moon, 1981, and Moon, et al., 1981) revealed evidence that streptococd
and leuconostocs initiate fermentation and are superceded by spedes of Lactobadlli

and Pediococd.

TABLE 1. Classification of Lactic Add Bacteria Important in Silage

 

(A) Hetemfermentatjve
99ers

Leuconostoc mesenteroides
Leuconostoc dextranicum
Leuconostoc cremoris

M

Lactobadllus brevis
Lactobacillus fermentum
Lactobadllus buchneri
Lactobadllus viridesceno

(B) W
QOOC‘UB

Streptococcus faecalis
Streptococcus faedum
Pediococcus addilactid
Pediococcus cerevisiae
Pediococcus pentosaceus

Red

Lactobadllus plantarum

Lactobadllus curvatus

Lactobacillus casei

Lactobacillus coryniformis subsp. coryniformis

 

McDonald, P. 1981

Table 2 illustrates the products of an anaerobic sugar fermentation by LAB
described by Whittenbury and coworkers (1967). Glucose and fructose are the most
common soluble sugars utilized by LAB, however LAB can also ferment pentoses,
xylose and arabinose, which are formed from the degradation of hemicellulose
(Dewar, et al, 1963) and amino adds (Rodwell, 1953).

2.1.3 Plant Proteolysis

The deamination of protein in silage is another process resulting from plant
enzyme activity. The breakdown of fresh plant material can be caused by plant
proteases (Bergen, et al., 1974; Ohshima and McDonald, 1978), however, most
proteolytic activity is a result of aerobic conditions inside the silo.

Figure 1 illustrates post-harvest nitrogen metabolism in ensiled plant material
from hay and cereal crops (Bergen, 1974). Fresh forage material contains 70-90%
of the total nitrogen in the form of protein while the remaining 10-30% is non-
protein nitrogen consisting of free amino adds, amides and small concentrations of
urides, amines, nucleotides, chlorophyll, low molecular weight peptides and amino
adds bound in non-protein form (Hegarty and Peterson, 1973). It is not uncommon
for 50-60% of the true protein nitrogen to be broken down into simpler non-protein
nitrogenous compounds in preserved forage (Whittenbury, 1967).

Amino acids resulting fiom proteolysis can be metabolized into ammonia
(deamination), amines (decarbouylation) and unidentified nitrogenous compounds
(Bergen, et al., 1974; Ohshima and McDonald, 1978). A good quality silage is
characterized by low concentrations of ammonia-N, amines and other compounds
produced from the break down of amino adds (Bergen, 1984). If aerobic conditions

remain in the silo it creates an environment which allows yeast and mold to

TABLE 2. Anaerobic Pathways of sugar Metabolism by Lactic Add Bacteria

 

W
1 glucose-mo 2 Lactic add
1 fructose-----> 2 Lactic add

1 pentose-----> 1 Lactic add + 1 Acetic add

W

1 glucose-

 

-> 1 Lactic add + 1 Ethanol + 1 Carbon dioxide
3 fructose-----> 1 Lactic add + 2 Mannitol + 1 Acetic add

1 Pentose-----> 1 Lactic add + 1 Acetic add

 

Whittenbury, et al., 1967

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l 0
multiply and increase the silage temperature (Bergen, 1984). Clostridial
fermentation is assodated with ammonia, butyric acid and a higher pH than that
found with lactic add bacteria. This results in an unstable and often unpalatable
silage. Butyric acid produced by sacchrolytic organisms which metabolize lactate
and sugars, (Table 3) often serves as an indicator of clostridial activity. The result
of this type of fermentation occurs at a high DM or a low pH (Whittenbury, et al.,
1967). Woolford (1984) suggested that clostridial activity is suppressed at a dry
matter above 31% and/or a pH below 4.5. Under ideal conditions, sufident
numbers of lactic add produdng bacteria occurring naturally, would produce a
drop in pH during day 2-5 of ensilement. Bergen and coworkers (1974) suggested
that DM of forage material at the time of ensilement is the most dedsive factor
influendng the amount of protein degradation which will occur during
fermentation. The lower the DM, the larger the amount of plant protein escaping
proteolysis. Thus, DM at the time of ensiling and rate at which the pH falls

during fermentation are factors one must consider during silage preservation.
2.1.4 Substrate Utilization During Silage Fermentation

The major water soluble carbohydrates (WSC) found in forage material are
glucose, fructose, sucrose and fructosans. The most available sugars for microbial
substrate are glucose and fructose, due to the continual hydrolysis of sucrose and
fructosans to glucose and fructose monomers (Whittenbury, et al., 1967).

The WSC content as well as the fructose/glucose ratio of green fodder plants
varies depending on spedes, weather conditions, stage of gowth, time of day,
wilting conditions and fertilizer application (Woolford et. al., 1982).

Soluble carbohydrates present in forage material after aerobic metabolism are

Jo!

11

TABLE 3. Biological Reactions Associated with Clostridial Fermentation

 

 

Organic Adds
2 Lactic add > 1 Butyric add + 200, + 2H,
Amino Adds
(A) Coupled oxidation-reduction reactions
1 Alanine + 2 Glydne--->3 Acetic add + 3NH3 + 100,
(B) De-amination
3 Alanine-mm) 2 Propionic add + 1 Acetic add + 3NH, + 100,
1 Valine-----> 1 lsobutyric add + 1 NH, + 1 CO,
1 Leudne-«--> 1 Isovaleric add + 1 NH, + 1 CO,U
(C) Decarboxylation

Histidine----> Histamine

Lysine------> Cadaverine
Arginine—------> Ornithine~--~~->Putresdne
Tryptophan»---> Tryptamine
Tyrosine-~«--> Tyramine

 

12

fermented by a variety of microorganisms, however, under ideal conditions LAB
ferment sugars and produce an intolerable addic environment for other
microorganisms (Whittenbury, et al., 1967). Lactic add bacteria utilize soluble
sugars through two fermentable pathways to produce lactate (Table 2,
Whittenbury, et al., 1967), as previously described. Homofermentative LAB are
the most desirable for they are more emdent in producing lactate than
heterofermentative LAB (produdng 2 moles of lactic add versus one mole), and
more efident in conservation of DM (McDonald, et al., 1973). One cannot predict
a final ratio of fermentation products, for it is possible to have 100% variation
occur in the amount of lactic add produced under two similar drcumstances.

In addition to phosphate, several organic adds also are commonly found in
fresh herbage and silage. These adds include malate, dtrate, and glycerate
(McDonald, 1979). Organic adds in combination with their salts comprise a
bufi'ering system in plants (Playne and McDonald, 1966). Legumes contain higher
amounts of add (0.6 to 0.8% of DM) than grasses (0.2 to 0.6% of DM), as well as
higher protein and more cations which contribute to a much greater bufi'ering
system.

Considerable interest has been given to those organic adds in silage which
bufi‘er within the pH range of 4-6. Early stages of fermentation are characterized
by the dissimilation of organic acids by LAB (Edwards and McDonald, 1978). The
main products of citrate and malate fermentation by LAB are shown in Table 4
(Whittenbury, et al.,1967). Products from these reactions include formation of
organic salts (lactate, acetate), neutral products (ethanol, acetone and 2,3
butanediol) and alkaline released cations (Whittenbury, et al., 1967). Other
substrates which can be fermented by LAB include amino adds (Rodwell, 1953).

13

TABLE 4. Fermentation of Organic Adds as Substrates by Lactic Add Bacteria

 

A. 1 Citric add------> 2 Acetic add + 1 formic add + 1 carbon dioxide
or

2 Citric add-mmo 2 Acetic add + 1 acetone (or 2,3 butanediol) + 4 carbon
dioxide.

or
2 Citric add-mm» 3 Acetic add + 1 lactic add + 3 carbon dioxide
B. 1 Malic add-----> 1 Acetone (or 2,3 butanediol) + 4 carbon dioxide

or
2 Malic add-----> 1 Acetone (or 2,3 butanediol) + 4 carbon dioxide

or

1 Malic add

 

--> 1 Acetic add (or ethanol) + 1 formic add + 1 carbon
dioxide

 

Whittenbury et al., 1967

14

Brady (1966) demonstrated that M and L. hrem’ can deaminate serine,

arginine, glutamine and aspargine.
2.1.5 Aerobic Stability of Silage

The most important factor in achieving high quality silage is rapid occurrence
of anaerobiosis in the silo. Other factors influendng aerobic deterioration include
quantity of substrate, DM of the ensiled crop, botanic origin and ambient
temperature (Woolford, 1990).

Aerobic deterioration of silage ultimately results in complete mineralization of
easily oxidized nutrients which are broken down into CO, and H,O, generating
heat and resulting in DM losses (Woolford, 1984). Studies have shown that DM
lossesoveraperiodof5-l5 dayscanbeasgreatas32%. Oncetheprocessof
aerobic deterioration commences, it is practically impossible to stop (Honig and
Woolford, 197 9).

Aningressofairassmallas 100tol50mgO,/ngMisadequatetomake
silage highly susceptible to aerobic deterioration (Woolford, et al. 197 9). Upon
exposure to oxygen, conditions become favorable for proliferation of aerobic
bacteria, yeasts and fungi (Moon et al., 1980 and Woolford et al., 1982). In most
silages, yeasts have the ability to increase in numbers from <102 to 1012 cfu/g DM
byday 3 ofaerobic exposure(Beck 1963, asdtedbyWoolford, 1990).

Yeasts involved in aerobic deterioration have been classified as add-utilizers
convened wmmmmmmdamnm and sugar-
ut'ilizers which are M sp. (Gross and Beck, 1970, as dted by Woolford,
1990; Moon and Ely 1979; Johnsson and Pahlow, 1984). A high population of

yeasts does not necessarily mean a silage will deteriorate. Instead, quantity of

l 5
lactate-utilizing yeasts deddes whether a silage will deteriorate or not upon
aerobic exposure (Johnnson and Pahlow, 1984).

Thermophilic filamentous fungi are also found in deteriorating silage, however
their growth is generally slower and thus have little afi'ect on silage as a feed.

Woolford and Cook (197 8) treated silage material with antibiotics that had
antibacterial and antifungal properties. Their studies revealed the involvement of
proteolytic bacteria from the genus Badllus. Bacteria appear to initiate
deterioration in maize silages, followed by yeasts (Woolford et al., 1978).
Deterioration in cereal crops and grass silages on the other hand, begins with
yeasts (Woolford et al., 1979). However, Woolford (1984) concluded that this
inconsistency concerning the identity of microbial groups responsible for the onset
of aerobic deterioration lies in the properties of ensiled material, spedfically DM
content rather than botanic origin.

Primary substrates of aerobic deterioration have been described as nitrogen
free extracts which included water soluble carbohydrates and organic adds (Honig
and Woolford, 1979). Woolford (1990) suggests that the organisms involved in
aerobic deterioration will use a wide range of substrates which include those found
in the original crop and others which are produwd by fermentation. Regardless of
the substrate utilized, deterioration in forage crops is always accompanied by a loss
of residual sugars and the evolution of ammonia and carbon dioxide. The latter
canbedirectlyequatedtoDMloss audits measurementcanbemdtomonitor
the progress of deterioration (Woolford, 1990). Fermentation acids (such as acetic
and lactic adds), amino adds and proteins are all used as substrates (Woolford,
1984). The pH increases with add depletion and tends to be greatest at the silage

surface where exposure to oxygen is greatest (Woolford, 197 8).

16

Aerobic deterioration occurs in all silages to some varying degree, except for
those undergoing an extensive secondary fermentation. This deterioration
depredates conservation efidency, causes nutritional losses and can even pose a
potential health hazard to livestock. Such management practices as rapid silo
filling, spedal cutting equipment for forage removal, rescaling between feed-outs
and use of an efi'ective inoculant at the proper application rate can minimize

aerobic deterioration.
2.1.6 Silage Inoculants

At the present time, there are several silage inoculants on the market. They
have been reported to influence the rate and extent of silage fermentation. Typical
ingredients found in inoculant may include enzymes, bacteria, molds,
micronuhientsformicroorganismsoramixtumsofaflthesetoinfluencefomge
respiration and fermentation rate (Parker, 1979). Bolsen (1978) has described
silage inoculants as ”those products that supply lactic add produdng
microorganisms and enzymes and/or microorganisms that increase the availability
of carbohydrates and other nutrients to lactic add produdng microorganisms”.

Commerdally available inoculants not only vary in ingredients but in type of
preparation (dried,-1iquid, fieszedried) and packaging (bottles, vacuum packs and
paper sacks). .

Whittenbury (as cited by Beck, 1978) described the requirements of a quality

silage microorganism as follows:

1. It must be fast growing and able to compete with and
dominate other microorganisms present in silage.

17

2. It must be homofermentative.
3. It must be add tolerant down to a silage pH of 4.0.

4. It must possess the ability to ferment glucose, fructose,
sucrose, and preferably fructosans and pentosans.

5. It should have no action on organic adds.
And in 1975, McCullough described the following as requirements of a cost
efi’ective quality inoculant:

1. The cost of the additive must be less than the silage lost
without the additive.

2. Addition of the additive must result in a more emdent
fermentation than occurs naturally.

3. The additive should produce a silage with a greater digestibility
energy and/or protein than untreated silage.

Several workers have shown varying results from inoculation, including
advantageous results (Rooke et al., 1985; Ohyama et al., 1975 and Owens, 1977)
and non-significant results (Ely et al., 1982; Moon et al., 1981 and Buchanan-

Smith and Yao, 1981).

2.2 Rumen Cellulolytic Bacteria and Their Role in Fiber Digestion
2.2.1 Rumen Microbial Fermentation and Digestion

The rumen is an ideal fermentation site. It makes up one-seventh of the total
mass of a ruminant’s body weight (Russell and Hespell, 1981). The rumen remains
at a constant temperature of 39°C and is well bufi‘ered by salivary secretions. The
microflora inhabiting the rumen is dense containing approximately 101° to 1011
bacterial and 10‘ protozoal cells per milliliter of rumen contents. There is an
extensive diversity and synergism in the ecosystem which contains more than 200
spedes of bacteria and over 20 spedes of protozoa (Bryant and Robinson, 1962).

During ruminal fermentation, feedstufi‘s are broken down and fermented into
short chain fatty adds through microbial metabolism and are used as the
ruminant’s energy source, while the animal relies heavily on the microbial mass as
a protein source. Methane, heat, and ammonia are formed as well, representing a
loss of energy and nitrogen to the animal. The balance of fermentation products
determines the efidency of nutrient utilization in ruminants. In turn, this balance

is ultimately controlled by the various microorganisms found in the rumen.
2.2.2 Plant Cell Wall Constituent

In ruminants the plant cell wall is extensively degraded and utilized as an
energy source by the rumen microflora. Plant cell walls are indigestible by animal

enzymes, however, gastrointestinal microflora partially degrade cell wall material.

18

19

The cell wall of plants is made up of an organic matrix of cellulose, hemicellulose,
lignin and other small fractions of pectins, gums mucilages, cutin, tannin, bound

cell wall protein and cell wall minerals.

Cellulose

First recognized by Payen in 1939 (Whistler and Smart, 1953), cellulose is the
most abundant carbohydrate in the world. Its recycling is dependent on microbial
activity which produces carbon dioxide during degradation. An enormous amount
of energy lies in these cellulosic carbohydrates, mahng them an excellent food
source for herbivores. Cellulose is the largest component of plant cell walls, thus
serving as a primary structural element. Linked at the C-1 and C-4 position
through glycosidic linkages, individual anhydrous glucose molecules make up the
linear polymer in a beta configuration. Glucan chains consist of 100 to 10,000 or
more units of glucose (Ott and Tennent, 1954), and are held together by tight
hydrogen bonds (Albersheim, 197 5) between the hydroxyl group of a sugar on one
chain and an oxygen atom of another. Chains are also held together by
VanderWaals forces.

Hemicellulose

Hemicellulose is the second largest constituent found in plant material
(Phillips, 1940). First named in 1891 by Schultz (Whistler and Richards, 1970),
hemicellulose has been defined as the polysaccharide in plant tissues other than
cellulose which is extracted with alkali and hydrolyzed in add (Collings, 1979).

Hemicellulose is a complex mixture of polysaccharides which constitute much of
the cell wall matrix (Bailey and Gaillard, 1965). It is a polybeta 1-4 D-

20

xylanopyranose based on a backbonehf xylose residues, with branches of arabinose,

glucose and/or galactopyranosides (Ahn and Barton, 1983).
lignin

Lignin is a polymer of phenylpropanoid units intimately assodated with
structural carbohydrates (Himmelsbach and Barton, 1980), and plays a major role
in redudng microbial attack on cell walls ( Ahn and Barton, 1983). Phenolic adds
such as p—coumaric add and ferulic add which are precursors of lignin can bind to
structural carbohydrates which inhibits carbohydrate degradation (Hartley et al.,
1974).

Other Constituents

Pectin is comprised of chains of galacturonic add, galactans and arabinans
(Aspinall, 1973). Pectins are not pure polysaccharides, but mixed and branched,
forming complex polysaccharide structures. It is found in intracellular spaces in
the cell wall and is assodated with cellulose in other cell layers (Esau, 1965).
Hemicellulose, pectin and lignin play an important role as matrix substances for
the cell wall.

Cowling (1976) demonstrated that crystallinity and lignification are the most
important factors in determining the susceptibility of cellulose to enzyme
degradation. It has been shown that spedfic enzymes which attack glucan bonds
in cellulose chains are incapable of attachng an intact plant fiber (Albersheim,
1975). Thus accessibility of cellulose to microbial enzymes and chemical magenta
depends on the arrangement of cellulose within the cell wall( Collings, 197 9).

Although some plant material is accessible and easily digested, the degradation

21

of fiber material in the rumen is a result of complex microbial processes (Cheng et
al., 1980). These processes include the digestion of plant cell walls, to yield
microbial cell growth and fatty adds and products, As with any ecological system,
the microorganism should be attracted to its nutrient substrate. It has been
demonstrated that plant material undergoing colonization and digestion by rumen
microorganisms includes the adherence of bacteria, protozoa and fungi, however,
bacteria are responsible for the majority of the digestion which takes place in the
rumen (Hungate, 1966). Ahn and Barton (1983) found through the use of the
scanning electron microscope (SEM) that plant cell wall digestion did not occur
unless rumen bacteria were closely assodated with or completely adhered to the
cell walls.

2.2.3 Rumen Cellulolytic Spedes

Based on relative numbers in the rumen of domestic ruminants and their
ability to attack various forms of cellulose in pure cultures, the major rumen
cellulolytic bacteria are W 11% (Sijpestein, 1951),
Mammalian (Hungate. 1957). andbeesrddes fibrobacter Masses
(Hungate, 1950). These are the three major spedes which obtain their energy for
growth solely through cellulose fermentation (Bryant, 197 3). L m will
digest cellulose to a lesser extent (Bryant, 1973; Hungate, 1966). Each of these
spedes except B, W are capable of utilizing hemicellulose-type
components fiom forage (Dehority and Scott, 1967). L W is the most
active cellulolytic, bacterium digesting the more resistent cellulose such as cotton

fibers and mature hay to a greater extent than W which are active,

22

but show much more variation between strains in ability to degrade more resistant
cellulose (Bryant, 1973).

Minato and coworkers (1966), noted that both Ruminm and B
W adhere to fiber during digestion, however, _B_, sgcdnggenes was firmly
attached to the cell wall. ‘A few other cellulolytic spedes of the genus Qlostridium
(Hungate, 1957; Shane et al., 1969) and Eubacterium gem (Bryant et al.,
1958; Van Gylswyck and Hoffman, 1971) have been found in the rumen
occasionally.

The largest numbers of cellulolytic bacteria are found when the ruminant is fed
a high roughage diet, however in ruminants fed cellulose as the total feed source,
cellulolytic bacteria only comprise 25% of the total rumen microbial population
(Slyter et al., 1971). Many non-cellulolytic bacteria found in the rumen are
responsible for the degradation of pectins and xylans. Numerous synergistic
interactions between cellulolytics and noncellulolytic spedes occur and has been
shown to enhance cellulose degradation (Dehority and Scott, 1967).

Rumen cellulolytics produce cellulose enzymes which hydrolyze insoluble
cellulose into soluble cellulodextrins or sugars, some of which they can absorb and
ferment to obtain energy for growth (Schaefer and King, 1965; Sheth and
Alexander, 1969).

End products of cellulose degradation include acetate, propionate, butyrate, CO,
methane, and microbial cells. This includes interacting populations of 1) rumen
cellulolytic bacteria, 2) carbohydrate fermenting spedes which can use products
hydrolyzed fi'om cellulose, 3) spedes which will degrade sucdnate, formate and any
lactate produced from microbes in 2 and 4) methanogenic bacteria which will

reduce CO, using H, or formate as an electron donor (Hungate, 1950).

23

All rumen cellulolytic bacteria require one or more B-vitamins for growth.
Biotin is the most common vitamin required by the cellulolytics. However, some
strains ofBJIngalsorequire pyridoxine. Afew strains ofthgmay require
folic add, riboflavin or thiamine (Bryant, 1973). The vitamins required by B,
W strains are similar to those required by BALM (Bryant and Robinson,
1961; Gill and King, 1958; Scott and Dehority, 1965), with some strains mquiring
pyridoxine and cobalamine which in some cases can be replaced by methionine
(Scott and Dehority, 1965). B W requires biotin, using this as its
primary B-vitamin. P-aminobenzoic acid has been shown to stimulate the growth
in some strains of B, M (Bryant and Robinson, 1961; Scott and Dehority, 1956).
B, W has a requirement for Na+ and a great demand for Ca2+ (Bryant et
al., 1959). The other cellulolytics have a lower demand for K‘, N a”, and Ca“.
Ferrous iron and Zn“ has been found to stimulate microbial activities even further
(Matturi, 1972).

All of the rumen cellulolytics have a requirement for sulfur. W
utilizes cysteine or sulfide, but not sulfate (Bryant et al, 1959). The W
grow well in media containing sulfide or sulfate (Bryant, 1973).

The main nitrogen source for cellulolytic bacteria is ammonia (Bryant and
Robinson, 1961; Bryant et al., 1959, Dehority, 1963). The ammonia is a product of
non-cellulolytic bacteria metabolism. This is just another example of co-existence
and cooperation between rumen spedes. Cellulolytic bacteria lack the ability to
use organic nitrogen sources for growth and though not established, it appears that
they probably lack the mechanism for transporting amino adds or peptides into the
cell (Pittmann et. al, 1967). Although W bacteria cannot use amino

adds if present, B, M will utilize the amide nitrogen from glutamine

24

and asparagine for growth and function (Bryant and Robinson, 1961).

Many strains of rumen cellulolytic bacteria require a carbon source beyond that
of the energy source. The source commonly used by these bacteria is CO, or
bicarbonate. B W and B, flgvgfadgg require large amounts of 00,,
which is fixed into pyruvate during glycolysis (Caldwell et al., 1969). Without 00,,
these bacteria are unable to obtain energy in the form of carbon, for growth
(Bryant, 1973). They also use CO, for biosynthetic purposes (Allison, 1969; Allison
197 0). B, ngg does not require large amounts of CO, for growth, but requires
small amounts for optimal growth and for biosynthetic processes (Bryant, 197 3).

Short chain fatty acids, better known as volatile fatty adds are essential for
growth of the three major rumen cellulolytics at 0.5-0.3mM in batch cultures
(Dehority and Scott, 1967). Carbon skeletons from these fatty adds are not
degraded, but incorporated into certain cellular constituents (Bryant, 1973 ).
W W is the only cellulolytic that requires the straight chain
valeric add, which can be replaced by longer chain adds (Wegner and Foster,
1963). The cellulolytic bacteria utilize the various branched chain fatty adds, such
as C14 and Cu from isobutyric, CH5 and 0,, from isovaleric, and anteisa C115 and C"
from 2-methyl-butyrate (Allison, et al., 1962; Wegner and Foster, 1963). These
branched chain fatty adds are also precursors for fatty aldehydes in these bacteria.
One or more of the above fatty adds are used for the biosynthesis of amino adds:
valine, leudne, and isoleudne respectively (Allison et al., 1962; Robinson and
Allison, 1969; Allison, 197 0) via reductive carboxylation reactions (Bryant and
Robinson, 1961; Allison, 1969).

25

2.2.4 Cellular Attachment and Digestion of Plant Material

There are many factors which influence the rate and extent of forage cell wall
digestion. Feeds containing fractions of cellulose and hemicellulose are relatively
insoluble in the rumen and are degraded slowly (Dehority, 1973; Van Soest, 1973).
Degradation is highly influenced by structural factors. Such factors would include
the close assodation of lignin with cellulosic materials, acting as a barrier against
bacterial cellulases (Russell and Hespell, 1981). Crystallinity also efi'ects digestion
(Bryant and Robinson, 1962). Russell and coworker (1981), showed that high
crystalline fibers were readily degraded by cellulases from certain cellulolytic
bacteria while fiber digestion was much slower for other cellulolytic spedes. Those
who have made extensive observations (Ahn and Amos, 1975; Ahn et al., 1974) of
mixed cultures of rumen bacteria have observed that many rumen bacteria appear
to adhere to plant cell walls by means of thin fibrous capsules. In many of these
observations, it has been noted that the bacteria digest plant cell wall material and
infiltrate the resultant cavities.

Cheng and coworkers (1977 ) found that bacteria in the rumen of cows fed corn
silage versus other forage based diets had the least bacterial slime formation, but
every bacterial cell showed some extracellular structure. Although some plant
material is accessible and easily digested, the process is. long and sequential (Ahn
and Amos, 197 5). Digestion begins with penetration through the stomata (Baker
and Harris 1947) and colonization on fiber macerations produced from mastication.
Dinsdale et al., (1978) in an i_n_vi1m study demonstrated that mixed populations of
rumen bacteria released 12 to 36% of the dry matter of damaged cells in legume
leaves. These organic nutrients are used to support enormous proliferations of

bacteria in intracellular space and at the leaf surface. Subsequently, plant cell

26

walls are ruptured by certain spedes of bacteria who digest cellulose in grasses
and cellulose and pectins in legumes (Dinsdale et al., 1978). Plant protoplasm
which remains to be digested supports a further proliferation of bacteria until
bacterial microcolonies fill plant cell wall compartments, while refractory cells

remain uncolonized (Ahn and Amos, 1975).

3.0 FERMENTATION CHARACTERISTICS AND NUTRITIVE VALUE
OF ALFALFA FORAGE ENSILED WITH AND WITHOUT ADDITION
OF A BACTERIAL INOCULANT

3.1 Introduction

Preservation of forage crops as silage has increased in popularity over the past
years due to excellent conservation of nutrients and the ability to obtain a higher
quality roughage. The success of ensiling forage relies on the presence of adequate
numbers of microorganisms, soluble sugars for use as substrates and an anaerobic
environment. Fulfillment of these conditions will allow a lactic add fermentation to
predominate (Whittenbury, et al., 1967). Kroulik, et al., (1955) reported that there
was a considerable variation in the numbers of bacteria found on green plants and cut
forages. Bacterial populations varied with the type of plant, anatomical location,
season, weather conditions and plant maturity.

Bacteria responsible for a rapid fermentation and production of a quality forage are
predominately lactic add producers (Kempton and Clement, 1959; Langston and
Bouma, 1960). The addition of m sp. to fresh forages has been
recommended for control of silage fermentation (Lesens and Shultz, 1968; McDonald,
et al., 1964). Previous efi'orts (Bolsen, 1978; Thomas, 1978) to utilize microbial
additions to silage have varied from no response to increased DM and protein
recovery.

As milk production increases, the requirement for total N for the lactating cow

27

28

increases. The intake of ruminally degradable N often exceeds the amount which is
converted into microbial protein. Consequently, protein nitrogen supply to the small
intestine may be limiting. Efidency of N utilization is improved as more rumen
undegradable protein is fed (Waldo and Glenn, 1984). Titgemeyer, et al., ( 1989)
evaluated amino add disappearance from the small intestine with four dietary protein
supplements. In their study, each protein supplement was inadequate in at least one
of the essential amino adds, thus suggesting that amino add requirements of
ruminants should be supplied by a combination of protein supplements.

The objectives of this study were to examine the ensiling characteristics of alfalfa
forage treated with or without the addition of a bacterial inoculant and to evaluate the
response of lactating Holsteins and crossbred beef heifers fed the silage in combination

with a slow or rapidly degradable rumen protein source.

3.2 Materials and Methods
3.2.1 Silo Filling and Sampling

Two hundred and sixty tons of 1/10th bloom first cutting alfalfa forage was
wilted to 45% dry matter (DM), chopped to .6 cm length and ensiled in two top
unloading upright concrete stave silos (4.3 x 18.3 M). One silo served as a control
silo, containing uninoculated forage material (C), while the other was inoculated (I)
with a commerdal inoculant (Ecosyl, CIL Inc., Ontario, Canada N6A 4L6). The
inoculant contained a strain of Lamhgfl 1i plantarum and was applied in liquid
form at the blower to provide 2.5 x 10‘ colony forming units cfu/g of chopped
forage. Each silo was filled in an alternate load sequence. Incoming loads of
forage were sampled for DM determination and composited based on whether they
were harvested in the AM or PM of each filling day. Samples were frozen (-10 °C)
for later laboratory analyses. Thermocouples positioned at the center and outer
perimeter of the silos. Two were placed at four elevations (1.5, 5.3, 9.1 and 12.9 m)
in each silo. Temperature changes were monitored over a 45 d post ensilement
period. Three nylon bags were buried near the thermocouples at each of the four
elevations in each silo. Upon retrieval, bags were emptied and the contents were
frozen for later laboratory analyses. Differences in DM weights in each bag before
and after ensiling were used to estimate DM recovery. Sampr of fermented
silage were taken with a Pennsylvania State Forage Sampler ( Nasco, Fort
Athnson, WI 53538) from ports in a door 1.5 m from the bottom of the silo on d O,
1, 2, 3, 5, 7, 10, 13 and 45 post ensiling for LAB enumeration and chemical

analyses. During feedout, samples of silage were taken twice weekly from each

29

3O

silo. Dry matter was determined, and samples were composited and frozen (-10 °C)

for later laboratory analyses.
3.2.2 Lactic Add Bacteria Enumeration

One hundred g of forage material were diluted with 900 ml of sterilized,
distilled water, placed in a Waring blender (Waring Products Inc., New York, NY),
and agitated for 30 s. The homogenate was strained through 2 layers of
cheesecloth. Serial dilutions (1:10 ml) were prepared using a 0.1% peptone (Difco,
Detroit, MI) medium. Microbial enumeration was determined on LBS (BBL,
Cockeysville, MD) agar plates inoculated with .2 ml of appropriate dilutions, using
a micropipetter. Plates were incubated aerobically for 45 hrs and colony forming

units were counted presumptively as lactic add produdng bacteria.
3.2.3 Aerobic Stability

Aerobic stability of inoculated and uninoculated forage was studied eight
monthspost-endhngtodeterminethequahtyofthesflageuponexposumtoair.
Approximately 1.3 kg of alfalfa silage from each silo was placed into each of 16
styrofoam containers (1600 cm“) and stored at room temperature (23 0C).
Temperature was monitored on a daily basis for 14 d. Duplicate containers were
emptied and subsamples obtained for both treatments on d 0, 1, 3, 5, 7, 10, and 14
ofairexposure. One hundredgofsilage were collectedbymixingthe entire
contents of each container and tahng random subsamples. These samples were
frozen (~10 °C) for future laboratory analyses.

31

Temperature, pH, DM, total N, lactic "add, ammonia N, soluble carbohydrate and

VFA’s served as indices of silage stability.

3.2.4 Preparation of Forage Samples

Fresh and fermented samples were removed from the freezer and minced
through a Hobart macerator. Approximately 100 g of material were placed in a
convection oven (60 °C) for 48 hrs, to determine DM (AOAC, 1984). Dried samples
were ground through a Cyclotec sample mill (Tecator Inc., Herndon, VA), for
further analyses. Dried plant material was ashed in a mums furnace (600 °C)
overnight to determine ash content (AOAC, 1984). Gross energy was determined
on the wet minced samples using an Automatic Adiabatic Bomb Calorimeter (Parr
Instrument Co., Moline, IL).

Neutral detergent fiber (NDF) and add detergent fiber (ADF) was determined
according to the procedures of Goering and Van Soest (1970).

A 10% homogenate was prepared by mixing 20 g of fresh or fermented forage
material with 180 g of distilled water and blended in a Sorvall Omnimixer (Ivan
Sorvall Inc., Newton, CT). The homogenate was strained through two layers of
cheesecloth, and allowed to stand for 15 min. before pH determinations were made.

Total N concentrations of fresh and fermented plant material was determined
by semi-micro Kjeldabl digestion followed by colorimetric N analysis (AOAC, 1984)
using a Technicon Autoanalyzer II (Technicon, Terryton, NY). The difference
between total N and N content alter protein predpitation with 50% sulfosalicylilic
add (SSA), 1 part SSA to 10 parts of 10% homogenate, and centrifuged at 15,000 x

g for 20 min., was used to represent soluble N. Add detergent insoluble nitrogen

3 2
was determined by Kjeldahl nitrogen analysis on the ADF residue. Ammonia-N
concentration was determined on 10% homogenates using the Technicon
Autoanalyzer II.

Lactic add concentration was determined using appropriate aliquots of water
soluble extract according to the procedure of Barker and Summerson (1941).

Soluble carbohydrate analysis (Dubois et al., 1956) was performed on the 10%
plant homogenates.

Volatile fatty add concentrations in fresh and fermented plant tissues were
determined by gas chromatography. Twenty ml of 10% homogenate was diluted
with 4 ml of 25% metaphosphoric add and centrifuged at 15,000 x g for 20 min.
Two ul of supernatant were injected into a Hewlett-Packard Gas Chromatograph
(5840A, Hewlett-Packard, Farmington Hills, MI 48024) with flame ionization
detector equipped with a 1.8 m x .2 mm stainless steel column (Supelco MR56559)
packed with 10% SP-1200 and 1% H,,PO4 on chromosorb WAW (80/100-Supelco
lnc., Bellefonte, PA).

3.2.5 Lactation Trial

Thirty-two Holstein cows were blocked according to calving date and parity. At
initiation of the trial, cows averaged 59 d post-partum. Cows were fed a 40:60
alfalfa silagezconcentrate total mixed ration ad libitum, along with five pounds of
alfalfahayperday. Attheendofthe 21 dpreliminaryperiod, cowsbegana56d
experimental period and were fed a ration comprised of 50% alfalfa silage and 50%
concentrate in sumdent quantifies to allow a 10% refusal. A 2 x 2 factorial

arrangement of treatments was utilized to differentiate differences in milk

33

TABLE 5. Diet Ingredients Fed to Holstein Cows During Lactation Trial

 

Rmen Degadahility

 

 

Ingredients Slow (SD) Rapid (RD)
% DM Basis
Alfalfa silage 50.00 50.00
High moisture corn 41.03 41.80
Soybean meal 2.05 8.20
Corn gluten meal 3.77 0.00
Blood and meat meal 2.05 0.00
Mono-dicaldum phosphate 0.00 0.41

Trace Mineral Salt 0.33 0.35

 

34

production by feeding one of two protein supplements containing difl‘erent levels of
rumen degradable protein with each alfalfa silage (Table 5). The protein
supplement with rapid rumen degradability (RD) contained primarily soybean
meal, whereas the second protein supplement contained a blend of 50% corn gluten
meal, 25% blood and meat meal and 25% soybean meal, which represented a
slowly degraded rumen protein source (SD). Total mixed rations were sampled
once a week for DM determination. Samples were composited and sent to a
commerdal laboratory (Ohio Agr. and Dev. Center, Wooster, OH) for nutritional
analyses. All four diets were balanced for 17.5% crude protein and ranged from
17.5 to 18.5% throughout the experimental period. Feed intake and milk yields
were recorded daily. Milk was sampled on two consecutive milhngs each week,
composited and taken to the Michigan Dairy Herd Improvement Assodation
(DHIA) Laboratory (East Lansing, MI 48823) for determination of total protein and

fat. Cows were weighed weekly.

3.2.6 Growth Trial

Seventy-one Hereford x Angus heifers (226 kg) were randomly assigned to eight
pens of nine head each with the exception of one pen containing eight head.
Animals were weighed on two consecutive d at 28 d intervals. Heifers were fed
once each day, with intakes and arts measured daily. A. one week adjustment
period was utilized to familiarize heifers with the 50:50 alfalfazcorn silage diet.

Following the preliminary period, each pen was randomly assigned to one of
four treatments (Table 6) which included control or inoculated alfalfa silage and
corn silage fed with one of two protein supplements used in the lactation trial.

Diets were formulated to contain 14.0% crude protein and fed for 104 d.

35

TABLE 6. Diet‘ Ingredients Fed to Beef Heifers During Growth Trial

 

Rumen Degr__aggbility

 

Ingredient Slow (SD) Rapid (RD)
% DM Bagig
Corn silage . 51.60 51.60
Alfalfa silage 45.20 45.20
Corn gluten meal 1.54 0.00
Soybean meal .77 3.07
Blood and meat meal .7 7 0.00

 

‘Formulated to contain 30,000 IU vitamin A/hd/d; 150 mg/hd/d monensin; .25%
T.M. salt; 1 ppm/hd/d Se; .6% K; .5% Ca; .3% P.

3.3 Statistical Analyses

Statistical analysis of fermentation parameters and the growth trial were
conducted with the General Linear Models Subroutine in SAS (SAS Institute, 1987).
least square means were generated to compare treatments. Mean comparisons were
made with Bonferroni’s T-test, as described by Gill (197 8). Initial weight of beef
heifers at the beginning of the trial was used as a covariate in the analysis. Results of
the lactation trial were analyzed as a repeat measurement design with cows blocked
according to calving date and parity. Milk production during the 21 d preliminary

period was used as a covariate in the analysis of the experimental period.

36

3.4 Results and Discussion
3.4.1 Silage Composition

Materials entering silos were similar in DM, pH and lactic add content, while
forage entering the inoculated silo had a greater ammonia N and water soluble
carbohydrate (W SC) content than that entering the control silo (Table 7). Water
soluble nitrogen (WSN) was greater for the material entering the control silo
versus the inoculated silo.

The pH of ensiled forage material is presented in Figure 2. A decline in pH
started immediately after ensilement and continued throughout 45 d post-
ensilement, with the lowest pH around d 5. This pattern reflects the changes in
lactobadlli population for control and inoculated silage (Table 8). Initial
population sire was similar on d 0, however inoculation caused a 3-fold increase in
lactate produdng organisms within 24 hours. Lactobadlli numbers in the control
silage were still increasing on day 13, but were still less than the number of
organisms present in the inoculated silo on day 3.

Inoculated silage had a greater overall average temperature by d 2 and
remained greater (p<.05) throughout the 45 d post-ensilement period (37.6 vs. 36.2
°C; Figure 3). This supports Woodford and Satters findings in which inoculation
increased silage temperature an average of .64 °C over a 14 d post-ensilement
period. Silage temperatures were significantly difi‘erent (p<.01) at the various
elevations within the silos (Table 9). Temperature means for the four elevations
were 36.75, 40.43, 37.79 and 32.51 °C for 1.5, 5.3, 9.1 and 12.9 m, respectively.

Temperature at the various elevations in the two silos are illustrated in Table
10. As one would expect temperatures were greatest in the middle of the silos with

the inoculated silage having a greater temperature at all locations, except 12.9 m
37

38

TABLE 7. Composition of Forage Material Placed Into the Silos

 

 

 

Control Inoculated

DM (95) 46.40 45.20

pH 6.20 6.20
Lactic Add‘ 0.08 0.05
Ammonia-N” 2.50 5.10
Water Soluble N“ 34.00 30.60
Water Soluble Carbohydratesc 8.80 10.10
‘Expressed as g/100 g DM.

I'Expressed as % of Total N.

“Expressed as % ofDM.

3.5

39

pH OF SILAGE

 

 

 

 

 

 

l I l l

 

 

-F—
“k
i—

0 1 2 3 6 7 10 13 46
LENGTH OF FERMENTATION (d)

‘h CONTROL ‘l— INOCULATED

Figure 2. Average pH of the Control and Inoculated
Silages During First 45 d Post-ensiling.

40

TABLE 8. Lactobadlli Numbers in Silage Material Post-Ensiling‘

 

 

 

Day Control Inoculated
0 1.6 x 10‘ 1.7 x 10“
1 2.6 x 10‘ 1.1 x 109
3 3.5 x 10' 3.2 x 10°
5 7.8 x 107 1.8 x 10’
13 2.8 x 10’ 9.6 x 107
45 -- --
'Expressed as cfu/g DM.

l'Fresh material entering silo before inoculation

46
43
41
39
37
36
33
31
29
27
26
23
21
19
17
16

41

TEMPERATURE (C)

 

 

 

 

: *‘II'I

L

L.

lllllllllllllllllJlLlJLL llllllllLllllllllllL

I l I '1 T T ’1 r l l l’ 1 l I l l l i T 1 PTT T

1 3 6 7 9111316171921232627293133363739414346

LENGTH OF FERMENTATION (d)
—‘— lNOOULATED + CONTROL + AMBIENT

Figure 3. Average Temperature of the Control and
Inoculated Silages During First 45 d
Post-ensiling.

42

TABLE 9. Mean Temperatures at the Various Elevations Within Each Silo

 

Mallow

1.5 36.75‘
5.3 40.43"
9.1 37.79‘
12.9 32.51':

 

‘A'Values within columns with unlike superscripts difl'er (p<.05)

TABLE 10. Effect of Elevation and Treatment on Silo Temperatures (0°)

 

ELEVATION (Meters)
1.5 _5_.§ 2.1 12.9
Control 35.4 39.4 37.2 32.5

Inoculated 38.1 41.9 38.5 ' 32.2

 

43

where the control silage had a slightly higher temperature.

Fermentation characteristics from ensilage in buried bags were similar for both
silos except for DM and gross energy (Table 11). The inoculated silage was
significantly lower (p<.05) in DM content and significantly higher (p<.05) in
energy.

Fermentation characteristics of silage post-ensilement are shown in Table 12.
Barnett (1954) subdivided silage fermentation into four phases; 1) plant
respiration; 2) acetic add production by aerobic bacteria; 3) lactic add and acetic
add production by lactobadlli and streptococd and 4) a relatively stable period
providing sufident fermentation has occurred. Post-ensilement parameters are
presented as phase 1-3 (14 d), phase 4 (5-21 d) and feedout (>100 d). Inoculated
silage supported a more active microbial population during the first three phases of
fermentation, which coindded with the faster rate of temperature increase. Similar
results have been reported by Kung et al., (1981) which demonstrated inoculation
increased microbial populations and lactic add concentrations prior to d 7 in
laboratory silos. Total LAB counts were significantly greater (p<.01) for the
inoculated silage during the first three phases. As a result, lactic add content was
greater (p<.01) for the inoculated silage through d 21 and during feedout (p<.05) as
compared to the control. Control silage required more than 21 d to accumulate
similar concentrations of lactic add as the inoculated material possessed by d 4.
Moon, et al. (1981) previously demonstrated the increased extent and rate of lactic
add accumulation that occurs with inoculation. The pH of both silages declined
over time however, the inoculated silage declined at a faster rate and had a lower
pH (p<.01) throughout the first 21 d as compared to the control silage. As lactic

add accumulated, pH decreased. Ammonia-N concentrations were lower (p<.01)

44

TABLE 11. Fermentation Characteristics of Alfalfa Silage in Buried Bags

 

 

Control SD Inoculated SD
DM 44.03; 6.06 38.8‘| 3; 5.40
pH 4.5 i 0.08 4.45 3; 0.19
LA' 3.1 _4; 1.00 3.40 5; 1.30
WSC‘ 4.77: 1.80 4.18 _-l; 1.24
TN' 3.00: 0.38 2.86 ;i-_ 0.21
WSN' (% of TN) 66.205; 2.48 64.6 + 6.60
NH,-N (% of TN) 12.173; 2.89 11.04 i: 2.69
NDF‘ 45.203; 4.35 47.10 + 2.64
ADF‘ 34.503; 0.82 34.8 + 2.22
ADIN‘ (% of TN) 7.40 _-I; 1.60 6.00 i. 0.90
ASH‘ 8.85 i: 1.00 8.45 1 0.43
Energy‘ 9.89“; 1.52 10.893; 2.19
DM Recovery (‘70) 93.55 i 4.75 93.41 i 3.42

 

‘ All values are %’s expressed on DM basis except DM and pH.
" Energy is expressed as Kcal/g DM.
“ Values within rows with unlike superscripts differ (P<.05).

45

TABLE 12. Characteristics of Fermentation During Ensiling for Inoculated

 

(I) and Control (0) Silage
Phase 1-3 Phase 4
__d_1-4___ ___§5-A__ M—
_C_ L _SLM. _£_ .1. _SE_. .2. _1_ M.

LAB'I 6.87‘ 9.26r 0.22 7.96 8.30 0.19 -- -- --
LA” 0.19‘ 2.45‘ 0.45 1.73‘ 5.32' 0.39 2.45‘ 3.04“ .16
WSC‘ 8.54 6.45 1.20 7.24‘ 3.00' 1.04 5.95‘ 6.04f .44
pH 5.96' 4.99r 0.23 5.64f 4.31‘ 0.20 4.60 4.49 .08
NHo-N'l 3.43‘ 1.89r 1.71 2.71“ 1.08f 1.48 12.08' 8.86f .63

 

'LAB = Lactic add bacteria, Log CFU/g wet forage.

I’LA = Lactic add, g/100 g DM.

‘WSC = Water soluble carbohydrate as % DM.

‘NH,-N= Ammonia nitrogen as % total N.

“Means within a phase with unlike superscripts differ (p<.01).
”Means within a phase with unlike superscripts differ (p<.05).

46

throughout ensiling for the inoculated silage.

During feedout, control silage had greater (p<.01) DM content and less (p<.05)
gross energy (Table 13). The greater gross energy concentration in the inoculated
silage would indicate less carbon loss occurred than with the control silage. The
other chemical indices measured were similar (p<.10) for both silage treatments.
Dry matter recovery estimates calculated from 12 buried bags were 93.55 and
93.41% for control and inoculated silage treatments, respectively. The estimates of
recovery from buried bags was greater than recoveries from the entire silos (93.5
vs. 81.0%). The 12% percentage unit difference may be attributed to more aerobic
losses on the exposed silage surfaces or weighing errors which would not have
occurred with the buried bags. The large percentage difl'erence in the DM recovery
between the silos is unknown. DM percentages did difi‘er between forage entering
the silos, however, this difference was also seen in the silage removed from the
silos.

3.4.2 Silage Aerobic Stability

Temperature and DM losses were similar for the control and inoculated silage
(Figure 4) throughout the first 9 d of aerobic exposure. However, on d 9 the
temperature began to increase in the inoculated silage, followed by an increase
beginning on d 10 for the control silage. By d 14, both silages had achieved similar
temperatures. Dry matter losses were evident by d 1 and continued at an equal
rate for both silages until d 10, at which time the rate of deterioration increased
for the inoculated silage. Dry matter losses occurred during the first nine d
without major increases in temperature.

Dry Matter, N, and pH all increased, while ammonia-N, lactic add and acetate

47

TABLE 13. Chemical Indices of Fermented Forage During Feedout‘

 

 

Control Inoculated SE

Dry matter, % 43.60” 42.40“ .28
Total nitrogen, % 2.91 2.92 .03
Water soluble nitrogen, % 64.20 63.27 7.55
NDF, % 45.20 44.60 1.56
ADF, % 34.60 34.80 .33
ADIN, % 6.88 7.48 .43
Ash, % 9.05 9.08 .39
Gross energy, kcal/g DM 9.96‘l 10.22‘ .06
Acetate, g/kg DM 29.855 28.755 1.657
Proprionate g/kg DM 1.184 1.834 0.452
Isobutyrate dkg DM 0.110 0.026 0.043
Butyrate g/kg DM 1.291 0.661 0.394
Isovalerate g/kg DM 0.1277 0.046 0.045
Valerate g/kg DM 0.010 .002 0.005
Dry matter recovery, % 93.55 81.0 1.42

 

'All means are expressed on a DM basis with the excep

recovery.

”Means with unlike superscripts difl'er (p<.01).
"Means with unlike superscripts differ (p<.05)

tion of DM, pH, and DM

34
32
30
28
26
24
22
20
13
16
14
12

48

 

 

 

 

TEMPERATURE (C) % DM RECOVERY
t. 4 _
_- “;\‘- j x
r- .\ —
_ ;‘J
_ ’ 1
L a
1 1 i 1 1 1 i i 1 1 i 1 1 i i
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
DAYS OF EXPOSURE
-"' CONTROL ‘1. DM CONTROL

+ INOCULATED

Figure 4.

‘9‘ DNIHNDCULATED

Temperature and Dry Matter Recovery for Control
and Inoculated Silage During Aerobic Exposure.

100

96

90

86

80

76

49

decreased as length of exposure increased (P<.001). Inoculated silage had a
significantly lower (p<.001) concentration of ammonia-N, WSC, acetate, isobutyrate
(p<.05) and isovalerate (p<.1) than the control silage. However, lactic add (p<.001)
and propionate (p<.05) concentrations were significantly greater for inoculated
silage throughout aerobic exposure. No difi‘erences were observed for DM, N, pH,
ammonia-N, WSC, propionate, butyrate and isovalerate between treatments on any
particular day (Table 14). Lactic add content was significantly greater (p<.05) on
day 0, 1, 3 (p<.10), 7 and 10 for inoculated silage.

3.4.3 Lactation Trial

Milk production of lactating dairy cows fed control or inoculated alfalfa silage
supplemented with different degradable proteins is presented in Table 15. Catt;e
fed the control silage supplemented with the more slowly degradable protein had
the lowest dry matter intake thus having the least weight gain throughout the
trial period. The largest weight gain was observed in cattle fed the inoculated
silage supplemented with the slow degradable protein source. This weight gain
can be attributed to the large dry matter intakes observed in this treatment group.

There was a significant interaction between silage treatment and protein
supplement. Cattle fed the more slowly degraded (SD) protein source with the
inoculated silage had an increase in 3.5% fat corrected milk (FCM) production by
2.1 kg/d (p<.05), as compared to SD added to the control silage, likewise they had
increased daily yields of fat and protein. This increased production of FCM
appeared to be the result of a 3.7 kg/d additional dry matter intake (DMI). Cows
produced similar milk yields with RD supplemented to either silage treatment.
Within the rapidly degraded protein supplement, FCM production was similar for

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52

TABLE 15. Response of Holstein Cows Fed Alfalfa Silage With or Without
the Addition of a Microbial Inoculant

 

_SleDe <11 M
C I C I

 

 

 

&
DM intake, kg/d 17.80 21.50 20.70 20.90 7.60
Weight change, kg/d 6.20 23.50 20.70 17.20 21.40
Milk production, kg/d 27.00” 31.80e 29.80” 29.90” 4.33
3.5% FCM', kg/d 27.50” 31.90c 29.50”- 29.80” 3.46
Fat, kg/d 0.97” 1.12c 1.03” 1.04'»e .12
protein, kg/d 0.87b 0.98” 0.95”, 0.98," .12
Lactose, kg/d 1.41 1.53 1.54 1.54 .12
Solids, kg/d 3.44 4.04 3.37 3.77 2.22

 

‘ Fat corrected milk.
l“Means within rows with different superscripts difi‘er (p<.05).

53

cows fed either silage. Similar results were reported by Gordon (1989) in which
lactating animals fed inoculated silage showed a 7% increase (P<.05) in FCM.
Grant and Colenbrander (1986) also reported an increase in FCM production with
inoculated alfalfa silage as compared to control silage. While Grant and
Colenbrander did not suggest a reason for an increase in milk production, Gordon
suggested the animal production response was consistent with the increase in the
metabolizable energy (ME) intake. Lactating cattle in this study showed a slight
increase in consumption of inoculated silages which seemed to follow FCM
production. These DM intake responses along with change in weight over the
study period were not significantly different. It has been demonstrated (NRC,
1985) that lactating dairy cattle fed alfalfa silage based diets supplemented with
slowly degradable protein in the rumen will produce more milk. This increased
FCM with supplementation of slowly degradable protein may not en'st with all
alfalfa silages.

The value of a protein source in produdng an increase in performance is
determined by its ability to 1) supply limiting amino adds (AA) to the small
intestine and 2) to supply N available for use by rumen microorganisms.
Titgemeyer and coworkers (1989) demonstrated that blood meal and corn gluten
meal supplied larger amounts of total AA and AA nitrogen to the duodenum than
soybean meal and feather meal. The addition of blood meal significantly (p<.05)
increased lysine, histidine, arginine and valine concentrations in the duodenum,
while corn gluten meal increased (p<.05) methionine, isoleudne, leudne and
tyrosine concentrations.

Perhaps this increase in AA to the lower gut with the small increase in energy

exhibited in the inoculated silage is responsible for the positive milk production

54

response seen in cattle fed the inoculated silage with slow degradable protein.
Cows in all treatment groups showed similar concentrations of lactose and milk

solids.
3.4.4 Growth Trial

Results of the beef heifer growth trial are shown in Table 16. Dry matter
intake was similar for all treatment groups. This is in agreement with the
findings of Kennedy and coworkers (1989) who showed no increase in DM intake
for finishing steers fed inoculated grass silage as compared to the control
treatment.

The heifers fed SD supplemented control silage gained more weight (P<.07)
than RD supplemented cattle fed control silage. Weight gains were similar for
both protein supplementation regimes with inoculated silage. Expression of
average daily gain (ADG) per unit of metabolic body size indicated that heifers fed
control silage with a slow degradable protein source gained faster than the other
three treatments. These results do not support data compiled from a six trial
summary in which Bolsen and Hinds (1984) found no significant differences in
performance between animals fed control or inoculated silage.

There are no explanations as to why similar results were not observed in both
animal production trials. Perhaps the higher nutrient demand and AA
requirements of lactating cows as compared to a growing heifer may explain the

difi'erent results between the two trials.

55

TABLE 16. Performance of Crossbred Heifers Fed Alfalfa Silage With and Without
the Addition of a Microbial Inoculant

 

 

CONTROL INOCULATED
RD §D RD SD SEM
No. of Animals 17 18 18 18 ---

Initial Weight, Kg 207.40 208.30 244.10 235.20

Final Weight, Kg 295.90c 309.80d 343.40‘ 346.90‘ 6.40
DM Intake, Kg/d 5.81 6.71 7.22 7.56 .51
DM Intake, Kg/wt'75/d .092 .104 .102 .106 .072
ADG, Kg/d .84‘ .97b .97” 1.01h .028
ADG, Kg/wt'75/d .013‘ .015d .014“ .014“ .0006
Gain/Feed .147 .146 .134 .134 .008

 

"" Values within rows with unlike superscripts differ (p<.07).
“"Values within rows with unlike superscripts differ (p<.10)

3.5 CONCLUSION

In summary, inoculation of alfalfa silage with a microbial inoculant resulted in
a three-fold increase in lactobadlli numbers within 24 hours. Lactic add content of
the inoculated alfalfa silage was greater and the pH lower throughout the first
three weeks of ensiling resulting in a fermentation with less protein degradation
and less gross energy loss. The favorable shift in fermentation pattern with
inoculation did not result in greater DM recovery.

Fat corrected milk production increased with SD supplementation of inoculated
silage, however a similar response was not evident in the control silage treatment.

Both silages tended to be stable under aerobic conditions through d 9.
Inoculation of alfalfa increased the rate of fermentation, however, DM recovery and
aerobic stability were not positively influenced. Cows fed the inoculated silage did
respond to slowly degradable protein supplementation. Inoculation appeared to
reduce proteolysis and energy losses which the high produdng dairy cow was able
to utilize for greater milk production. The higher gross energy concentration of the
inoculated silage would be advantageous for high produdng dairy cows since DM
intake generally limits production. The ability of the cows to respond to rumen
undegradable protein in this trial may be a result of the added energy provided by
the alfalfa silage.

56

4.0 EFFECT OF A BACTERIAL SILAGE INOCULANT ON FIBER DIGESTION
AND RUMEN CELLULOLYTIC SPECIES

4.1 Introduction

Currently, several commerdal microbial inoculants are available for use on
ensiled forages. Most are marketed on the premise that epiphytic lactobadlli
populations are often too low to support a rapid fermentation, and consist either of
a single Mg strain or a mixture of selected Lactobadlli Strepmggd and

 

m strains. Recently, sdentists have reported that silage inoculation
improves dry matter (DM) and add detergent fiber (ADF) digestibility of ensiled
forage material in ruminants (Harrison, 1989; Hooper, 1989; Harrison et.al., 1989).
These experiments were not designed to evaluate the mechanism of the observed
increase in digestibility. Further research is needed to determine the chemical or
physical change in the inoculated silage and its effect on rumen microbes during
digestion.

Whether the effects of microbial inoculants on fiber degradation are direct or
indirect is unknown. A direct effect might include inoculant bacteria having the
capadty to degrade or utilize fiber components released from alfalfa degradation,
such as dextrins, pectins, cellobiose, xylose, arabinose and oligomeric fragments.
Such spedes of lactobadlli have been isolated from the rumen. Sharpe et al.
(1973) isolated a spedes of lactobadllus from the bovine rumen characterized as
being able to use cellobiose with similar morphological characteristics as
W plantarum. The organism was named Lactobadllus rumings. A

continual consumption of end products from cellulolytic digestion could stimulate a

57

58

higher rate of fiber digestion by these bacteria. A second direct effect could involve
lactobadlli interacting with cellulolytics in colonization of alfalfa particles,
providing a sticky matrix for an immediate attachment to alfalfa which would
fadlitate attachment of cellulolytic bacteria to alfalfa.

An obvious indirect effect of inoculant stimulation of fiber digestion by rumen
cellulolytic bacteria involves the production of some major growth factor within the
treated silage material which is required by the microbes. Thus the lactobadlli
inoculant itself would not be involved directly in increasing the fiber degradation
but supply the growth factor. Interactions between rumen cellulolytic spedes and
epiphytic bacteria or silage inoculant bacteria have not been investigated as of the
present time.

The objectives of these studies were: (1) To determine if selected microbial
inoculants and isolated epiphytic strains improve the digestibility of forages and (2)
To determine if their effect is directly on forage degradation as scavengers or

indirectly by their metabolic interactions with cellulolytic spedes.

4.2 Materials and Methods

4.2.1 Digestion Trial

Seven Holstein steers (269 kg) were utilized in a crossover design to determine
the digestibility of control (C) and inoculated (I) silages. Steers were housed in
individual metabolism pens with slotted floors and fed a total alfalfa silage diet for
a one week adaptation period. After the adaptation period, steers were randomly
assigned to one of two diets. Diets included C and I silage fed at 2.5% of body
weight on a dry matter (DM) basis (Table 17) Minerals and vitamins were
supplemented according to recommendations of the National Research Coundl for
beef cattle (NRC, 1984). Cross-over periods were 14 d long, with steers receiving
chromium oxide Cr,O, at .5% of body weight. Chromium oxide was used as a
digestion marker to measure the percent digestibility of CP, ADF and NDF. It was
administered orally in a gelatin capsule at the same time each day, beginning on d
1 of the study. On d 9-14 of each period, fecal samples were collected from the
rectum of each steer four times daily (6 h intervals) and composited.

Fecal material was analyzed for DM, ADF, NDF and CP as previously
described in Chapter 3 of this manuscript Chromium content was determined by
the procedure of Fenton and Fenton (1979) with modifications. Fecal samples were
dried in a convection air oven at 60°C for 48 h and ground through a Wiley mill
equipped with a 1 mm screen. Approximately .5 g of mound dry feces was digested
using 40 ml of nitric add and 7 ml of 70% perchloric add. Samples were heated
until oxidation was complete and diluted to a volume of 100 ml with deionized

distilled water. Chromium content of the diluted sample was measured on a

59

60

TABLE 17. Characteristics of Alfala Silage Fed to Holstein Steers During The

 

Digestibility Trial
anml In (1
DM (%) 50.30 47.25
pH 4.38 4.38
Lactic Add‘ 3.50 4.30
WSC‘ 5.90 5.48
TN‘ 3.23 3.07
WSN (% of TN) 61.90 67.15
Ammonia-N (% of TN) 8.13 5.35
NDF‘ 45.15 45.25
ADF‘ 34.15 33.90
ADIN (% of TN) 4.95 5.15
ASH‘ 8.30 8.40
Energy” 8.28 8.91

 

‘Values expressed as a percentage of DM.
l'Energy is expressed as Kcal/g.

61

leeman Ion Coupled Plasma Spectrophotometer 40 (ICP) using the National
Institute of Safety and Health (NIOSH) method 7300.

Percent digestibility of NDF, ADF and CP was calculated using the method
described by Church (1983) as shown in Figure 5.

Feed intake and orts were measured on a daily basis and silage samples were
taken weekly, composited and analyzed for DM, pH, lactic add, water soluble
carbohydrates (W SC), water soluble nitrogen (WSN), total nitrogen (N), ammonia-
N, ash, energy, neutral detergent fiber (NDF), add detergent fiber (ADF), and add

detergent insoluble nitrogen (ADIN).
4.2.2 Electron Microscopy

Cultures of two major rumen cellulolytic bacteria Rumingms 911mg 7 and
W 11% FD-l, were individually grown to mid-exponential phase
in media shown in Table 24. Rather than glucose, alfalfa was used as a energy
substrate so that after cultures were mixed with B, m there would be no
transfer of glucose for use as a substrate by the lactobadlli. B, m was
isolated from a commerdal silage inoculant (#1174) manufactured by Pioneer
Hibred International (Des Moines, Iowa), and grown to mid-log phase in LBS
medium (Table 24). Then .1 ml of each cellulolytic and .1 ml of lactobacilli were
mixed in co-culture in 9.8 ml of media shown in Table 21. Each strain of bacteria
was also transferred individually into 9.9 ml of this media. In each tube small
transverse sections of fresh alfalfa leaves (2 mm x 5 mm) were placed and
saturated 2 h before inoculation. Three tubes of each bacterial treatment were
placed in an incubator at 39°C for 36 h. Tubes were shaken every 6 hours. All

media was anaerobic and remained so until leaves were removed.

62

FIGURE 5. Calculation for Digestibility Using Accumulation of Chromium
Concentration in Feces.

 

‘ Indicator consumed (g/d)
Fecal output (g nutrient/d) =

 

Indicator conc. in feces (g/g nutrient)

% Indicator in feed % Nutrient in feces
Digestibility = 100 100 " "

 

% Indicator in feces % Nutrient in feed

 

Church, D.C., 1983.

63

Alfalfa leaves were removed and were fixed in 5% glutaraldehyde in 0.1 M
phosphate bufi‘er on ice for 2 h. Following fixation, samples were washed with 0.1
M phosphate buffer and were dehydrated in a graded ethanol series (25%, 50%,
75%, 95% and 100%). Samples were then critical point dried and adhered to 10
mm aluminum stubs with double sided tape. A small line of graphite was drawn
from the sample to the outer perimeter of the stub. Each specimen was than gold
coated in a Film Vac sputter coater and viewed at 15 hlovolts in a JEOL, JSM-35
CF scanning electron microscope at Michigan State University’s Center for

Electron Optics.

4.2.3 Growth Enhancements

Inoculated and control silages were obtained from the appropriate silos and 500
g of each silage along with 1000 ml of water were blended using a Waring blender.
Contents were strained through cheesecloth and filtered through a sterile millipore
filter. This filtrate was autoclaved and 2.7 ml was added to sterile test tubes
containing 6.3 ml of sterile GCS-RF media (Table 29). Each tube was inoculated
with .1 ml of B, my}, 13.-W and B, W. Cellulolytic cultures
were also inoculated at .1 ml to sterile test tubes containing 9 ml GCS-RF media.
Mono cultures of individual cellulolytics acted as a control for comparison of growth

curves. Cultures O.D.’s were read hourly at 600 nm.

4.2.4 I_n_ m Digestion

Samples of C and I silages were obtained from the appropriate silos were
ground with dry ice through a Wiley mill using a 3 mm screen. This silage

material along with rumen fluid from a fistulated Holstein cow maintained on a

64

alfalfa hay diet was use to determine i_r; £1112 dry matter digestibility (IVDMD)
according to the two stage method described by Tilly and Terry (1963). Silage was
not dried prior to digestion due to concern for altering any factors which may
increase silage digestibility, thus .5 g was used instead of .25 g as a sample weight.
Twenty-eight tubes were used for each treatment, with 4 being emptied at each of
0, 4, 8, 16, 24, 36 and 48 b. Two tubes were also prepared as blanks, containing no

silage material and were emptied at similar time endpoints.

4.3 Statistical Analysis

Statistical analysis of the digestion trial were conducted using the General
Linear Models Subroutine in SAS (SAS Institute, 1987 ). Least square means were
generated to compare digestibility of the two treatments. Mean comparisons were
made with Bonferroni’s T-test as described by Gill ( 197 8).

One animal was eliminated from the data set because of extreme illness and
injury which required antibiotic treatment.

I_n_ my; dry matter digestibility data was analyzed using the General Linear
Models Subroutine in SAS. Least square means were generated for silage

treatments for each hour and LSD was used for mean comparisons.

4.4 Results and Discussion
4.4.]. Digestion Trial

There were no statistical differences observed in initial weight, final weight,
ADG and feed eficiency (Table 18). The Holstein steers tended to gain more
weight while being fed the inoculated silage as compared to the control, thus
having a better gain to feed ratio. However, this was not significant.

Table 19 illustrates the digestibility of the alfalfa silage. Crude protein
content, NDF and ADF digestibility of the two silages were very similar. The
inoculated silage had a numerically lower CP digestibility and a slightly greater
NDF and ADF digestibility. These differences however were not statistically
different. This data does not support the work of Harrison et. al. (1989).
Percentage of ADF and BM in their study was significantly increased with
inoculation. The bacterial inoculant applied in their study however contained
other strains of microbes including pediococci and streptococci. These microbes
could possibly have afl‘ected the digestibility of the inoculated material. Perhaps
the one strain of lactobacilli contained in the inoculant used in this study does not
alter the physical or chemical properties needed to cause the increase seen by
Harrison et.al., (1989) and prer (1989). The climatic conditions during the time
of this trial could also have exhibited an efi'ect on digestibility. The weather was
extremely hot and humid. The air ventilation system in the metabolism room at
The Beef Cattle Research Center did not maintain adequate air flow. This in turn
could have been a reason for the one animal becoming sick and eliminated from
the trial.

66

67

TABLE 18. Performance of Holstein Steers fed Alfalfa Silage With and Without
The Addition of a Microbial Inoculant

 

ngtml Inoculated SEM
No. of Animals 6 6
Initial Wt., kg 279.26 275.85 11.53
Final Wt, kg 280.40 282.67 10.94
DM intake, kg/d 7.164 7.47 .243
ADG, kg/d 0.08 0.405 .220

Gain/Feed 0.0117 0.0553 .0298

 

68

TABLE 19. Digestibility of Alfalfa Silage With and Without the Addition
of a Microbial Inoculant

 

QM MM

Crude protein‘ 52.2 51.64 2.13
Neutral deterg. fiber“ 36.2 38.26 2.44
Acid detergent fiber“ 33.3 34.69 2.37

 

‘Percentages on a DM basis.

4.4.2 Electron Microscopy

Figure 6 and 7 illustrate what a normal alfalfa leaf looks like when observed
under a scanning electron microscope before and after digestion by rumen
cellulolytic microbes. These micrographs have been included to exhibit the
difi'erence in alfalfa leaves before and after digestion.

The leaf exhibited in Figure 6 through scanning electron microscopy (SEM)
reveals large bundles of mesophyll and parenchyma bundle sheath undigested,
thus leaving large pits where nutrient solubles appear throughout the leaf.

A fiesh transverse section of an alfalfa leaf undigested is shown in Figure 7. Open
cells revealing inside nutrients are exposed for rumen cellulolytic colonization and
digestion.

Figure 8 shows 3, M in monoculture attached to an alfalfa leaf after 24 h of
digestion. 3, gm tended to form large clusters around the leaf solubles.
Although & QILILS will adhere to its nutrient substrate, it did not produce clear
defined zones of erosion in the leaf, however it appears to degrade the readily
available inter-cell nutrients. Extracellular enzymes have been isolated and
defined in I}; albus as well as studies revealing that anywhere from 0 to 49% of the
strains will attach to their nutrient substrate. I; _a_lb;1_s_ in this culture which was
strain 7, appeared to have little problem in attaching and degrading part of the
alfalfa leaf. Figure 9 and 10, however, reveal i M in co-culture with L,
m. L, plantarum is not present in these micrographs because none of
these organisms attached to the alfalfa leaf. R_.a1_b;u_s tended to gather around the
stomata (Figure 9) of the leaf. This supports the findings of Baker and Harriss
(1947), who suggested that digestion begins with penetration through the stomata.

Several clusters consisting of 6 to 20 cocci of I; alblg were observed around leaf
69

7o

 

Figure 6. Digested Alfalfa leaf in
rumen fluid. 24h. 1800K.

 

Figure 7. Undigested Alfalfa Leaf. Cut
surface of transverse leaf section
exposed. 2000K.

71

 

W»

Figure 8. Ruminococcus albus in monoculture,
attached to a degraded alfalfa leaf.
24h. 3000K.

 

72

 

Figure 9. E. albus attached to an alfalfa
leaf stomata while in co-culture
with L. plantarum. 24h. 4000K.

 

 

Figure 10. R. albus in co-culture with L.

 

'Elantarum (not present) attached
to an alfalfa leaf. 24h. 7000K.

73

stomates. There was no penetration through the waxy cuticle on the leafs surface
or pitting, as often observed during cellulolytic fiber digestion (Akin, 1980).

The lack of attachment of ; plantarum and large numbers of I_L M when
placed in co-culture could result from L. plantarum using plant sugars as a
substrate and driving the pH down with large amounts of lactic acid being
produced as its primary end product. Stewart (1977) and Stewart et al. (1979)
have shown that the reduction of rumen pH from 7.0 to 6.0 has a profound effect
on the activity of cellulolytic bacteria, specifically affecting its attachment to cell
wall materials.

When alfalfa leaves that had been exposed in monoculture and co-culture with
I; flgvefaciens and L_. plantarum were viewed by SEM (Figures 11 and 12), they
revealed many of the same observations seen with B_._ albus. _IL flavefacieng has
been known to exhibit a pronounced capsule (Akin and Rigsby, 1985), however, on
the heavily colonized leaf (Figure 12) this physical feature was not observed.
Collings (1979) reported string like projections on _11, flgvefaciens during cell wall
digestion, observed under SEM. These features failed to be present on g
M both in monoculture and co-culture with L; plantarum. B_., flavgfaciegs
did readily digest parts of the plant cell wall and its components when in a pure
monoculture. ; plantarum failed in attaching itself to any part of the alfalfa leaf
and not one bacterium was located using SEM (Figure 11). B; flavefgcigns formed
long chains when in co-culture versus clumping in monoculture. Perhaps this
clumping of bacterium could result or contribute to the pit formation often seen in
digestion of plant material by Ruminococcus sp. (Cheng et al., 1983).

Often synergistic effects among species appear to influence fiber digestion

(Miura et al., 1983). In this experiment the effect tended to be negative.

74

 

Figure 11. 3. flavefaciens in co-culture
with L. plantarum (not shown)
attached to an alfalfa leaf.
24h. 16000X.

 

Figure 12. R. flavefaciens in mono-culture

attached to an alfalfa leaf.
24h. 3000K.

75

An SEM study done by Brazle and Harbers ( 197 7) on the digestion of hay revealed
that the leaf cuticle and epidermis were sloughed after 24 h of digestion, causing
extensive mesophyll degradation, with only the cuticle, abaxial hairs and partially
hydrolyzed vascular tissues remaining. The 24 h digestion period i_1_1_ m should
have Men sufficient time to allow considerable digestion of alfalfa leaves. This
proved to be true when cellulolytics were in monoculture however when in co-
culture, the pH may have had a chance to rise when available substrate for L
PM; was depleted and lactic acid was no longer produced. A greater number
of cellulolytics might have attached to the alfalfa leaves, however, the possibility of

L, Dim becoming directly associated with the leaf is very unlikely.
4.4.3 Growth Enhancements

Rumen cellulolytic growth curves with and without the addition of extract
obtained fiom inoculated silage is shown in Figures 13 through 15. _Ph 2114;;
(Figure 13) and & flavefaciens (Figure 14) tended to grow faster throughout the
entire exponential phase without the addition of the silage extract in the media.
However, L, succinogenes’ growth rate (Figure 15) was stimulated with the
addition of silage extract. This increase was observed beginning 1 h post-transfer
of the culture and continued throughout most of the exponential growth phase.
This suggests that a growth factor provided by the silage extract is stimulating the
growth of I_3_._ gug'noggnes. It has been demonstrated that _B_. succinogenes is the
most active rumen cellulolytic species, digesting the more resistant cellulose
(Bryant, 1973), as well as attaching itself more firmly to fiber particles than the
Ruminococcus sp. (Minato et.al., 1966). _B_. succinoggnes is the only cellulolytic

which has a requirement for valeric acid. An elevated concentration of valeric acid

76

optical density (600nm)

 

 

 

 

 

11
Hours

*7 +7+IE +7+CE

Figure 13. Growth of Ruminococcus albus 7 With and
Without Silage Extract.

 

77

optical density (600nm)

 

1.2
1.1 '

0.9 ‘
0.8 -
0.7 '
0.6
0.5
0.4

T

r

0.2
0.1

I

 

Figure 14.

 

 

Hours

—‘—FD-1 +FD-1+|E ‘9‘FD-1+CE

Growth of Ruminococcus flavefaciens FD-l With
and Without Silage Extract.

 

 

11

78

optical density (600nm)

 

 

 

 

 

11
Hours

"h 8-86 + 8-86 + IE ‘9' 8-85 + CE

Figure 15. Growth of Bacteroides succinogenes S-85 With
and Without Silage Extract.

 

79

in the inoculant supernatant could cause this increase in growth response.
Unfortunately at the time the electron microscOpe was being used, a clean
culture of _B_, succinogenes was not available in our lab. The interaction of these

bacterium with L plantarum might have been detected.
4.4.4 Q Vitro Digestibility

There were no significant differences observed in in yi_tr_q DM digestibility
between the two silages (Table 20). The percentage of DM disappearance is very
similar for both silage treatments following a similar pattern throughout the 48
hour period (Figure 16.). The inoculated silage exhibited a greater amount of DM
digestibility at 8 and 16 h, however this difi‘erence was very small. This supports
the data fi'om the Holstein steer experiment reported earlier in this chapter, in
which CP, ADF and NDF digestibility did not differ between treatments.
Digestibility of DM peaked at 24 h and remained elevated through 48 h.

80

TABLE 20. n1, Vim Digestibility of Dry Matter (IVDMD) of Control

 

and Inoculated Silage
Treatment=
Hog ontrol Inoculated §EM
0 0.00 0.00 1.5
4 29.35 29.65
8 49.83 55.25
16 57.48 61.03
24 69.80 69.20
36 69.85 68.73
48 69.20 70.10

 

80
75
70
55
50
55
50
46
4O
35
30
25
20
15
10
5

‘1) IVDMD

81

 

I I I

I

I I I I r r I

I

I

 

Figure 16.

 

«v

 
 
 
 
  
 
 
 
 
 
 
 
   

l l l l

10 20 30 40
Hours

—‘— Control + inoculated

In Vitro Digestibility of Dry Matter (IVDMD)
of C and I Silages.

ii.

 

50

4.5 Conclusion

The result of this study indicated that there are no differences in the
percentage of crude protein, ADF and NDF digestibilities in inoculated versus
control silage. The conclusion drawn from the 48 h IVDMD study is similar to the
in _v_it_r_q animal digestibility trial with no differences detected. The difi‘erences
exhibited in other studies resulting in an increase in digestibility could be due to
the fact that the silage inoculant contained more than one strain of microorganism.
Perhaps a synergistic effect amongst these microbes in the silage itself could cause
an increase in digestion, or a combination of metabolic products produced by them
during fermentation.

Electron microscopy revealed no direct interactions between 35%
species and L, W- The interaction had a negative efi’ect with fewer
numbers of cellulolytic organisms attaching to alfalfa leaf particles in the presence
of L plantarum. A more eficient and perhaps effective way to reveal these
interactions would be to run this experiment i_n_ m.

There were no indirect efl‘ects of microbial interactions observed in the growth
enhancement study. Growth of I_L al_b_u_s and IL flavefaciens was not enhanced with
the addition of silage extract from either the control or inoculated silage, however
L W did exhibit a greater growth rate when grown in media containing
inoculant supernatant.

More studies are needed to identify the mechanism causing this increase in
digestibility as well as more studies providing data supporting the hypothesis that
silage inoculation increases the digestibility of the silage. Studies in the future

should involve singular as well as multi-species bacterial inoculants.

82

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Akin, DE. and LL. Rigsby. 1985. Degradation of Bermuda and orchard grass by
species of ruminal bacteria. Appl. and Environ. Microbial. 50:825.

Akin, D.E., Burdick, D. and GE. Michaels. 1974. Rumen bacterial interrelations with
plant tissue during degradation revealed by transmission electron microscopy. Appl.
Microbial. 27:1149.

Akin, DE. and HE. Amos. 1975. Rumen bacterial degradation of forage cell walls
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Akin, D.E., E.L. Robinson, F.F. Barton II, and BS. Himmelsbach. 1977. Changes
with maturity in anatomy, histochemistry, chemistry and tissue digestibility of
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Akin, DE. 1979. Microscopic evaluation of forage digestion by rumen
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Akin, DE. and FE. Barton 11. 1983. Rumen microbial attachment and degradation of
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Albersheim, P. 1975. The walls of growing plant cells. Sci. Amer. 232:80.

Allison, M.J. 1969. Biosynthesis of amino acids by ruminal microorganisms. J. Anim.
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Allison, M.J. 1970. In Physiology of Digestion and Metabolism in the Ruminant, Ed:
T. Phillipson. Newcastle-upon-tyne: Oriel. p. 456.

Allison, M.J., M.P. Bryant, and RN. Doetsch. 1962. Studies on the metabolic function
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of isovalerate into leucine. J. Bacterial. 83:523.

Allison, M.J., M.P. Bryant, I. Katz, and M. Keeney. 1962. Metabolic function of

brached-chain volatile fatty acids, growth factors for W. J. Bacteriol.
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Allison, M. J. and J. L. Peel. 1971 The biosynthesis of valine from isobutyrate by
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American Association of Analytical Chemists. 1980. Oficial Methods of Analysis
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Aspinall, GO. 1973. Carbohydrate polymers of plant cell walls. Laewus, F. ed.
Biogenesis of plant cell wall poilysaccharides. New Yarszcademic; p. 95.

Bailey, RW. and 8D. Gaillard. 1965. Carbohydrates of the rumen ciliate Epidinig
m Biachem. Journal 95:758.

Barker, F. and ST. Harriss. 1947. Microbial digestion in the rumen (and caecum),
with special reference to the decomposition of structural cellulose. Nutr. Abstr. Rev.
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Baker, SD. and W.H. Summerson. 1941. The colorimetric determination of lactic acid
in biological materials. J. Biol. Chem. 138:535.

Barnett, A.G. 1954. Silage Fermentation. Academic Press, Inc., Landon. p. 107.

Barton, F.E. II and DE. Akin. 1977. Digestibility of delignified forage cell walls. J.
Agric. Food Chem. 25 (6). P. 1299.

Beck, T. 1978. The microbiology of silage fermentation. Fermentation of silage - A
review. National Feed Ingredients Assoc. p. 63.

Bergen, W.G. 1984. Protein conservation in silage management. National Feed
Ingredients, p. 113.

Bergen, W.G., E.H. Cash and HE. Henderson. 1974. Changes in nitrogenous
compounds of the whole corn plant during ensiling and subsequent efi'ects on dry
matter intake by sheep. J. Anim. Sci. 69 (3):629.

Bolsen, KK. 197 8. The use of aids to fermentation is silage production. In
Fermentation of Silage-a Review. Ed. M.E. McCullough. Natl. Feed Ingred. Assoc.
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APPENDIX

ADF ADIN

NDF

ASH

Control:

 

TABLE 21. Composition of alfalfa forage entering silos before treatment'.:

W'OND
Damian”:
a a a s a a a
MQQMMV

V—unnd'at
inmate—no
0 O O O 0 O 0
010650150
MCI-tank"
OPIQQ'OO
O O O O 0 O O
NGOOON

V5001 a
Flo-ONO h
0 C C O O O I
MMNNMN

mm mm
mc ow

ddmmmm
unvaum

('60-:de
Vin???

X

Inoculated

%

WGQUNOQ’

.DQhOl-nib

nmvnmmfi

meemmm
888883

NMNQHN

n mahm'
v vvvv

most-aqua:
moisocasoat

RKSSS

”09:60:”:

gMIflVIflN
DONDN

s a o o a s
OOHOQNO
"fl H

and QND
v NO”
0

s s s a a o
ufinlflmfim

gommom'
NMMQM

s o o s s o a
mmmmmm
GMOQQ'QN
s s a a

O O O
on ems
cw vm¢v

W

11 values are expressed as a percent of dry setter. except pH. on and energy.

tactic acid is expressed as 911009 DH.

Energy is expressed as Keel/90H.

a A
b
c

 

 

TABLE 122 Composition of samles bored from parts 1.5 I fro-l botto- af each silaa.

 

 

HNNF‘MHSO
misnomer O
”fid—i—i 'uiqi 'ci
2?
n.33353333
V'. '10 U

 

'9

"SC

 

 

”0‘ fl
aggmocfim
a s a s o a a a a
mmmmmmmm

.YQNTRRQR
83338333

 

”'66'665n
| mesaeees

pt ('11:! atom
a: N hue-0Q

Anln

 

IQ

ADF

 

NDF

 

ASH

 

"f .

Ene

Alumnia II
C

 

96

.fiRRRfiRRR
SSESSSSS

vmvmacmo
"646'6Kd
:vvv vet

“ml-0'01”“!!!
o a s o o

:scsiisé

NQNDUQMM
C O I 0 O O O I O
OQOQQQOQ

QNMOn—tvm
s s o a s s o

 

ll values are expressed as a percent of dry clatter, except 011. pit and E.

Lactic acid is expressed as g/lOOgDH.

Energy is expressed as Kcal/gofl.

a A
b
C

.3323 an connotes. up 33 333 a
in a... .3 annexe 63qu be .6 access; a, no unmask—.8 En mos—2. Z: a

 

a
uuhummmmumm a

\D

97
we ~
' mvwv amass
O O O O O

NDMMFUMNUN

LlPILILIILlPIILIlHI Lllulu u

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ea.n ~—.n -.m o—.u oc.v ea." mc.v c¢.v v.9v «.mv «N
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99

TABLE 24. Medium Used to Grow Rumen Cellulolytic Bacteria to Mid-

 

Exponential Phase

Ingr_edient Amgunt gr 309 ml
Ground alfalfa 1.5 g
Starch .2 g
Yeast Extract .6 g
Trypticase 1.5 g
Rumen fluid 60.0 ml
Mineral #1‘ 11.2 ml
Mineral #2‘ 11.2 ml
Resazurin 0.3 ml
N aCO, 15.0 ml
Cysteine-HCl 6.0 ml
Distilled HQO 193.6 ml

 

‘Compositian of mineral mixes are in Table 25.

100

TABLE 25. Mineral Mixes Used in Rumen Cellulolytic and Digestion Media‘

 

Min #1
Inggdient Agagg
K,PO, .6%
Distilled H,O 1000 ml
Min #2
Inggdignt Agggt
KH,PO, .6%
(NI-1,),SO4 .6%
N aCl 1.2%
MgSO, 7H,O 245%
CuCl2 2H’O .159%
Distilled H,O 1000 ml

 

‘Ingredients were dissolved in H,O and media is autoclaved at 15 psi for 20
minutes.

101

TABLE 26. Lactobacilli (LBS) Medium

 

Inggdignt Am t r 1
Trypticase l g
Yeast Extract .5 g
Dextrose .6 g
Monopotassium phosphate .2 g
Ammonium citrate 2 g
Tween 80 .l g
Sodium Acetate 2.5 g
Magnesium Sulfate (MgSO,) .0575 g
Manganese Sulfate (M0,) .012 g
Ferric Sulfate (FeSO,) .0
NaCO8 5.0 ml

Cysteine HCl 2.0 ml

 

102

TABLE 27. Medium Used In Digestion ofAlfalfa LeafWith individual

 

and Co-cultures

Lam; gaunt Par 1m ml
Trypticase 0.3 g
Yeast extract 0.2 g
Rezasurin 0.1 ml
Mineral #1‘ 7.5 ml
Mineral #2‘ 7.5 ml
VFA” 0.3 ml
FeSO4 7H,O 1.0 ml
CoCl, 6H,O 1.0 ml
Cysteine-H01 (2.5%) 2.0 ml
Na,CO, (8.0%) 5.0 ml

 

‘Compositian of mineral mixes are shown in Table 25.
"Composition of VFA mixture shown in Table 28.

103

TABLE 23. Volatile Fatty Acid Mixture Used for Digestibility Medium

 

In ‘ r1 Agngaat
Acetic acid ‘ 17 ml
Propionic acid 6 ml
N-butyric acid 4 ml
lsobutyric acid 1 ml
DL-d-Methyl N butyric acid 1 ml
N-valeric acid 1 ml
Isovaleric acid 1 ml

Phenylacetic acid 1 g

 

104

TABLE 29. GCS-RF Medium

 

In ' n Amount par 3m ml
Glucose 0.2 g
Cellobiose 0.2 g
Starch 0.2 g
Yeast Extract 0.6 g
Trypticase 1.5 g
Rumen fluid 60.0 ml
Mineral #1‘ 11.2 ml
Mineral #2‘ 11.2 ml
Resazurin 0.3 ml
Distilled I-I,O 193.6 ml
Cysteine-H01 6.0 ml
NaCOa 15.0 ml

 

‘Composition of mineral mixtures are shown in Table 25.