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thesis entitled
CHEMICAL COMPOSITION AND ENSILING CHARACTERISTICS
OF WHOLE CROP WHEAT, OATS, AND BARLEY HARVESTED
IN THE MILK AND DOUGH STAGES OF MATURITY

presented by
Todd Martin Byrem

has been accepted towards fulfillment
of the requirements for

Mdegree in Animal Science

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CHEMICAL COMPOSITION AND ENSILING CHARACTERISTICS
OF WHOLE CROP WHEAT, OATS, AND BARLEY HARVESTED IN THE
MILK AND DOUGH STAGES OF MATURITY

BY

Todd Martin Byrem

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1988

ABSTRACT

CHEMICAL COMPOSITION AND ENSILING CHARACTERISTICS
OF WHOLE CROP WHEAT, OATS, AND BARLEY HARVESTED IN THE
MILK AND DOUGH STAGES OF MATURITY

BY
Todd Martin Byrem

Wheat, oats, and barley were harvested for silage in
the milk and dough stages of maturity to determine whether
late maturation has adverse effects on silage quality.
Whole plant composition data revealed lower water content,
water soluble carbohydrate (WSC), and buffering capacity in
the dough stage of maturity (P<.05). Fiber fractions were
not effected by stage (P>.05) in wheat and barley but were
lower for dough stage oats (P<.05). Total nitrogen was not
different (P>.05) among species or stage of maturity.

During fermentation in experimental silos, milk stage
silages exhibited faster pH declines (P<.05), greater WSC
utilization (P<.05), and greater lactic acid production
(P<.05), indicating a more significant role of fermentation
in the preservation of earlier harvests. Volatile fatty
acid analysis indicated all silages were of excellent
quality. Protein degradation was more rapid in milk stage
silages (P<.05), and higher throughout the duration of

fermentation.

ACKNOWLEDGMENTS

I am ever indebted to Dr. Werner G. Bergen, committee
chairman, for the opportunity to continue my education
beyond the Bachelor's degree. The guidance and assistance
he provided in the preparation of this work will not be
forgotten. To my other committee members, Dr. Milo Tesar
and Dr. Margaret Benson, I would like to express my
sincere appreciation for the lasting influence they have
bestowed upon me. I regret now the benefit of their
knowledge and practicality may not have been realized to
it's full potential.

I would like to mention Dr. Robert Merkel, a voice in
the background but never out of mind, for during times of
question his sound advice brought timely relief. I would
also like to thank Elaine Fink for the technical support
without which my results would still be awaiting. Never
could someone find a more patient and unselfish person
devoting both time and space to this project.

It is not possible to sufficiently acknowledge my
parents Alva and Vera Byrem, however, at this moment, I
find myself pondering over the endless array of sacrifices

they have made for my future. To them I dedicate this

ii

thesis. Also, I would like to put in words my sincere
gratitude for Jim and Pat Zurek, my father and mother in-
law, for their continual support and encouragement that has
inevitably allowed me to succeed in this pursuit.

Finally, I wish to extend my deepest feelings of love,
respect, and appreciation to my wife Theresa and my son
Matthew. In addition to the assistance given in completing
this program, they' gave ‘me 'unending' happiness and. the
motivation to continue. They represent a new meaning to
the word "life" and it is both for and with them I advance

from day to day, month to month, year to year.

iii

TABLE OF CONTENTS

EQQQ
LIST OFTABIES00000000000000.00000000000000000000000 v

LIST OF FIGURES..................................... Vi
LIST OF APPENDIX TABLES.............................viii
1.0 INTRODUCTION............. ........... . .......... l
2.0 REVIEW OF LITERATURE........................... 4
2.1 Silage Microbiology.................. ..... 4
2.2 Silage Chemistry.... ...................... 11
2.3 Fermentation Quality. ..................... 19
2.4 Stage of Maturity. ..... . ................ .. 23
3.0 MATERIALS AND METHODS....... ................... 29
3.1 Harvest....... ....... ..................... 29
3.2 Fermentation........... ...... . ............ 30
3.3 Silage Analysis...................... ..... 31
3.4 Statistical Analysis......... ............. 36
4.0 RESULTS................ ..... .. ............... .. 37
5.0 DISCUSSION ......... ........... ................. 63
6.0 SUMMARY..... ......... . ............... .......... 84
7.0 BIBLIOGRAPHY... ..... ............... ............ 87

APPENDIX0000.0000000000000000000 ...... 00000000000000 95

iv

 

10

LIST OF TABLES

Title Page

 

Dry Matter Yields and Buffering Capacities
upon Harvest of Wheat, Oats, and Barley at
the Milk and Dough Stages of Maturity ........... 38

Influence of Stage of Maturity and Ensiling
on Dry Matter, Total Nitrogen, and Ash in
Wheat, Oats, and Barley...... ................... 39

Influence of Stage of Maturity and Time of
Ensiling on pH in Wheat, Oats, and Barley.. ..... 42

Influence of Stage of Maturity and Time of
Ensiling on Lactic Acid as a Percent of Dry
Matter in Wheat, Oats, and Barley.......... ..... 45

Influence of Stage of Maturity and Time of
Ensiling on Acetic Acid as a Percent of Dry
Matter in Wheat, Oats, and Barley...............47

Influence of Stage of Maturity and Time of
Ensiling on Water Soluble Carbohydrates as

a Percent of Dry Matter in Wheat, Oats, and
Barley.. ............... . ................. . ...... 50

Influence of Stage of Maturity and Time of
Ensiling on Water Soluble Nitrogen as a

Percent of Total Nitrogen in Wheat, Oats,

and Barley................................ ...... 53

Influence of Stage of Maturity and Time of
Ensiling on Ammonia Nitrogen as a Percent
of Total Nitrogen in Wheat, Oats, and Barley....56

Influence of Stage of Maturity and Time of
Ensiling on Acid Detergent Insoluble

Nitrogen as a Percent of Total Nitrogen in

Wheat, Oats, and Barley.................... ..... 58

Influence of Stage of Maturity and Ensiling

on Neutral Detergent Fiber, Acid Detergent

Fiber, and Hemicellulose in Wheat, Oats,

and Barley.... .............. ...... .............. 62

V

LIST OF FIGURES

Figgre Title Page

1 Flow Diagram of Laboratory Analysis Conducted
on Silage Samples........ ............... . ...... 32

2 Changes in pH of Wheat, Oats, and Barley at
the Milk and Dough Stages of Maturity During
EDSiling0000000000 00000 000000000000 00000 0 000000 43

3 Changes in Lactic Acid (% of DM) of Wheat,
Oats, and Barley at the Milk and Dough Stages
of Maturity During Ensiling .................... 46

4 Changes in Acetic Acid (% of DM) in Wheat,
Oats, and Barley at the Milk and Dough Stages
of Maturity During Ensiling....................48

5 Changes in Water Soluble Carbohydrates (% of
DM) in Wheat, Oats, and Barley at the Milk
and Dough Stages of Maturity During Ensiling...51

6 Changes in Water Soluble Nitrogen (% of TN)
in Wheat, Oats, and Barley at the Milk and
Dough Stages of Maturity During Ensiling.......54

7 Changes in Ammonia Nitrogen (% of TN) in
Wheat, Oats, and Barley at the Milk and Dough
Stages of Maturity During Ensiling ...... . ...... 57

8 Changes in Acid Detergent Insoluble Nitrogen
(% of TN) in Wheat, Oats, and Barley at the
Milk and Dough Stages of Maturity During
Ensiling.......................................59

9 Changes of Major Fermentation Parameters
During Ensiling of Milk Stage Wheat.... ........ 78

10 Changes of Major Fermentation Parameters
During Ensiling of Dough Stage Wheat..... ...... 79

11 Changes of Major Fermentation Parameters
During Ensiling of Milk Stage Oats.............80

vi

LIST OF FIGURES (Cont.)

Figgre Title Page

 

12 Changes of Major Fermentation Parameters
During Ensiling of Dough Stage Oats.... ........ 81

13 Changes of Major Fermentation Parameters
During Ensiling of Milk Stage Barley...... ..... 82

14 Changes of Major Fermentation Parameters
During Ensiling of Dough Stage Barley..........83

vii

 

LIST OF APPENDIX TABLES

it e Page

Dry Matter Means for Wheat, Oats, and Barley
at the Milk and Dough Stages of Maturity
During EnSiling0000000000000000.00000000000000 95

Total Nitrogen Means for Wheat, Oats, and
Barley at the Milk and Dough Stages of
Maturity During Ensiling ..... ........... ..... . 96

Ash Means (% DM) for Wheat, Oats, and Barley
at the Milk and Dough Stages of Maturity
During Ensiling ..... ........ ..... ...... ....... 97

Neutral Detergent Fiber Means (% DM) for
Wheat, Oats, and Barley at the Milk and Dough
Stages of Maturity During Ensiling ....... ..... 98

Acid Detergent Fiber Means (% DM) for Wheat,
Oats, and Barley at the Milk and Dough Stages
of Maturity During Ensiling..... ....... . ...... 99

Hemicellulose Means (% DM) for Wheat, Oats,

and Barley at the Milk and Dough Stages of
Maturity During Ensiling.... .................. 100

viii

1 . 0 INTRODUCTION

The primary goal in any forage conservation program is
to maximize the preservation of nutrients found in the
original crop upon harvesting. Haymaking and ensiling are
the most widely accepted practices used today. In the
past, hay was the principal conserved forage utilized for a
number of reasons. The basic skepticisms toward silage
were: 1) large dry matter losses involved during ensiling,
2) inconsistency associated with the ensiling process and
its endproduct, 3) low production by animals fed silage and
4) due to its immobility, silage is rarely a marketable
commodity.

As the understanding of the ensiling process
progressed so too did the use of silage as a conservation
alternative. Even though silage remains a relatively
unmarketable commodity, most other drawbacks have been
alleviated through continued research. When harvested
properly, silage retains more dry matter than its
counterpart hay, and animal performance has not been
different when silage is fed in a completely balanced diet.
Today, with mechanized feedbunks so prevalent, silage can
be fed more efficiently than was possible before. It
became evident that when particular attention is paid to

1

2
such things as plant maturity, moisture content, and
compaction, a consistently high quality feed could be
produced with negligible losses from the ensiling process.

These and similar findings have given the livestock
producer additional options with respect to forage
management. Today it's possible to successfully ensile
virtually any crop for use at a later date. Double
cropping is an option that enables the midwest farmer to
grow and harvest two crops in one year or three silage
crops in two years. This type of system makes use of a
cool season annual small grain which is harvested as silage
by early summer. The increasing use of double, even triple
cropping has prompted the greater use of small grains,
(wheat, oats, rye, and barley) for ensiling. The optimal
stage of maturity at which these grains should be harvested
as silage is still under debate however.

Traditionally it has been recommended that small
grains be harvested for silage at a stage of growth
associated with maximal protein concentrations and minimal
fiber content. This situation occurs during early
development when water is also present in high
concentrations. A great deal of research was directed
toward determining which of the earlier stages of growth
were more conducive to successful fermentation. Meanwhile,
studies by Bishnoi et a1. (1978) and Bolsen and Berger
(1976) have observed better feedlot performance when

animals were fed the more mature milk and dough stage

3
silage. Higher yields, intakes, protein digestibility, and
live weight gains with dough stage silage have prompted the
practice of delaying the harvest of these small grains, but
accompanying maturation is a loss of nutrients required for
efficient fermentation. Ashbell et al. (1985) concluded
the greatest loss of nutrients occurred during transition
of wheat from the milk to dough stage which provided some
insight into the difficulty in ensiling the more mature
small grain crops. There have been few studies measuring
the fermentation parameters of small grains approaching
maturity and the effects of associated nutrient losses on
these parameters. This study was developed to assess the
chemical composition of late stage wheat, oats, and barley,
and to determine how compositional changes that accompany
maturation from the milk to the dough stage of maturity

effect the subsequent fermentation upon ensiling.

2.0 REVIEW OF LITERATURE

2.1 Silage Microbiology

Silage is the product of anaerobic fermentation of a
moist forage. The micro-organisms associated with silage
ferment available carbohydrates and amino acids to organic
acids which inhibit further microbial activity, hence
nutrient destruction. Of most importance to successful
ensiling are the lactic acid bacteria. These facultative
anaerobes are scarce on intact plants (Stirling and
Whittenbury, 1963), and it is believed they arise from farm
machinery involved in the harvest (Gibson et a1. 1961:
Woolford and Wilkins, 1974; Beck, 1978). Obtaining their
name from the primary end product they produce, the lactic
acid bacteria are divided into two broad categories,
homofermentative and heterofermentative.

In silages where lactic acid bacteria dominate the
activity, there is a microbial shift from a homolactic to
heterolactic dominance (Beck, 1972; cited by McDonald,
1981). On the fourth day of ensiling Beck found the most
predominate homofermentative species, Lactobacillus
curvatus and Lactobacillus plantarum, comprised 85% of the
total lactic acid bacteria. By the tenth day of the

4

5

ensiling period, W 229211211 and Lactobacillus
bre is, major heterolactics, dominated the fermentation.
Other genera of lactic acid species found in silage are
streptococci and leuconostocs, which. are. significant in
earlier stages of ensiling, and pediococci, more prevalent
in the later stages. The most widely accepted theory
behind the microbial shift was published by Gibson and
Stirling (1959). They attribute the periodical domination
by certain micro-organisms to their power of survival; this
survival referring to acid tolerance. As the pH decreases,
the less acid tolerant homolactics give way to the
heterolactics. Beck (1978) supports the tolerance theory,
however, believes the shift may be due to the increasing
tolerance of acetate in particular, arising from
‘mitochondrial respiration and bacterial fermentation.
Another possibility can be explained on the basis of
antibiosis. The release of unidentified substances (other
than lactate or acetate) which are inhibitory to other
organisms cannot be totally excluded (Singh and
Laxminarayana, 1973).

Elevated pH, butyrate, and ammonia in silage indicate
the activity of bacteria belonging to the genera
clostridia. Clostridia are responsible for silage of poor
quality, therefore it is of primary interest to minimize
their activity. Because of their presence in soil and lack
of on green plant material (Gibson et al., 1961), it is

generally' agreed their' presence: in. silage :results from

6

contamination by soil and farm equipment. Early studies of
these spore forming, acid intolerant, facultative anaerobes
by Allen and Harrison (1937) led to their division into two
main physiological groups. Those which utilize
carbohydrates as their main energy source are saccharolytic
Clostridia while those utilizing proteins were termed
proteolytic clostridia.

Gibson (1961) identified the common species of
Clostridia found in silage. Q1. butyrigum, Q1.
paraputrificum, and §_. tyrobutyrigum are saccharolytic,
but also have the ability to utilize lactate as a
substrate. These organisms have a greater acid tolerance
than the proteolytic Clostridia, and if given a chance, can
convert strongly acidic lactate to weaker acids and neutral
alcohols (Wood, 1961). This counteraction of acidification
provides an environment suitable for proteolytic
Clostridia, mainly 9;. bifermentans and 9;. sporogenes
(Gibson, 1961). These bacteria acquire their energy from
amino acids, arising from plant enzyme based protein
degradation, through three types of fermentation pathways
summarized by Barker (1961) and Mead (1971). Briefly they
are deamination, decarboxylation, and Stickland reactions
which liberate ammonia and organic acids, carbon dioxide
and amines, and fatty acids, respectively. Other
Clostridia found in silage are (Q1. sphenoides and (Cl.
perfringen§_ which have been shown to be highly

saccharolytic and proteolytic.

7

Another common group of bacteria occurring in silage
are from the family enterobacteriaceae. Gibson et al.
(1958) identified. Klebsiella sp., Escherichia. 991;, and
Bacterium herbicola as those found in the greatest
concentrations. Commonly called coliforms or acetic acid
bacteria, their existence in silage is limited to the early
stages of ensiling provided acidic conditions develop
(Langston and Conner, 1962). Considered weakly
proteolytic, they ferment mainly carbohydrates to acetate,
lactate, and carbon dioxide, and in some situations are
known to produce formate, ethanol, 2,3-butanediol,
succinate and H2. For this reason, Stirling (1951)
concluded that coliform bacteria were undesirable since
they compete with lactic acid bacteria for available
carbohydrates.

Fungi are eukaryotic micro-organisms, the majority of
which require oxygen in order to grow. With ensiling being
a predominantly anaerobic process, their significance was
overlooked until 1964 when Beck and Gross found a
correlation. between the existence of“ yeasts and. silage
instability upon feedout. However, Henderson et a1. (1972)
found 105 of these organisms per gram of silage raising the
question of their contribution to ensiling itself. It is
generally agreed that being very acid tolerant, the fungi,
including yeasts and molds, merely survive fermentation and
exert their effects by respiring away available nutrients

when exposed to air. The more fungi present when the silo

8
is opened, the greater the extent of aerobic deterioration
(Ohyama and Masaki, 1975).

From the time the silo is filled until the time the
silage is consumed, definitive changes take place with
respect to the microbial population. The majority of
microbes found on the original plant are aerobes and their
dominance lasts until oxygen is depleted (Gibson and
Stirling, 1959). During this stage they compete with plant
enzymes for oxygen and nutrients, primarily available
carbohydrates. According to Ruxton et al. (1975) , rapid
achievement of anaerobic condition is crucial. Respiration
of carbohydrates produces heat, carbon dioxide and
indirectly more aerobes, none of which contribute to
successful ensiling. As a result up to 50% of the
fermentable sugar can be lost. Once oxygen is depleted,
plant and microbial respiration cease and the anaerobes,
mainly coliforms, clostridia, and lactic acid bacteria,
begin to proliferate. At this time, the forage medium is
suitable for all types of anaerobes present and the success
or failure of ensiling may be determined in the next few
hours.

In a quality silage there was initially enough soluble
carbohydrates to sustain a lactic acid fermentation. The
coliform bacteria usually predominate until day three at
which. time the accumulation of lactic acid favors the
growth of lactic acid bacteria. The dominate species of

lactic acid bacteria thereafter depends on the pH (Beck,

9
1978). Once a pH of 4.2 has been established, the activity
of other microbes is assumed negligible (Gibson and
Stirling, 1959).

When a stable pH has not been attained, two pursuant
fermentations may develop depending on the carbohydrate
supply. Acetate silages have been described by Henderson
and McDonald (1975). They found that some silages produced
under laboratory conditions had undergone fermentation by
coliform bacteria. Due to human handling in these cases,
insufficient inoculation with lactic acid bacteria resulted
in adequate preservation but by high acetate
concentrations. However, when the available carbohydrates
are exhausted before a stable pH is reached, clostridia
became active.

It has been known for some time that clostridia are
extremely sensitive to water availability. The critical
pH, below which clostridial growth is inhibited, was
observed by Wieringa (1958) when the water availability was
adjusted by either wilting or sodium chloride addition. In
both cases, the critical pH increased as dry matter
increased and when wilted to 30% dry matter, clostridial
growth is restricted irrespective of pH. In cases where
they do develop, the saccharolytic clostridia are first to
appear fermenting lactate to butyrate (Stirling, 1954; as
cited by Woolford, 1984). As butyrate replaces strongly
acidic lactate, the pH increases to a point where

proteolytic clostridia can proliferate depleting the silage

10
of valuable nitrogen constituents. Thus, the quality of
silage is very dependent on the dominating species which is
principally governed by the rate and extent of

acidification.

11

2.2 Silage Chemistry

It must be recognized that silage cannot be
nutritionally superior to the original forage ensiled. By
its very nature fermentation is a destructive process and
nutrients in the original crop undergo changes yielding
products of equal or diminished value. Some reactions
involve the evolution of gases with a consequent loss of
dry matter altogether. These reactions are catalyzed by
enzymes endogenous to the plant material and those found in
the relevant micro-organisms. The majority of reactions
during ensiling involve carbohydrates, organic acids, and
proteins.

Water-soluble carbohydrates are the primary source of
energy for those organisms which are responsible for silage
fermentation. The most abundant of these are the readily
available sugars (glucose, fructose, and sucrose) and the
storage carbohydrates (fructosans and starch). The
disaccharide sucrose was found by Raguse and Smith (1966)
and Smith (1973) to account for 20-56 g kg'1 DM in legumes,
nearly twice that observed for the monosaccharides glucose
and fructose. Fructose is generally found in greater
concentrations than glucose. Fructosans, found primarily
in the stem of grasses by Mackenzie and Wylam (1957), are
usually in the range of 50-90 g kg"1 DM, increasing with

age. Starch, used for storage by legumes and grains,

12

accumulates throughout growth attaining concentrations as
high as 280 g kg'1 DM in corn (Smith, 1973). Sucrose,
fructosans, and starch yield their respective monomers,
mainly glucose and fructose, upon acid hydrolysis or
enzymatic cleavage. Of the structural carbohydrates, only
hemicellulose makes a significant contribution to the
fermentation process. The products released from
hemicellulose degradation are mainly the pentoses,
arabinose and xylose.

The level and proportion of the individual
constituents which are collectively regarded as
carbohydrates are influenced by numerous factors. Edwards
et al. (1968) working with barley cultivars and Waite and
Gorrod (1959) with three species of grass, observed peak
concentrations of water-soluble carbohydrate in the milk
stage of maturity. Accompanying maturation was a steady
decline in free monosaccharides and a concomitant increase
in both the storage forms of soluble carbohydrates and the
insoluble structural carbohydrates with the latter
accounting for the greater proportion. Other variables
affecting carbohydrate content in green plants include
nitrogen fertilization 0Jones, 1970), weather' conditions
(King et al., 1984), plant density, leaf-to-stem ratio and
time of day (Smith, 1973). Once harvested, silage is
exposed to an initial aerobic phase where plant enzymes
continue to oxidize available carbohydrates. The practice

of wilting the crop in the field prior to ensiling

13

potentiates this problem even though photosynthesis is
still active (Brady, 1973). The oxidation of
monosaccharides through the citric acid cycle via the
glycolytic pathway occurs at the expense of sucrose,
fructosans, starch, and hemicellulose. It was shown by
Wylam (1953) that during a 4 hour wilt, levels of sucrose
and fructosan had fallen by 23% and 26%, respectively,
while concentrations of glucose and fructose remained
constant. Amylase, a starch hydrolysing enzyme, has been
identified in various plant families by Gates and Simpson
(1968). Carpintero et al. (1979) compared four wilting
regimes against the original herbage and found no
significant difference in water-soluble carbohydrates.
They attributed this to hemicellulase activity on insoluble
hemicellulose yielding the soluble sugars, xylose,
arabinose, galactose and glucose (Dewar et al. 1963).

Oxidative reactions stop once anaerobic conditions
have been established inside the silo and surviving
carbohydrates are available to the developing microflora.
Wood (1961) found that the homolactic bacteria used the
glycolytic pathway to produce two moles of lactate per mole
of glucose or fructose. IHeterolactics use the hexose
monophosphate pathway which yields one mole each of
lactate, C02, and ethanol when glucose is fermented and
lactate, acetate, C02 and 2 moles of mannitol when 3 moles
of fructose are fermented. The production of carbon

dioxide constitutes a loss of dry matter and is one reason

14

why homolactic fermentation is preferred. Another reason
raised by Whittenbury et al. (1967), is the efficiency of
lactate production. Since lactate is a stronger acid than
acetate, the more efficient homolactic fermentation results
in a steeper decline in pH which is beneficial to
preservation. The fermentation of pentoses to a mole each
of lactate and acetate is similar in both types of lactic
acid bacteria (Wood, 1961).

Quantitatively, the two most important organic acids
present in herbage are citric and malic acids (Hirst and
Ramstad, 1957) . Other acids they found in appreciable
amounts were malonate, succinate, and glycerate. Interest
in organic acids increased when Playne and McDonald (1966)
found these acids accounted for 68%-80% of the buffering
capacity in herbage. Alfalfa and clover, noted for their
difficulty in ensiling, contain twice the concentration of
organic acids than ryegrass. Playne et al. (1967) studied
the role of plant enzymes in the metabolism of organic
acids. When aseptically-grown timothy was ensiled with and
without bacterial inoculum, the inoculated silage exhibited
a 65% degradation of malic acid while only minimal
degradation was observed in microbe-free silage.

The metabolism of organic acids by bacteria during
ensiling is rapid and limited to the lactic acid bacteria.
The pathways of citrate and malate fermentation, described
by Edwards and McDonald ( 1978) , are numerous and involve

pyruvate as a common intermediate. A. wide range of

15
products are formed. depending on ‘the. pH; they include
lactate, acetate, formate, 2,3-butanediol, <ethanol, C02,
and alkaline released cations. Gunsalus and Campbell
(1944) reported that as the pH was adjusted toward 7, the
predominant product changed from lactate to formate and
acetate.

An excellent review on the role of nitrogenous
compounds in silage has been jpublished by' Ohshima and
McDonald (1978). Proteins contain 75%-90% of the total
nitrogen found in fresh herbage, and their amino acid
composition is relatively constant between all forage crops
examined (Wilson and Tilley, 1965; Gerloff et al., 1965).
The amino acids arginine and glutamate were found in the
greatest concentrations, ranging between 12% and 14% of the
total protein nitrogen, while glycine, lysine, alanine, and
aspartate ranged. between 6% and 8%. Lyttleton (1973)
identified stage of maturity and fertilization as primary
factors influencing forage protein content. The remaining
nitrogen found in plant tissue is termed non-protein
nitrogen, and relevant constituents include free amino
acids, amides, amines, nitrate, and variable length
peptides. Low levels of ammonia (1.5% of total nitrogen)
have been detected by Brady (1960) and are assumed to occur
from nitrate reduction.

Protein degradation to water soluble nitrogen, mainly
amino acids, ammonia, and amides, by plant proteinases is

considered rapid and extensive (Bergen et al., 1974).

16

Ohshima and McDonald (1978) reported values of soluble
nitrogen as high as 60% of the total nitrogen. Brady
(1960) observed a three-fold increase in soluble nitrogen
during a 48 hour period of ensiling and limited degradation
thereafter. He also examined the fractions of resultant
water soluble nitrogen and found a substantial increase in
amino nitrogen and minor amounts of ammonia and amide
nitrogen. Using microbe free grass, Kemble (1956)
confirmed enzymatic activity on amino acids as well, but
only to a limited extent. The majority of enzymes involved
in protein hydrolysis are known to be acid labile and in
silages of sufficiently low pH (4.3), hydrolysis levels off
by day 5 of ensiling (Bergen et al., 1974).

Even in silage undergoing vigorous lactic acid
fermentation, bacterial degradation of amino acids will
occur. Among many accounts of both deamination and
decarboxylation of amino acids by lactic acid bacteria,
Brady (1966) produced evidence showing deamination of
serine, arginine, glutamine, and asparagine by L. plantgrgm
and L. brevis. However, it is generally agreed these
activities are minimal compared to clostridial fermentation
of amino acids. Clostridial activity results primarily in
ammonia which can contain 60% of the total nitrogen in
silages where clostridia dominate the microflora (Kemble,
1966). Sources of ammonia are deamination and Stickland
reactions along with the reduction of nitrate. Amines,

which are products of decarboxylation, are commonly found

17
in elevated concentrations in butyrate silages and have
been associated with toxicity in animals (Dain et al.,
1955). Decarboxylation also evolves carbon dioxide again
constituting a loss of dry matter. In any extent, the
fermentation of amino acids results in a significant loss
of nutritive value.

Another undesirable nitrogen transformation is a non-
enzymatic browning reaction commonly called the Maillard
reaction. These reactions typically involve a
condensation between aldose sugars and amino compounds,
later polymerizing to form compounds unavailable to
enzymatic hydrolysis during rumen fermentation or small
intestinal digestion (Hodge, 1953). Prior to
polymerization, intermediates in this process, termed
premelanoidins, are soluble in water and included in the
water soluble nitrogen fraction. Bergen et al. (1974) and
Brady (1960) separated the water soluble nitrogen fraction
into amino acid nitrogen, including small peptides, ammonia
nitrogen, and an undetermined fraction constituting as much
as 48% of the total. This later third may contain the
premelanoidins but once polymerization has occurred,
melanoidins become insoluble accumulating in the fiber
fraction. Thomas et al. (1982) identified heat exposure
and moisture level as the major factors influencing
nitrogen transformation in silage as measured by acid

detergent insoluble nitrogen formation. Other factors

18
included aeration and near neutral pH, characteristic of

poor ensiling conditions.

19

2.3 Fermentation Quality

There have been many attempts to characterize the
entire silage fermentation. process with. quick: and. easy
determinations of single fermentation parameters. This may
well be useful to the farmer who has limited access to
facilities capable of extensive chemical analysis.
However, the effectiveness of preservation is a function of
many interrelated factors. Alone or in combination,
knowledge of these factors are instrumental in determining
what actually occurred in the silo.

The loss of dry matter (DM) during fermentation has
been. difficult to determine because of the problem of
separating these from losses in the initial aerobic phase.
IMcDonald et al. (1973) using calculations based on
fermentation. pathways, figured. a DM loss of 4.2% when
heterolactic bacteria dominated the fermentation. This
agrees well with actual losses of 2.7% to 10% observed by
Anderson and Jackson (1970) for a wide range of dry
matters. McDonald et al. (1973) calculated a complete
recovery of DM with homolactic carbohydrate fermentation
whereas heterolactic fermentation results in losses of 24%
and 5% when substrates are glucose and fructose,
respectively. Losses associated with clostridial
fermentation are much higher. The production of butyrate

from lactate liberates 51% of the DM as carbon dioxide, and

20
amino acid degradation is a significant source of loss no
matter what pathway (deamination, decarboxylation, or
Stickland) is undertaken. Although the loss of dry matter
is a very crude measure of fermentation quality, it can
give some insight about the dominant fermentation.

Silage pH has been widely used as a measure of silage
quality and it is generally thought that a pH of 4.5 or
less is indicative of good fermentation. However, Geasler
(1970) found that as the mean dry matter increased from 21%
to 43%, mean critical pH increased from 3.52 to 4.65 with
one sample achieving stability at pH 5.63. As mentioned
earlier, clostridia's acid tolerance is highly dependent on
water availability with wetter silages requiring more acid
to inhibit clostridial growth. Rather than the amount of
acid produced, a more descriptive measure would be the rate
of acid production. Responsible for excessive nutrient
destruction, undesirable micro-organisms including yeasts,
coliforms, and clostridia, are active during the initial
stage of ensiling, the extent of which is positively
correlated to pH days above 4.00 (Gibson et al., 1958). In
addition, most plant enzymes responsible for protein
degradation are acid labile and their activity ceases as
the pH nears 4.3 (Kemble, 1956; Brady, 1960; MacPherson and
Violante, 1966; Bergen et al., 1974). Decreasing the
length of time above pH 4.3 will retard protein

degradation. Therefore, a rapid decline in pH represents a

21
lower degree of nutrient destruction compared to more
gradual acidification.

Woolford (1984) stated, "Chemical assessments of the
principal fermentation products provide an unequivocal
basis on which to judge quality." In 1938, Flieg developed
a system to evaluate fermentation based on the production
of lactic, acetic, and butyric acids. This system was
revised by Zimmer (1966) as cited by McCullough (1978) into
the most recognized method in use today. Briefly, points
are awarded based on each acid's contribution to the total
acid concentration, with maximum points indicating quality
fermentation. Greater scores are received for elevated
proportions of lactic acid and minimal concentrations of
acetate and butyrate.

McDonald et al. (1962) produced a clostridial silage
yielding a comparatively low level of butyrate, with 29%
ammonia nitrogen the only sign of amino acid degradation.
These results prompted investigators (Carpintero et al.,
1969) to include volatile nitrogen, ammonia being the major
constituent, when classifying fermentation quality. The
production of ammonia during ensiling is due primarily to
proteolytic clostridia (WOod, 1961), and positively
correlated to amino acid deamination (Gibson et al., 1958).
Well preserved silages have been found to contain up to 12%
of the total nitrogen. as ammonia, greater* amounts are
indicative of clostridial activity (Carpintero et al.,

1969).

22

Enhanced formation of acid detergent insoluble
nitrogen (ADIN) requires heat and oxygen usually present
during the initial aerobic phase after ensiling (Thomas et
al., 1982). In the event of an inordinant amount of air
entrapped in the silo, heat from plant and microbial
respiration can produce temperatures in excess of 60°C.
This is evidenced by a dark brown, caramel smelling product
and is quantified by ADIN analysis. ADIN values of less
than 10% of the total nitrogen are deemed desirable,
indicating minimal heat damage to forage proteins (Goering
et al., 1972).

Taken together, these factors reveal. a. great. deal
about the activities occurring in the silo. Researchers
use these parameters to compare the ability of various
treatments to influence silage fermentation, hence, silage

quality.

23

2.4 Stage 0f Maturity

Annual small grains are generally harvested for silage
at four stages of growth. Oltjen and Bolsen (1978)
described these stages as follows:

Bg_t. IHead, remaining inside stem, visibly
distends sheath of flag leaf. Head of
main stem usually enters boot stage first,
followed by the tillers. Stage lasts
about 10 days.

Elgwering. iHead is fully emerged with anthems
shedding pollen. Fertilization and
initial grain development occur. Plant is
green but lower leaves have begun to die.
Stage lasts about 7 days.

Milk. ‘White, milk-like fluid occupies the

 

kernel, made up of water and many starch

granules. More leaves die; embryo
develops fully; Stage lasts. 10 ‘to 14
days.

131191.1- Water content of kernel decreases to a
dough consistency. Leaves are dying;
plant changes from green to yellow. Stage
lasts 10 to 14 days.

The effect of maturity on silage fermentation is

related to the content of moisture, water soluble

24

carbohydrates, and buffering constituents at the time of
harvest. Water content being the most important, it has
also been directly related to effluent loss (Miller and
Cliftin, 1965) and silage intake (Johnston et al., 1970).
As with most plant species, the dry matter of cereal grains
increases as maturity progresses. While ensiling whole
crop barley, Edwards et al., ( 1968) reported dry matter
percentages of 20, 26, 31, and 37 for boot, flowering,
milk, and dough stages, respectively. Depending on
variety, results of Bolsen and Berger (1976) show similar
dry matters at various stages of growth for wheat, oats,
and barley.

Lactate, the most. beneficial acid.‘with. respect ‘to
silage production, is the dominant product of fermentation
when the moisture content of the ensiled crop is correct.
A range of 55% to 70% moisture for cereal crops is
recommended, with 60% to 65% the optimum (Oltjen and
Bolsen, 1978). As the moisture increases beyond 70%, a
given hydrogen ion concentration becomes less inhibitory to
undesirable micro-organisms. MacGregor and Edwards (1968)
ensiled barley at various growth stages with dry matters
ranging from 19.1% to 42%. silages harvested at boot and
flowering stages contained the greatest amount of
fermentation acids, yet. had the. highest pH; Butyrate
concentrations reached 1.95% of the DM for' the wetter
silages while only trace amounts were found at the milk and

dough stages. Lactate to acetate ratios were 1.24, 1.50,

25
3.22, and 1.63 for boot, flowering, milk, and dough stages,
respectively. The greater extent of fermentation in the
low dry matter silages also suggests higher fermentation
losses.

Ensiling excessively dry crops has been reviewed by
Gordon (1967) . Larger amounts of oxygen are entrapped
within the silage mass increasing the possibility of
heating and molding. These silages usually exhibit
elevated acid detergent insoluble nitrogen and pH due to
minimal anaerobic bacterial activity.

Water soluble carbohydrates (WSC) being the primary
fermentable substrate are needed in quantity to allow
sufficient lactic acid production. Compared to legumes
which average 5% to 10% WSC on a dry matter basis and
remain relatively constant throughout growth, cereal grain
crops have greater WSC concentrations which are very
dependent on the stage of maturity. Ashbell et al., (1985)
measured the WSC concentration in wheat at the four common
stages of maturity. They found WSC concentrations of 20,
19, 28, and 13% in the boot, head, milk, and dough stages,
respectively. A mutual peak concentration during the milk
stage has also been observed in oats by Sutoh et al.,
(1972). lMacGregor and Edwards (1968) followed the
individual components comprising the WSC fraction during
the maturation of barley and concluded that varying
fructosan levels were responsible for the peak (31.9%) and

subsequent decline (14.7%) into the dough stage.

26
Fructosans are believed to be temporary storage compounds
for available carbohydrates before conversion into starch.

It is generally assumed that WSC concentrations of at
least 10% will result in satisfactory preservation
irrespective of dry matter content. In cereal grain
silages, soluble carbohydrates are rarely a limiting factor
of preservation (Oltjen and Bolsen, 1978). In fact, they
have been extensively used in combination with both legumes
and. grasses to insure sufficient substrate. availability
during ensiling. However, in work reported by Sutoh et
al., (1972), resultant silage from green oats harvested at
boot, flowering, milk, and dough stages were all
unsatisfactory as evidenced by Flieg scores of 37, 58, 33,
and 27, respectively. The WSC concentrations never reached
9% in that study (Sutoh et al., 1972) and no explanation
was given for the unusually low levels of carbohydrates
observed.

Organic acids, being the prominent buffering
constituent in plants, dictates to some extent the relative
ensilability of a crop in terms of the amount of acid
necessary to achieve anaerobic stability. MacGregor and
Edwards (1968) reported a linear decline in the
concentrations of malate and citrate, the most abundant
organic acids in barley, during maturation. Total organic
acids accounted for 6.6% of the dry matter in the boot

stage while only 1.1% was found in the dough stage.

27

Proteins can contribute to the buffering capacity
associated with plant material and their concentration is
known to decrease with maturity in corn (Johnson et al.,
1966), wheat (Ashbell et al., 1983), oats (Sutoh et al.,
1972), and barley (Edwards et al., 1968). However, because
of the relatively low crude protein content as compared to
legumes, the contribution of proteins to the buffering
capacity has been overlooked in cereal grain silage (Oltjen
and Bolsen, 1978).

The effect of maturity on proteolysis during ensiling
is believed to be related to the plant's dry matter
content. Fermentation is known to diminish as the dry
matter percentage increases as evidenced by lower
concentrations of fermentation acids and high pHs. Hawkins
et al., (1970) ensiled freshly chopped and wilted alfalfa
of the same maturity and concluded the extent of
proteolysis decreases with increasing dry matter content.
Geasler (1970) ensiled whole crop corn at various dry
matters and reported reductions in water soluble nitrogen
and ammonia of 43.7% and 51%, respectively, as the dry
matter increased from 22% to 42%. These results were
confirmed by Bergen et al., (1974). Reports of proteolysis
during the ensiling of small grains are conflicting. When
expressed as a percentage of total nitrogen, ammonia
production increased with dry matter content in oats (Sutoh
et al., 1972), while Ashbell et al., (1985) observed a

decrease when ensiling wheat at more mature and dryer

28
growth stages. Water soluble nitrogen was not presented in
either study. However, the effects of moisture on
proteolysis in small grain silage would be expected to be
similar to those observed for other type silages.

In summary, the earlier stages of growth are
associated with high moisture and buffering contents, and
comparatively low amounts of water soluble carbohydrates.
Taken together, the result. is a. material with. a high
probability of protein degradation and clostridial
fermentation unless wilted prior to ensiling. Optimal
ensiling characteristics are observed during the milk stage
of maturity, however, the more mature stages have
substantially lower protein concentrations. Although the
high dry matter percentage in the dough stage lessens the
necessity of fermentation, water soluble carbohydrates may
be a limiting factor of successful preservation. Also,
diminishing protein contents observed during these latter
stages may be offset by lower rates of protein degradation

during initial stages of fermentation.

3.0 MATERIALS AND METHODS

Frankenmuth wheat, Corwood oats, and Bowers barley
were harvested for silage at the milk and dough stages of
maturity. Fermentation was carried out in small
experimental silos and opened for analysis at predetermined

times after sealing.

3.1 Harvest

In 1986, wheat was harvested on June 24, and July 2,
oats and barley on July 2, and July 14, for the milk and
dough stages, respectively. Due to greater than average
rainfall during the spring, wheat was harvested later than
planned. On all harvest dates, a small flail-type
harvester was used to cut 5 successive 3' x 25' paths and
weights were recorded for average yield analysis. Milk
stage wheat was further chopped to .5 inch on a paper
cutter in the field. When it appeared that the paper
cutter would not suffice for multiple harvestings on July
2, the remaining samples were transferred into burlap bags
and transported to the Beef Cattle Research Center where
they were hand fed into a typical tractor-drawn forage
harvester adjusted for a chop length of .5 inch. After
chopping, the samples were immediately packed into silos.

29

30

3.2 Fermentation

The experimental silos consisted of 2 quart Mason
canning jars equipped with lids modified with automatic gas
release valves. Valves were constructed by attaching
rubber policemen onto 15 gauge hyperdermic needles which
were inserted through rubber stops sealed into the lids.
Tiny slits were cut into the policemen with an Exacto knife
to allow the escape of gasses without the re-entry of air
once the silos were sealed.

Before the silos were filled, 10g of solid carbon
dioxide was placed in the bottom of the silo to promote
evacuation and anaerobiosis. For each harvest, sixteen
silos were filled with approximately 1.5kg of chopped plant
material and compressed by hand. Between 4 and 6 hours had
elapsed from the time of harvest until the last silo was
sealed. Silos were sealed and incubated at 30°C for 3
days: then allowed to set at room temperature (20°C) for
the remaining time period. In addition to two samples of
fresh material taken from each field, two silos of each
grain were opened after .5, 1, 2, 4, 8, 16, 32, and 64 days
from the average time of ensiling. The contents of the
opened silos were mixed thoroughly and a representative
sample from each silo was frozen at -20°C until subsequent

analyses.

31

3.3 Silage Analysis

A Flow diagram. of analyses conducted is shown in
Figure 1” Immediately after thawing, the silage samples
were initially ground through a Hobart chopper for two
minutes to reduce the particle size and facilitate mixing.
Total nitrogen was directly determined by sulfuric acid
digestion in the presence of a copper sulfate based
catalyst (Pope Kjeldahl mixtures, P.O. Box 903, Dallas,
Texas 75221). The analysis of free ammonia nitrogen was
conducted on a Technicon Auto Kj eldahl System calibrated
with 100 and 200 ppm. nitrogen standards from ammonium
sulfate.

Dry matters were determined by drying 100 grams of
ground sample at 55°C in a forced air oven (verified by
Geasler, 1970). Dried samples were ground in a Wiley mill
through a 1.ndllimeter screen and later analyzed for ash,
neutral detergent fiber (NDF), acid detergent fiber (ADF),
and acid detergent insoluble nitrogen (ADIN) . Ash was
determined by heating to 500°C for 3 hours in a muffle
furnace and weighing the residue. NDF and ADF was analyzed
according to Goering and Van Soest (1970). Amylase
digestion according to Goering and Van Soest (1970) was
necessary during NDF analysis because of the high
concentrations of starch expected in the samples.

Hemicellulose was calculated as the difference between NDF

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 2
Sample
Total Nitrogen
Dry Matter
Adi.___
EXTRACT
NDF‘———-
Buffering Capacity
ADF
H
ADIN p
Meta-phosphoric Acid Suliosalacylic Acid
Soluble Nitrogen
VFA’s
Lactic Acid
NH 3-Nitrogen
Soluble Carbohydrate's
FIGURE 1

Flow Diagram of Laboratory Analysis
Conducted on Silage Samples

 

33
and ADF. ADIN was analyzed by micro-Kjeldahl digestion of
the ADF residue in concentrated sulfuric acid and Pope
Kjeldahl catalyst. Samples were quantified on the
Technicon Auto Analyzer.

Silage extracts were prepared by homogenizing a 20
gram aliquot of chopped sample with 200 milliliters of
chilled distilled and deionized water. Homogenization was
conducted with a Sorvall Omnimixer at maximum speed for two
minutes in an ice bath. The resultant slurry was strained
through four layers of cheesecloth and portioned into three
fractions of 100, 22.5, and 20 milliliters kept chilled
until further preparation.

The pH was determined immediately on each 100
milliliter aliquot with an Orion (model 801A) digital
ionalyzer equipped with a glass electrode. Time 0 samples
were acidified to pH 3 with .1 normal hydrochloric acid and
the buffering capacity was calculated as the
milliequivalents of sodium hydroxide necessary to raise the
pH from 4 to 6. Buffering capacity is expressed as
milliequivalents per 100 grams of dry matter.

The 22.5 milliliter portion was deproteinized with 2.5
milliliters of 50% sulfosalicylic acid (SSA) and
centrifuged at 15,000 x g for fifteen minutes. The
supernatant was poured into a sterilized blood dilution
‘vial and frozen at -20°C for later analysis. Colorimetric
procedures of Barker and Summerson (1941) and Dubois et

al., (1958) were used for the determination of lactic acid

34

and water soluble carbohydrates (WSC) , respectively. Both
were quantified by linear regression on a standard curve
constructed by varying the concentrations of lactic acid
and glucose around the range expected in the silage
samples. Water soluble non-protein nitrogen (WSN) was
determined by digesting 5 milliliters of the SSA extract in
concentrated sulfuric acid and Pope Kjeldahl catalyst and
analyzed on the Technicon Auto Analyzer.

The remaining 20 milliliter portions were vortexed for
thirty seconds with 4 milliliters of 25% meta-phosphoric
acid and allowed to set at room temperature for 30 minutes.
After deproteinization, the samples were centrifuged at
15,000 x g for fifteen minutes. The supernatant was then
transferred to sterilized blood dilution vials and frozen
at -20°C until further analysis. Volatile fatty acids were
analyzed by a Hewlett-Packard gas-liquid chromatograph
(Model 5840A) fitted with a 183cm x .32cm stainless steel
Chromosorb column (10% SP 1200 and 1% phosphoric acid
80/100 WAW mesh, Supelco Inc., Bellefone, PA.) and with the
following conditions programmed into the chromatograph:
column temperature, 120°C, injection temperature, 170°C,
flame ionization detector temperature, 200°C and carrier
gas flow (Helium) 50ml/min. A standard containing the
three primary volatile fatty acids was used to determine
the concentrations of acetate, propionate, and butyrate
present in the silage samples. Ammonia nitrogen was

determined with the Technicon Auto Analyzer on the meta-

35
phosphoric acid extract. The standards (100 and 200 ppm)
were prepared from a stock solution of ammonium phosphate
at a concentration of one milligram nitrogen per
milliliter. It was recognized that free amino acids are
not precipitated by meta-phosphoric acid, and certain amino
acids are known to react with automated Kjeldahl reagents
and recorded by the analyzer. Weber (1983) deammoniated
duodenal fluid samples by boiling at 100°C of one hour.
After deammoniation, he added 100 and 200 ppm equivalents
of ammonium phosphate. When compared against standards
prepared from distilled water, the duodenal based standards
averaged 2 to 3 ppm more ammonia nitrogen equivalent
presumably due to free amino acids. This 1% difference was
considered insignificant to warrant the adjustment of

standards.

36

3.4 Statistical Analysis

Statistical analysis was performed on the .MSTAT
program developed by Michigan State University's Crops and
Soil Science Department (East Lansing, Michigan).
Fermentation parameters measured over time (dry matter,
total nitrogen, ash, pH, lactate, acetate, WSC, WSN, ADIN,
ADF, NDF, and hemicellulose) were analyzed as a two factor
completely randomized design with forage and stage
combinations as one factor (A) and days of fermentation (B)

the other. The model statement was as follows:
Yijk = mean + A1 + Bj + (AB)ij + error<ij)k

Yield and buffering capacity were subjected to one-way
analysis of variance in a completely randomized design,
again combining forage and stage combinations as
treatments. In all sets of analyses, treatment combination
means were separated by Tukey's honestly significant
difference test (HSD) as described by Gill (1978). Where
interaction between factors was not significant, overall

means were separated as with treatment combination means.

4 . 0 RESULTS

Dry matter yield and buffering capacity of the fresh
herbage are presented in Table 1. In both the milk and
dough stages, wheat yielded approximately twice the dry
matter (P<.05) as either oats or barley. In general,
yields were not different between oats and barley, but
harvesting these grains at the dough stage resulted in
significantly greater yields (P<.05) compared to those
obtained in the milk stage harvest. No difference in dry
matter yield was observed between these two stages for
wheat. At both growth stages, wheat had the lowest
buffering capacity (P<.05) of the three species evaluated
in this study while no difference was found between oats
and barley. In each small grain, the buffering capacity
decreased with maturation from the milk to the dough stage,
however, only the decrease in barley's buffering capacity
was significant (P<.05).

Values for dry matter, total nitrogen, and ash of the
ensiled small grains are presented in Table 2. Since
average time effects were not significant (P>.07), only
initial and final observations are shown here; complete
data sets can be found in the appendix. Overall means
across time include observations from all sampling days.

37

38

 

 

 

Table 1. Dry Matter Yields and Buffering
Capacities upon Harvest of Wheat,
Oats, and Barley at the Milk and
Dough Stages of Maturity.
DM Yield1 Buffering Capacity2
Species Stage Ton/acre mEZlOOg DM
Milk 3.97A 17.45AD
Wheat
Dough 3.90A 16.60A
Milk 1.43B 25.10BC
Oats
Dough 1.90CD 22.30BE
Milk 1.65BC 28.45C
Barley
Dough 2.21D 20.80DE

 

ABCDEMeans within columns with unlike superscripts

differ (P<.05).

1Tabular entries represent averages of five
observations and have a standard error of the
mean (S.E.M.) of .10.

2Tabular entries represent averages of two fresh
samples and have a S.E.M. of 1.41. Values are
expressed as milliequivalents of base required to
raise the pH from 4 to 6.

39

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As expected, greater dry matter percentages were
observed as maturity progressed from the milk to dough
stage (P<.05) for all small grain crops. Due to the
delayed harvest, the dry matter content was higher in wheat
than in oats and barley within both growth stages (P<.05).
Dry matters were different (P<.05) between oats and barley
in the milk stage but similar in the dough stage. Except
for milk stage wheat, dry matter percentages were unchanged
throughout fermentation.

Comparisons of total nitrogen within time and stage of
maturity indicate milk stage barley as having the highest
crude protein content (total nitrogen x 6.25) on Day 0
(P<.05). No differences were observed between the
remaining species and stage combinations on Day 0, and all
species and stage combinations were similar through Day 64.
Nonsignificant interactions between treatments and time
allowed the comparison of treatment means across time.
Overall, there was a decrease in total nitrogen into the
dough stage in oats and barley (P<.05) while no change was
observed in wheat.

Although not significant, ash, as a percent of dry
matter, increased 'with fermentation. in. all species. and
stage combinations. Averaged together, ash increased 13%,
from 7.7% of DM at harvest to 8.7% of DM on Day 64. Growth
stage effects within species were not significant. The ash

content of wheat was significantly lower (P<.05) than oats

41
and barley in the milk stage and oats, but not barley, in
the dough stage.

The pH means are reported in Table 3 and shown
graphically in Figure 2. At harvest, barley had higher pH
values than wheat and oats within the milk stage and oats
within the dough stage (P<.05). The only difference
between stage of maturity at harvest was found in barley
(P<.05). After .5 days, the pH of milk stage oats and
barley had declined to a greater extent than their dough
stage. counterparts (P<.05). ‘This relationship remained
throughout fermentation as the pH continually decreased
through Day 16. In contrast, dough stage wheat had
significantly lower (P<.05) pH values during initial
fermentation but, by Day 8 and thereafter, the pH of wheat
was lower in the milk stage as ‘with oats and. barley
(P<.05). In all silages, the critical pH of 4.5 had been
surpassed by the second day of fermentation and oats and
barley in the milk stage had reached this value between .5
and 1 days. All species and stage combinations plateaued
around Day 16 except for milk stage oats, which continued
to decrease throughout the duration of the experiment
(P<.05). Across species, milk stage silages had lower
stable pH values than silages made from the dough stage
harvest (P<.05).

Initial lactic acid concentrations were not different
between treatment combinations (Table 4 and Figure 3), even

though small amounts were found initially in dough stage

42

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mm.v mamm.e ummfl.e o¢s~.o ammo.e cocon.¢ ocmn.e v
sn.¢ o<so.e oooo~.e oomon.e oo-.e om<¢¢.v o<se.¢ a
no.4 o<mm.e come.¢ omoo.o ooo¢.¢ omoo.e ocmo.e a
oa.m nmsm.m nomo.e nom~.m noeo.v ammo.m n<Hs.m m.
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coo: Boson xaaz aoooo as“: noooo ea“: son

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43

6.1

6.. i.
5.9

 

 

 

Y
5.8 a
507 h :0.
5.6 -
5.5 . ': .
P E ' Nheat --Milk Stage
504 '- ..o .0
- 2:. : ' Oats ""- Dougb Stage
5.3 l- :: :
_ 3.0. o: vBarley
5-2 - °-.'~. °-.
5.1 b ':
5.0 - i ': '-
pH
4.9 C
P
4.8 - l
4.7 b
4.6 b
4.5 L
4.4 b
r
403 '-
4.2 -
L- 0......
4.0 . \
309 '
308 '-
1 l 1 L i L l L
0 .5 1 2 4 8 16 32 64

Days of Fermentation

Figure 2. Changes in pH of Wheat, Oats, and Barley at the

Milk and Dough Stages of Maturity During
Ensiling.

44

oats and barley while lactate was absent among the other
combinations. Lactate concentrations increased
significantly by .5 days of ensiling in milk stage oats and
barley (P<.05) whereas increases of this same magnitude
were not noticed until 1 day in all species at the dough
stage and not until 2 days in milk stage wheat silage
(P<.05). Milk stage oat and. barley silages had. more
lactate than the other silages throughout fermentation but
the differences were not always significant (P>.05). Mean
concentrations across specie and stage combinations
increased continually, leveling off around Day 32 attaining
a maximum concentration of 7.74% of DM on Day 64.
Individually, milk stage silages plateaued later than their
dough stage complements (P<.05) . As can be seen from
Figure 3, at any given time dough stage silages generally
contained less lactate than milk stage silages except for
wheat silage made at the milk stage which did not differ
from any of the dough stage silages.

Analyses were performed for the volatile fatty acids,
acetate, propionate, and butyrate, but no appreciable
amounts of propionate or butyrate were found in any of the
samples under study. Acetate means are presented in Table
5 and displayed graphically in Figure 4. Mean acetate
concentrations increased sharply within time until Day 4
and gradually thereafter through Day 64. The comparison of
individual specie and stage combinations show

nonsignificant but higher initial acetate concentrations in

45

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46

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a". 0
o. . o...-O..°o
.0. 0. ° ..'o:'o
.0 . ‘0
o o
0'..... 0..
. .0 .0
0.... .-
0. o. o.

.5 1 2 4 8 16 32 64
Days of Fermentation

I Wheat -—Milk Stage
0 Oats «on Dough Stage
v Barley

Changes in Lactic Acid (% of DM) of Wheats,
Oats, and Barley at the Milk and Dough Stages
of Maturity During Ensiling.

47

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48

 

 

Figure 4.

.5 1 2 4 8 16 32 64

Days of Fermentation

I Wheat -—Milk Stage
' Oats "-"Dough Stage
v Barley

Changes in Acetic Acid (% of DM) in Wheat,
Oats, and Barley at the Milk and Dough Stages
of Maturity During Ensiling.

49

dough stage silages. During fermentation, acetate
increased more rapidly in milk stage silages than in their
dough stage complements (P<.05). By the end of the
fermentation period, milk stage wheat and barley silage had
the greatest concentration of acetate (P<.05) while :no
differences were observed between the other combinations.
Overall means across time show higher average acetate
concentrations for milk stage silages in wheat and barley
but equal concentrations between these two stages in oats.

Initial water soluble carbohydrate (WSC)
concentrations were different (P<.05) between milk and
dough stages in all cereal grains under investigation
(Table 6 and Figure 5). Losses of 39%, 43%, and 54% of the
initial WSC were observed with maturation from the milk to
the dough stage in wheat, oats, and barley, respectively.
Wheat had higher WSC concentrations than oats and barley
within both growth stages (P<.05) and this relationship
persisted through the end of the fermentation period.
Figure 5 indicates the relationship between oats and barley
within each growth stage was very similar. Dough stage
wheat silage responded just as the other dough stage
silages except was consistently higher in WSC
concentrations (P<.05). Excluding wheat, the disappearance
of WSC was significant (P<.05) throughout fermentation in
milk stage silages whereas in dough stage silages extensive
WSC utilization occurred only during the first four days of

ensiling. WSC concentrations on Day 64 were not different

50

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51

 

P 19
e 18
r
c 17 "
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n
t 15 ‘
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u '.
b 11 '- ..
l 10 L- .
e '.
9 I- CC. 00....
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a _ '0. .0.
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0 ' ...'.O.':: - . ......-....'00.0000
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z 1 I. 000.00 M ' :ztzzzz; :.h$::'
I I l 1 1 1 l I 'T
0 .5 1 2 4 8 16 32 64
Days of Fermentation
I Wheat -— Milk Stage
0 Cats "on Dough Stage
v Barley
Figure 5. Changes in Water Soluble Carbohydrates (% of

DM) in Wheat, Oats, and Barley at the Milk and
Dough Stages of Maturity During Ensiling.

52
between growth stages in oats and barley. This observation
coupled with higher initial concentrations in the milk
stage resulted in greater losses of WSC during fermentation
in milk stage silages. Milk stage wheat silage responded
unlike other silages exhibiting WSC disappearance only
between Day .5 and Day 1 (P<.05).

Water soluble non-protein nitrogen (WSN) means are
presented in Table 7 and displayed graphically in Figure 6.
Interactions were found to be nonsignificant (P>.07) and
permitted the comparison of both treatment and time means.
Within species, average WSN was higher in milk stage
silages than in dough stage silages (P<.05). The
comparison of time means show a two-fold increase of WSN
within Day 1, no changes between Days 2 and 16, followed by
a slight increase thereafter (P<.05). Individually there
were no differences between specie and stage combinations
in the fresh herbage. Compared with their dough stage
complements, WSN increased more rapidly (P<.05) in oat and
barley milk stage silages but concentrations had
equilibrated by the sixteenth day of fermentation. No
differences were observed between the two growth stages in
wheat at all times sampled. Final WSN concentrations were
higher for all milk stage silages on. Day’ 64 but the
differences were not significant.

Comparison of treatment by time combinations shows few
significant differences in ammonia nitrogen between specie

and stage combinations within time (Table 8). Differences

53

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68
66
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62
60
58
56
54
52
50
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46
44
42
40
38
36
34
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30
28
26
24
22
20
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ml—Io‘r—‘Hom «Scoot-(mtg

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Figure

 

54

    

.-:""-:"""
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- 0.. a... V... .. 0
_ .looooooofloo'::::: 00".
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: 000-6......'......'
’- I Wheat _Milk Stage
.- . Cats 00... Dough stage
' V Barley
I I ' ' ' I I l
'5 1 2 4 8 16 32 64

Days of Fermentation

Changes in Water Soluble Nitrogen (% of TN) in
Wheat, Oats, and Barley at the Milk and Dough
Stages of Maturity During Ensiling.

55

(P<.05) were noted only within Days 1, 4, and 64 due
primarily to excessive ammonia production in milk stage
wheat silage. Examination of Figure 7 points out the close
relationship of individual combinations as fermentation
proceeded. Insignificant interactions (P>.1) between
specie and stage combinations and time allowed general
comparisons of aggregate means. Significant increases
(P<.05) were noticed after .5, 1, and 4 days of
fermentation and not again until Day 64, resulting in a
four-fold accretion of ammonia nitrogen. Across time, a
significant difference (P<.05) between milk and dough
stages was observed only within wheat where higher ammonia
concentrations were associated with milk stage wheat
silage. Oat silages generally contained the lowest
percentage of ammonia nitrogen (P<.05), followed by barley
and then wheat.

Changes in acid detergent insoluble nitrogen (ADIN)
for each specie and stage combination are depicted in Table
9 and can be visualized in Figure 8. Interactions were not
significant (P>.10) which. enabled. direct. comparisons. of
treatment and time means. Although there was a difference
between initial and final means averaged across all silages
(P<.05), differences among time means were not consistent
and 'visual interpretation of Figure 8 shows a general
straight line trend across time. Averaged over time, dough
stage silages contained larger amounts of ADIN than milk

stages silages in all three small grains (P<.05). The

56

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Figure 7.

57

 

.S 1 2 4 8 16 32 64

Days of Fermentation

0 Wheat —Milk Stage
0 Cats "-o- Dough Stage
v Barley

Changes in Ammonia Nitrogen (% of TN) in
Wheat, Oats, and Barley at the Milk and Dough
Stages of Maturity During Ensiling.

58

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Figure 8.

I I

 

59

 

 

L.
h-
b
I Wheat —- Milk Stage
__ 0 Oats “"0 Dough Stage
5 v Barley
L.
n u n n u u n u t
O .5 1 2 4 8 16 32 64
Days of Fermentation
Changes in Acid Detergent Insoluble Nitrogen (%

TN) in Wheat, Cats, and Barley at the Milk and
Dough Stages of Maturity During Ensiling.

60

largest increase associated with maturation occurred in
barley as dough stage barley had the highest initial
concentration of ADIN (P<.05), nearly 60 %more than in the
milk stage. Oats and barley in the milk stage produced
silages with the lowest amount of ADIN (P<.05). Reviewing
individual combination means reveals lower values prior to
ensiling although not always significant.

The means for neutral detergent fiber (NDF), acid
detergent fiber (ADF), and hemicellulose are reported in
Table 10. Only initial and final observations are included
in this table since the effects of time within specie and
stage combinations were inconsistent and often misleading:
Complete tables can be found in the appendix. Overall time
effects were not significant (P>.09) in the analysis of NDF
means. Only milk stage wheat silage had different NDF
concentrations (P<.05) by’ the end of fermentation.
Initially, as maturation proceeded from the milk to dough
stage, NDF increased, decreased, and remained the same in
wheat, oats, and barley, respectively (P<.05). The drop in
NDF in barley was consistent but not significant (P>.05).
NDF concentrations were higher in milk stage oats (P<.05)
and lower in milk stage wheat, while the other combinations
centered around 50% NDF. Interactions in ADF analysis were
not significant (P>.09) and differences between overall
specie by stage means were similar to NDF as anticipated.
ADF percentages increased with wheat maturation (P<.05) and

remained the same in barley. During fermentation,

 

61
increased ADF concentrations were observed in three of six
forage combinations resulting in a significant (P<.05)
overall increase of 8.5% over the 64 days of fermentation.
As in NDF, higher ADF concentrations were found in milk
stage oats (P<.05) with the lowest concentration in milk
stage wheat. Hemicellulose decreased in all forage
combinations during fermentation but significance (P<.05)
could only be attributed to the difference in milk stage
barley silage. Decreases in hemicellulose were associated
with the growth stage advancement within oats and barley
(P<.05) but a nonsignificant increase was observed within
wheat. In general, fiber fractions tended to increase as
wheat progressed from the milk to dough stage whereas they
decreased in oats and barley during the same period.
Fermentation had very little affect on these fractions over

the 64 days under observation.

62

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5 . 0 DISCUSSION

All silages evaluated in this study were of excellent
quality according to Flieg scores as modified by Zimmer
(1966). With a score of 100 being ideal, values of 98,
100, 100, 100, 100, and 100 were calculated for milk and
dough stage wheat, milk and dough stage oats, and milk and
dough stage barley silages, respectively. Mason jars
equipped with modified lids and the packing scheme employed
provided excellent conditions, assuring minimal effects of
oxygen inclusion and aerobic microbial activity. Evidence
acquired from the fermentation parameters observed suggests
small grains harvested at the milk stage undergo a higher
degree of both fermentation and nutrient destruction than
when harvested at the dough stage. Furthermore, values
obtained for the more mature stages in this study are
similar to corn silage data alleviating concern over the
substitution of these silages for one another in feeding
regimes.

Figures 9 thru 14 represent the major fermentation
parameters compiled for individual specie and stage
combinations. Comparing Figure 9, milk stage wheat, with
the others reveals a delayed onset of fermentation in milk
stage wheat silage. First cutting wheat (milk stage) was

63

64

chopped by hand with a paper cutter, decreasing the extent
of laceration and microbial inoculations associated with
farm equipment used for the remaining harvests. Diminished
release of plant solubles and lower initial bacterial
counts may have resulted in an additive effect to postpone
bacterial growth and subsequent acid production. Gibson et
al. (1961) studied microbial reproduction over numerous
degrees of laceration and found a positive correlation
between bacterial populations and degree of chopping.
Stirling and Whittenbury (1963) followed the changes in
bacteria numbers from the field to the silo and found
significant numbers of lactic acid bacteria at the silo on
herbage lacking these bacteria in the field. They
concluded that inoculation must have occurred through the
chopper during harvest. Consequently, comparisons between
fermentation rates involving milk stage wheat must be done
with discretion since differences may be due, in part, to
harvesting methods.

Ashbell et al. (1985) , working with wheat, reported
dry matter yields of 4.9 tons/acre and 5.3 tons/acre for
milk and dough stages, respectively. The yield from oats
and barley are generally lower than wheat at similar stages
of growth, averaging 4 tons/acre as reported by Miller et
al. (1967) and Bishnoi et al. (1978). These investigators
found little differences in dry matter yields between milk
and dough stages with the latter averaging slightly higher

yields. Results from this trial show wheat yielding nearly

65

twice the dry matter as did oats and barley however, all
yields were below average and the production of oats and
barley was extremely poor when compared to past
observations (Miller et al., 1967; Bolsen and Berger, 1976;
and Bishnoi et al., 1978). Since yield was not the
objective of this study, plots set aside for production
were small and their maintenance was unlike those for
larger scale production studies. Although production was
not comparable to yields observed by other investigators,
average increases due to maturity were noted, complying
with results of Bishnoi et al. (1978).

Literature on buffering capacities for cereal grain
crops is largely limited to corn. Reported as
milliequivalents (ME)/100g DM, Wilkinson (1978), as cited
by McDonald (1981), observed buffering capacities of 22.5,
18.0, and 14.9 for whole crop corn progressing through the
dough, dent, and glaze stages of maturity, respectively.
MacGregor and Edwards (1968) reported buffering capacities
for barley silages of various maturation stages but these
values are irrelevant since these materials had already
been ensiled. In the same study, initial organic acids
comprising the majority of buffering constituents in fresh
herbage accounted for 6.6% of the dry matter in the head
stage and continually decreased to 1.1% of the dry matter
in the dough stage, implying an inverse relationship
between maturity and buffering capacity. In the present

experiment, the buffering capacities were similar to values

66

obtained for corn, averaging 22 ME/lOOg DM and decreased
with growth stage advancement in all three species under
investigation. Although wheat was harvested a week late
due to weather conditions, this could not account for all
the difference observed between wheat, with the lowest
capacities, and the other two small grain crops. Depressed
buffering capacities have been associated with ease of
ensiling observed in grasses and corn when compared to
legumes (Playne and McDonald, 1966). Buffering capacity
would rarely be a concern when ensiling these small grain
crops during later stages of maturity, especially in the
dough stage and wheat in particular.

Compositional changes in plants during maturation have
been studied in great detail and similar trends during
maturation are observed in all crop species (Heath et al.,
1973). In a preliminary study by Edwards et a1. (1968),
the chemical composition of whole-crop barley was analyzed
at seven successive stages of growth. During development,
dry matter increased steadily, ash decreased until the
dough stage, crude protein decreased through the milk stage
and increased with seed development and crude fiber
increased initially but decreased through the later stages
of maturity. Water soluble carbohydrates peaked in the
milk stage, decreasing to their lowest levels at the ripe
stage. Ashbell et al. (1985) assessed dry matters of wheat
in the milk and dough stages of 35% and 43%, respectively.

These values are in agreement with those of Bolsen and

67

Berger (1976) who also reported lower values of 28% and 35%
for oats and barley in milk and dough stages, respectively.
Oltjen and Bolsen (1978) concluded wheat reached comparable
stages of maturity at a higher dry matter percentage than
other small grain crops. Dry matters in this experiment
are well within the range found for the milk and dough
stage by other investigations. The higher dry matters
observed for wheat are due in part to the delayed harvest,
however, all cereal crops ensiled in this study were
assumed to be at comparable growth stages.

Protein concentrations of cereal crops average between
5% and 12% of the dry matter, much lower than forages such
as grasses and legumes which may contain up to 22% crude
protein. Delaying the harvest of cereal crops until the
milk or dough stage reduces crude protein content by as
many as 10 percentage units, expanding the differences
between forage and cereal crops (Bishnoi et al., 1978). Of
wheat, oats, barley, and triticale, they found oats
contained higher concentrations of protein at all growth
stages investigated. Oltjen and Bolsen ( 1978) reported
protein reductions of 40% as wheat and barley matured from
the boot stage (15%) to the dough stage (9%) with the
largest reduction occurring between flowering and milk
stages. Both Edwards et al. (1968), working with barley
and Ashbell et al. (1985) working with wheat concluded that
reductions in protein content had plateaued by the milk

stage with slight increases observed with seed development

68

in the dough stage. In contrast to the results of Bishnoi
et al. (1978), oats had the lowest crude protein content in
the present study (7.8%) , compared to wheat (8.3%) and
barley (8.5%) . Protein concentrations in this experiment
are typical for more mature cereal crops, including corn
(Geasler, 1970). Slight decreases were noted as maturity
progressed from milk to dough stages in oats and barley
which might have resulted from poor soil maintenance in
these small experimental plots. Wheat dry matter yields
were far better than for either oats or barley, suggesting
better soil conditions, and no protein reductions were
observed in wheat. Although protein reductions were
significant, proportionally they accounted for lesser
losses compared to those occurring during earlier
development.

Reports of crude fiber concentrations in maturing
cereal crops are conflicting. Miller et a1. (1967) found a
direct relationship between maturity and crude fiber in
barley. A year later studying eight varieties of barley,
Edwards et al. (1968) found an inverse relationship with
32% crude fiber in the head stage and only 19.5% in the
dough stage. They also found organic matter
digestibilities had increased with maturity, in contrast to
results of Miller et al. (1967), supporting their
conclusion that seed development dilutes crude fiber
percentages. Ashbell et al. (1985) found similar results

between milk and dough stages in wheat as did Sutoh et al.

69

(1972) with oats: however increasing fiber contents were
associated with earlier growth stage advancement. Assuming
higher digestibilities result from lower fiber
concentrations, results of Bolsen and Berger (1976), Bolsen
et al. (1976), and Oltjen and Bolsen (1978) show a drop in
crude fiber between the milk and dough stages in wheat,
oats, and barley. In the present experiment, parallel
changes in neutral detergent fiber (NDF) and acid detergent
fiber (ADF) concentrations reveal a similar downward trend
in oats and barley. In wheat, fiber concentrations
increased into the dough stage which may be the result of
grain losses associated with harvesting a dryer cereal
crop. Values of NDF, ADF, and hemicellulose obtained in
this study were similar across species and comparable to
those found in corn (Ensminger and Olentine, 1978).

Waite (1957) believed the concentration of water
soluble carbohydrates (WSC) was largely dependent on the
rate of protein synthesis; higher rates of protein
synthesis would indirectly utilize more of these readily
available energy containing compounds, diminishing existent
WSC concentrations. This relationship exists in cereal
crops as increases in WSC concentrations coincide with
decreasing protein concentrations during early growth and
visa-versa towards maturation as seed development involves
both protein and starch synthesis (Edwards et al. 1968;
Geasler, 1970; Sutoh et al., 1972; and Ashbell et al.,

1985). In addition, results of MacGregor and Edwards

70

(1968) and Ashbell et al. (1985) show water soluble
carbohydrates comprise between 20% and 30% of ‘the, dry
matter in wheat and barley during the milk stage,
decreasing by as much as 50% in the dough stage. Although
WSC concentrations never reached 20% DM in this trial,
significant reductions occurred between milk and dough
stages as expected. Poor soil conditions may have caused
the consistently depressed WSC concentrations, explaining
the close relationship between dry matter yield and WSC
content in this study. Wheat, which yielded twice the dry
matter as oats and barley, also contained significantly
higher amounts of soluble carbohydrates, the primary energy
substrates during plant development.

Ash concentrations observed during this study were not
affected by stage of maturity, agreeing with results of
Edwards et a1. (1968) for more mature growth stages.
Concentrations of ash were higher than those of Edwards et
al. (1968), but lower than those of Sutoh et al. (1972) at
similar growth stages. In this study higher ash
concentrations in oats and barley when compared to wheat
are not surprising since WSC were also depressed in these
two examples, and a greater percentage of the dry matter
would have been assumed by the inorganic ions.

Fermentation patterns for individual grain and stage
combinations can be visualized in Figures 9 thru 14. The
final pH of silage is largely dependent on moisture content

provided available carbohydrate is present (Barnett, 1954).

71

In low dry matter silages extensive fermentation must take
place before a sufficiently low pH value is obtained to
preserve the material (Wieringa, 1958) . In the present
study, milk stage silages had lower pH values in all three
species under investigation. Steeper pH declines were also
observed in milk stage oat and barley silages while in
wheat, delayed acidification was most likely due to the
method of chopping. All silages were well preserved as
evidenced by low stable pH values attained during
fermentation.

Lactic acid accumulation, the primary end-product of
lactic acid bacterial metabolism, is mainly responsible for
pH declines observed in adequately preserved silages
(Whittenbury et al., 1967). This is well illustrated in
the present data by noting the close inverse relationship
between lactic acid content and pH ih.Figures 9 thru 14.
Further observation of these figures reveals a more rapid
rate of fermentation occurring in milk stage silages. The
intersection between lactic acid content and pH took place
28 days earlier in milk stage oat and barley silage and 12
days earlier in milk stage wheat despite the delayed onset
of fermentation in the latter silage. Edwards et al.
(1968) and Ashbell et al. (1985) observed nearly twice the
amount of lactic acid in milk stage silages suggesting a
greater extent of fermentation in these less mature
silages. Similar ratios were observed in this study in

oats and barley, however total lactic acid concentrations

72

far exceeded those of previous researchers for identical
growth stage silages. This is hard to explain since WSC
concentrations were abnormally low for cereal crops.
Lactic acid bacteria must have completely dominated the
microbial population since propionate and butyrate, end-
products of undesirable microbial activity, were absent
from the majority of samples analyzed.

Provided sufficient fermentation by lactic acid
bacteria, acetate production arising from
enterobacteriaceae activity prior to acidification would be
minimal (Langston and Conner, 1962). But, heterolactic
bacteria produce acetate when fermenting fructose and
excess acetate concentrations have been found in silages
undergoing vigorous fermentation (Edwards and. McDonald,
1978). Ashbell et a1. (1985), when ensiling wheat, found
greater concentrations of acetate in wetter silages that
also had the highest concentrations of lactate. Geasler
(1970) calculated a correlation coefficient of +.70 between
lactate and acetate in corn silage implying higher acetate
productions are a consequence of normal fermentation in low
dry' matter silages. .Acetate jproduction in this study
responded in a similar fashion in wheat and barley
resulting in higher acetate concentrations in the milk
stage silages. Milk and dough stage silages made from oats
contained similar concentrations of acetate throughout the
ensiling period which may have been due to limited amounts

of fructose available for fermentation. The low overall

73
levels of acetate found in these silages indicate efficient
use of available carbohydrates by the developing lactic
acid bacteria.

WSC concentration necessary for adequate preservation
are influenced by dry matter content, buffering properties,
initial respiration, and bacterial species dominating the
fermentation (Woolford, 1984). The fermentation of cereal
crops ensiled while in the milk stage utilize a greater
percentage of the initial WSC than dough stage silages
(MacGregor and Edwards, 1968; Geasler, 1970; Ashbell et
al., 1985). This would be expected since larger amounts of
volatile fatty acids were produced during the ensiling of
milk stage crops. In the present study, milk stage silages
lost more WSC through 64 days of fermentation than dough
stage silages, presumably’ due to greater microbial
activity. WSC utilization was similar within growth stage
for the three species in this study except for milk stage
wheat silage where utilization ceased after 1 day of
fermentation even though lactic acid accumulated through
the sixteenth day of fermentation (Figure 9). Either WSC
concentrations were replenished through hydrolysis of
storage and structural carbohydrates or the developing
microflora preferred organic acids over carbohydrates as
their energy source. The actual substrate source of lactic
acid fermentation in milk stage wheat silage could not be
assessed from the analyses performed in this study. The

only specific carbohydrate fraction analyzed was

74
hemicellulose which was not utilized to any great extent
during fermentation.

Bergen et al. (1974) concluded that enzymes endogenous
to the ensiled plant material were mainly responsible for
protein degradation to water soluble nitrogen (WSN) during
ensiling. Enzymes being acid labile prompted investigators
to test methods of hastening the reduction of pH to reduce
protein. degradation (Henderson et al., 1982). Geasler
(1970) and Bergen et al. (1974) produced conclusive
evidence that increasing dry matter content by wilting or
delaying harvest would also substantially decrease WSN
concentrations in resultant silages. Results obtained here
suggest dry matter content is more influential than pH in
reducing the activity of plant proteases. Although milk
stage silages exhibited rapid pH declines and lower final
pH's, protein conversion to WSN was faster and more
extensive in these silages (Figures 9 thru 14) . Dough
stage silages reached a pH considered inhibitory to protein
degradation 12 days later than milk stage silages yet lower
WSN concentrations were associated with silages made from
the dryer, more mature cereal crops. Lack of moisture may
shield proteins from enzymatic degradations explaining how
silages exhibiting longer periods of protease activity have
less protein degradation. Within growth stage, differences
between. species *were :negligible, and.‘WSN' concentrations
were comparable to concentrations found in corn silage at

similar growth stages and dry matters (Geasler, 1970).

75

Ammonia concentrations obtained by the Technicon Auto
Analyzer may be overestimated as evidenced by extreme
concentrations found in the fresh cereal samples. Micro-
organisms on dead and decaying leaves produce the majority
of ammonia in fresh plants, and elevated amounts of ammonia
are expected in older' plants that have 'more perishing
leaves (Brady, 1960). Even in advanced stages of maturity,
the quantity of ammonia nitrogen is usually less than 1.5%
of the total nitrogen (Brady, 1960; Bergen et al., 1974).
Initial values greater than 2.5% in this study may have
been caused by background color in the homogenate which was
not subjected to digestion prior to analysis. Relating
these concentrations to those obtained through other
methods would not be legitimate.

As proteolytic clostridia are assumed to be the major
source of ammonia nitrogen in silage, it is surprising that
Significant amounts of ammonia were found despite evidence
against the presence of clostridia in these silages. It is
known, however, that some ammonia can come from other
sources such as the reduction of nitrates and nitrites, and
the action of plant enzymes and Enterobacteriaceae (Brady,
1960: Seale et al., 1986). In accordance, observations
here show’ the rate of ammonia production ‘was greatest
during the first four days of ensiling, but gradual
increases in ammonia-N concentrations were noted
thereafter. This secondary accumulation of ammonia may

arise from lactic acid bacteria which are capable of amino

76

acid fermentation (Brady, 1966). There were no conclusive
differences in ammonia production between milk and dough
stage silages which agreed with findings of Sutoh et al.
(1972) and Ashbell et al. (1985). Lower ammonia
concentrations observed in oat silages are of minor
practical importance since all silages in this trial
contained ammonia concentrations typical of quality
silages.

Rates of acid detergent insoluble nitrogen (ADIN)
accumulation were negligible and not different between
specie or stage combinations investigated here. Slight
increases in, ADIN concentrations were :noted. in initial
stages of fermentation, undoubtedly the result of
incubation at 30°C for three days. Maillard reactions
responsible for ADIN production during ensiling are
nonenzymatic, spontaneous and occur to some degree in well
made unheated silages (Yu and Veira, 1977). Maturation of
the three cereal species in this study increased the
initial content of ADIN which persisted throughout
fermentation. Both Thomas et al. (1982) and Yu and Veira
(1977) consistently found highly negative correlations
between nitrogen digestibilities and ADIN. The lower
nitrogen digestibilities assumed in these dough stage
cereal silages do not agree with results of Bolsen and
Berger (1976) who reported higher nitrogen digestibilities
in lambs fed silages made from dough stage cereal crops.

ADIN was not assessed by Bolsen and Berger (1978) and,

77

contrary to the present study, silages made from early out
crops generally produce more ADIN due to higher levels of
moisture at earlier growth stages (Thomas et al., 1982).

Dry matter losses could not be assessed with any
appreciable accuracy since differences were rarely
significant throughout fermentation. Losses greater than
7% would have shown significance but taking into account
that lactic acid bacteria dominated the microflora, losses
would rarely exceed 7% in such cases (McDonald et al.,

1973).

78

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Figure 9. Changes of Major Fermentation Parameters During
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Figure 10. Changes of Major Fermentation Parameters During

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65
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Figure 11. Changes of Major Fermentation Parameters During

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81

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Figure 12. Changes of Major Fermentation Parameters During
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82

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6.0 SUMMARY

Whole plant wheat, oats, and barley were harvested,
chopped, and ensiled at. the 'milk; and dough stages of
maturity. Equipped with gas release valves, Mason jars
combined with the addition of solid carbon dioxide prior to
packing provided ideal ensiling conditions as all silages
produced were of superior quality. Chemical analyses prior
to and during fermentation of these small grains revealed
compositional changes upon development and their effects on
fermentation.

Dry matter yields were below those established for
small grain crops in this area, however a truly
representative estimation could not be obtained due to the
size and location of the experimental plots. Dry matter
percentages were higher during the dough stage which
resulted in greater yields for oats and barley, but no
change was observed for wheat.

Chemical analyses prior to ensiling revealed only
minor differences in ash, protein, and fiber contents
between the small grains investigated here. Developmental
changes of these constituents are more pronounced during
early rather than late growth. and. their' concentrations
would be expected to remain relatively stable over the two

84

85

weeks between harvests. Acid detergent insoluble nitrogen
(ADIN) concentrations were similar among species in the
milk stage and rose slightly in the dough stage in wheat
and cats but doubled in barley. Substantial differences
were noted in buffering capacities and water soluble
carbohydrates (WSC) with wheat having the lowest buffering
capacity and highest concentration of WSC. Although WSC
concentrations fell sharply in the dough stage, the
buffering capacity decreased as well and existing
carbohydrates appeared sufficient for acid production.

During ensiling a classic example of inoculation by
farm equipment was observed. In milk stage silages, lactic
acid appearance (production) was faster except in milk
stage wheat which was chopped by hand on a paper cutter
while the remaining harvests were chopped through a forage
harvester. WSC were utilized to a greater extent in milk
stage silages reflecting the higher concentrations of acids
found. Hemicellulose did not contribute to acid production
as few differences were found in its concentration during
ensiling. Acetate production was negligible in all
silages, however higher concentrations were noted in milk
stage wheat and barley.

Milk stage silages exhibited more rapid declines in pH
but also had more protein degradation. Results of this
investigation show protein degradation in silages to be
more responsive to moisture content than acidification.

Ammonia production was significant but similar in all

86
silages despite the apparent domination, by lactic 1acid
bacteria providing some evidence of amino acid deamination
(utilization) by these bacteria. ADIN concentrations were

not different as a result of fermentation.

7.0 BIBLIOGRAPHY

Allen, L.A. and Harrison, J. 1937. Anaerobic
sporeformers in grass silage. Ann. appl. Biol.
24:148.

Anderson, B.K. and Jackson, N. 1970. Conservation of

wilted and unwilted grass ensiled in air-tight metal
containers with and without the addition of molasses.
J. Sci. Food Agric. 21:235.

Ashbell, G., Theune, H.H. and Sklan, D. 1983. Changes in
the amino acid compounds of whole crop wheat during
ensiling and after fermentation. J. Sci. Food Agric.
34:321.

Ashbell, G., Theune, H.H. and Sklan, D. 1985. Ensiling
whole wheat at various maturation stages: Changes in
nutritive ingredients during maturation and ensiling
and upon aerobic exposure. J. Agric. Food Chem. 33:1.

Barker, H.A. 1961. Fermentation of Nitrogenous Organic
Compounds. In "The Bacteria" Vol II (I.C. Gunsalus &
R.Y. Stanier, eds) Academic Press, New York.

Barker, S.D. and Summerson, W.H. 1941. The colorimetric
determination of lactic acid in biological materials.
J. Biol. Chem. 138:535.

Barnett, A.J.G. 1954. Silage Fermentation. Academic
Press, Inc., New York.

Beck, T. 1978. The microbiology of silage fermentation.
Fermentation of Silage-A Review. National Feed
Ingredients Assoc.

Bergen, W.G., Cash, E.H. and Henderson, H.E. 1974.
Changes in nitrogenous compounds of the whole corn
plant during ensiling and subsequent effects on dry
matter intake by sheep. J. Animal Sci. 39:629.

Bishnoi, U.R., Chitapong, P., Hughes, J. and Nishimuta, J.

1978. Quantity and quality of triticale and other
small grain silages. Agron. J. 70:439.

87

88

Bolsen, K.K. and Berger, L.L. 1976. Effects of type and
variety and stage of maturity on feeding values of
cereal silages for lambs. J. Anim. Sci. 42:168.

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95

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