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thesis entitled

CORN GRAIN ENDOSPERM TYPE AND THE BROWN
MIDRIB 3 MUTATION IN CORN SILAGE: EFFECTS ON

NUTRIENT DIGESTION. FEEDING BEHAVIOR. MILK YIELD,

AND RUMINAL FERMENTATION OF LACTATING DAIRY
COWS

presented by

CHRISTINA CHARLENE TAYLOR

has been accepted towards fulfillment
of the requirements for the

M. S. degree in Animal Science

flaflfl,

Wfldfessor’s Signature
/ 2/4 / o 5

Date

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6/01 c:/ClFIC/DaleDue.p65-p.15

 

CORN GRAIN ENDOSPERM TYPE AND THE BROWN MIDRIB 3 MUTATION
IN CORN SILAGE: EFFECTS ON NUTRIENT DIGESTION, FEEDING
BEHAVIOR, MILK YIELD, AND RUMINAL F ERMENTATION OF
LACTATING DAIRY COWS

By

Christina Charlene Taylor

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

2003

ABSTRACT

CORN GRAIN ENDOSPERM TYPE AND THE BROWN MIDRIB 3 MUTATION
IN CORN SILAGE: EFFECTS ON NUTRIENT DIGESTION, FEEDING
BEHAVIOR, MILK YIELD, AND RUMINAL F ERMENTATION OF
LACTATIN G DAIRY COWS

By

Christina Charlene Taylor

Interactions of corn silage hybrids varying in fiber lignification and corn grain hybrids
varying in endosperm type on nutrient digestion, feeding behavior, milk yield, and
ruminal fermentation of lactating dairy cows were investigated. Eight ruminally and
duodenally cannulated cows were fed diets containing corn silage (bm3 or isogenic
normal) and corn grain (floury or vitreous endosperm) in a 2 x 2 factorial arrangement of
treatments. Floury endosperrn grain decreased dry matter intake (DMI) by 1.9 kg/d when
in control corn silage diets but did not affect DMI when combined with bm3 corn silage.
Effects on DMI occurred primarily because floury endosperrn decreased DM meal size
when combined with control corn silage, but increased DM meal size when combined
with bm3 corn silage. DMI was not different between endosperrn types combined with
bm3 corn silage because of greater meal frequency for bm3 corn silage. Main treatment
interactions occurred for 3.5 % fat-corrected milk yield (FCM) and reflected treatment
effects on DMI. Floury corn grain increased ruminal starch digestibility dramatically
compared to vitreous corn grain. Corn grain endosperm type affects site of starch
digestion, which can interact with fiber source to affect feeding behavior, feed intake, and

milk yield.

For my parents, who taught me the love of learning

iii

ACKNOWLEDGEMENTS

I am grateful to Dr. Allen, for the mentoring and support he has provided me over
the past two years. By teaching me the basics of ruminant nutrition to explaining the
nuances of the scientific process, I have learned more in the past years than I thought
possible. He has taught me to question everything and look at the world in a different
light. I appreciate his willingness to present some of my research after my scientific
attempt to see which was stronger: a curb or my ankle. Thank you for having the time
and the patience to teach me at least some of your knowledge.

My graduate advisory committee, Dr. Dale Romsos, Dr. Nathalie Trottier, and Dr.
Thomas Herdt, deserve many thanks. They have not only provided guidance during this
degree process, but have also taught me much about nutritional metabolism. Their
insights and contributions not only have increased my knowledge about my field, but also
made me think globally, which has made me more well rounded as a graduate student.
Also thanks to many others not on my committee, especially Dr. Dave Beede, Dr. Mike
VandeHaar, and Dr. Herb Bucholtz, for enlightening conversations in seminars and
around the lunch table.

I would like to thank Purina, Inc., and Syngenta Seeds, Inc. for financial support
of this thesis research. Without their support, this experiment could not have occurred. I
am grateful to Dr. Kent Ames for performing duodenal and ruminal cannulations of
experimental animals as well as giving me the opportunity to experience large animal
surgery. I also am grateful to Dr. Ames for fixing a Christmas Day cannula emergency.

Many thanks also go to the crew of the Michigan State University Teaching and Research

iv

Dairy Farm. The crew was supportive of all the extra work and changing their schedule
to accommodate my experiment, and they never complained, except to demand more
cookies. They deserve much of the credit for this experiment.

Truly, this experiment could not have occurred without the help of the Dairy
Nutrition Lab. It is difficult to express how thankful I am to everyone who has helped me
throughout the collection process, analysis, and writing periods. Thanks to Dave Main,
for bending over backwards and running analyses instantly. Dewey Longuski, thank you
for the entertainment and the VFA analyses. Thanks to Jackie Ying for help with so
many analyses (cobalt, feeding behavior, valerate) and for checking all my calculations.
Jenn Voelker, thanks for the answers to all my “How did you do this?” questions, but also
being a fiend to talk to at all times. Although we may not have gotten as much work
done, it’s been much more fun sitting behind you in the office. Steve Mooney, thanks for
showing me the ins and outs of the AA. Kevin Harvatine, thanks for all the help and the
antagonism; the past years wouldn’t have been nearly as fun without you. Thanks to
Barry Bradford for running hormone and plasma assays. Masahito Oba, thank you for all
the advice and knowledge. Thanks to our lab undergraduates, Heather Wieczorak, Amy
Nash, Allison Rober, Jessica Main, and many others, for all the hours of grinding,
weighing, and washing; I’d be here another year if I didn't have your help.

Finally, I’d like to thank my parents, my sister Val, Dave, and many of my
friends, both at Michigan State and other places. Each of you has encouraged and
supported me, and really have as much a hand in this thesis as I have. Thank you for

being a shoulder to cry on, a fiend to laugh with, and above all, someone to love me.

TABLE OF CONTENTS

 

LIST OF TABLES ------ - - _ - - 3 ............................................................ x
LIST OF ABBREVIATIONS .................................................................................. xii
INTRODUCTION ...................................................................................................... 1
CHAPTER 1

A REVIEW OF LITERATURE ................................................................................ 3

SECTION I: IMPORTANCE OF NUTRIENT DIGESTIBILTIY
Fiber Digestibility ................................................................................. 3
Starch Digestibility ............................................................................... 7

SECTION II: INTERACION OF FIBER AND STARCH DIGESTIBILITY

SITE OF NUTRIENT DIGESTION ............................................................... 11
Changing Nutrient Supply ................................................................... 13
Compensatory Digestion ..................................................................... 15

REGULATION OF INTAKE ......................................................................... 17
Physical Regulation of Intake .............................................................. 17
Metabolic and Hormonal Regulation of Intake .................................... 19
Site of Nutrient Supply ........................................................................ 19
Timing of Nutrient Supply .................................................................. 22

MICROBIAL PROTEIN SUPPLY ................................................................. 23
Effects of Ruminal pH on Fiber Digestion and Microbial Growth ....... 24
Effects of Ruminal Turnover Rate on Microbial Growth ..................... 28

SUMMARY ................................................................................................... 30

vi

CHAPTER 2 _
Effects of corn grain endosperm type and brown midrib corn silage on ruminal

kinetics and site of digestion in lactating dairy cows

ABSTRACT ................................................................................................... 32
INTRODUCTION .......................................................................................... 33
MATERIALS AND METHODS
Cows and Treatments .......................................................................... 35
Data and Sample Collection ................................................................ 37
Sample and Statistical Analysis ........................................................... 38
RESULTS AND DISCUSSION
DM and OM Digestion ........................................................................ 43
Site of Starch Digestion ....................................................................... 43
Fiber Digestion ................................................................................... 49
CONCLUSIONS ............................................................................................ 5 1
CHAPTER 3

Effects of corn grain endosperm type and brown midrib corn silage on feeding

behavior, milk production, and energy balance of lactating dairy cows.

ABSTRACT ................................................................................................... 59
INTRODUCTION .......................................................................................... 60
MATERIALS AND METHODS
Cows and Treatments .......................................................................... 62
Data and Sample Collection ................................................................ 63
Sample and Statistical Analysis ........................................................... 65

vii

RESULTS AND DISCUSSION

DM1 and Meal Patterns ....................................................................... 67

Chewing Activity ................................................................................ 7O

Ruminal Nutrient Pools ....................................................................... 72

Milk Yield and Composition ............................................................... 73

Energy Balance and Plasma Metabolites ............................................. 74

CONCLUSIONS ............................................................................................ 75
CHAPTER 4

Effects of corn grain endosperm type and brown midrib corn silage on ruminal

fermentation and microbial efficiency in lactating dairy cows

ABSTRACT ................................................................................................... 81
INTRODUCTION .......................................................................................... 82
MATERIALS AND METHODS
Cows and Treatments .......................................................................... 84
Data and Sample Collection ................................................................ 85
Sample and Statistical Analysis ........................................................... 86
RESULTS AND DISCUSSION
Ruminal pH ......................................................................................... 89
Ruminal Fermentation Acids ............................................................... 92
Nitrogen Flow ..................................................................................... 93
Partitioning of Duodenal N ................................................................. 97
CONCLUSIONS ............................................................................................ 98

viii

CHAPTER 5

 

CONCLUSIONS AND IMPLICATIONS ............................................................. 103
APPENDIX A ......................................................................................................... 107
REFERENCES ....................................................................................................... 112

ix

LIST OF TABLES

CHAPTER 2

Table 1. Nutrient compositions and physical characteristics of corn silages used to
formulate experimental diets ...................................................................................... 52

Table 2. Nutrient compositions and physical characteristics of corn grains used to
formulate experimental diets ...................................................................................... 53

Table 3. Ingredient and nutrient compositions of experimental diets (% of dietary DM)..

................................................................................................................................... 54
Table 4. Effects of corn grain endosperm type and brown midrib 3 corn silage on
digestion of DM and OM ........................................................................................... 55
Table 5. Effects of corn grain endosperm type and brown midrib 3 corn silage on
digestion of starch ...................................................................................................... 56
Table 6. Effects of corn grain endosperm type and brown midrib 3 corn silage on
digestion of total NDF and potentially digestible NDF ............................................... 57
Table 7. Effects of corn grain endosperm type and brown midrib 3 corn silage on ruminal
digestion kinetics ........................................................................................................ 58
CHAPTER 3

Table 1. Effects of corn grain endosperm type and brown midrib 3 corn silage on feeding
behavior ..................................................................................................................... 76

Table 2. Effects of corn grain endosperm type and brown midrib 3 corn silage on
chewing behavior ....................................................................................................... 77

Table 3. Effects of corn grain endosperm type and brown midrib 3 corn silage on
ruminal pools ............................................................................................................. 78

Table 4. Effects of corn grain endosperm type and brown midrib 3 corn silage on milk
yield and components ................................................................................................. 79

Table 5. Effects of corn grain endosperm type and brown midrib 3 corn silage on energy
balance ....................................................................................................................... 80

Table 6. Effects of corn grain endosperm type and brown midrib 3 corn silage on plasma
metabolite and hormone concentrations ...................................................................... 80

CHAPTER 4

Table 1. Effects of corn grain endosperm type and brown midrib 3 corn silage on ruminal
pH ............................................................................................................................ 100

Table 2. Effects of corn grain endosperm type and brown midrib 3 corn silage on ruminal
VFA profiles and concentrations .............................................................................. 101

Table 3. Effects of corn grain endosperm type and brown midrib 3 corn silage on nitrogen
metabolism ............................................................................................................... 102

APPENDIX B

Table 1. Effects of corn grain endosperm type and brown midrib 3 corn silage on
additional measures of feeding behavior and chewing behavior ................................ 109

Table 2. Effects of corn grain endosperm type and brown midrib 3 corn silage on ruminal
nutrient concentration and turnover time .................................................................. 110

Table 3. Effects of corn grain endosperm type and brown midrib 3 corn silage on amount
of ruminal VFA and ruminal pH during meal and rumination bouts .......................... 111

Table 4. Effects of corn grain endosperm type and brown midrib 3 corn silage on net
energy for maintenance ............................................................................................ 111

xi

LIST OF ABBREVIATIONS

AA

BCS
bm3
BW
CP
DIM
DM
DMI
FCM
GIP
GLP-l

iNDF

MNE
MUN
NAN
NANMN
NDF

N EL

NEM

Amino Acid

Acid Detergent Fiber

Body Condition Score

Brown Midrib 3 Mutation in Corn Silage
Body Weight

Crude Protein

Days in Milk

Dry Matter

Dry Matter Intake

Fat-corrected Milk

Gastric Inhibitory Peptide
Glucagon-like Peptide 1

Indigestible NDF

Metabolizable Energy

Microbial Nitrogen Efficiency

Milk Urea Nitrogen

Non-Ammonia Nitrogen
Non-Ammonia, Non-Microbial Nitrogen
Neutral Detergent Fiber

Net Energy of Lactation

Net Energy of Maintenance

xii

OM
pdNDF
TCA
TRDOM

VFA

Organic Matter

Potentially Digestible NDF

Tricarboxylic Acid

Truly Ruminally Degraded Organic Matter

Volatile Fatty Acids

xiii

INTRODUCTION

Energy intake is an important determinant of milk production, and therefore
maximizing energy intake of high producing dairy cows is a primary objective for
nutritionists. Cows in peak lactation can produce greater than 50 kg of milk per day, and
often consume more than 25 kg of feed per day to support production. However, body
reserves are frequently mobilized because the cow cannot consume enough feed in a day
to meet the energy demands of milk production. Increasing ruminal fiber digestibility of
forage in the diet can potentially increase dry matter intake (DMI) by reducing physical
limitations to feed intake (Allen, 1996). Another strategy to potentially increase energy
intake is to increase the energy density of the diet by adding cereal grains. Ruminal
starch degradability of grains varies widely and can influence site of starch digestion and
regulation of DM1 (Allen, 2000). Understanding how fiber and starch digestibility
interact to affect DMI might lead to a better understanding of how feed intake is
regulated in high producing dairy cows.

Ruminal digestion characteristics can affect production of microbial protein in the
rumen. Microbial protein is the primary source of amino acids to the cow and it is
important to maximize microbial protein flow so that amino acid availability does not
limit milk production. Interactions of starch and fiber digestibility on DMI might change
efficiency of microbial protein production. Rapid ruminal fermentation can decrease
efficiency of microbial growth (Russell, 1998), but microbial growth can be limited by
energy supply in the rumen (Clark et al., 1992), so moderation of ruminal fermentation is

critical for maximal efficiency of microbial protein production. Because starch and fiber

digestibility probably interact to affect both DMI and nutrient digestibility, interactions of
starch and fiber digestibility probably also occur to affect microbial growth and
fermentation.

The objective of this thesis research was to evaluate interactions of endosperm
type of corn grain and the brown midrib mutation in corn silage on site of nutrient
digestion, feeding behavior, milk yield, ruminal fermentation, and microbial protein

production.

CHAPTER 1

A REVIEW OF THE LITERATURE

SECTION I: IMPORTANCE OF NUTRIENT DIGESTIBILITY

Fiber Digestibility

Energy intake is a primary limitation on productivity of high producing cows. As
the genetic potential of dairy cows increases, greater energy intake is required to meet
energy demands for milk production. Increasing energy density of the diet helps support
greater milk yield, and dietary fiber concentration is ofien reduced in favor of more
energy dense concentrate feeds. However, it has been recommended that to maintain a
healthy rumen environment, neutral detergent fiber (NDF) should comprise 25% of diet
dry matter (DM) with at least 19% of NDF coming from forage sources (NRC, 2001).
This recommendation, while realizing that ruminants require a certain amount of NDF,
does not take into account the wide range of NDF digestibility of various forages. In
vitro NDF digestibility can range from less than 20% to greater than 60% over a variety
of forages (Allen and Oba, 1996), but diets are generally formulated based upon NDF
concentration alone. However, NDF digestibility could have significant impact on animal
productivity because DMI and milk yield are positively related to NDF digestibility (Oba
and Allen, 1999b).

NDF digestibility is determined by the fraction of NDF that is potentially

digestible, the rate of NDF digestion in the rumen, and the rate of passage from the rumen

(Allen and Mertens, 1988). Potentially digestible NDF (pdNDF) is a laboratory measure
of the absolute extent of NDF digestion by ruminal microorganisms. Increasing
proportion of pdNDF and decreasing the indigestible NDF fraction (iNDF) could result in
greater fiber digestibility. However, in vivo NDF digestibility is also a function of the
competing processes of rates of digestion and passage; increasing rate of digestion or
decreasing rate of passage from the rumen could also increase NDF digestibility.
Predicting NDF digestibility is difficult because rates of digestion and passage can be
affected by physical structure of the plant, microbial attachment, enzyme activity, particle
size reduction, buoyancy, and ruminal motility (Allen, 1996). The least practical method
of increasing digestibility of the pdNDF fraction is by reducing rate of passage from the
rumen; lower rate of passage could increase digestibility of pdNDF by increasing ruminal
retention time but might depress DMI because of greater ruminal distension.

The most practical methods of increasing NDF digestibility lie primarily with
increasing amount and rate of pdNDF digestion in forages. Grasses ofien have a greater
proportion of pdNDF to iNDF than legume forages and higher in vitro NDF digestibility,
but rate of digestion of legume pdNDF is faster (Smith et al., 1972) and could increase
total amount of NDF digested per day in vivo. Additionally, within a forage type,
immature plants generally have higher NDF digestibility than mature plants, because as
the plant matures the indigestible NDF fraction increases and rate of NDF digestion
decreases (Smith et al., 1972). Replacing early vegetative alfalfa hay with late bud or full
bloom alfalfa reduced total tract NDF digestibility and DMI (Llamas-Lamas and Combs,
1990). Another potential method to increase pdNDF digestibility is by the use of genetic

mutations in forage crops that reduce iNDF and increase pdNDF fraction of the plant.

The first brown midrib mutation in corn, aptly named bml, was discovered at the
University of Minnesota in 1924 (Jorgenson, 1931). Since then, three other mutations
have been found in corn, named bm2, bm3, and bm4. The various brown midrib
mutations, so named because of accumulation of reddish-brown phenolic lignin
monomers in the midrib of the leaf, reduce lignin content of corn because of alterations or
deletions of genes coding for enzymes involved in lignin biosynthesis. The gene
mutation in bm3 com occurs for caffeic acid-O-methyl transferase (Vignols et al., 1995).
Lignin is an indigestible polymer in plants that is important to maintain structural
integrity of plant tissue. Although lignin comprises little of the total structural
carbohydrate system in plants, it has been recognized as the primary component of the
cell wall that limits digestion (Jung and Deetz, 1993). Negative effects on digestion
occur because lignin forms strong covalent bonds with hemicellulose and may also
physically block enzymatic access to digestible structural carbohydrate. Reduced lignin
synthesis lessens the amount of crosslinking that occurs among lignin and digestible
structural carbohydrates to increase digestibility.

It was not until the 1970’s that research on the nutritional aspects of brown midrib
hybrids occurred. Of the brown midrib mutations, hybrids containing the bm3 mutation
have been most widely used in nutritional experiments because it consistently reduces
lignin content and increases in vitro DM digestibility (Colenbrander et al., 1973). The
bm3 mutation decreased lignin and increased in vitro DM digestibility more than other
bm hybrids (me) or normal corn hybrids (Muller et al., 1971). Reduced lignin synthesis
and greater in vitro DM digestibility occurred in all parts of the bm3 corn plant, including

the leaf, stem, and ear (Weller et al., 1984). As bm3 corn plants mature, lignin content

increases similar to normal hybrids, but bm3 hybrids had consistently lower lignin at all
stages of maturity (El-Tekriti et al., 1976). Based on in vitro data, bm3 corn hybrids had
the potential to increase in vivo NDF digestion and possibly performance, but little was
known about the effects of feeding bm3 hybrids to animals.

Early in vivo research with bm3 hybrids agreed with in vitro results. Muller et a1.
(1972) performed a classical study investigating the feeding value of a bm3 corn silage
hybrid versus a high yielding commercial hybrid. Two adjoining plots were planted with
either bm3 corn for silage or a normal hybrid, and ears were removed from both plant
sections so that differences between hybrids could not be attributed to grain concentration
differences. When fed to lambs, bm3 corn silage improved apparent total tract
digestibility of cell wall constituents and cellulose versus the normal corn hybrid. Similar
to that study, replacing normal silage with bm3 silage in diets for lactating cows
increased apparent total tract digestibilities of cell-wall constituents and cellulose by 33
and 44%, respectively (Weller and Phipps, 1986).

Perhaps the more important benefit of bm3 corn silage was the observation that
animals ofien increased feed intake. Brown midrib 3 corn silage increased voluntary
DMI by lambs by 29% compared to a normal hybrid (Muller et al., 1972). When fed to
cows in early lactation, bm3 corn silage increased DMI by 11% versus a normal hybrid
(Block et al., 1981). However, some studies observed no increase in DMI when bm3
corn silage replaced a normal hybrid (Frenchick etal., 1976; Keith et a1. 1979). Lack of
treatment effect on DMI when bm3 corn silage replaces control corn silage could occur if
DMI is not limited by ruminal fill. A more recent study found a relationship between

pretrial milk yield and DMI response to bm3 silage (Oba and Allen, 1999a); cows

producing more milk in the pretrial period increased DMI to a greater extent when fed
bm3 silage than cows yielding less milk. The authors concluded that ruminal fill is more
limiting to intake for higher yielding cows, and increasing NDF digestibility of forage by
feeding bm3 corn silage might increase DMI to a greater extent in higher producing
cows.

A statistical analysis of the literature found that for cows in early or mid-lactation
(41 to 154 days in milk [DIM]), a one unit increase in NDF digestibility was associated
with a 0.17 kg increase in DMI and 0.25 kg increase in 4% fat corrected milk (FCM; Oba
and Allen, 1999b). Because the bm3 mutation in corn silage can potentially increase
NDF digestibility and might reduce limitations to DMI by physical fill, more research is
needed to determine how high producing animals respond to bm3 corn silage and how

this response might be affected by other dietary factors, such as starch digestibility.

Starch Digestibility

Because energy intake is such a limitation on milk production, increasing the
energy density of diets by replacing fibrous forages with starchy grains is common. Diets
are generally formulated based on starch concentration alone, but ruminal starch
digestibility ranges from 42 to 96% over a variety of grain sources (N ocek and
Tamminga, 1991). This wide range of starch digestibility can affect whether starch is
digested primarily in the rumen or intestines. Site of starch digestion can considerably
influence DMI and microbial protein production (Oba and Allen, 2003 a, c). Therefore,
changing starch digestibility can have a direct impact on energy intake and milk

productivity.

Starch digestibility can be manipulated by processing and conservation methods.
Physical processing such as cracking, grinding, flaking, or rolling generally increases
ruminal starch digestibility. This processing disrupts the outer coating of the kernel and
makes the starch more accessible to microbial attack. Grinding corn increased ruminal
and intestinal starch digestion compared to whole corn in steers (Galyean et al., 1979),
and decreasing corn grain size from coarsely to finely ground increased ruminal starch
digestibility from 35.2 to 70.1% (Callison et al., 2001). F laking and rolling substantially
disrupts the kernel structure and increases ruminal digestibility versus whole or cracked
grains (Firkins et al., 2001).

Physicochemical processing methods also increase starch digestibility of grains.
Starch granules consist of multiple starch chains arranged in highly organized and less
organized sections, called crystalline and amorphous areas, respectively. These chains of
starch are associated by a significant amount of hydrogen bonding, but application of heat
and water swells the amorphous and crystalline regions of the starch bonds and breaks
down interconnecting hydrogen bonds (Rooney and Plugfelder, 1986). This process,
known as gelatinization, increases area available for microbial penetration and enzyme
access. Steam treatments are often combined with physical processing such as flaking to
increase ruminal and total tract starch digestibility. Steam flaking increased total tract
starch digestibility by approximately 19 units compared to dry rolled (Plascencia and
Zinn, 1996). Similarly, replacement of dry rolled corn with steam flaked corn linearly
increased ruminal and total tract starch digestibility (Crocker et al., 1998).

Ensiling grains can increase starch digestibility because the ensiling process

partially ferments the grain and weakens the physical structure of the cereal. Conserving

grains as high moisture grain applies both hydration and ensiling processes to increase
starch digestibility. High moisture grains are readily fermentable and are a common feed
source used to increase energy density of dairy cow rations. High moisture corn
increased ruminal starch digestibility by 19 units (Knowlton et al., 1998a) and 24 units
(Oba and Allen, 2003b) in high starch diets for lactating cows.

Physical properties of grains can play a substantial role in starch digestibility.
When cereal grains are ground, ruminal starch digestibility of wheat, oats, and barley is
greater than corn, which is greater than sorghum (Herrera-Saldana et al., 1990).
Replacing corn grain with barley in the diet of lactating cows linearly increased ruminal
and total tract starch digestibility (Overton et al., 1995). Apparent total tract starch
digestibility was greater for steam flaked corn than steam flaked sorghum (Santos et al.,
1999). However, even within a single grain source, ruminal starch digestibility can range
widely; ruminally degraded starch ranged from 51 to 93% for corn grain (N ocek and
Tamminga, 1991).

Wide variation of ruminal starch digestibility within a grain source is because of
the physical and chemical structure of the grain kernel. Cereal kernels are composed of
three distinct parts; the germ, the outer pericarp, and the endosperm, which comprises
most of the total kernel area (Kotarski, 1992). Physical processing disrupts the pericarp
and exposes starch granules to digestion, but a protein matrix within the endosperm
surrounds starch granules and slows digestion. Starch granules in vitreous endosperm are
imbedded in an insoluble protein matrix, whereas granules in floury endosperm are more
loosely associated with a soluble protein matrix (Kotarski, 1992). The protein matrix of

vitreous endosperm is composed primarily of zein proteins, whereas the more digestible

protein matrix of floury endosperm is comprised of glutelin proteins (Philippeau et al.,
2000). Zein proteins are very resistant to ruminal degradation, but glutelin proteins are
much more digestible (Romagnolo et al., 1994). Much of the variability of starch
digestibility within a grain type is because of the physical and chemical structure of this
protein matrix.

Many hybrids of corn grain are commercially available in the U. S., but effects of
chemical structure of these hybrids on starch digestibility have been largely overlooked.
Vitreousness can range widely across hybrids; 6 commercially available hybrids grown in
3 states varied in Vitreousness from 4 to 62% (Allen et al., 2003). Within a hybrid,
Vitreousness increases as grain matures (Philippeau and Michalet-Doreau, 1997). The
potential impact that Vitreousness can have on digestibility is tremendous; indeed,
averaged across hybrids, Vitreousness explained 86% of the variation in ruminal starch
degradability (Philippeau and Michalet-Doreau, 1997). In steers, replacing dent corn
with a more vitreous flint hybrid decreased ruminal starch digestibility 43% and
increased small intestinal starch digestibility (as a percent of starch intake) by 98%
(Philippeau et al., 1999a). Although these results indicate that Vitreousness of corn grain
can have a significant impact on starch digestion, to date only two studies have been
published investigating the effects of corn grain endosperm type on in vivo performance.
Furthermore, both of these studies were conducted in steers; no information is available
on how corn grain endosperm type affects productivity and nutrient digestibility in high

producing dairy cows.

10

SECTION II: INTERACTIONS OF FIBER AND STARCH DIGESTIBILITY

Fiber and starch digestibility could play an important role in energy intake and
productivity of dairy cows. The bm3 mutation in corn silage increases fiber digestibility
considerably, and endosperm type of corn grain can have significant effects on starch
digestibility. Interactions of endosperm type of corn grain and the bm3 mutation in corn
silage could affect site of nutrient digestion, voluntary DMI, and microbial protein

production.

SITE OF NUTRIENT DIGESTION

The rumen has evolved to digest fibrous feeds, and this ability to utilize fiber is a
distinct evolutionary advantage, but the disadvantage of this system is the long retention
time required for fiber digestion. As the genetic potential of dairy cattle increases, higher
milk yields require greater DMI, but voluntary DMI is negatively associated with
retention time in the rumen. Thus, a strategy used in diets for high producing dairy cows
is to increase nutrient digestibility and energy density. Greater DMI increases rate of
nutrient passage from the rumen, which can reduce ruminal nutrient digestibility and shift
some digestion postruminally in high producing dairy cows. Additionally, greater
ruminal digestibility of fiber and starch might interact to shift site of nutrient digestion
from the rumen to other sites in the gastrointestinal tract.

Increasing ruminal starch fermentation can affect the ability of ruminal
microorganisms to grow and to digest fiber, as discussed in detail in a later section.

Replacing beet pulp with high moisture corn linearly reduced ruminal pdNDF

11

digestibility from 67.3 to 46.1%. (Voelker and Allen, 2003a). While greater ruminal
starch digestion is associated with reduced ruminal fiber digestibility in multiple studies
(McCarthy et al., 1989; Crocker et al., 1998; Callison etal., 2001), it is not always
associated with reduced total tract NDF digestibility (Crocker et al., 1998; Callison et al.,
2001). In the experiment of Crocker et a1. (1998), postruminal NDF fermentation linearly
increased with greater ruminal starch digestibility but total tract NDF digestibility was
not affected. Replacing dry corn with high moisture corn reduced ruminal NDF
digestibility, and shifted so much NDF digestion postruminally that hindgut fermentation
of NDF contributed 53% of total tract NDF digestion (Oba and Allen, 2003b). However,
total tract digestion of NDF was not different between diets containing dry or high
moisture com. This indicates that ruminal starch digestibility can have significant effects
on site of fiber digestion without reducing total tract fiber digestibility.

Additionally, greater fiber digestibility of bm3 corn silage could potentially shift
site of starch digestion from the rumen to the intestines. Replacing normal corn silage
with bm3 silage decreased ruminal starch digestibility by 10% but increased postruminal
starch digestibility 13 % (Oba and Allen, 2000c). The authors suggested that greater
DMI of bm3 silage might have increased passage rate of starch from the rumen and
shifted site of starch digestion to the intestines. Another experiment found that ruminal
starch digestibility was 36% lower for bm3 versus normal corn silages, but differences in
total tract starch digestibility were small, indicating compensatory postruminal starch
digestion (Greenfield etal., 2001). Greater DMI could explain greater rate of starch
passage from the rumen in the Oba and Allen (20000) experiment, but no differences in

DMI were observed for the Greenfield et a1. (2001) experiment. Greater starch passage

12

in diets containing bm3 corn silage could be the result of other factors; for example, the
ruminal fiber mat formed by bm3 corn silage fiber might be less effective at retaining
corn grain particles. Feeding bm3 silage can affect site of starch digestion, which could

play an important role in production response to bm3 silage.

Changing Nutrient Supply

Starch yields different end products depending on site of digestion. As amount of
ruminally fermented starch increases, propionate production increases relative to acetate
to shift the volatile fatty acid (VFA) profile in the rumen. Although propionate is a
primary glucose precursor, postruminal starch digestion by amylase yields glucose
directly and is theoretically more efficient. In theory, greater postruminal starch
digestion could provide more glucose for milk lactose synthesis, but in reality milk yield
response to greater postruminal starch digestion is inconsistent.

Several experiments have investigated animal response to postruminal infusions
of starch and glucose. Reynolds et al. (2001) found that long term (14 d) infusions of
wheat starch into the abomasum of late lactation dairy cows did not affect milk yield but
tended to increase energy balance because of a 93% increase in tissue energy retention.
Additionally, duodenal glucose infusion increased energy balance of late lactation dairy
cows but did not affect milk yield (Lemosquet et al., 1997). In contrast, a cubic increase
in milk production was noted for cows in early lactation receiving increasing amounts of
corn starch infused into the duodenum (Reynolds et al., 2001). These data show that

response to postruminal starch digestion is probably dependent on stage of lactation and

13

could explain inconsistent effects across the literature for milk yield response to
postruminal starch digestion.

Animal response to increased postruminal starch digestion might depend on
changes in plasma hormone secretion in response to glucose. In particular, duodenal
glucose infusion increased plasma insulin of lactating dairy cows (Lemosquet et al.,
1997). Insulin increases glucose uptake by adipose and skeletal muscle (McGrane,
2000), but glucose transporters expressed by the mammary gland are not insulin-sensitive
(Zhao et al., 1996), so greater plasma insulin concentrations might not affect glucose
uptake at the mammary gland, but could reduce glucose availability if other insulin-
sensitive tissues increased glucose uptake. Competition for glucose availability among
tissues might explain why postruminal infusions of glucose or starch might not increase
milk production but can increase tissue retention (Lemosquet et al., 1997; Reynolds et al.,
2001). Why does this effect appear to occur in cows that are later in lactation, while
cows in early lactation can respond by increasing milk production? Cows in late
pregnancy and early lactation have reduced expression of the insulin-responsive glucose
transporter in muscle and adipose tissue, which decreases glucose utilization by
peripheral tissues and increases glucose available for milk synthesis (Bell and Bauman,
1997). The lack of response of peripheral tissues to insulin is markedly reduced through
early lactation, and recovers as the animal progresses through lactation (Bell and
Bauman, 1997).

Greater body weight (BW) gain has been observed in several experiments where
bm3 corn silage replaced normal corn silage (Frenchick et al., 1976; Sommerfeldt et al.,

1979; Weller and Phipps, 1986), and this effect might occur if bm3 corn silage shifts site

14

of starch digestion to the intestines. Perhaps most striking was the experiment conducted
by Block et al (1981); feeding cows bm3 corn silage from weeks 3 to 10 postpartum
increased DMI by 2.2 kg per day and although milk yield was numerically increased, the
greatest effects of treatment occurred on BW change. Cows consuming bm3 corn silage
gained 10.3 kg over the 8 week period, whereas cows consuming normal corn silage lost
24.6 kg over the same period. Oba and Allen (1999a) found that compared to a control
corn silage, bm3 corn silage increased energy balance of lactating cows by 2.1 Mcal per
day. Greater concentrations of metabolizable energy (ME) in bm3 corn silage diets fed
ad libitum resulted in more ME partitioned toward tissue energy gain instead of milk
energy (Tine et al., 2001). Brown midrib 3 corn silage increases ruminal propionate and
shifts a substantial portion of starch digestion to the intestines (Oba and Allen, 20000), so
greater glucose availability in bm3 corn silage diets might increase plasma insulin
concentration and tissue energy retention, but effects of bm3 corn silage on hormone

profiles have not been investigated.

Compensatory Digestion

Although greater passage rate of starch in bm3 corn silage diets reduces ruminal
starch digestibility, bm3 corn silage reduced total tract starch digestibility by only 1.5
units (Oba and Allen, 20000) and 1.7 units (Greenfield et al., 2001), and total tract starch
digestibility was still greater than 91% in both experiments. This indicates that
substantial compensatory digestion occurred in the intestines. While much of this
digestion probably occurs in the small intestine, some fermentation probably occurs in

the hindgut.

15

Abomasal infusions of corn starch increased ileal digesta concentrations of
glucose and starch (Kreikemeier et al., 1991). Additionally, replacing dent corn grain
with a flinty corn grain in the diets of beef steers reduced ruminal starch digestibility, but
increased small intestinal and hindgut digestion (Philippeau et al., 1999a). In that study,
flint corn increased small intestinal digestibility from 8.9 to 17.6% compared to dent
corn, but hindgut fermentation of starch was increased 110% versus dent corn. Infusion
of increasing amounts of corn dextrin into the abomasum of steers linearly increased
VF A concentration in ileal digesta (Kreikemeier et al., 1991). To some extent, VFA
produced from hindgut fermentation can be absorbed in the large intestine and used by
the animal as an energy source, but microbial organic matter (OM) cannot be digested or
absorbed and loss of microbial OM in the feces reduces efficiency of energy utilization
by the cow.

Changing site of starch digestion can considerably change the nutrient supply to
the animal. Additionally, DMI and microbial protein production can be affected by site
and extent of starch and NDF digestion, as discussed in later sections. Because
endosperm type of cereal grains is a substantial determinant of starch digestibility,
research on effects of endosperm type on site of starch and NDF digestion needs to be
conducted. Com silage with the bm3 mutation has been shown to interact with site of
starch digestion. The interactions of fiber and starch digestibility on site of starch
digestion could significantly change nutrient partitioning and DMI, and should

demonstrate the importance of nutrient digestibility on performance of dairy cattle.

l6

REGULATION OF INTAKE

Dry matter intake is a primary determinant of milk yield of lactating dairy cows.
Given this, understanding how intake is regulated is critical to obtain maximal
production. Conrad et a1. (1964) proposed that feed intake was determined by physical
fill at low digestibility, and as digestibility increased, metabolic factors determined
intake. Although overly simplistic, the concept of physical versus metabolic regulators of
intake is the basis for many current models of feed intake. In reality, feed intake is
probably determined by the integration of a variety of physical, metabolic, and hormonal

factors.

Physical Regulation of Intake

Physical regulation of intake can occur when physical fill in the gastrointestinal
tract limits feed intake. A significant amount of research has been conducted on factors
affecting physical regulation of intake (Allen, 1996; Allen, 2000). The primary cause of
physical limitation on intake is long retention time of the fibrous fraction of the diet.
Although fiber is crucial to maintaining a healthy rumen environment, digestion of the
fibrous feed fraction is slow and can increase ruminal retention time if particles cannot be
broken down and passed from the rumen. Limitations to flow from the rumen have been
reviewed (Allen, 1996), and include particle size and density, which are closely
associated with ruminal digestibility. Ruminal digestion of fibrous feed increases particle
fragility and makes particles more susceptible to breakdown during chewing (Chai et al.,

1984). Additionally, as ruminal digestion of fiber occurs, particle buoyancy decreases

l7

and particles sink (Sutherland, 1988). With greater rate of NDF digestion, particles are
probably broken down faster and sink faster to increase rate of passage from the rumen
and decrease the filling effect of the diet. Brown midrib 3 corn silage increases rate of
NDF digestion compared to normal corn silage (Ying and Allen, 1995), and because bm3
corn silage also increases rate of NDF passage from the rumen, bm3 corn silage might
reduce physical limitations to intake and allow greater DMI.

Filling effect of dietary NDF can be increased by greater ruminal starch
digestibility. The presence of starch has been demonstrated to reduce rate of in vitro
NDF digestion (Grant and Mertens, 1992). Increased ruminal starch digestion may also
indirectly impede rate of fiber digestion by reducing ruminal pH. In vitro NDF digestion
occurs at a slower rate as pH decreases (Grant and Mertens, 1992), and in the rumen this
might increase retention time and could potentially limit DMI. Brown midrib 3 corn
silage reduces ruminal pH compared to normal corn silage (Oba and Allen, 20000), and
the authors speculated that bm3 corn silage reduced ruminal digestion rate of pdNDF
because of lower pH in bm3 silage diets. Although in vitro pdNDF digestibility was
greater for bm3 corn silage compared to isogenic control, reduced pdNDF digestion rate
and increased passage rate in bm3 corn silage diets could explain why bm3 corn silage
does not always increase NDF digestibility in vivo. Greater ruminal starch digestion and
lower ruminal pH might reduce rate of pdNDF digestion, and could contribute to physical

limitation of intake if it increases ruminal retention time.

18

Metabolic and Hormonal Regulation of Intake

As physical limitations to feed intake decrease, metabolic and hormonal controls
become more limiting to DMI. The liver has been suggested to be one of the primary
sites of integration of metabolic and hormonal regulatory factors because of its central
anatomical position and because it is the first organ to interact with nutrients absorbed
from the gut (Langhans, 1996). Interest in the role of the liver in controlling feed intake
has led to many experiments to determine which nutrients exert hypophagic responses.
These studies show that metabolites extensively metabolized by the liver have
hypophagic effects, whereas those that are not metabolized by the liver do not (Langhans
et al., 1985). Langhans et al. (1985) found that hypophagic effects of metabolic fuels
occur when they are oxidized in the tricarboxylic acid (TCA) cycle in the liver.
Oxidation of metabolic fuels could be a primary regulator of feed intake in dairy cattle
(Allen, 2000), and because ruminal and postruminal starch digestion produces different
metabolic fuels, site and timing of starch digestion could contribute to metabolic

regulation of feed intake in ruminants.

Site of Nutrient Supply

Depression of DMI has been observed to occur in some diets containing high
proportions of ruminally degraded starch (Allen, 2000). Propionate is a primary end
product of starch fermentation by ruminal microorganisms. Infusions of propionate
reduced meal size and length to a greater extent than equimolar infusions of sodium
chloride or acetate, suggesting that effects of propionate on feeding behavior were due to

the substrate rather than effects on osmolality (Choi and Allen, 1999). The liver could be

19

central to effects of propionate on feeding behavior because hepatic vagotomy reduced
hypophagic effects of propionate (Anil and Forbes, 1980). Based on this and other
research, Allen (2000) proposed that oxidation of propionate in the liver could regulate
feed intake in dairy cattle. Ruminal infusions of propionate linearly reduced 12 h
cumulative DMI by reducing meal size (Oba and Allen, 20030). Increased ruminal starch
fermentation increases ruminal propionate concentrations and is probably why increasing
amount of ruminally degraded starch can sometimes depress DMI in dairy cows.

If DMI depression can occur because of greater starch fermentation in the rumen,
then it might be beneficial to shift site of starch digestion to the intestines to relieve
hypophagic effects of propionate. Shifting site of starch digestion to the small intestine
allows mammalian amylase to digest starch to glucose. Glucose can be absorbed across
the enterocyte into the portal blood, partly metabolized to lactate before entering the
portal, or fully oxidized by the enterocyte itself. Because the liver is probably the
primary integrator of metabolic satiety signals, glucose or lactate from intestinal starch
digestion would have to be absorbed by the liver to directly affect feed intake. Allen
(2000) discussed effects of glucose on feed intake, and concluded that glucose is
probably not a metabolic regulator of feed intake in ruminants. Partial metabolism of
glucose to lactate in the enterocyte can occur, and although 60 to 90% of luminal glucose
is metabolized to lactate in rat intestine (Hanson and Parsons, 1976), the extent to which
glucose is metabolized to lactate in ruminant intestine is unknown. Infusions of lactate
into the jugular or mesenteric veins has inconsistently decreased intake (Baile and
Forbes, 1974), and Allen (2000) suggested that inconsistent hypophagic effects of lactate

on DMI are probably dependent on hepatic carbon balance and redox state, which affects

20

lactate removal by the liver (Reynolds, 1995). Additionally, lactate also does not appear
to be taken up by the liver in appreciable quantities in the fed state (Reynolds and
Maltby, 1994), and so might not directly affect feeding behavior.

However, shifting site of starch digestion to the intestine might cause satiety by
hormonal factors. Duodenal glucose infusion increased plasma insulin in dairy cows
(Lemosquet et al., 1997). Although insulin probably does not control short-term feed
intake (Langhans, 1996), increased plasma insulin: glucagon ratio might decrease
gluconeogenesis and increase oxidation of metabolic fuels in the liver (Watford and
Goodridge, 2000) and could induce satiety indirectly. Additionally, gut peptides may
affect feed intake; cells in the intestinal tract release glucagon-like peptide 1 (GLP-1) and
gastric inhibitory peptide (GIP) in response to absorbed nutrients. The presence of
duodenal carbohydrate increased plasma concentrations of insulin, GIP, and GLP-1
(Lavin et al., 1998). GLP—l may directly signal satiety, because intravenous GLP-1
administration suppressed energy intake in humans (Flint et al., 1998), or may have
effects on feed intake by inducing secretion of insulin. GLP-1 and GIP are both potent
insulin secretagogues, and greater than 50% of insulin release after a glucose meal may
be mediated by GIP and GLP-1 (Tseng et al., 1999). This research indicates that
intestinal starch digestion could affect feed intake by inducing secretion of insulin, GIP,
and GLP—1. However, little is known about the effects of gut peptides on intake behavior
in ruminants, and little research concerning dietary effects on gut peptide secretion in

ruminants exists.

21

Timing of Nutrient Supply

Although substantial evidence exists that propionate can depress DMI in
ruminants (Oba and Allen, 2003c), greater ruminally degraded starch decreased DMI in
only 3 of 10 experiments (Allen, 2000). Why does increasing the proportion of ruminally
degraded starch decrease DMI in some but not all experiments? Oba and Allen (2003d)
proposed that inconsistent hypophagic responses to propionate were due to its fate, which
might be determined by temporal supply of propionate relative to its use for
gluconeogenesis. They found that hypophagia in response to propionate infusion was
positively related to plasma glucose concentration. As blood glucose concentration
increased, propionate induced a greater depression in DMI. They speculated that sating
effects of propionate might occur if propionate could not be used toward gluconeogenesis
and was instead oxidized in the TCA cycle. As glucose demand increases, more
propionate might be used for gluconeogenesis and less could be oxidized, thus delaying
satiety. If propionate flux is too fast for the capacity of the gluconeogenic enzymes,
oxidation of propionate might induce satiety. This effect is purely temporal; increased
starch digestion to propionate might not limit DMI if propionate flux to the liver is not
more than the capacity of the gluconeogenic enzymes to utilize it for glucose. The
concept that propionate can induce satiety in a temporal pattern relating to
gluconeogenesis has not been specifically tested, but current research in our laboratory is
investigating this proposal further.

DMI is probably the most important limitation to milk production in dairy cows.
For this reason, it is important to understand factors affecting feeding behavior.

Regulation of feed intake is complicated, and DMI is the determined by integration of

22

physical, metabolic, and hormonal factors. Additionally, DMI is a function of meal size
and frequency, as determined by satiety and hunger, respectively (Allen, 2000), so it is
important to understand how regulators of feed intake can affect feeding behavior and not
just cumulative DMI. Endosperm type of corn grain can substantially change site of
starch digestion, which can profoundly affect nutrient profile absorbed by the animal and
possibly feeding behavior. Fiber digestibility can affect feeding behavior via physical
regulation, but because bm3 corn silage can shift site of starch digestion, regulation of
DMI could occur by metabolic and hormonal factors as well. Interactions of starch and
fiber digestibility might occur to affect ruminal fermentation and DMI, which could

potentially influence microbial protein supply.

MICROBIAL PROTEIN SUPPLY

The primary source of nitrogen to ruminants is supplied by flow of ruminal
microorganisms to the duodenum. Microbial protein has a high biological value because
of its amino acid (AA) profile and high digestibility. To reduce limitations to milk yield
by AA availability, it is important to maximize microbial protein flow. DMI and nutrient
digestibility can play substantial roles in microbial fermentation and efficiency. Energy
availability often limits microbial growth, so increasing grain intake can increase
substrate available for microbial protein production (Clark et al., 1992). However,
increased ruminal starch digestion can result in reduced pH, depressed fiber digestibility,

and reduced microbial growth efficiency.

23

Effects of Ruminal pH on Fiber Digestion and Microbial Growth

Grain is relatively inexpensive and abundant in the US, and is commonly used to
increase energy density of diets. However, many nutritionally related diseases (acidosis,
bloat, liver abscesses) result from use of grains in ruminant diets. These symptoms are
related to reduced ruminal pH, associated with feeding rapidly fermented cereal grain
diets. Low ruminal pH has been associated with depression in ruminal NDF digestibility.
Feeding a highly fermentable grain source, such as steam rolled barley, increased ruminal
starch digestibility but reduced ruminal pH and ruminal NDF digestibility (Overton et al.,
1995). Additionally, feeding a more fermentable forage source like bm3 corn silage can
reduce ruminal pH (Oba and Allen, 20000; Greenfield et al., 2001) and might reduce
ruminal NDF digestibility (Oba and Allen, 20000).

To understand how reduced ruminal pH can decrease fiber digestion, effects of
low pH on ruminal cellulolytic bacteria has been studied extensively. Chow and Russell
(1992) investigated the response of a predominant cellulolytic bacterium, F ibrobacter
succinogenes, to reduction in external pH. They noted at pH less than 6.0, F.
succinogenes, which maintains intracellular pH at approximately 7.0, was unable to grow
or utilize glucose. As extracellular pH decreased, the pH gradient across the bacterial
membrane increased to the point that F. succinogenes was unable to maintain the pH
gradient and intracellular pH decreased. Ruminococcus albus, another predominant
cellulolytic bacteria, allows intracellular pH to decline with environmental pH to avoid
increasing the pH gradient across the bacterial membrane (Thurston et al., 1993). This
method of 00ping with low external pH is the primary method of acid-resistant bacteria,

but the key difference is that acid-resistant bacteria have intracellular enzymes that are

24

active at lower pH. For F. succinogenes and R. Albus, internal enzyme systems are not
able to maintain function at lower pH, and grth is inhibited at pH < 6.0 (Chow and
Russell, 1992; Thurston et al., 1993).

Although research suggests that cellulolytic bacteria cannot digest cellulose under
pH 6.0 (Russell and Wilson, 1996), ruminants consuming high grain diets often have a
ruminal pH below 6.0. How does cellulose digestion occur if cellulolytic bacteria cannot
grow under environments created by typical dairy cow rations? F errnentative capability
of mixed ruminal bacteria continued, albeit at a lower rate, as pH declined below 7, and
was not inhibited until pH reached 5.3 (Mourifio et al., 2001). Calsamiglia et al. (2002)
investigated the effects of varying pH on nutrient digestibility in a continuous culture
system. They kept pH constant at 6.4 or 5.7, or fluctuated pH in cycles of 4 hours at pH
6.4 and 4 hours at pH 5.7, or maintained pH at 6.4 except for two cycles where pH
dropped to 5.7 for 1 hour, then slowly recovered over the next 3 hours. True NDF
digestibility was depressed only when pH was constantly maintained at 5.7. In practical
feeding situations, pH is probably not maintained at a constant level; raw data from Dado
and Allen (1995), as presented in Allen (1997), shows significant fluctuation of ruminal
pH in a dairy cow fed a 35% NDF diet twice daily. The work by Calsamiglia et al.
(2002) indicates that the pH fluctuations that occur in normal dairy cattle rations probably
do not inhibit fiber digestibility like some in vitro work might suggest.

Most research investigating mechanics of cellulose digestion has been conducted
using pure cultures of ruminal bacteria. However, cocultures of cellulolytic and
noncellulolytic strains removed more cellulose in vitro than pure cultures of cellulolytic

bacteria (Mourifio et al., 2001). The cellulase enzyme complex of ruminal cellulolytic

25

bacteria hydrolyzes cellulose into smaller cellodextrin and cellobiose pieces. A
cellobiose analog, thiocellobiose, inhibited cellulose utilization by F. succinogenes; the
authors suggested that cellobiose acted as an inhibitor of the cellulase enzyme complex
and that microbial consumption of cellobiose was necessary for cellulase complex
activity (Maglione et al., 1997). Noncellulolytic strains, such as Prevotella ruminicola
and Selenomonas ruminantium cannot hydrolyze cellulose but can use cellodextrins
(Russell, 1985), and this endproduct removal by noncellulolytic strains may increase
cellulase enzyme activity. Cross-feeding activity in the rumen may also explain why in
vivo NDF digestibility is not reduced to the extent that in vitro pure culture work often
predicts.

Increased ruminal starch digestibility might indirectly affect fiber digestion by
reducing ruminal pH, but may also directly reduce fiber digestion. In vitro work
examined the effects of buffer pH and addition of corn starch on digestion kinetics of
NDF in bromegrass hay, alfalfa hay, and corn silage (Grant and Mertens, 1992).
Interestingly, for alfalfa hay, reducing pH from 6.8 to 5.8 reduced rate of NDF digestion
only slightly. However, addition of corn starch to the media reduced NDF digestion by
75 to 78% for all pH. For bromegrass hay and corn silage, reducing media pH depressed
NDF digestion, but similar to alfalfa hay, addition of corn starch further decreased rate of
NDF by 75 to 78%. Presence of starch may decrease rate of NDF digestion because
some organisms preferentially utilize starch before cellulose (Hungate, 1966).
Depression of fiber digestion in diets containing highly ruminally degradable starch
sources might occur because of low ruminal pH, but may be highly related to availability

of starch itself.

26

Efficiency of microbial growth can be reduced by low environmental pH.
Microbial growth efficiency is defined as grams of microbial protein flow to the
duodenum per kg of truly fermented organic matter (TRDOM). Energy availability is
often the primary limitation on microbial growth (Clark et al., 1992), so generally any
increase in fermented OM would increase microbial growth. However, Clark et al.
(1992) found a quadratic relationship between TRDOM (kg per day) and efficiency of
microbial protein production. Under conditions of energy limitation, increasing amount
of TRDOM improves microbial efficiency, but as amount of TRDOM continues to
increase microbial efficiency declines. Replacing dry corn with high moisture corn
increased ruminal starch fermentation and amount of TRDOM but reduced efficiency of
microbial growth, and the authors speculated that a process is known as energy spilling or
uncoupling occurred (Oba and Allen, 2003c). Energy spilling is the use of ATP toward
non-growth functions, and appears to be a common method of handling excess
carbohydrate in ruminal bacteria (Russell, 1998). During periods of energy limitation,
bacteria ferment substrate to provide maximal ATP; however, as fermentable substrate
increases and energy availability exceeds the ability of the bacteria to use it for growth,
ATP is used toward non-growth functions (Russell, 1998). Several processes can
contribute to this wasteful energy use. Organisms can switch from VFA to lactate
production because lactate generates fewer ATP per mol hexose fermented (Russell,
1998). Rapid increase in heat production by microbial cultures can occur after a pulse
dose of glucose, possibly due to increased maintenance energy and futile cycling
(Russell, 1986). Membrane ion maintenance is an important component of the

maintenance energy of bacteria, and might be used to increase futile cycling. An

27

inhibitor of the membrane-bound ATPase abolished energy spilling, and a protonophore
that increased proton flux across the membrane doubled energy spilling (Russell and
Stroebel, 1990). Futile cycles of maintaining proton balance across the microbial
membrane might reduce ATP available for growth, and might also indicate why low pH

can reduce microbial efficiency.

Effects of Ruminal Turnover Rate on Microbial Growth

Microbial growth efficiency is also affected by turnover rate in the rumen. F eed
intake and flow of microbial N to the duodenum are positively related (Clark et al.,
1992), so effects of starch and fiber digestibility on DMI are expected to affect microbial
protein production. Additionally, increasing rate of passage from the rumen probably has
dramatic effects on increasing microbial growth efficiency by decreasing microbial
turnover (Wells and Russell, 1996). Feeding bm3 corn silage increased microbial
efficiency, possibly because of increased ruminal passage rate of NDF, despite a
reduction of ruminal pH by bm3 silage (Oba and Allen, 20000). Microbial efficiency was
positively correlated with ruminal passage rate of starch and pdNDF (Voelker and Allen,
2003b), probably from reduced microbial turnover. Microbial turnover in the rumen
occurs both by autolysis and predation. Mechanisms of autolysis are not well studied, but
might contribute considerably to intraruminal N recycling when ruminal retention times
are long. Protozoal predation can have significant effects on microbial efficiency and is
often considered the primary cause of bacterial turnover in the rumen. Research with

defaunated animals has shown that absence of protozoa increased duodenal microbial N

28

flow and bacterial efficiency, and reintroduction of protozoa subsequently decreased
these variables (Koenig et al., 2000).

Protozoa were not believed to have direct advantage for the host animal because
they reduce bacterial efficiency and do not contribute significantly to duodenal N flow
because they are selectively retained in the rumen (Williams and Coleman, 1988).
However, reduced OM and NDF digestibility in defaunated animals suggests that
protozoa probably do contribute to ruminal fermentation (Koenig et al., 2000). A
substantial portion of ruminal fiber digestion is due to protozoal degradation (Orpin,
1984). Additionally, protozoa engulf starch particles and digest them more slowly than
do ruminal bacteria, thus potentially modulating ruminal pH (N agaraja et al., 1992).
Although the presence of protozoa may reduce bacterial efficiency by predation, the
absence of protozoa may reduce bacterial efficiency by low ruminal pH.

Changing starch and fiber digestibility could influence efficiency of microbial
protein production and ruminal fermentation. Endosperm type of corn grain can affect
ruminal starch digestibility, which could affect ruminal pH, NDF fermentation, and
microbial nitrogen efficiency. Additionally, bm3 corn silage has been demonstrated to
reduce ruminal pH but increase rate of passage, so effects of bm3 corn silage on
microbial efficiency are not predictable. Interactions of starch and fiber digestibility on
DMI might also influence microbial efficiency by affecting microbial turnover. Research
on the interactions of endosperm type of corn grain and the bm3 mutation in corn silage

on microbial efficiency is warranted.

29

SUMMARY

Fiber digestibility ranges considerably, and the ability to change the extent of
lignification and fraction of pdNDF by a single gene mutation (bm3 mutation in corn
silage) provides a model to study NDF digestibility. Starch digestibility is highly related
to Vitreousness of corn grain endosperm and grain hybrids can range from 100% floury
endosperm to highly (> 60%) vitreous endosperm. Individually, fiber and starch
digestibility play an important role in the utilization of nutrients for production, but no
research has directly examined how starch and fiber digestibility can interact to affect
animal productivity.

Corn grain with floury endosperm and bm3 corn silage might reduce ruminal pH,
NDF digestibility, microbial fermentation, and microbial growth efficiency, although
greater passage of bm3 corn silage might reduce negative effects of pH on microbial
growth efficiency. Reduced ruminal NDF digestibility from rapid starch fermentation
might shift more NDF digestion to the hindgut, but might reduce total tract NDF
digestibility if hindgut fermentation cannot compensate. Additionally, interactions of
fiber and starch digestibility are expected to affect DMI; greater ruminal starch
fermentation might reduce DMI by oxidation of metabolic fuels in the liver, but bm3 corn
silage might reduce filling effects of the diet and increase DMI.

Shifting site of starch digestion to the small intestines could occur by feeding corn
grain with vitreous endosperm. Although the evidence is scant, bm3 could affect starch
digestion by increasing ruminal passage rate and shifting site of starch digestion to the

intestines. Additionally, if bm3 silage shifts site of starch digestion to the intestines,

30

hypophagic effects of propionate from ruminal starch fermentation might be removed,
but digestion of starch in the small intestine could also potentially limit intake by other
metabolic or hormonal factors. Greater glucose absorption in the small intestine might
increase milk yield or tissue retention, or might not affect either. Greater rate of NDF
passage for bm3 silage might increase microbial efficiency by decreasing microbial

turnover in the rumen, but greater hindgut fermentation could reduce microbial yield.

There is sufficient evidence to suggest that bm3 corn silage and endosperm type of corn
grain might interact to affect digestion and productivity of lactating dairy cows. We
hypothesized that corn grain with floury endosperm is more ruminally fermentable than
vitreous corn grain, and will decrease rate of digestion of pdNDF to a greater extent for
bm3 corn silage compared to control silage. Greater ruminal starch fermentation of
floury corn grain would have greater effect at reducing meal size and possibly DMI when
combined with bm3 corn silage than control silage. Finally, corn grain with floury
endosperm reduces microbial efficiency because of more rapid starch digestion, and
might interact with corn silage type because bm3 silage reduces microbial efficiency by
low ruminal pH but increases microbial efficiency by increasing rate of ruminal passage.
Therefore, the objective of this thesis was to evaluate the interactions of corn grain
endosperm type and the brown midrib 3 mutation in corn silage on site of nutrient
digestion, feeding behavior, milk production and energy balance, ruminal fermentation,

and microbial protein efficiency in high producing dairy cows.

31

CHAPTER 2

Interactions of corn grain endosperm type and brown midrib 3 corn silage on

ruminal kinetics and site of digestion in lactating dairy cows

ABSTRACT

Interactions of endosperm type of corn grain and the brown midrib 3 mutation in corn
silage on ruminal kinetics and site of nutrient digestion of lactating dairy cows were
evaluated. Eight ruminally and duodenally cannulated cows (72 :9; 8 DIM; mean :1: SD)
were used in a duplicated 4 x 4 Latin square design experiment with a 2 x 2 factorial
arrangement of treatments. Treatments were com grain endosperm type, floury or
vitreous, and corn silage type, bm3 or isogenic normal. Diets contained 26% neutral
detergent fiber (NDF) and 30% starch. Interactions of treatments were not observed for
any measure of digestibility, but digestion kinetics of starch and fiber interacted to affect
digestible OM intake by affecting DM intake. Rate of ruminal starch digestion was faster
and rate of ruminal starch passage tended to be slower in diets containing corn grain with
floury endosperm, resulting in a mean increase for ruminal starch digestibility of 22.1
units. Although compensatory postruminal starch digestion decreased differences among
treatments for total tract starch digestibility, starch entering the duodenum was more
digestible for grain with floury endosperm compared to vitreous grain, resulting in
greater total tract starch digestibility for floury corn grain compared to vitreous corn

grain. Fermentation rate of potentially digestible NDF was not affected by either bm3

32

corn silage or greater ruminal starch digestion of floury grain. Brown midrib corn silage
increased total tract NDF digestibility versus control by numerically increasing ruminal
and postruminal digestibility of NDF. Endosperm type of corn grain greatly influences

starch digestion and should be considered when formulating diets.

INTRODUCTION

Ruminants have dietary requirements for forage to maximize production and
ruminal health. However, because fiber is digested more slowly and is retained longer in
the rumen than other nutrients, it can potentially limit DMI and milk yield. The brown
midrib 3 (bm3) mutation in corn silage increases potential digestibility of fiber and
allows for greater feed intake while providing forage fiber (Oba and Allen, 2000a).
Although increased fiber digestibility can reduce the filling effect of the fibrous feeds and
might allow greater feed intake, bm3 corn silage reduces ruminal pH and might reduce
ruminal digestibility of potentially digestible NDF (pdNDF; Oba and Allen, 20000).
Feed intake could also be altered by manipulating site of starch digestion (Allen, 2000);
site of starch digestion can be affected by grain conservation method (Oba and Allen,
2003b) and method of processing (Callison et al., 2001). Additionally, corn silage with
the bm3 mutation reduced ruminal starch digestibility compared to the isogenic corn
silage without the bm3 mutation (Oba and Allen, 2000c). The authors attributed reduced
ruminal starch digestibility in bm3 corn silage diets to greater passage rate of starch from
the rumen. Endosperm type of corn grain also can affect site of starch digestion; flint

corn, which has a higher proportion of vitreous endosperm, was digested more slowly

33

and to a lesser extent than dent corn in the rumen of beef steers (Philippeau et al., 1999a).
Endosperm type of corn grain varies widely across commercially available hybrids, but is
not considered when formulating diets for lactating dairy cows.

Controversy exists as to the benefits of ruminal versus postruminal starch
digestion. Ruminal starch digestion is needed to provide substrate for microbial growth
and propionate as a glucose precursor for milk synthesis, but can reduce ruminal pH and
inhibit fiber digestibility if starch fermentation is too rapid. Rapid flux of propionate to
the liver might limit DMI if it is oxidized instead of being used for gluconeogenesis (Oba
and Allen, 2003c). Shifting starch digestion to the intestines can theoretically provide
more glucose to the animal, but infusion studies have suggested that increasing small
intestinal glucose absorption may not increase glucose available for milk production
(Knowlton et al., 1998b; Arieli et al., 2001). Instead, increased glucose may be used for
tissue retention (Reynolds et al., 2001) or may be oxidized to C02 (Knowlton et al.,
1998b). It is important to understand how chemical and structural aspects of dietary
ingredients can affect starch and fiber digestibility and how they interact to change
ruminal kinetics and site of nutrient digestion. We hypothesized that com grain with
floury endosperm is more rapidly degraded in the rumen compared to corn grain with
vitreous endosperm, and will decrease rate of digestion of pdNDF to a greater extent for
bm3 corn silage compared to control corn silage. Greater rate of passage of pdNDF and
indigestible NDF (iNDF) from the rumen in diets containing bm3 corn silage will
increase ruminal passage rate of starch. The objective of this experiment was to evaluate
interactions of the brown midrib 3 mutation in corn silage and corn grain endosperm type

on ruminal digestion kinetics and site of nutrient digestion of lactating dairy cows.

34

MATERIALS AND METHODS

Cows and Treatments

Experimental procedures were approved by the All University Committee on
Animal Use and Care at Michigan State University. Eight multiparous Holstein cows (72
1 8 DIM; mean :1: SD) from the Michigan State University Dairy Cattle Teaching and
Research Center were randomly assigned to duplicate 4 x 4 Latin squares balanced for
carry-over effects. Cows were randomly assigned to treatment sequence. A 2 x 2
factorial arrangement of treatments was used with main effects of corn grain endosperm
type (floury or vitreous) and bm3 mutation (present or absent). Treatment periods were
21 (1, consisting of an 11 d diet adaptation period followed by 10 d of collection. Surgical
preparation of ruminally and duodenally cannulated cows was performed after dry-off,
approximately 50 (1 prior to calving. Duodenal cannulas were soft gutter type made of
tygon and vinyl tubing (Crocker et al., 1998). For each animal, the duodenum was
fistulated distal to the pylorus region prior to the pancreatic duct, and the cannula was
placed between 10th and 11th ribs as described by Robinson et al. (1985). Surgery was
performed at the Department of Large Animal Clinical Science, College of Veterinary
Medicine, Michigan State University. At the beginning of the experiment, empty body
weight (ruminal digesta removed) of cows was 531.8 :I: 43.9 kg (mean 1 SD).

Two corn hybrids, 6208FQ and 657 (Cargill Hybrid Seeds, Minneapolis, MN),
were planted for silage in Spring of 2001 at the Michigan State University Research
Farm. The hybrids are isogenic except that Cargill 657 contains the bm3 mutation.

Cargill 6208F Q corn forage was harvested at 30.6% whole plant dry matter and chopped

35

to 11 mm theoretical length of cut. Cargill 657 com forage was harvested at 32.2% whole
plant dry matter and chopped to 10 mm theoretical length of cut. The chop lengths of the
two hybrids differed in order to achieve a similar particle size distribution as measured
using a Penn State Particle Size Separator (Lammers et al., 1996). Both hybrids were
ensiled in 12.43 rn diameter AgBags® oriented in a west to east direction in order to
minimize the effects of wind on silage DM. Nutrient compositions and physical
characteristics of the corn silage treatments used in the experiment are shown in Table 1.

Two corn hybrids (1 floury and 1 vitreous) were planted for grain in Spring of
2001 at the Michigan State University Research Farm. The floury and vitreous hybrids,
SL53 (Crow’s Hybrid Corn Company, Kentland, IN) and Z75W (Wilson Genetics,
Harlan, IA) were selected based upon their high and low in vitro starch digestibility,
respectively. Grains were harvested after field drying to ~20% DM, and were
commercially dried at low temperature (< 32 °C) to ~14% DM. The corn was ground
and bagged for use in the experiment. Vitreousness (% of endosperm) for floury and
vitreous corn grain was 3.0 and 67.2%, respectively (Table 2). Nutrient compositions
and physical characteristics of the corn grain treatments used in the experiment are shown
in Table 2.

Experimental diets contained dry ground corn treatments (floury or vitreous), corn
silage (bm3 or isogenic normal), alfalfa silage (10% of diet dry matter), whole linted
cottonseed (7% of diet dry matter), a protein supplement premix (soybean meal, distillers
grains, and blood meal), and a premix of minerals and vitamins. Experimental diets were

fed as total mixed rations, and were formulated to contain 27% NDF, 18% crude protein

36

with adequate metabolizable protein, and minerals and vitamins to requirements.

Ingredient and nutrient compositions of the experimental diets are shown in Table 3.

Data and Sample Collection

Throughout the experiment, cows were housed in tie-stalls and fed once daily
(1100 h) at 110% of expected intake. Amounts of feed offered and orts were weighed for
each cow daily during the collection period. Samples of all diet ingredients (0.5 kg) and
orts from each cow (12.5%) were collected daily on (1 12-18 and combined into two
samples per period, representing the digestibility (CI 12-14) and feeding behavior ((1 15-
18) sub-periods.

Chromic oxide was used as a marker to estimate nutrient digestibility in the
rumen and in the total tract. Gelatin capsules (1.5 oz., Tropac Inc., Airfield, NJ)
containing 5 g of Chromic oxide and approximately 4 g of ground spelt hulls were dosed
through the ruminal cannula at 0400, 1200, and 2000 h (total of 15 g 0203 /d) from 7 to
14 d with a priming dose of 3X on d 7. Duodenal (1,000 g), fecal (500 g), and rumen
fluid samples (100 mL) were collected every 9 h from d 12 to d 14 so that 8 samples were
taken for each cow each period, representing every 3 h of a 24-hour period to account for
diurnal variation. Digesta from 5 sites in the rumen was combined and strained to obtain
rumen fluid. All digesta and fecal samples were immediately frozen at -20°C until
processing.

Effect of treatment on rate of liquid passage was measured on d 19 using a pulse
dose of cobalt EDTA (Allen et al., 2000). Cobalt EDTA was dosed two hours after

feeding. Rumen fluid was sampled immediately before dosing and at 0.5, 1, 1.5, 2, 2.5,

37

3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8 h after dosing. Samples were immediately
frozen.

Ruminal contents were evacuated manually through the ruminal cannula at 1500 h
(4 h after feeding) on d 20 and at 0900 h (2 h before feeding) on d 21 of each period.
Total ruminal content mass and volume were determined. During evacuation, 10%
aliquots of digesta were separated to allow accurate sampling. Aliquots were squeezed
through a nylon screen (1 mm pore size) to separate into primarily solid and liquid
phases. Samples were taken from both phases for determination of nutrient pool size, and
an additional liquid sample was taken to measure VFA concentration. All samples were

frozen immediately at —20°C.

Sample and Statistical Analysis

Diet ingredients and orts were dried in a 55°C forced-air oven for 72 h and
analyzed for DM concentration. All samples were ground with a Wiley mill (1mm
screen; Authur H. Thomas, Philadelphia, PA). Fecal samples were processed similarly,
and dried, ground fecal samples were combined on an equal DM basis into one sample
per cow per period. Rumen liquid and solid sub-samples were lyophilized (Tri-Philizer’"
MP, FTS Systems, Stone Ridge, NY), ground, and recombined according to the original
ratio of solid and liquid DM. Duodenal samples were thawed, combined, and filtered
into primarily solid and liquid phases using nylon mesh (1 mm pore size) to minimize
sampling errors due to segregation of samples into solid and liquid phases. Both phases

were weighed, and sub-samples were taken from each phase. Liquid and solid sub—

38

samples were lyophilized, ground, and recombined by weight according to the original
ratio of solid and liquid DM.

Samples were analyzed for ash, NDF, indigestible NDF (iNDF), crude protein
(CP), and starch. Ash concentration was determined after 5 h oxidation at 500°C in a
muffle furnace. Concentrations of NDF were determined according to Van Soest et a1.
(1991, method A). Forage samples were analyzed for acid detergent fiber (ADF) and
sulfuric acid lignin content (Van Soest et al., 1991). Indigestible NDF was estimated as
NDF residue after 240-h in vitro fermentation (Goering and Van Soest, 1970). Rumen
fluid for the in vitro incubations was collected from a non-pregnant dry cow fed only
alfalfa hay. Fraction of potentially digestible NDF (pdNDF) was calculated by difference
(1.00 - iNDF). Crude protein was analyzed according to Hach et al. (1987). Starch was
measured by an enzymatic method (Karkalas, 1985) after samples were gelatinized with
sodium hydroxide; glucose concentration was measured using a glucose oxidase method
(Glucose kit #510; Sigma Chemical Co., St. Louis, MO), and absorbance was determined
with a micro-plate reader (SpectraMax 190, Molecular Devices Corp., Sunnyvale, CA).
Concentrations of all nutrients except DM were expressed as percentages of DM
determined by drying at 105° C in a forced-air oven for more than 8 h.

Corn silage particle size was determined on wet silage samples using the Penn
State Particle Size Separator (Lammers et al., 1996). Wet corn silage samples were
homogenized in distilled water to determine pH and concentrations of major VFA and
lactate by HPLC (Waters Corp., Milford, MA) as described by Oba and Allen (2003a).
Corn grain was dry sieved (Sieve apertures: 4750, 2360, 1180, 600, 300, 150, 75 um and

bottom pan) using a sieve shaker (model RX-86, W. S. Tyler Inc., Gastonia, NC) for

39

approximately 20 minutes until the bottom pan weight was constant, and the mean
particle size of the corn grain was calculated (ASAE, 1968). For determination of corn
grain Vitreousness, 10 whole kernels from each hybrid were weighed, and kernels were
dissected into germ, pericarp, and vitreous endosperm sections and individually weighed.
Total (floury and vitreous) endosperm weight was determined by subtracting germ and
pericarp weight from whole kernel weight, and Vitreousness was calculated by dividing
vitreous endosperm weight by total endosperm weight.

Rumen fluid samples taken for measurement of rate of liquid passage were
analyzed for cobalt concentration by flame atomic absorption spectrophotometry
according to manufacturers recommendations (SpectrAA 220/F S, Varian Australia Pty.
Ltd., Mulgrave, Victoria, Australia). Rate of cobalt disappearance was determined by
non-linear regression (JMP® Version 4, SAS Institute, Cary, NC) of its decline in
concentration in rumen fluid over time after dosing using a one-pool, first-order model
after accounting for background cobalt concentration.

Diet ingredients, duodenal digesta, and feces were analyzed for concentrations of
chromium. Samples were digested with phosphoric acid (Williams et al., 1962), and
chromium was quantified by flame atomic absorption spectrometry (SpectraAA 220,
Varian, Victoria, Australia) according to manufacturer's recommendation. Although we
intended to use chromic oxide as an external marker in this study, an apparent liquid
phase subsampling problem occurred (Appendix A), and iNDF was chosen as a duodenal
flow marker. Subsampling problems would not be expected in fecal samples but iNDF
was used as a fecal flow marker for consistency. Duodenal and fecal DM flows

calculated using chromic oxide were highly correlated to flows calculated using iNDF as

40

a marker (r2 = 0.60 and r2 =0.79, respectively). Nutrient intake was calculated using the
amounts and compositions of feed offered and refused. Duodenal flow of microbial OM
was determined as described by Oba and Allen (2003c), and true ruminally degraded OM
(TRDOM) was calculated by subtracting duodenal flow of non-microbial OM from OM
intake. True ruminally degraded starch was calculated by subtracting duodenal flow of
non-microbial starch from starch intake. Ruminal pool sizes (kg) of OM, NDF, iNDF,
pdNDF, and starch were determined by multiplying the concentration of each component
by the ruminal digesta DM mass (kg). Turnover rate in the rumen, passage rate from the
rumen, and ruminal digestion rate of each component (%/h) were calculated using the

following equations:

Turnover rate in the rumen (%/h) =

100 x (Intake of component / Ruminal pool of component) / 24

Passage rate from the rumen (%/h) =

100 x (Duodenal flow of component / Ruminal pool of component) / 24; and

Digestion rate in the rumen (%/h) =

Turnover rate in the rumen (%/h) - Passage rate from the rumen (%/h).
All data was analyzed using the fit model procedure of JMP® (Version 4, SAS

Institute, Cary, NC) according to the following model:

Yijkl = Ii + Ci + Pj + Tk + PTjk + eijkl

41

where

u = overall mean,

C, = random effect of cow (j = 1 to 8),

Pj = fixed effect of period (k = 1 to 4),
Tk = fixed effect of treatment (I = 1 to 4),

PTJ-k =interaction of period and treatment, and

eijkl = residual

A reduced model without period x treatment interactions was used when this
effect was not significant (P > 0.15). Orthogonal contrasts were used to determine main
effect of corn silage type, main effect of corn grain type, and the interaction of corn silage
type and endosperm type of corn grain. Pearson correlation coefficients were determined
between cow-period observations for some parameters. Main treatment effects and
correlations were declared significant at P < 0.05, and tendencies were declared at P <
0.10. Interactions between treatments were declared significant at P < 0.10, and
tendencies were declared at P < 0.15.

Data from one cow was removed from all statistical analysis due to clinical
mastitis during the first period. This cow was replaced with a spare animal for the
remaining three periods. Another cow developed pneumonia during the diet adaptation
period of period 2, and was recovering during the first day of the collection period. Data
from this cow was omitted from the digestibility sub-period of period 2, but was utilized

from the feeding behavior sub-period as she had recovered sufficiently.

42

RESULTS AND DISCUSSION

DM and OM Digestion

No interactions between main treatment effects were found for any measure of
DM or OM digestibility, and replacing control corn silage with bm3 corn silage did not
affect DM or OM digestibility. Total tract DM digestibility was higher for floury versus
vitreous corn grain (P < 0.01; Table 4). Although floury corn grain increased apparent
ruminal OM digestibility 9.5 percentage units versus vitreous grain (P < 0.04), slightly
higher duodenal flow of microbial OM in vitreous diets reduced the differences between
endosperm types so that TRDOM did not differ among treatments (Chapter 4). TRDOM
averaged 14.5 kg/d and is among the highest reported in literature, primarily because
animals in this experiment averaged 22.6 kg/d OM intake and were producing 40 kg/d
milk. Postruminal OM digestibility did not differ among treatments, but increases of
ruminal OM digestion in diets with floury endosperm resulted in greater total tract

apparent OM digestion (P < 0.01).

Site of Starch Digestion

Contrary to our hypothesis, no interactions of main treatment effects occurred to
affect starch digestion. Although previous research from our laboratory found that bm3
corn silage reduced ruminal and total tract starch digestibility (Oba and Allen, 20000), in
the present experiment bm3 corn silage did not affect ruminal starch digestibility and
only tended to reduce total tract starch digestibility compared to control corn silage (P <

0.08; Table 5). However, endosperm type of corn grain caused substantial changes in site

43

of starch digestion. Apparent ruminal digestibility of starch increased from 35.0% to
57.0% when floury endosperm grain replaced vitreous corn grain (P < 0.01). A previous
experiment showed that apparent ruminal starch digestibility was increased from 34.8 to
60.8 % when dent corn replaced flint corn in diets of steers (Philippeau et al., 1999a).
Corn grain with floury endosperm also increased true ruminal starch digestibility
compared to vitreous grain (P < 0.001 ), but variation was greater for apparent versus true
ruminal starch digestibility because of the greater contribution of microbial starch to
duodenal starch in the diet containing corn grain with vitreous endosperm and control
corn silage compared to other diets (Chapter 4). As expected, vitreous grain shifted the
primary site of starch digestion to the intestines; postruminal starch digestibility as a
percent of intake was greater for vitreous versus floury corn grain (56.8 versus 39.3 %; P
< 0.03). However, as a percent of duodenal starch flow, starch digestibility was lower for
vitreous grain compared to floury corn grain (P < 0.03). This indicates that corn grain
with vitreous endosperm is not only more indigestible than floury grain in the rumen, but
the starch that passes out of the rumen is also less digestible. Although apparent ruminal
starch digestibility was reduced by 39 % for vitreous grain diets, total tract starch
digestibility was only reduced 5 % because of substantial postruminal compensatory
digestion.

Ruminal starch digestibility was greater for floury corn grain treatments because
starch in floury endosperm was digested at a rate of 21.9 %/h versus 12.9 %/h for
vitreous endosperm (P < 0.01; Table 7). Philippeau et al. (1999b) reported that dent
hybrids (51.4% vitreous) degraded faster than flint (71.8% vitreous) hybrids in situ (P <

0.0001). Floury endosperm is associated with a digestible protein matrix and is easily

broken down by ruminal bacteria (Kotarski et al., 1992). In contrast, starch granules in
vitreous endosperm are embedded in a protein matrix that can resist enzyme hydrolysis
(Rooney and Plugfelder, 1986). Vitreous protein matrix is more resistant to digestion
because ruminal bacteria digest zein proteins more slowly than glutelin proteins
(Romagnolo et al., 1994), and Vitreousness of corn grain is positively correlated with
concentration of zein protein and negatively correlated with true glutelin protein
concentration in corn grain (Philippeau et al., 2000). Because the protein matrix in floury
endosperm is more easily hydrolyzed, greater microbial penetration of the starch granule
occurs to increase rate of starch digestion. In this study, Vitreousness (% of total
endosperm) of corn grain was 3.0 and 67.2 % for floury and vitreous hybrids,
respectively (Table 2). Additionally, the corn grains used were selected based on their
wide range of in vitro starch degradability, which was 7.7 and 1.8 %/h for the floury and
vitreous hybrids, respectively (Table 2). The floury and vitreous endosperm corn grains
used in this experiment represent the two extreme endosperm compositions that are
commercially available.

Corn grain with vitreous endosperm tended to increase ruminal passage rate of
starch (21.2 vs. 16.2 %/h; P < 0.10; Table 7). We expected floury corn grain might
disperse in the liquid fraction and possibly increase rate of starch passage from the
rumen. However, mean ruminal liquid passage rate was approximately 19.9 %/h, and
rate of starch passage from the rumen and ruminal liquid passage rate were not correlated
across cow period means (r2 = 0.03; P < 0.86). Faster ruminal starch passage rate in this
experiment might be because of greater density of vitreous corn grain; vitreous flint corn

grain was more dense than less vitreous dent corn grain (Philippeau et al., 1999b), and

45

greater particle density decreased mean ruminal retention time (Lechner-Doll et al.,
1991). Faster rate of starch digestion and a tendency for slower rate of ruminal starch
passage for floury corn grain versus vitreous grain resulted in a lack of treatment effects
on ruminal starch turnover rate.

It is unclear when shifting starch digestion from the rumen to the intestines is
beneficial for milk production. Small intestinal digestion of starch to glucose is
theoretically more efficient than ruminal fermentation to VF A (Owens et al., 1986), and
while glucose availability might increase with greater intestinal starch flow, milk
production does not necessarily increase (Knowlton et al., 1998b; Arieli et al., 2001;
Reynolds et al., 2001). As greater amounts of concentrate are included in ruminant diets,
understanding limits to starch digestion and absorption in the intestines of ruminants has
become a question of interest. A linear response in net portal glucose absorption was
observed for infusions of glucose into the abomasum, but a quadratic response in net
portal glucose absorption was observed for infusions of corn starch in to the abomasum;
diminished absorption occurred with greater amounts of corn starch infusion
(Kreikemeier et al., 1991). This suggests that limitations to small intestinal starch
utilization are from starch digestion and not glucose absorption. Huntington (1997)
suggested that limitations to small intestinal starch digestion are because of less secretion
of effective pancreatic amylase with increasing duodenal starch flow. However, Oba and
Allen (2003b) found that postruminal starch digestibility (% of duodenal flow) increased
as duodenal starch flow increased (r2 = 0.49; P < 0.05). They suggested that limitations
to starch utilization by pancreatic enzyme activity would be expected to decrease

intestinal starch digestibility as duodenal flow of starch increased, and concluded that

46

lower digestibility of starch escaping the rumen was because of differences in the
physical and chemical characteristics of the starch particles. In this experiment,
relationship between postruminal starch digestibility and duodenal starch flow was not
significant, but similar to Oba and Allen (2003b) this relationship was not negative (12 =
0.23; P < 0.20). Primary limitations to starch digestion in the small intestine may more
dependent on characteristics of starch particles escaping ruminal digestion.

Ruminal and postruminal starch digestibility ranges widely for a variety of
feedstuffs, but generally intestinal starch digestion can compensate for reduction in
ruminal starch digestion so that variation in total tract starch digestibility is relatively
small (Huntington, 1997). However, greater intestinal starch flow may decrease
efficiency of energy use for productive purposes. Abomasal infusion of increasing
amounts of corn starch increased ileal concentrations of starch and glucose (Kreikemeier
et al., 1991). Greater starch flow to the intestines might increase large intestinal
fermentation of starch to VF A. Some of this energy could be absorbed by the cow, but
microbial OM from large intestinal fermentation would be lost in the feces. Additionally,
even if increasing starch flow and intestinal digestion results in greater uptake of glucose
by the gut, the energy might not be used toward milk production but instead might be
oxidized or used toward tissue retention if the animal is in positive energy balance
(Lemosquet et al., 1997; Knowlton et al., 1998b; Reynolds et al., 2001). Knowlton et al.
(1998b) observed that infusing starch hydrolysate in the abomasum versus the rumen
increased blood C02 from glucose by 93 % (P < 0.001). Duodenal glucose infusions
increased energy balance without affecting milk yield because of increased plasma

insulin (Lemosquet et al., 1997). The shift of starch digestion from the rumen to the

47

intestines observed in vitreous corn grain diets tended to increase plasma insulin:
glucagon ratio (P < 0.07; Chapter 3), which might decrease the rate of gluconeogenesis
and increase oxidation of fuels, reducing glucose supply.

Inconsistencies with shifting digestion to the intestines coupled with requirements
of microbes for fermentable carbohydrate have led many to conclude that extensive
ruminal starch fermentation is often more beneficial than intestinal digestion
(Huntington, 1997). Ruminal fermentation of starch can increase ruminal propionate
production and theoretically could increase substrate available for gluconeogenesis.
However, DMI can also be limited by propionate; Oba and Allen (2003d) speculated that
if temporal flux of propionate to the liver is greater than glucose demand, DMI
depression could occur by oxidation of propionate and other metabolic fuels in the
tricarboxylic acid (TCA) cycle. Corn grain with floury endosperm was fermented faster
and to a greater extent in the rumen, and depressed DMI when combined with control
corn silage possibly because of oxidation of fuels, but did not limit DMI when combined
with bm3 corn silage probably because of a concurrent increase in milk production
(Chapter 3). Ruminal fermentation of starch is less likely to benefit production if it
reduces DMI.

It is unlikely that optimal site of starch digestion is constant across all diets and
animals. Postruminal starch digestion could theoretically increase glucose availability
but in reality may decrease gluconeogenesis and increase tissue retention. Ruminal
starch digestion can increase glucose precursors but can limit DMI if fuels are oxidized in
the liver. Microbial protein production is also integrally tied with starch digestion, and

probably often plays an important role in animal response to site of starch digestion. The

48

response of any given animal is also likely to vary considerably, and will depend on
energy balance, stage of lactation, hormone responsiveness, and nutrition. Therefore, the
optimum site of starch digestion for lactating dairy cows depends on a variety of factors

and response is difficult to predict.

Fiber Digestion

Contrary to our hypothesis, no interactions of main treatment effects were
observed for digestibility of NDF, pdNDF (Table 6), or ruminal digestion kinetics of
pdNDF (Table 7). Corn grain with floury endosperm and bm3 corn silage were expected
to reduce rate of ruminal pdNDF digestion by reducing ruminal pH, but pdNDF digestion
rate was not affected by either floury endosperm grain or bm3 corn silage (Table 7).
Although mean pH was lower for both corn grain with floury endosperm and bm3 corn
silage (Chapter 4), pH might not have been low enough to substantially affect pdNDF
digestion. Some in vitro work has suggested that substantial rates of cellulose digestion
can occur at pH less than 6.0, and it is not until pH drops below 5.3 that lysis or
detachment of cellulolytic bacteria occurs (Mourifro et al., 2001). Additionally, although
rate of pdNDF digestion can be inhibited by the presence of starch regardless of pH
(Grant and Mertens, 1992), previous work in our laboratory reported no relationship
between the digestion rate of pdNDF and amount of ruminally degraded starch (Oba and
Allen, 2003b). Similarly, increased rate of starch digestion did not affect NDF digestion
rate in situ (Callison et al., 2001). Starch digestion may inhibit fermentation of pdNDF in
batch cultures where products are not removed, but constant removal of fermentation

products from the rumen may reduce any direct effects of starch on pdNDF fermentation.

49

Interestingly, ruminal starch digestibility and ruminal pdNDF digestibility were
positively correlated across cow period means in this experiment (r2 = 0.49; P < 0.007).
Extensive interaction among ruminal microorganisms occurs, and it is possible that this
positive relationship between starch and fiber digestibility represents microbial synergy
and cross-feeding. Mixed cultures of cellulolytic and non-cellulolytic bacteria increased
cellulose disappearance in vitro more than pure cultures of three predominant species of
ruminal cellulolytic bacteria (Mourifio et al., 2001). Highly fermentable, high starch diets
in vivo may not be as detrimental to microbial fiber digestion as in vitro studies indicate.

Unlike a previous experiment from our laboratory (Oba and Allen, 20000),
ruminal passage rate of iNDF was not increased by bm3 corn silage compared to control
(Table 7). Other experiments reported no effect on ruminal passage rate of iNDF for bm3
corn silage (Tjardes et al., 2000) or brown midrib sorghum (Aydin et al., 1999).
Differences among these studies are probably because of treatment effects on DM1 and
are probably related to the extent that ruminal distension limits intake. In this study,
ruminal pool size mirrored treatment effects on DMI (Chapter 3), which indicates that
ruminal fill was probably not limiting intake. Additionally, range of DMI in this study
was 1.9 kg per day and may not have been large enough to significantly affect iNDF
passage.

Consistent with the lack of treatment effects on rates of digestion and passage, no
treatment effects were observed for turnover rate of pdNDF or iNDF. Additionally,
although in vitro NDF digestibility was 12.6 units higher for bm3 silage than control
silage (Table 1), bm3 corn silage did not increase ruminal or postruminal pdNDF or NDF

digestibility in vivo and only slightly increased total tract NDF digestibility (P < 0.02;

50

Table 6). Similarly, Oba and Allen (20000) reported bm3 corn silage did not increase
ruminal or total tract NDF digestibility. The authors speculated that lack of treatment
effects from bm3 corn silage occurred because of greater rate of iNDF passage and
reduced rate of pdNDF digestion; however, in the present experiment, bm3 corn silage
did not affect either rate of iNDF passage or rate of pdNDF digestion. These results
suggest that although in vitro NDF digestibility can be improved by bm3 corn silage, in

vivo response is dependent on other factors.

CONCLUSIONS

Floury endosperm improved total tract DM and OM digestibility because of
increased starch digestibility. Contrary to our hypothesis, no interaction of treatments for
any measure of starch or fiber digestion occurred. Vitreous corn grain fermented more
slowly and passed from the rumen faster, resulting in decreased ruminal starch
digestibility. Compensatory postruminal starch digestion in diets containing vitreous
corn grain resulted in relatively small differences in total tract starch digestion compared
to grain with floury endosperm. Greater ruminal starch digestion in floury grain diets and
lower ruminal pH for both floury grain and bm3 corn silage did not affect ruminal fiber
digestion kinetics, and a positive relationship between ruminal starch and pdNDF
digestibility could suggest substantial interactions between microbial populations in the
rumen. Endosperm type of corn grain can affect digestion kinetics and site of starch

digestion.

51

Table 1. Nutrient compositions and physical characteristics of corn silages used to
formulate experimental diets.1
Control bm32 SEM

DM,%(ovendried at55°C) 30.5b 31.58 0.3

 

 

— % of DM—

NDF 41.2 a 38.9 b 0.5
ADF 24.4 a 22.2 b 0.3
Lignin 2.25 a 1.22 b 0.1
Starch 25.8 26.6 0.5
CP 7.70 8.30 0.3
Ether Extract 3.52 3.01 0.23
Ash 2.96 3.32 0.2
Acetate 1,49 3 1.29 b 0.04
Propionate 0.12 0.10 0.01
Lactate 5,67 b 595 a 0.07
Ethanol 1.89 1.99 0.2
1v TDMD’ 79.5 b 87.1 a 0.26
1v NDFD, % of NDF" 54.0 b 66.6 a 0.69
pH 3.78 3.77 0.01
Particle size (% of DM)5

Top 6.87 6.60 1.3

Middle 69.9 69.0 0.84

Bottom 23.0 24.1 0.85

 

I Nutrient compositions of corn silages sampled each period (11 = 8 for each silage) were
compared by ANOVA. Means for nutrient composition in the same row followed by different
superscript letters differ (P < 0.05).

2 Brown midrib 3 mutant for corn silage.

3 IV TDMD: in vitro true dry matter digestibility estimated after 30 h of incubation.

4 IV NDFD: in vitro NDF digestibility estimated after 30 h of incubation.

5 Determined with Penn State Particle Size Separator (Lammers et al, 1996).

52

Table 2. Nutrient compositions and physical characteristics of corn grains used to
formulate experimental diets.1

F loury Vitreous SEM

 

DM, % 86.8 87.0 0.1
— % of DM —

Starch 73.4 72.1 0.80
NDF 8.69 a 8.43 b 0.02
CP 8.19 b 9.07 a 0.14
Ether Extract 4.81 5.02 0.15
Ash 1.28 1.24 0.04
IVSD, %/h2 7.7 1.8
Vitreousness, % of endosperm 3.0 b 672 a 1.9

Percent retained
Sieve aperture (pm)

4750 0.1 0.3 0.1
2369 4.5 5.5 0.4
1180 51.7b 66.53 1.1
600 25.7 a 18.5 b 1.8
300 11.2 7.5 1.2

150 4.6 1.6 1.3

75 2.0 0.0 1.1

pan 0.1 0.0 0.1

Mean particle size (um)3 1377 b 1594 a 12.7

Standard deviation particle size 655 b 744 a 11.2

' Nutrient compositions of corn grains sampled each period (11 = 4 for each grain) were compared
by ANOVA. Means for nutrient composition in the same row followed by different superscript
letters differ (P < 0.05).

2 In vitro starch digestibility estimated after 24 h of incubation.

3 Determined using sieve shaker (model RX-86, W. S. Tyler Inc., Gastonia, NC).

 

53

Tablel 3. Ingredient and nutrient compositions of experimental diets (°/o of dietary
DM).

 

 

 

Control bm32
Floury Vitreous Floury Vitreous SEM
Ingredient
Normal corn silage 37.5 37.3 — — —
Brown midrib 3 corn silage — — 39.6 39.2 '—
Floury ground corn 22.7 — 20.7 — —
Vitreous ground corn — 23.7 — 23.7 —
Protein min3 17.8 17.0 17.9 17.0 —
Alfalfa silage 9.9 9.9 9.8 9.9 —
Whole linted cottonseed 7.2 7.2 7.1 7.2 —
Vitamin mineral min4 4.9 4.9 4.9 4.9 —

Nutrient Composition

DM 48.0 48.2 48.0 48.2 0.2
OM 93.9 94.0 93.8 93.8 0.1
Starch 29.7 30.0 29.1 29.4 0.3
NDF 26.0 25.8 25.7 25.5 0.3
ADF 11.92‘ 11.98 11.5b 11.5b 0.1
Lignin 1.43 a 1.42 a 1.06 b 1.05 b 0.03
Indigestible NDF5 8.91 “ 8.89a 7.04 b 7.02 b 0.2
CF 17.3 17.2 17.5 17.4 0.1
Ether extract 4.71 4.77 4.48 4.54 0.1
Forage NDF 72.0 72.0 72.4 72.4 0.3
Treatment corn grain starch, 55,7 3 565 a 519 b 52.9 b 0.5
% of dietary starch
Treatment corn silage starch, 32.7 b 32.2 b 36.3 a 35.6 a 0.5
% of dietary starch

 

I The actual nutrient composition of the experimental diets was calculated for each period (n = 8
for each treatment) and compared by ANOVA. Means for nutrient composition in the same
row followed by different superscript letters differ (P < 0.05).

2 Brown midrib 3 mutant for corn silage.

3 Protein mix contained 74.1% soybean meal (48%), 22.2% corn distillers grain, and 3.8% blood
meal.

4 Vitamin mineral mix contained 59.9% dry ground corn, 17.3% limestone, 10.7% dicalcium
phosphorus, 8.7% trace mineral and salt premix, 2.1% magnesium oxide, 0.4% Vitamin A,
0.4% selenium, 0.3% Vitamin D, and 0.1% Vitamin E.

5 Indigestible NDF: estimated after 240 h of in vitro ruminal fermentation.

54

Table 4. Effects of corn grain endosperm type and brown midrib 3 corn silage on

 

 

 

 

d_igestion of DM and OM.
Control bm3 P
Floury Vitreous Flog Vitreous SEM S1 G2 S x G3
DM
Intake, kg/d 23.6 25.5 25.2 24.9 1.0 0.40 0.18 0.07
Apparent total tract digestion
kg/d 16.8 17.5 18.1 17.2 0.64 0.30 0.83 0.11
% 73.2 70.4 74.8 71.1 1.3 0.35 0.01 0.73
OM
Intake, kg/d 21.5 23.4 22.8 22.8 1.0 0.53 0.15 0.14
Digestible OMI, kg/d 17.9 19.5 19.5 19.5 0.8 0.15 0.16 0.16
Apparent ruminal digestion
kg/d 9.62 7.43 9.13 7.98 1.1 0.98 0.11 0.61
% 44.8 31.4 40.7 35.2 4.4 0.97 0.04 0.36
True ruminal digestion
kg/d 14.3 14.7 15.0 13.8 1.0 0.92 0.53 0.18
% 66.7 63.1 66.2 60.5 3.3 0.59 0.11 0.71
Passage to duodenum, kg/d 11.9 16.1 13.7 14.8 1.2 0.81 0.04 0.21
Apparent postruminal digestion
kg/d 6.42 9.31 8.07 8.34 1.0 0.74 0.14 0.21
% of intake 29.7 40.3 35.1 37.0 4.3 0.79 0.13 0.28
% of duodenal passage 53.7 56.2 59.4 56.3 3.0 0.27 0.90 0.29
Apparent total tract digestion
kg/d 16.0 16.7 17.2 16.3 0.62 0.41 0.85 0.12
% 74.5 71.6 75.8 72.1 1.2 0.45 0.01 0.76

 

rS = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.

55

Table 5. Effects of corn grain endosperm type and brown midrib 3 corn silage on

 

 

 

 

digestion of starch.
Control bm3 P
Floury Vitreous Floury Vitreous SEM S1 02 S x G3
Intake, kg/d 6.90 7.63 7.15 7.28 0.3 0.79 0.04 0.15
Apparent ruminal digestion
kg/d 4.26 2.28 3.75 3.04 0.7 0.82 0.03 0.27
% 61.5 29.0 52.5 40.9 8.6 0.84 0.01 0.17
True ruminal digestion
kg/d 4.89 4.04 4.54 3.81 0.56 0.33 0.01 0.84
% 70.8 52.0 63.2 51.3 6.0 0.30 0.001 0.40
Passage to duodenum, kg/d 2.65 5.36 3.40 4.23 0.66 0.76 0.01 0.14
Apparent postruminal digestion
kg/d 2.43 4.74 3.07 3.57 0.67 0.66 0.03 0.15
% of intake 35.4 63.2 43.1 50.4 8.8 0.73 0.03 0.18
% of duodenal passage 90.9 83.5 90.6 83.6 3.6 0.96 0.01 0.91
Apparent total tract digestion
kg/d 6.69 7.02 6.82 6.63 0.27 0.47 0.70 0.17
% 96.9 92.1 95.6 91.3 0.78 0.08 <.0001 0.66

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.

56

Table 6. Effects of corn grain endosperm type and brown midrib 3 corn silage on

d_igestion of total NDF and potentially digestible NDF.

 

 

 

Control bm3 P
F loury Vitreous F loury Vitreous SEM Sl G2 S x G3

NDF
Intake, kg/d 5.74 6.21 6.09 6.00 0.3 0.71 0.29 0.13
Apparent ruminal digestion

kg/d 2.14 2.22 2.45 2.39 0.21 0.21 0.97 0.72

% 37.4 34.8 41.0 40.2 3.3 0.20 0.61 0.79
Passage to duodenum, kg/d 3.60 4.01 3.64 3.62 0.27 0.51 0.45 0.42
Apparent postruminal digestion

kg/d 0.52 0.57 0.70 0.54 0.18 0.68 0.77 0.57

% of intake 8.95 9.96 11.5 9.60 3.0 0.71 0.88 0.62

% of duodenal passage 14.2 14.2 19.7 14.9 4.2 0.44 0.55 0.54
Apparent total tract digestion

kg/d 2.66 2.80 3.15 2.90 0.13 0.04 0.69 0.16

% 46.3 45.3 52.4 49.7 2.1 0.02 0.37 0.68
Potentially digestible NDF
Intake, kg/d 3.75 4.03 4.45 4.40 0.19 0.001 0.38 0.22
Apparent ruminal digestion

kg/d 2.14 2.22 2.45 2.39 0.21 0.21 0.97 0.72

% 57.1 53.0 55.8 54.8 4.7 0.97 0.60 0.75
Passage to duodenum, kg/d 1.61 1.84 2.00 2.00 0.23 0.25 0.61 0.63
Apparent postruminal digestion

kg/d 0.52 0.57 0.70 0.54 0.18 0.68 0.77 0.57

% of intake 13.6 15.4 15.6 13.0 4.4 0.95 0.92 0.60

% of duodenal passage 30.8 28.6 36.1 25.1 7.4 0.90 0.33 0.51
Apparent total tract digestion

kg/d 2.66 2.80 3.15 2.90 0.13 0.04 0.69 0.16

% 70.7 69.0 71.3 67.4 2.8 0.85 0.31 0.68
Indigestible NDF
Intake, kg/d 1.99 2.18 1.64 1.59 0.10 <.0001 0.23 0.07

 

T S = Main effect of corn silage hybrid.
2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.

57

Table 7. Effects of corn grain endosperm type and brown midrib 3 corn silage on

ruminal digestion kinetics.

 

 

 

 

Control P
Floury Vitreous Floury Vitreous SEM SI 02 S x G3

Ruminal digestion rate, %/h

pdNDF4 3.30 3.00 2.79 3.11 0.38 0.48 0.98 0.30

Starch 24.8 11.9 19.0 13.8 3.6 0.55 0.01 0.25
Ruminal passage rate, %/h

pdNDF4 2.58 2.58 2.37 2.68 0.52 0.92 0.77 0.78

iNDF’ 3.26 3.01 3.29 3.52 0.27 0.27 0.94 0.34

Starch 16.6 24.0 15.7 18.3 3.1 0.27 0.10 0.43

LiquidII 18.0 20.4 20.6 20.6 2.0 0.46 0.53 0.54
Ruminal turnover rate, %/h

DM 10.3 9.44 9.58 10.3 0.80 0.92 0.93 0.33

OM 9.84 9.1 1 9.19 9.91 0.80 0.92 0.99 0.35

NDF 4.57 4.32 4.46 4.93 0.44 0.54 0.78 0.39

pdNDF‘I 5.88 5.58 5.16 5.88 0.64 0.72 0.72 0.39

iNDF5 3.26 3.01 3.29 3.52 0.27 0.27 0.94 0.34

Starch 41.4 36.3 34.8 31.9 3.6 0.14 0.28 0.76

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.

3 Interaction of corn silage hybrid and ground corn grain endosperm type.

‘ Potentially digestible NDF.
I Indigestible NDF.
6 Measured using Cobalt-EDTA.

58

CHAPTER 3

Interactions of corn grain endosperm type and brown midrib 3 corn silage on

feeding behavior, milk production, and energy balance of lactating dairy cows

ABSTRACT

Interactions of endosperm type of corn grain and the brown midrib 3 mutation in corn
silage on feeding behavior, productivity, energy balance, and plasma metabolites of
lactating dairy cows were evaluated. Eight ruminally and duodenally cannulated cows
(72 :1: 8 DIM; mean :1: SD) were used in a duplicated 4 x 4 Latin square design experiment
with a 2 x 2 factorial arrangement of treatments. Treatments were com grain endosperm
type, floury or vitreous, and corn silage type, bm3 or isogenic control. Diets contained
26% NDF and 30% starch. Floury endosperm grain decreased DMI 1.9 kg/d compared to
vitreous grain when combined with control corn silage but did not affect DMI when
combined with bm3 corn silage. This interaction of treatments occurred because of
changes in meal size; floury endosperm grain decreased meal size in control silage diets
but increased meal size in bm3 corn silage diets. Ruminal pool sizes reflected DMI
differences among diets, suggesting that ruminal fill was not the primary limitation on
intake. Brown midrib 3 corn silage reduced rumination time per day and number of
rumination bouts per day. Floury endosperm grain decreased 3.5% FCM 1.2 kg/d when
combined with control silage but increased 3.5% FCM 2.1 kg/d when combined with

bm3 corn silage. Starch and fiber digestibility interact to affect feeding behavior and

59

milk production; additionally, production response to bm3 corn silage might depend on

the grain source that is fed.

INTRODUCTION

Energy intake often limits milk yield for high-producing dairy cattle, and
strategies to maximize energy intake are critical to maximize production. Although diets
are often formulated based on nutrient concentration alone, an analysis of the literature
determined that one unit increase in NDF digestibility is related to increases in DMI of
0.17 kg/d and 4% FCM of 0.25 kg/d (Oba and Allen, 1999b). The brown midrib 3 (bm3)
mutation in corn silage decreases lignin content and increases in vitro NDF digestibility
of forages (Chemey et al., 1991). Feeding brown midrib hybrids has improved DMI and
milk production in dairy cows (Grant et al., 1995; Oba and Allen, 2000a), and increases
in DMI are a result of changes in meal patterns (Oba and Allen, 2000a). Brown midrib
hybrids can affect DMI by changing meal patterns, but how interactions of bm3 corn
silage and other dietary components affect feeding behavior are unknown.

Variation in starch digestibility is often not considered in diet formulation.
Ruminal starch digestibility for a variety of feedstuffs ranged from 42 to 96% (Nocek and
Tamminga, 1991). Starch digestibility is dependent on several factors, including grain
type, processing methods, and physical characteristics of the grain. Starch granules in
vitreous or flinty endosperm are surrounded by an insoluble protein matrix that resists
digestion; in contrast, floury or opaque endosperm has a soluble protein matrix that is
easily digested by ruminal microorganisms (Kotarski et al., 1992). Grain Vitreousness is

dependent on both hybrid and maturity (Philippeau and Michalet-Doreau, 1997), but is

negatively (r2 = -0.86) correlated with in situ ruminal starch disappearance across hybrids
(Philippeau et al., 1999b). Flint endosperm decreased ruminal starch digestibility 26
percentage units (34.8 vs. 60.8, P < 0.001) compared to a dent genotype when fed to
steers (Philippeau et al., 1999a).

Increased ruminal starch degradability has significantly depressed DMI in some
studies but not others (Allen, 2000). Feed intake is a function of both meal size and meal
frequency, determined by satiety and hunger, respectively (Allen, 2000), so meal patterns
might be influenced by ruminal starch digestion. Indeed, diets high in ruminally
degraded starch depressed DMI by decreasing meal size (Oba and Allen, 2003a), and
linear addition of refined corn starch to the diet linearly decreased meal length but tended
to increase the number of meals (Krause et al., 2003). Because endosperm type can
dramatically change ruminal starch digestibility, research is needed to specifically
examine the effect of corn endosperm type on feeding behavior of lactating dairy cows.

Meal patterns and DMI are affected by both changes in ruminal starch and fiber
digestibility. The interactions between varying digestibility of fiber source and
endosperm type of corn grain on intake and production of dairy cows have not been
investigated. We hypothesized that interactions exist between fiber and starch
fermentability of diets. Highly fermentable grain would have greater effect at reducing
meal size and possibly DMI when combined with bm3 corn silage than control. The
objective of this experiment was to evaluate effects of the brown midrib 3 mutation in
corn silage and corn grain endosperm type on feeding behavior, DMI, productivity, and

energy balance of lactating dairy cows.

61

MATERIALS AND METHODS

Cows and Treatments

Experimental procedures were approved by the All University Committee on
Animal Use and Care at Michigan State University. Eight multiparous Holstein cows (72
a: 8 DIM; mean :1: SD) from the Michigan State University Dairy Cattle Teaching and
Research Center were randomly assigned to duplicate 4 x 4 Latin squares balanced for
carry-over effects. Cows were randomly assigned to treatment sequence. A 2 x 2
factorial arrangement of treatments was used with main effects of corn grain endosperm
type (floury or vitreous) and bm3 mutation (present or absent). Treatment periods were
21 (1, consisting of an 11 d diet adaptation period followed by 10 d of collection. Surgical
preparation of ruminally and duodenally cannulated cows is reported in Chapter 2. At the
beginning of the experiment, empty body weight (ruminal digesta removed) of cows was
531.8 :1: 43.9 kg (mean 1 SD).

Two corn hybrids, 6208FQ and 657 (Cargill Hybrid Seeds, Minneapolis, MN),
were planted for silage in Spring of 2001 at the Michigan State University Research
Farm. The hybrids are isogenic except that Cargill 657 contains the bm3 mutation.
Harvesting conditions of both silages were reported in Chapter 2. Nutrient compositions
and physical characteristics of treatment corn silages used in the experiment are reported
in Table 1 of Chapter 2.

Two corn hybrids (1 floury and 1 vitreous) were planted for grain in Spring of
2001 at the Michigan State University Research Farm. The floury and vitreous hybrids,

SL53 (Crow’s Hybrid Corn Company, Kentland, IN) and Z75W (Wilson Genetics,

62

Harlan, IA) were selected based upon their high and low in vitro starch digestibility,
respectively. Harvesting conditions of both corn grains were reported in Chapter 2.
Vitreousness (% of endosperm) for floury and vitreous corn grain was 3.0 and 67.2%,
respectively (Chapter 2). Nutrient compositions and physical characteristics of the corn
grain treatments used in the experiment are shown in Table 2 of Chapter 2.

Experimental diets contained dry ground corn treatments (floury or vitreous), corn
silage (bm3 or isogenic normal), alfalfa silage (10% of diet dry matter), whole linted
cottonseed (7% of diet dry matter), a protein supplement premix (soybean meal, distillers
grains, and blood meal), and a premix of minerals and vitamins. Experimental diets were
fed as total mixed rations, and were formulated to contain 27% NDF, 18% crude protein
with adequate metabolizable protein, and minerals and vitamins to requirements.
Ingredient and nutrient compositions of the experimental diets are reported in Table 3 of

Chapter 2.

Data and Sample Collection

Cows were housed and fed as described previously (Chapter 2). Cows were
milked twice daily in their stalls during the feeding behavior monitoring phase (d 15-18)
and in a milking parlor during the remainder of each period. Milk yield was measured
and milk was sampled at each milking on CI 15-18. Empty body weight was measured
after evacuation of ruminal digesta on the day immediately prior to the start of the first
period, and on d 21 of each period. Body condition score (BCS) was determined on the
same day by three trained investigators blinded to treatments (Wildman et al., 1982; five-

point scale where 1 = thin and 5 = fat).

63

Feeding behavior was monitored from d 15 through d 18 (96 h) of each period by
a computerized data acquisition system (Dado and Allen, 1993). Data of chewing
activities, feed disappearance, and water consumption were recorded for each cow every
5 sec. When chewing equipment malfunctioned for an individual cow during a 24-h
period (1100h to 1100h), chewing behavior was deleted for that cow during that 24-h
period. The system successfully collected 79.0% of the total chewing behavior data
(average 3.1 (1 per cow per period).

Blood was collected from a coccygeal vessel into two evacuated tubes
(Vacutainer, Becton Dickinson, Franklin Lakes, NJ), one containing sodium heparin and
another containing potassium oxalate and sodium fluoride. Samples were collected every
9 hours from (1 12-14, starting at 1400 h on d 12, so that samples represented 3-h intervals
of a 24-hour period in order to account for diurnal variation. Blood was centrifuged at
2,000 x g for 15 min immediately after sample collection, and plasma was harvested and
frozen at -20°C until analysis.

Ruminal contents were evacuated manually through the ruminal cannula at 1500 h
(4 h after feeding) on d 20 and at 0900 h (2 h before feeding) on d 21 of each period.
Total ruminal content mass and volume were determined. During evacuation, 10%
aliquots of digesta were separated to allow accurate sampling. Aliquots were squeezed
through a nylon screen (1 mm pore size) to separate into primarily solid and liquid
phases. Samples were taken from both phases for determination of nutrient pool size, and
an additional liquid sample was taken to measure VFA concentration. All samples were

frozen immediately at —20°C.

64

Sample and Statistical Analysis

Diet ingredients and orts were dried and ground as described previously (Chapter
2). Rumen liquid and solid sub—samples were lyophilized (Tri-Philizer'“ MP, FTS
Systems, Stone Ridge, NY), ground, and recombined according to the original ratio of
solid and liquid DM. All dried samples were analyzed for DM, ash, NDF, indigestible
NDF (iNDF), potentially digestible NDF (pdNDF; l- iNDF), CP, and starch as described
in Chapter 2.

A commercial kit was used to determine plasma concentration of insulin (Coat-A-
Count, Diagnostic Products Corporation, Los Angeles, CA), pancreatic glucagon (Linco
Research, Inc., St. Charles, MO), glucose (Glucose kit #510; Sigma Chemical Co., St.
Louis, MO), NEFA (N EFA C-kit; Wako Chemicals USA, Richmond, VA), and B-
hydroxybutyrate (BHBA; Procedure No. 2440; Stanbio Laboratory, Boerne, TX). Milk
samples were analyzed for fat, true protein, and lactose with infrared spectroscopy and
urea nitrogen by chemical methodology based on a modified Berthelot reaction
(ChemSpec 150 Analyzer, Bentley Instruments, Chaska, MN) by Michigan DHIA (East
Lansing).

For analysis of feeding and chewing behavior data, daily means were calculated
for number of meal bouts per day, interval between meals, meal size, eating time,
ruminating time, and total chewing time. Daily means for each response variable were
averaged over the number of successful collection days for each period, and for statistical

analysis were weighted according to the number of successful collection days.

65

Energy values for net energy for lactation (N E) and net energy for maintenance (NEM)

were calculated as follows:

NEL intake = DMI (kg) x (0.0245 x TDN %) (NRC, 1989)

DM digestibility for calculation of TDN % was measured as reported in Chapter 2

Milk NEL (Meal/kg) = 0.0929 x (Fat %) + 0.0563 x (True Protein%) + 0.0395 x
(Lactose%) (NRC, 2001);

NEM = 0.080 x BW 0” (NRC, 2001); and

NEL balance = NEL intake — NEM — NEL (Meal/d).

Ruminal pool sizes (kg) of OM, NDF, iNDF, and starch were determined by multiplying

the concentration of each component in DM by the ruminal digesta DM weight (kg).

All data was analyzed using the fit model procedure of JMP® (Version 4, SAS

Institute, Cary, NC) according to the following model:

where

Yijkl = 11 + Ci + Pj + Tk + PTjk + eijkl

u = overall mean,

C, = random effect of cow (j = l to 8),

Pj = fixed effect of period (k = 1 to 4),
T1, = fixed effect of treatment (1 = 1 to 4),

PTJ-k =interaction of period and treatment, and
0in = residual

A reduced model without period x treatment interactions was used when this

effect was not significant (P > 0.15). Orthogonal contrasts were used to determine main

effect of corn silage type, main effect of corn grain type, and the interaction of corn silage

66

type and endosperm type of corn grain. Pearson correlation coefficients were determined
between cow-period observations for some parameters. Main treatment effects and
correlations were declared significant at P < 0.05, and tendencies were declared at P <
0.10. Interactions between treatments were declared significant at P < 0.10, and
tendencies were declared at P < 0.15.

Data from one cow was removed from all statistical analysis due to clinical
mastitis during the first period. This cow was replaced with a spare animal for the
remaining three periods. Another cow developed pneumonia during the diet adaptation
sub-period of period 2, and was recovering during the first day of the collection period.
Data from this cow was omitted from the digestibility sub-period of period 2, but was

utilized from the feeding behavior sub-period as she had recovered sufficiently.

RESULTS AND DISCUSSION

DMI and Meal Patterns

A significant interaction of main effects was detected for DMI (P < 0.07; Table
1). Specifically, endosperm type had no effect on DMI within bm3 corn silage diets, but
within control corn silage diets, floury endosperm grain decreased intake 1.9 kg/d
compared to vitreous grain. This interaction of treatments was caused by changes in
meal intake patterns; floury endosperm grain decreased DM meal size 0.29 kg compared
to vitreous corn when combined with control corn silage, but had the opposite effect,
increasing DM meal size 0.20 kg compared to vitreous grain for diets containing bm3

corn silage. Diets containing vitreous corn grain and bm3 silage did not reduce DMI

67

because cows consuming bm3 silage tended to eat a greater number of meals per day
(10.6 vs. 11.7, P < 0.10). Control of meal size and frequency can be affected by
numerous factors related to physical distension, metabolism of fuels, or hormonal effects.
Because diets were low in NDF and ruminal pool size responded in a similar fashion to
DMI (Table 3), physical factors were probably not limiting DMI. Therefore, the focus of
this discussion will be on metabolic and hormonal factors limiting DMI.

Increased ruminal starch degradability has been shown to decrease DMI in some
but not all cases (Allen, 2000). Inconsistent effects of ruminal starch fermentation on
DMI might be the result of temporal pattern of fuel supply relative to the rate of
utilization for milk yield and body tissues. Oxidation of variety of metabolic fuels in the
liver has been shown to depress feed intake (Langhans, 1996). Because a primary fuel
utilized by ruminants for gluconeogenesis is propionate, this VFA has garnered
significant attention. Infusions of propionate into the rumen linearly decreased DMI and
DM meal size in lactating cows, and extent of hypophagia induced by propionate infusion
was positively related plasma glucose concentration (Oba and Allen, 2003b). Oba and
Allen (2003b) speculated that propionate from rapid ruminal fermentation might be
oxidized in the liver, causing satiety in animals that are not able to utilize propionate for
gluconeogenesis. As milk production increases, increased glucose demand should result
in decreased plasma glucose concentrations. In the present experiment, plasma glucose
was also negatively correlated with DM meal size across cow period means (1'2 = -0.37; P
< 0.04). Oxidation of metabolic fuels may explain meal size differences within floury
endosperm diets. In diets with floury corn grain, rapid fermentation of starch probably

occurred, resulting in greater influx of metabolic fuels (including propionate) during

68

meals; floury endosperm increased ruminal propionate concentration (P < 0.001; Chapter
4). Because milk production (and glucose demand) was not increased in diets containing
floury endosperm and control corn silage, these fuels might have been oxidized in the
liver and reduced meal size. Conversely, in floury endosperm, bm3 corn silage diets,
milk production and glucose demand were increased, and fuels were possibly directed
toward gluconeogenesis, thus reducing oxidation of fuels and prolonging meal size.
Although vitreous corn, when combined with bm3 silage, reduced DM meal size,
meal frequency tended to increase to compensate. Oba and Allen (2000a) reported that
cows consuming bm3 corn silage ate smaller meals more frequently when compared to
control corn silage in low (29%) NDF diets, and speculated that more rapid VFA
absorption might decrease meal size but increase feeding frequency. Although valerate
absorption rate (a measure of VFA absorption rate) was not different among treatments,
acetate: propionate ratio was greater in vitreous diets containing control silage versus
bm3 silage (Chapter 4). Infusions of propionate reduced meal size and length to a greater
extent than equimolar infusions of acetate or sodium chloride, indicating hypophagic
effects of propionate on feeding behavior were because of the substrate and not
osmolality (Choi and Allen, 1999). Greater ratio of ruminal acetate to propionate in diets
containing vitreous corn grain with control versus bm3 corn silage might indicate that
less propionate was produced, which may have increased meal size. Additionally,
insulin: glucagon ratio tended to be higher for vitreous grain (P < 0.07; Table 6),
primarily because the diet containing vitreous corn grain and bm3 silage increased plasma
insulin and reduced glucagon concentrations compared to other treatments. Higher

plasma insulin: glucagon ratio is expected to result in increased oxidation of fuels in the

69

liver (Watford and Goodridge, 2000), and if more propionate were available in this diet
because of the lower ruminal acetate: propionate ratio, increased oxidation of propionate
may have resulted in satiety sooner and smaller meal size. It must be stressed that
oxidation of metabolic fuels is a temporal effect, and although it may decrease meal size,
meal frequency may increase to compensate, as appears to have occurred for vitreous
corn grain, bm3 corn silage diets.

In addition to oxidation of metabolic fuels, gut peptides could have influenced
meal patterns because of the significant changes in site of starch digestion (Chapter 2).
Intraduodenal glucose infusion strongly elicited increases in plasma insulin, gastric
inhibitory polypeptide (GIP) and glucagon-like peptide-l (GLP-l) in humans (Lavin et
al., 1998), and although GIP and GLP-1 appear to play a role in signaling satiety (Havel,
2001), this has not been definitively established, and little work has been conducted on
the role of gut peptides in regulation of DMI for ruminants. Understanding how feed
intake is controlled is difficult because many factors act synergistically to control meal
size and frequency, and predicting feed intake precisely may never be possible for this
reason. Additionally, dietary factors can interact to affect meal size and frequency
without changing total DMI, which is why it is important to investigate the effects of
diets on feeding behavior rather than just DMI to understand the physiology of intake

regulation.

Chewing Activity

Chewing time (total and eating) was similar between endosperm types in control

corn silage, but floury endosperm grain tended to increase chewing time (total and eating)

70

versus vitreous grain in bm3 diets (interaction P < 0.12 and P < 0.03, respectively; Table
2). These interactions occurred because of the interaction of main treatment effects for
DMI combined with greater chewing for the floury corn grain and control corn silage
diet. Greater chewing for the diet containing floury grain and control silage also resulted
in treatment effects of endosperm type on chewing (total, eating, and ruminating) per kg
DMI. Although rate of starch digestion was highest in this diet (Chapter 2), cause and
effect is not apparent. Ruminating time per day was positively related to rate of starch
digestion across cow period means (1'2 = 0.37; P < 0.05). Greater mastication increased
starch digestion rate of several cereals (Beauchemin et al., 1994), and rate of starch
digestion is faster as corn particle size decreases (Callison etal., 2001). Greatest rate of
starch digestion in the diet containing floury endosperm grain and control silage could be
related to greater chewing per kg DMI; however, the cause of greater chewing is
unknown.

Total ruminating time per day, rumination bout length, and ruminating chews per
bout and per day were greater for diets containing control versus bm3 corn silage (Table
2). These results suggest that bm3 silage may not be as effective as control corn silage in
stimulating rumination. In contrast, Oba and Allen (2000b) reported ruminating time per
day was not different between bm3 and control corn silages, and concluded that bm3 corn
silage was as effective as normal corn silage in stimulating chewing activity. Similar to
our study, brown midrib sorghum silage tended to decrease chewing per kg NDF intake
when compared to normal sorghum silage (Grant et al., 1995). Enhanced fiber
degradability and increased particle fragility of brown midrib hybrids could cause brown

midrib silages to be slightly less effective at stimulating chewing than normal com

71

silages. Brown midrib 3 corn silage stimulated chewing as well as control in the
experiment by Oba and Allen (2000b) but not this experiment, possibly because 30-h in
vitro NDF digestibility of the bm3 hybrid used in this study was 10.7 units higher than
the bm3 hybrid used in the study by Oba and Allen (20003). Because bm3 hybrids might
not be quite as effective at stimulating rumination as some other forages, it may be
advisable to increase forage NDF concentration when formulating diets containing bm3

hybrids.

Ruminal Nutrient Pools

Significant interactions of main treatment effects were detected for ruminal pools
of DM (P < 0.03) and OM (P < 0.03; Table 3). Similar to DMI, floury endosperm
decreased DM and OM pool size when combined with control silage but increased pool
sizes when in bm3 silage diets. Significant interactions of treatments for ruminal pools of
NDF (P < 0.03), pdNDF (P < 0.08), and iNDF (P < 0.02) were also detected. These
pools reflected DMI differences among diets, but also indicate greater pool sizes of
pdNDF and less iNDF in diets containing bm3 silage. The ability of the pool size to
reflect changes in DMI indicates that intake was not likely limited by ruminal fill in these
animals. Starch ruminal pool size tended (P < 0.07) to be greater for vitreous endosperm
versus floury, because ruminal turnover rate of starch was numerically greater for floury
versus vitreous corn (Chapter 2). Wet weight, volume, and density of ruminal contents

did not differ among treatments.

72

Milk Yield and Composition

In control corn silage diets, floury corn grain decreased 3.5% FCM 1.2 kg/d, but
increased F CM by 2.1 kg/d when combined with bm3 corn silage (P < 0.10; Table 4). A
similar trend occurred for both milk yield (P < 0.22) and milk fat % (P < 0.11). Milk fat
% was similar between diets containing floury grain regardless of silage type, but
compared to floury grain, vitreous endosperm corn grain tended to increase milk fat % in
control diets but decrease milk fat % in bm3 silage diets. Milk fat % was positively
correlated with ruminal acetate: propionate ratio (r2 = 0.67; P < 0.0001); as ruminal
acetate concentrations increased, greater substrate availability might have increased fatty
acid synthesis in the mammary gland. Protein, lactose, and SNF composition of milk
were not affected by any treatment. Although a significant main effect of silage (P <
0.005) was detected for milk urea nitrogen (MUN), differences were small (< 1.5 mg/dl).

Several studies document increased milk production when brown midrib hybrids
replace normal silage, probably due to increased DMI (Grant et al., 1995; Oba and Allen,
2000a; Tine et al., 2001). However, there have been inconsistent results regarding
ruminal starch degradation and milk production. Plascencia and Zinn (1996) noted a 2.6
kg/d increase in milk yield when steam-flaked corn replaced dry rolled corn, probably
because of a 1.1 kg increase in DMI. Replacing corn with steam rolled barley decreased
DMI 3.2 kg/d and milk yield by 4.3 kg/d (Overton et al., 1995). In contrast, changing
apparent ruminal NSC digestibility 38.2 units had no effect on either DMI or milk
production (Callison et al., 2001). Increased ruminal starch digestibility usually
decreases milk yield in cases where DMI is depressed. In this experiment, differences in

milk production reflect patterns of feeding behavior and DMI. F loury endosperm grain

73

decreased DMI by decreasing meal size when combined with control silage, but when
combined with bm3 corn silage did not decrease DMI because fuels from rapid ruminal

starch fermentation were probably utilized for increased milk yield.

Energy Balance and Plasma Metabolites

An interaction of treatments was detected (P < 0.08) for NEL intake (Table 5);
floury corn grain decreased NEL intake 0.8 Meal/d in control silage diets but increased it
4.3 Meal/d in bm3 silage diets. This interaction reflects the changes in DMI observed but
to a greater extent because it accounts for greater digestibility of corn grain with floury
endosperm. Milk NEL (Meal/kg) was increased for grain with vitreous endosperm versus
floury with control silage but decreased when combined with bm3 corn silage (interaction
P < 0.05) because of the similar treatment effects on milk fat yield. A tendency for an
interaction of treatments was detected for NEL balance (P < 0.15). NEL balance was
similar between endosperm types in control silage diets, but was greater for floury
endosperm than vitreous within bm3 silage diets.

Vitreous endosperm corn grain tended to increase partitioning of NE, intake
toward milk NEL compared to floury endosperm grain (P < 0.10; Table 5). Insulin:
glucagon ratio tended to be higher for cows consuming vitreous diets (P < 0.07; Table 6)
because vitreous endosperm numerically increased plasma insulin concentration. Greater
plasma insulin concentration might be expected to increase intake NEL partitioned toward
tissue retention and not milk NEL, but other factors influence energy partitioning,
including substrate availability and metabolic hormone secretion. Additionally, because

these samples represent only every 3-hr over a 24-hr day but metabolic hormones act in a

74

much shorter time frame, these data can still be accurate in representing basal hormonal
profiles, but pulsatility of insulin and glucagon plays a significant role in whether glucose
is used toward tissue energy retention versus milk production. Because more frequent
sampling is required to identify insulin and glucagon peaks and nadirs, this study cannot
examine how endosperm type affects pulsatility, but there is evidence that differences
between hybrids might exist. More research is needed to examine the interactions of

starch and fiber digestibility on hormone pulsatility and energy balance.

CONCLUSIONS

Endosperm hybrid and bm3 mutation in corn silage interact to affect DMI; floury
endosperm grain decreased meal length and size in control silage diets, but increased
meal length and size in bm3 silage diets. Total DMI was not reduced in diets containing
vitreous corn grain and bm3 corn silage because bm3 silage tended to increase meal
frequency per day compared to control corn silage. Ruminal pool sizes reflected
treatment effects on DMI, indicating that DMI was probably not limited by physical fill.
Differences in feeding behavior could have been caused by a number of regulatory
factors including, but not limited to, temporal supply of nutrients, oxidation of metabolic
fuels, gut peptides, and metabolic hormone profiles. Changing diet fermentability affects
milk production primarily by affecting DMI. Additionally, production responses to bm3
corn silage are probably dependent on grain source. Starch and fiber fermentability can
interact to affect feeding patterns and production, but animal response to changes in diet

fermentability is complex and predicting how animals will respond is unlikely.

75

Table 1. Effects of corn grain endosperm type and brown midrib 3 corn silage on

feeding behavior.
Control bm3

 

Floury Vitreous Floury Vitreous

DMI, kg/d 23.6 25.5 25.2 24.9
Meal bouts, #/d 10.8 10.4 11.2 12.1
Meal length, min 28.0 28.4 27.0 24.0
DM meal size, kg 2.18 2.47 2.25 2.05

Interrneal interval, min 95.5 98.0 90.3 93.1
I S = Main effect of corn silage hybrid.
2 G = Main effect of ground corn grain endosperm type.

P

 

SEM s' 02 SxG'I
1.0 0.40 0.18 0.07

0.8 0.10 0.74 0.31
1.3 0.02 0.23 0.12
0.13 0.11 0.67 0.03
6.9 0.32 0.59 0.97

3 Interaction of corn silage hybrid and ground corn grain endosperm type.

76

Table 2. Effects of corn grain endosperm type and brown midrib 3 corn silage on
chewing behavior.

 

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI G2 S x G3

Chewing time, min/d

Total 755 745 728 680 20.3 0.001 0.02 0.12

Eating 259 258 266 242 6.4 0.36 0.02 0.03

Ruminating 496 486 462 43 8 20.9 0.002 0.16 0.53
Total Chews

/ kg DMI 2241 2006 1984 1961 95 0.002 0.005 0.02

/ d 50,796 50,432 48,316 45,649 1446 0.002 0.15 0.26
Eating chews

/ meal 1907 1958 1828 1650 111 0.08 0.56 0.30

/ kg DMI 880 802 816 801 44 0.14 0.05 0.16

/ (1 19,939 19,886 19,966 18,660 782 0.30 0.25 0.28
Ruminating chews

/ bout 2218 2073 1988 1945 177 0.03 0.23 0.51

/ kg DMI 1361 1204 1168 1160 84 0.006 0.04 0.06

/ (1 30,854 30,375 28,344 26,981 1584 0.001 0.25 0.57
Ruminating

Bouts, #/d 14.3 14.9 14.5 14.3 0.6 0.62 0.63 0.28

Bout length, min 36.1 33.4 32.8 32.0 2.5 0.03 0.11 0.39

Interval, min 65.2 62.1 63.4 62.1 2.9 0.74 0.40 0.74
Water Intake, L/d 94.4 90.5 92.9 92.0 4.2 0.99 0.33 0.53

 

I S = Main effect of corn silage hybrid.
2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.

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Table 3. Effects of corn grain endosperm type and brown midrib 3 corn silage on
ruminal pools.

 

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI G2 S x GII

Wet weight, kg 75.0 83.0 74.9 76.4 3.7 0.27 0.12 0.28
Volume, L 89.1 96.9 87.3 90.0 4.9 0.28 0.20 0.53
Density, kg/L 0.85 0.86 0.84 0.85 0.02 0.58 0.54 0.93
Rumen pool, kg

DM 10.2 12.3 11.6 10.9 0.68 0.95 0.20 0.02

OM 9.37 1 1.18 10.62 9.92 0.66 0.99 0.29 0.03

NDF 5.41 6.34 5.95 5.37 0.43 0.49 0.59 0.03

pdNDF‘I 2.75 3.16 3.75 3.44 0.27 0.004 0.78 0.08

iNDF5 2.67 3.18 2.20 1.93 0.19 <.0001 0.42 0.02

Starch 0.74 0.95 0.89 0.98 0.08 0.24 0.07 0.42

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.

I Interaction of corn silage hybrid and ground corn grain endosperm type.
4 Potentially digestible NDF.

5 Indigestible NDF.

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Table 4. Effects of corn grain endosperm type and brown midrib 3 corn silage on

milk yield and components.

 

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI G2 S x G3

Yield, kg/d

Raw milk" 39.8 40.6 42.5 40.6 2.1 0.21 0.59 0.22

3.5 % FCM 39.7 40.9 42.2 40.1 2.2 0.40 0.62 0.10

Fat 1.40 1.45 1.48 1.40 0.09 0.69 0.69 0.07

Protein 1.18 1.20 1.28 1.19 0.07 0.28 0.44 0.19

Lactose 1.95 1.99 2.09 1.98 0.1 1 0.22 0.58 0.20

SNF 3.50 3.57 3.78 3.56 0.19 0.23 0.52 0.21
MUN, mg/dl 16.5 16.6 15.0 15.8 0.58 0.005 0.24 0.29
Composition, %

Fat 3.51 3.62 3.49 3.44 0.20 0.04 0.51 0.11

Protein 2.95 2.95 3.00 2.93 0.05 0.50 0.24 0.28

Lactose 4.88 4.89 4.90 4.88 0.04 0.71 0.78 0.18

SNF 8.79 8.80 8.87 8.77 0.08 0.52 0.30 0.19
3.5% FCM / DMI 1.68 1.61 1.67 1.67 0.05 0.20 0.08 0.11

 

I S = Main effect of corn silage hybrid.
2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.
4 Milk yield not measured during digestibility and nunen evacuation sub-periods.

79

Table 5. Effects of corn grain endosperm type and brown midrib 3 corn silage on
energy balance.

 

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI G2 S x G3
BW change, kg/21 d 12.7 12.3 16.9 12.1 4.4 0.62 0.51 0.58
BCS change, /21 d 0.04 0.12 0.14 0.32 0.06 *
NEL intake, Meal/d 42.9 43.7 46.1 41.8 1.6 0.65 0.22 0.08
Milk NEL
Meal/kg 0.68 0.70 0.69 0.68 0.02 0.09 0.87 0.05
Meal/d 27.3 28.0 29.1 27.5 1.5 0.37 0.54 0.11
NEL balance, Meal/d 5.46 5.26 6.79 3.11 1.2 0.73 0.11 0.15
Milk NEL, % of Intake 63.5 64.4 62.9 68.5 2.6 0.36 0.10 0.22

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.

3 Interaction of corn silage hybrid and ground corn grain endosperm type.
* Significant (P < 0.15) period by treatment interaction.

Table 6. Effects of corn grain endosperm type and brown midrib 3 corn silage on
plasma metabolite and hormone concentrations.

 

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI G2 S x G3

Glucose, mg/dl 59.6 58.3 58.8 58.6 0.92 0.62 0.16 0.32
Insulin, uIU/mL 13.6 13.7 12.4 14.7 1.5 0.90 0.24 0.26
Glucagon,pg/ml 120 119 118 115 9.7 0.50 0.54 0.84
Insulin: Glucagon 0.11 0.12 0.10 0.13 0.01 0.75 0.07 0.18
NEFA, 11qu 57.0 58.9 55.2 67.7 4.3 *
B-hydroxybutyrate, mg/dl 6.47 6.77 6.46 6.72 0.39 0.91 0.31 0.94

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.

3 Interaction of corn silage hybrid and ground corn grain endosperm type.
* Significant (P < 0.15) period by treatment interaction.

80

CHAPTER 4

Interactions of corn grain endosperm type and brown midrib 3 corn silage on

ruminal fermentation and microbial efficiency in lactating dairy cows

ABSTRACT

Interactions of endosperm type of corn grain and the brown midrib 3 mutation in corn
silage on ruminal fermentation and microbial efficiency of lactating dairy cows were
evaluated. Eight ruminally and duodenally cannulated cows (72 z 8 DIM; mean :1: SD)
were used in a duplicated 4 x 4 Latin square design experiment with a 2 x 2 factorial
arrangement of treatments. Treatments were com grain endosperm type, floury or
vitreous, and corn silage type, bm3 or isogenic normal. Diets contained 26% NDF and
30% starch. Increasing ruminal starch digestibility by replacing vitreous corn grain with
floury grain reduced mean and minimum ruminal pH. Brown midrib 3 corn silage
reduced mean and minimum ruminal pH and increased total VFA concentration.
Ruminal pH was positively associated with rate of valerate absorption. Although floury
endosperm reduced acetate: propionate ratio in both control and bm3 corn silage diets, it
had a greater effect on reducing acetate: propionate ratio for control silage compared to
bm3 corn silage. Non-ammonia N (NAN) flow to the duodenum did not differ among
treatments, and because variability was high, differences were not significant for

microbial N and non-ammonia, non-microbial N (N AN MN) flow. More research is

81

needed to determine how starch and fiber digestibility interact to affect microbial protein
production and efficiency.

INTRODUCTION

Maximum milk production is commonly limited in high producing animals by
both energy and metabolizable protein supply. Metabolizable protein is derived from
microbial protein, feed protein that bypasses ruminal digestion, and endogenous proteins.
Of these sources, microbial protein produced in the rumen is the primary source of amino
acids to the cow. Microbes are a high quality, highly digestible protein source, yet
predicting microbial protein flow to the duodenum is difficult. Increasing diet
digestibility has the potential to increase microbial growth, which is often limited by
energy availability (Clark et al., 1992). However, diet fermentability can have significant
impact on the efficiency by which microbes use energy from ruminally fermented feed
toward growth (defined as g microbial N flowing to the duodenum per kg OM truly
ruminally fermented [TRDOM]). Predicting how diet fermentability will affect
microbial N efficiency (MNE) is difficult because factors such as pH, retention time, and
energy and nitrogen availability all interact to change the efficiency of microbial growth
(Clark etal., 1992; Russell, 1998).

Increasing ruminal starch digestibility of high starch diets has inconsistent effects
on MN E; greater ruminally degraded starch increased (Plascencia and Zinn, 1996),
decreased (Oba and Allen, 2003c), and did not affect MN E (Overton et al., 1995).
Microbial efficiency might be increased as ruminal starch digestibility increases if energy
availability is limiting microbial growth. However, when fermentation occurs faster than

the ATP produced can be used toward cellular growth, MNE can be reduced if ATP is

82

used toward non-growth functions in energy spilling or uncoupling reactions (Russell,
1998). Energy spilling can occur in situations of low ruminal pH (Stroebel and Russell,
1986) and lack of available N (Van Kessel and Russell, 1996).

Microbial efficiency might be reduced in diets with long ruminal retention times.
As retention time increases, microbial autolysis and predation by protozoa are increased
and MNE could be reduced (Wells and Russell, 1996). Despite a decrease in ruminal pH,
greater MN E for brown midrib 3 (bm3) corn silage was attributed to a faster ruminal
passage rate, which might have reduced microbial turnover (Oba and Allen, 20000).
Lower ruminal pH might have reduced MN E for bm3 corn silage compared to control
silage when DMI, and presumably rate of passage, were not affected by treatment
(Greenfield et al., 2001). Because feeding bm3 corn silage often reduces ruminal pH, its
potential to increase MNE might depend on the extent to which ruminal passage rate is
increased relative to the negative effects of low pH on MNE.

We hypothesized that com grain with floury endosperm reduces MN E because of
more rapid starch digestion, and might interact with corn silage type because bm3 corn
silage reduces MNE by low ruminal pH but increases MNE by increasing rate of ruminal
passage. The objective of this experiment was to determine interactions of endosperm
type of corn grain and the bm3 mutation in corn silage on ruminal pH, VFA profile, and

microbial N efficiency.

83

MATERIALS AND METHODS

Treatments and Cows

Experimental procedures were approved by the All University Committee on
Animal Use and Care at Michigan State University. Eight multiparous Holstein cows (72
1 8 DIM; mean 1 SD) from the Michigan State University Dairy Cattle Teaching and
Research Center were randomly assigned to duplicate 4 x 4 Latin squares balanced for
carry-over effects. Cows were randomly assigned to treatment sequence. A 2 x 2
factorial arrangement of treatments was used with main effects of corn grain endosperm
type (floury or vitreous) and bm3 mutation (present or absent). Treatment periods were
21 d, consisting of an 11 d diet adaptation period followed by 10 d of collection. Surgical
preparation of ruminally and duodenally cannulated cows is reported in Chapter 2. At the
beginning of the experiment, empty body weight (ruminal digesta removed) of cows was
531.8 1 43.9 kg (mean 1 SD).

Two corn hybrids, 6208FQ and 657 (Cargill Hybrid Seeds, Minneapolis, MN),
were planted for silage in Spring of 2001 at the Michigan State University Research
Farm. The hybrids are isogenic except that Cargill 657 contains the bm3 mutation.
Harvesting conditions of both silages were reported in Chapter 2. Nutrient compositions
and physical characteristics of treatment corn silages used in the experiment are reported
in Table 1 of Chapter 2.

Two corn hybrids (l floury and 1 vitreous) were planted for grain in Spring of
2001 at the Michigan State University Research Farm. The floury and vitreous hybrids,
SL53 (Crow’s Hybrid Corn Company, Kentland, IN) and Z75W (Wilson Genetics,

Harlan, IA) were selected based upon their high and low in vitro starch digestibility,

84

respectively. Harvesting conditions of both corn grains were reported in Chapter 2.
Vitreousness (% of endosperm) for floury and vitreous corn grain was 3.0 and 67.2%,
respectively (Chapter 2). Nutrient compositions and physical characteristics of the corn
grain treatments used in the experiment are shown in Table 2 of Chapter 2.

Experimental diets contained dry ground corn treatments (floury or vitreous), corn
silage (bm3 or isogenic normal), alfalfa silage (10% of diet dry matter), whole linted
cottonseed (7% of diet dry matter), a protein supplement premix (soybean meal, distillers
grains, and blood meal), and a premix of minerals and vitamins. Experimental diets were
fed as total mixed rations, and were formulated to contain 27% NDF, 18% crude protein
with adequate metabolizable protein, and minerals and vitamins to requirements.
Ingredient and nutrient compositions of the experimental diets are reported in Table 3 of

Chapter 2.

Data and Sample Collection

Cows were housed and fed as described previously (Chapter 2). Duodenal (1,000
g), fecal (500 g), and rumen fluid samples (100 mL) were collected every 9 h as
previously described (Chapter 2). Digesta from 5 sites in the rumen was combined and
strained to obtain rumen fluid for determination of VFA, lactate, and ammonia
concentrations. Additional ruminal fluid (400 ml) was collected near the reticulo-omasal
orifice at the same time as duodenal and fecal samples for the determination of microbial
OM, starch, nitrogen, and purine concentrations. All digesta and fecal samples were

immediately frozen at -20°C until processing.

85

Effect of treatment on relative rate of VF A absorption and rate of liquid passage
was measured on d 19 using a pulse dose of valeric acid and cobalt EDTA (Allen et al.,
2000). Valerie acid and cobalt EDTA was dosed two hours after feeding. Rumen fluid
was sampled immediately before dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,
6.5, 7, 7.5, and 8 h after dosing. Samples were immediately frozen.

Ruminal pH was monitored from d 15 through d 18 (96 h) of each period by a
computerized data acquisition system (Dado and Allen, 1993). Ruminal pH data were
recorded for each cow every 5 sec. Electrodes for ruminal pH determination were
checked daily and calibrated as needed, and ruminal pH data were deleted for the
previous 24 h if readings had changed more than 1 0.05 unit at pH 7 or pH 4. The system
successfully collected 58.9% of the total ruminal pH data (average 2.4 11 per cow per

period).

Sample and Statistical Analysis

Processing methods for diet ingredients, orts, digesta, and fecal samples were
reported previously (Chapter 2). Microbial pellets were obtained from combined rumen
fluid samples for each cow by differential centrifugation (Overton et al., 1995) and were
lyophilized (Tri-Philizer'" MP, FTS Systems, Stone Ridge, NY). Microbial pellets were
ground with a mortar and pestle and analyzed for ash, CP, and starch as described in
Chapter 2.

Rumen fluid was analyzed for concentration of VF A and lactate by HPLC
(Waters Corp., Milford, MA) as described by Oba and Allen (2003a). Duodenal digesta

was analyzed for purines and ammonia to estimate microbial N flow and non-ammonia

86

non-microbial N flow to the duodenum. Purine concentration was used as a microbial
marker, and purine to microbial N ratio was estimated by analysis of microbial pellets.
Total purines were measured by spectrophotometer (Beckman Instruments, Inc.,
Fullerton, CA) at 260 nm (Zinn and Owens, 1986). Ammonia concentration was
determined for centrifuged duodenal and rumen fluid samples according to Broderick and
Kang (1980).

Rumen fluid samples collected to measure rate of valerate absorption were
analyzed for valerate concentration by HPLC (Waters Corp., Milford, MA) and for Co
concentration by atomic absorption spectrophotometry according to manufacturers
specifications (SpectrAA 220/F S, Varian Australia Pty. Ltd., Mulgrave, Victoria,
Australia). Rates of valerate and cobalt disappearance were determined by non-linear
regression of the decline in their respective concentrations in rumen fluid over time after
dosing using a one-pool, first-order model after accounting for background (JMP®
Version 4, SAS Institute, Cary, NC).

For analysis of pH data, daily means for pH mean, minimum, maximum, range,
standard deviation, time below pH 6.0, 5.8, and 5.5, and area below pH 6.0, 5.8, and 5.5
were calculated. Daily means for each response variable were averaged over the number
of successful collection days for each period and for statistical analysis were weighted
according to the number of successful collection days.

All data was analyzed using the fit model procedure of JMP® (Version 4, SAS
Institute, Cary, NC) according to the following model:

Yijkl = 11 + Ci + Pj + Tk + PTjk + eijkl

where

87

11 = overall mean,

C1 = random effect of cow 0 = l to 8),
P; = fixed effect of period (k = 1 to 4),
Tk = fixed effect of treatment (1 = 1 to 4),

PTjk =interaction of period and treatment, and

cum = residual

A reduced model without period x treatment interactions was used when this
effect was not significant (P > 0.15). Orthogonal contrasts were used to determine main
effect of corn silage type, main effect of corn grain type, and the interaction of corn silage
type and endosperm type of corn grain. Pearson correlation coefficients were determined
between cow-period observations for some parameters. Main treatment effects and
correlations were declared significant at P < 0.05, and tendencies were declared at P <
0.10. Interactions between treatments were declared significant at P < 0.10, and
tendencies were declared at P < 0.15.

Data from one cow was removed from all statistical analysis due to clinical
mastitis during the first period. This cow was replaced with a spare animal for the
remaining three periods. Another cow developed pneumonia during the diet adaptation
period of period 2, and was recovering during the first day of the collection period. Data
from this cow was omitted from the digestibility sub-period of period 2, but was utilized

from the feeding behavior sub-period as she had recovered sufficiently.

88

RESULTS AND DISCUSSION

Ruminal pH

No interactions of main treatment effects were observed for any measure of
ruminal pH. Floury endosperm grain reduced mean and minimum ruminal pH (P < 0.03
and P < 0.02, respectively; Table l), but pH maximum, range, and standard deviation
were not affected by endosperm type. F loury endosperm grain tended to increase fraction
of time spent under pH 6.0 compared to vitreous grain (P < 0.09). Greater ruminal starch
(and OM) digestibility decreases ruminal pH by increasing fermentation acid production,
and although ruminally fermented OM was negatively correlated to ruminal pH for a
database of treatment means in the literature (Allen, 1997), effects of fermentable OM on
ruminal pH have not been consistent. Replacing ground shelled corn with rolled barley
increased ruminal starch digestibility from 41.9 to 74.4% but linearly decreased ruminal
pH (Overton et al., 1995). In contrast, replacing dry corn with high moisture corn
increased rate and extent of ruminal starch digestion but did not effect any measure of
ruminal pH in another study (Oba and Allen, 2003b, 0). Ruminal pH is a function of
hydrogen ion production from fermentation and removal by absorption, passage,
buffering, and neutralization. Ruminal pH decreases only when hydrogen ion production
exceeds removal from the rumen and ruminal buffering capacity. Corn grain with floury
endosperm increased ruminal starch and OM digestibility (Chapter 2) and hydrogen ion
removal was not rapid enough to offset VFA production, resulting in lower ruminal pH.

Brown midrib 3 corn silage reduced mean and minimum ruminal pH compared to

its isogenic control corn silage (P < 0.03 and P < 0.04, respectively). Brown midrib 3

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corn silage tended to increase hours under pH 6.0 (P < 0.09), but other measures of
ruminal pH did not differ between corn silage types. Lower ruminal pH for bm3 corn
silage treatments corresponded with higher total VFA concentration (P < 0.03; Table 2).
Brown midrib 3 corn silage decreased ruminal pH in several experiments (Block et al.,
1981; Oba and Allen, 2000a; Greenfield et al., 2001). Total DMI was not affected by
bm3 corn silage, and although greater ruminally degraded OM might increase
fermentation acid production and reduce ruminal pH, TRDOM was not affected by bm3
corn silage treatment. Brown midrib 3 corn silage did not affect rate of valerate
absorption (Table 2) or liquid passage rate (Chapter 2) compared to control silage, so
treatment effects on removal of VF A by absorption or passage from the rumen did not
occur. Cows fed diets containing bm3 corn silage spent less time ruminating per day and
chewed less during rumination per day than cows consuming control corn silage (P <
0.002 and P < 0.001; Chapter 3). Chewing stimulates saliva flow, which contains
bicarbonate and phosphates that buffer and neutralize fermentation acids produced in the
rumen (Allen, 1997). A negative relationship existed between mean ruminal pH and
daily rumination time, but silage type also affected this relationship. Mean pH was
negatively correlated with both rumination time for control corn silage (r2 = -0.5 5; P <
0.03) and bm3 corn silage (1'2 = -0.58; P < 0.02), and although the slopes were not
different (P = 0.59), the intercept of the regression line was lower for bm3 than control
corn silage (P = 0.02). When rumination time is 450 minutes per day, cows consuming
bm3 corn silage had a mean ruminal pH of 5.99 while cows consuming control corn

silage had a mean ruminal pH of 6.22. Brown midrib corn silage might reduce ruminal

90

pH both by stimulating rumination less effectively than control silage and reducing
ruminal motility and removal of VFA from the rumen, as discussed below.

Rate of valerate absorption is used as a general measure of VFA absorption across
the ruminal epithelium. Mean valerate absorption rate did not differ among treatments
and averaged 44 % per h. Because VFA are absorbed across the rumen wall in the
undissociated form, valerate absorption rate might be expected to increase as pH
decreases. This was demonstrated using the washed rumen technique by Dijkstra et al.
(1993); fractional absorption rates of propionate and butyrate increased as pH decreased
from 7.2 to 4.5. In contrast, rate of valerate absorption was positively correlated with
mean pH across cow period means in the present experiment (1'2 = 0.53; P < 0.003). A
previous study from our laboratory also reported a positive relationship between rate of
valerate absorption and mean pH (Voelker and Allen, 2003b). VFA absorption rate is
dependent on several factors other than form of VF A, including blood flow, ruminal
consistency, surface area, and ruminal motility. In particular, ruminal motility may play
a significant role in rate of VFA absorption. Ruminal motility was not measured in this
study, because most quantitative measurements of motility interfere with ruminal
function. Although no relationship between valerate absorption rate and iNDF passage
rate was detected across cow period means in the present experiment, these variables
were positively correlated in a previous experiment (12 = 0.43; P = 0.02; Voelker and
Allen, 2003b), which may indicate that VFA absorption rate increases as ruminal motility
increases. As ruminal motility increases, VFA molecules are expected to be replenished

at the rumen wall more rapidly, increasing the concentration gradient across the ruminal

91

epithelium and rate of VFA absorption. Reduction of ruminal pH may decrease ruminal
motility and VFA absorption rate.

Across cow period means, valerate absorption rate was positively correlated with
both 3.5% F CM (1'2 = 0.39; P < 0.03) and the proportion of intake energy used toward
milk production (Milk NEL, % of NEL intake; r2 = 0.59; P < 0.001). Greater partitioning
of intake energy toward milk probably occurred because of dilution of maintenance
energy with greater milk yield. The positive relationship between VFA absorption rate
and milk yield might occur by either greater VFA supply or demand. Greater VFA
absorption, perhaps by increased ruminal motility, might increase VFA available for
production and milk yield. Conversely, as milk production increases, greater utilization
of VFA for production could create a greater concentration gradient across the ruminal
epithelium and thus increase VFA absorption rate. Although it is difficult to establish
cause and effect between VFA absorption rate and milk production, these data suggest
that understanding dynamics of rumen function and VFA absorption might provide

practical methods to increase milk yield.

Ruminal Fermentation Acids

No interactions of main treatment effects occurred for concentration or profile of
ruminal fermentation acids. Although floury corn grain treatment increased ruminal
starch digestibility, there was no effect of endosperm type of corn grain on total VFA
concentration. Compared to floury corn grain, vitreous corn grain tended to increase
molar proportion of acetate more in diets with control corn silage than bm3 corn silage

(interaction P < 0.13; Table 2). Floury grain increased molar proportion of propionate

92

compared to vitreous corn grain (P < 0.001). An interaction of treatment effects occurred
for ratio of acetate to propionate. Compared to vitreous grain, floury endosperm reduced
acetate: propionate ratio to a greater extent in control corn silage than bm3 corn silage
(interaction P < 0.07). Additionally, bm3 corn silage reduced acetate: propionate
compared to control corn silage (P < 0.05). Increasing proportion of starch digested in
the rumen often decreases the acetate: propionate ratio (Overton et al., 1995; Crocker et
al., 1998). Lactate did not differ among treatments. Control silage increased and vitreous
grain tended to increase branch chain VFA (P < 0.007 and P < 0.08; respectively), and
although the main effect of silage type is strong, the differences among treatments are

probably not enough to be biologically significant.

Nitrogen Flow

Nitrogen intake, ruminal ammonia N, and ammonia flow to the duodenum did not
differ among treatments (Table 3). NAN flow to the duodenum was not different among
treatments with a mean of 625 g/d and 91.6% of N intake across treatments. These
values are high relative to previous reports in the literature, but DMI is also relatively
high and NAN flow to the duodenum would increase with DMI. NAN flow was
positively correlated with DMI across cow period means (1'2 = 0.64; P < 0.0001). This
increase in NAN flow as DMI increased was not associated with greater passage of feed
and endogenous N (N ANMN) but rather with greater microbial N flow to the duodenum
(r2 = 0.55; P < 0.002). A review by Clark et al. (1992) reported the relationship between

OM intake and passage of NAN as (NAN = 30.15 [OM intake] — 33.15 [r2 = 0.83]).

93

Substituting our observed mean OM intake of 22.6 kg/d OM would predict NAN flow to
be 648 g/d, which is consistent with mean NAN flow of 625 g/d observed in this study.

NANMN flow to the duodenum was not different among treatments, but the mean
of 64.4 g/d seems very low. NANMN averaged only 10.1% of N intake and 11.3% of
total duodenal NAN flow. High proteolytic activity in the rumen could decrease
NANMN flow to the duodenum. Fulghum and Moore (1963) reported that 38% of
isolated ruminal bacteria demonstrated proteolytic activity. Proteolytic species of
bacteria in the rumen include Ruminobacter amylophilus, Butyrivibriofibrisolvens, and
Prevotella ruminicola (Wallace and Brammall, 1985). Proteolytic activity appears to be
constitutively expressed in isolated ruminal bacteria and differences in proteolytic
activity in response to diet is probably more related to changes in size and composition in
the microbial population than enzyme activity per se (Wallace and Brammall, 1985).
Indeed, as diets increase in cereal grain concentration, proteolytic and amylolytic activity
increases because of proliferation of the amylolytic population of bacteria (Hristov et al.,
2002). Additionally, although bacteria have a higher proteolytic activity than protozoa
(Brock et al., 1982), protozoa can be more important in the fermentation and digestion of
less soluble particulate proteins (Hino and Russell, 1987).

Microbial N flow was similar among endosperm types in bm3 silage diets, but in
control silage diets, vitreous corn tended to increase microbial N flow 185 g/d versus
floury grain (interaction P < 0.17; Table 3). The proportion of duodenal NAN
contributed by microbial N averaged 88.7% and is at the high end of the range of 34 to
89% reported in a review by Clark et al. (1992). Greater DMI increases DM passage

rate, and because most rmninal microbes are attached to feed particles, greater DMI is

94

expected to increase microbial flow to the duodenum. In this experiment, microbial N
flow tended to be positively correlated with iNDF passage rate (r2 = 0.34; P < 0.07) but
was not correlated with liquid passage rate (1'2 = -0.12; P < 0.53) across cow period
means. F irkins et al. (1986) reported that microbial flow was highly correlated with fluid
dilution rate and speculated that this was because small particles pass largely with the
fluid phase, but that experiment was conducted with steers consuming low amounts of
finely chopped hay. Similar to our experiment, Voelker and Allen (2003b) found
microbial N flow in high producing dairy cows to be more related to passage of iNDF
from the rumen rather than liquid passage. In animals consuming fibrous diets at 3 to 4
times maintenance energy, particulate passage rate may become more critical in
determining microbial flow.

Microbial efficiency was numerically higher for diets containing vitreous corn
grain (41.3 versus 34.5 g/kg TRDOM; P < 0.12). F loury endosperm corn grain might
have reduced MNE by uncoupling growth from energy supply. F loury corn grain
reduced mean and minimum ruminal pH, which increases maintenance requirements of
microbes to maintain ion balance across the cell membrane (Russell, 1998). Across cow
period means, MN E was negatively correlated with ruminal starch digestibility (r2 = -
0.47; P < 0.01) and positively correlated with mean ruminal pH (r2 = 0.53; P < 0.006).
Similar to our study, dent corn decreased microbial efficiency when compared to more
vitreous flint corn in steers (Philippeau et al., 1999a). Greater ruminal starch digestion
could reduce MN E by increasing substrate availability more than needed for maximum
growth rate, increasing maintenance energy from low pH, or increasing cell lysis. MNE

was positively related to rate of starch passage from the rumen across cow period means

95

(r2 = 0.46; P < 0.01). This relationship has been reported previously (Oba and Allen,
2003c; Voelker and Allen, 2003b), and is probably related to decreased microbial
turnover with greater passage from the rumen (Wells and Russell, 1996). Greater MNE
in vitreous grain diets could be the result of higher ruminal pH and faster passage rate of
starch from the rumen.

NAN postruminally digested (g/d) tended to be less for floury versus vitreous
corn grain in control silage diets but greater for floury versus vitreous grain in bm3 corn
silage diets (interaction P < 0.12). Amount of NAN digested postruminally reflects
differences in DMI that occurred among diets, so postruminal NAN digestibility as a
percent of NAN intake did not differ among treatments. Additionally, reductions in
NANMN digestion were compensated for by increases in microbial N digestion so that
NAN digestibility (% of N intake) did not differ. A significant interaction of treatment
effects occurred for postruminal NAN digestibility (% of duodenal flow; interaction P <
0.05). F loury endosperm corn grain reduced postruminal NAN digestibility (% of
duodenal flow) versus vitreous endosperm in control silage diets but increased
postruminal NAN digestibility (% of duodenal flow) versus vitreous grain in bm3 corn
silage diets. Amount of N apparently digested in the total tract did not differ among
treatments with a mean of 507 g/d. Vitreous corn grain tended to reduce apparent total
tract N digestibility compared to floury endosperm grain (76.0 versus 73.6%; P < 0.06).
Across cow period means, total tract N digestibility was negatively related with ruminal
iNDF passage rate (1'2 = -0.45; P < 0.01). Greater rate of passage could increase substrate

supply to the hindgut and might increase microbial growth and loss in the feces.

96

Additionally, limitations to N digestion may occur with greater passage rate because of

reduced time for enzymatic hydrolysis and digestion.

Partitioning of Duodenal N

Based on analysis of the literature, partitioning of NAN flow into NANMN and
microbial N fractions appears to be too low and high, respectively. Clark et al. (1992)
determined relationships between OM intake, NANMN flow, and microbial N flow from
multiple experiments reporting these variables. Using OM intake as an input into the
prediction equations of Clark et al. (1992), NANMN flow in this experiment is predicted
to be 301 g/d for cows consuming 22.6 kg/d OM, but our observed value averaged 64.4
g/d (Table 3). Extensive proteolysis by amylolytic bacteria could have reduced the flow
of feed N, as discussed previously. However, it is surprising that animals with high DMI
and adequate dietary protein (17.4%) would not have considerable NANMN flow to the
duodenum. Conversely, microbial N appears to be very high in this study; observed
mean microbial N flow was 564 g/d, whereas prediction equations based on OM intake
(Clark et al., 1992) predicted microbial N flow to be 354 g/d for cows consuming 22.6
kg/d OM. Few studies exist reporting duodenally cannulated cows with high intakes and
milk production, and because microbial N flow is correlated with OM intake (1'2 = 0.62;
Clark et al., 1992), it is possible that very high microbial N flow occurred in this study.

Although values obtained for NANMN and microbial N flow are within the range
reported in the literature (Clark et al., 1992), we do not have complete confidence in
partitioning of N between NANMN and microbial N in this experiment. Partitioning

duodenal NAN into microbial and non-microbial fractions is problematic.

97

Overestimation of microbial N to duodenal NAN flow could occur if feed N
contamination increased microbial pellet N concentration and N: purine ratio. However,
microscopic examination of microbial pellet did not reveal gross contamination by feed
particles, and any feed N contamination would have to be exceptionally high in N
concentration to significantly affect N: purine ratio, but few feeds have a higher N
concentration than microbial protein. Loss of purines in microbial pellet samples but not
duodenal digesta samples might overestimate microbial protein contribution to duodenal
NAN flow. However, both sample types were lyophilized to minimize effects of heat,
and purine analysis was conducted on microbial pellet samples at the same time as the
corresponding duodenal digesta sample, so consistent loss of purine in microbial pellet
samples but not duodenal samples is unlikely. Additionally, compounds in duodenal
samples that absorb at 260 nm will cause overestimation of microbial N concentration in

duodenal samples.

CONCLUSIONS

F loury endosperm grain reduced ruminal pH compared to vitreous corn by increasing
ruminal fermentation of OM and starch compared to vitreous corn grain. Brown midrib 3
corn silage reduced ruminal pH and increased total VFA concentration compared to
control corn silage, probably by reducing amount of neutralization and buffering of
fermentation acids. Valerate absorption rate decreases as ruminal pH decreases, and
could be the result of reduced ruminal motility. Low ruminal pH for floury endosperm

might have reduced MNE by uncoupling energy fermentation from microbial cell

98

production, while greater passage rate of starch for vitreous grain increased MNE
possibly because of reduced microbial turnover in the rumen. However, greater passage
of substrate from the rumen in vitreous grain diets may have increased hindgut

fermentation and reduced apparent total tract N digestibility.

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Table 1. Effects of corn grain endosperm type and brown midrib 3 corn silage on

 

 

 

 

ruminal pH.
Control bm3 P
Floury Vitreous Floury Vitreous SEM SI G2 S x G3

Mean 6.01 6.23 5.92 6.01 0.09 0.03 0.03 0.37
Minimum 5.48 5.66 5.36 5.49 0.08 0.04 0.02 0.67
Maximum 6.56 6.74 6.53 6.55 0.07 0.12 0.14 0.22
Range 1.07 1.07 1.15 1.07 0.06 0.45 0.41 0.45
Standard deviation 0.29 0.29 0.32 0.29 0.02 0.42 0.24 0.37
Hours under

pH 6.0 10.71 7.10 12.75 10.66 2.0 0.09 0.09 0.64

pH 5.8 6.97 4.67 8.01 6.85 1.8 0.27 0.24 0.70

pH 5.5 2.47 1.25 2.68 2.95 1.0 0.28 0.59 0.40
Area under the curve

pH 6.0 411 257 473 434 114 0.21 0.30 0.54

pH 5.8 200 116 225 226 69 0.25 0.46 0.47

pH 5.5 36 18 41 53 19 0.22 0.85 0.35

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.

3 Interaction of corn silage hybrid and ground corn grain endosperm type.

100

Table 2. Effects of corn grain endosperm type and brown midrib 3 corn silage on
ruminal VFA profiles and concentrations.

 

 

 

 

Control P
Floury Vitreous Floury Vitreous SEM SI G2 S x G3
Total VFA, mmol 138 136 142 139 2.6 0.03 0.14 0.72
VFA, % of total VFA
Acetate - 53.6 56.3 53.5 54.6 1.2 0.07 0.001 0.13
Propionate 28.3 25.2 28.3 26.7 1.1 0.18 0.001 0.19
Butyrate 12.7 13.4 13.2 13.3 0.5 0.57 0.23 0.35
Branch chain VFA 2.64 2.75 2.31 2.53 0.2 0.007 0.08 0.55
Lactate 0.51 0.46 0.48 0.80 0.3 0.53 0.60 0.48
Acetate: Propionate 1.92 2.29 1.91 2.07 0.1 0.05 0.001 0.07
Rate of valerate absorption,
%/h‘I 0.43 0.47 0.41 0.46 0.04 0.67 0.25 0.97

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.

4 Measured using Co-EDTA and valerate.

101

Table 3. Effects of corn grain endosperm type and brown midrib 3 corn silage on

nitrogn metabolism.

 

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI G2 S x G3
TRDOM, kg/d‘I 14.3 14.7 15.0 13.8 1.0 0.92 0.53 0.18
N intake, g/d 643 684 699 690 30 0.15 0.43 0.24
Ruminal Ammonia, mg/dl 9.8 10.3 9.4 10.1 0.5 0.46 0.20 0.78
Flow to duodenum
Ammonia N, g/d 15.8 17.5 16.4 18.6 1.6 0.49 0.12 0.87
NAN5
g/d 555 676 635 633 49 0.68 0.19 0.18
% ofN intake 86.5 98.5 90.1 91.2 5.3 0.74 0.23 0.31
NANMN‘I
g/d 98.6 33.1 70.7 55.3 26 0.95 0.37 0.57
% ofN intake 14.7 5.6 11.4 8.6 7.8 0.98 0.37 0.64
% duodenal NAN 16.9 4.5 13.2 10.8 8.4 0.85 0.29 0.47
Microbial N
g/d 457 642 575 579 74 0.68 0.16 0.17
% duodenal NAN 83.1 95.5 86.8 89.2 8.4 0.85 0.29 0.47
g/kg TRDOM4 32.2 42.1 36.8 40.5 4.2 0.72 0.12 0.47
NAN postruminal digestion
g/d 395 498 470 444 41 0.79 0.34 0.12
% of N intake 61.4 72.6 67.0 64.1 5.2 0.77 0.42 0.17
% of duodenal flow 71.0 73.2 74.3 69.6 1.8 0.94 0.46 0.05
N total tract apparent digestion
g/d 483 508 534 502 21 0.24 0.86 0.13
% 75.0 74.2 76.9 72.9 1.3 0.79 0.06 0.21

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.

I Truly ruminally digested OM.

5 Non-ammonia N.

6 Non-ammonia non-microbial N.

102

CHAPTER 5

CONCLUSIONS AND IMPLICATIONS

Interactions of endosperm type of corn grain and the bm3 mutation in corn silage can
affect feeding behavior and feed intake. Compared to vitreous corn grain, floury
endosperm corn decreased DM meal size in control corn silage diets but increased DM
meal size in bm3 corn silage diets. However, DMI was not different among endosperm
types in bm3 corn silage diets because cows consuming bm3 corn silage with vitreous
corn grain increased meal frequency to compensate for reduced DM meal size. These
interactions of main effects on feeding behavior may have occurred because of the type
and temporal pattern of metabolic fuel supply to the animal. Floury endosperm increased
rate and extent of ruminal starch fermentation and molar proportion of propionate in the
rumen. Lower DM meal size and DMI for diets containing floury endosperm with
control corn silage might have been because the rate of propionate production and
absorption exceeded its rate of utilization for gluconeogenesis, resulting in oxidation in
the liver and a satiety signal. However, DM meal size and DMI may have been greater
for floury endosperm when combined with bm3 corn silage because greater milk yield
increased glucose demand and flux of propionate through gluconeogenesis, delaying
satiety.

In diets containing vitreous corn grain, greater DM meal size for control versus
bm3 corn silage might be a result of a greater ratio of acetate to propionate produced in

control versus bm3 corn silage, as indicated by a greater ratio of acetate to propionate

103

concentrations in the rumen. Less hypophagic effects of acetate versus propionate might
reduce satiety signaling in diets containing vitreous corn grain and control corn silage and
might act to prolong meal length. Additionally, greater plasma insulin: glucagon ratio in
the diet with vitreous corn grain and bm3 corn silage might have increased oxidation of
metabolites in the liver to signal satiety. Numerically higher plasma insulin
concentrations might have occurred in vitreous grain diets because greater rate of passage
of starch from the rumen effectively shifted the primary site of starch digestion to the
intestines. Although digestion of starch to glucose in the small intestine could
theoretically increase plasma glucose, greater intestinal starch digestion in vitreous diets
did not increase milk yield per se. Additionally, greater passage rate of starch to the large
intestine might have increased microbial growth in the hindgut and reduced total tract N
digestibility.

Ruminal pH was positively associated with rate of valerate absorption, and
suggests that low ruminal pH may reduce ruminal motility and VFA absorption, and
could ultimately limit supply of metabolites for milk production. Floury endosperm grain
reduced mean ruminal pH, which might have caused the tendency for lower microbial
efficiency in floury grain diets by increasing energy spilling. Greater ruminal starch
fermentation in diets containing floury endosperm did not reduce ruminal fiber
digestibility; on the contrary, ruminal starch and fiber digestibility were positively
related. This suggests synergy and cross-feeding between ruminal amylolytic and
cellulolytic populations, so high proportions of ruminally degraded starch in diets of
lactating cows may not be as detrimental to fiber digestibility as previously indicated

with in vitro experiments.

104

Several important areas of research have been highlighted by results from this
experiment. Although ruminally degraded starch is associated with DMI depression in
some studies, DMI is not always reduced (Allen, 2000), as occurred in the diet containing
floury endosperm grain and bm3 corn silage. Understanding how DMI is regulated by
both metabolic and hormonal factors is important in order to maximize energy intake and
milk yield. Additionally, milk yield is determined by supply of metabolites from both the
rumen and the intestines, which can change dramatically based on site of nutrient
digestion. Although VFA absorption from the rumen can significantly affect substrate
supply, little is known about how ruminal motility and pH might affect VFA absorption
rate. Finally, understanding how DMI and the ruminal environment interact to affect
microbial protein production will allow nutritionists to maximize microbial protein yield
and utilization by the animal to reduce environmental N.

This experiment proves that digestion characteristics of starch and fiber can
interact to affect feeding behavior and ultimately production. However, animal response
to changes in diet fermentability involves understanding interactions between physical,
metabolic, and hormonal regulation of feed intake, and predicting how animals will
respond is unlikely. Because floury endosperm grain increased DMI and milk yield when
combined with bm3 corn silage but vitreous corn did not, production responses to bm3
corn silage are probably dependent on grain source. The results from the present
experiment suggest that diets should not be formulated based upon starch and NDF
concentrations alone, but that consideration of the fermentability of individual ingredients

and their interactions is important to maximize DMI and milk yield.

105

APPENDICES

106

APPENDIX A

Use of Markers in Assessing Site of Digestion

External and internal markers to estimate duodenal flow are well known for being
imprecise. Cr203 was originally closed and used in this experiment as an external marker
to determine digesta flow. However, the absolute values of DM flow appeared high,
even considering that DMI was exceptionally high for surgically prepared animals.
Indigestible NDF (iNDF) was consequently used as the final flow marker in this study. It
is important to note that DM flow determined using Cr203 was highly correlated with
DM flow using iNDF. Also, because this study is investigating treatments and is not
trying to characterize animal response to one particular diet, it is the relative differences
among treatments rather than the absolute values of nutrient flows that are important.

Incomplete recovery of Cr203 will overestimate duodenal DM flow. Sampling
digesta before marker concentration reaches steady state will underestimate marker flow
and overestimate DM flow (Owens and Hanson, 1992). It is estimated that marker
concentration reaches steady state in approximately 5 to 7 d for cattle with constant feed
intake (Owens and Hanson, 1992). Additionally, in dairy cattle with high feed intake,
time to steady state of Cr203 outflow would be reached faster than in animals with lower
intakes and passage rates. In the present experiment, chromic oxide was closed for 7 days
with a prime dose on the first day, so it is not likely that steady state had not yet been
reached. Diurnal variation of marker flow can occur even after steady state has been
reached (Corbett et al., 1959). However, frequent (3 times daily) dosing and collection of

8 digesta samples, representing every 3-hr over a 24-hr period, would minimize effects of

107

diurnal variation on DM flow. Zinn et al. (1980) determined that 8 samples, representing
every 6-hr over a 48-hr period was sufficient to account for diurnal variation, and there
was no difference between DM flow calculated using 020; as a marker or total
collection. Previous research using this method (Oba and Allen, 2003b) has indicated
that 8 samples are adequate. Using chromic oxide as a marker has been criticized
because it may incompletely mix with ruminal contents when dosed in gelatin capsules
(Corbett et al., 1959). However, ground spelt hulls were included in the gelatin capsules
with Cr203 to facilitate dispersal in the rumen. Furthermore, there was no evidence that
incomplete mixing occurred because during digesta sampling no Cr203 was seen in either
the rumen or duodenal contents. Incomplete recovery of Cr203 while subsampling may
have occurred during this study. Eight digesta samples are separated into primarily solid
and liquid phases using a nylon mesh and are composited into one solid and one liquid
sample per cow-period. Mixing and subsampling of solid phase of duodenal contents
was performed by hand for several minutes and taking small samples from numerous
locations. It is unlikely that settling of Cr203 would occur in a solid subsample, but the
liquid sample, although well mixed, would be expected to exhibit some settling of Cr203.
It is possible that the liquid subsample did not contain a representative amount of marker
because of settling to the bottom of the container, and subsequent analysis for Cr in the
subsample underestimated actual Cr concentration and overestimated DM flow at the
duodenum. Fecal flow was not overestimated to the same degree because fecal samples
were composited on a wet weight basis, and also would not expect to exhibit Cr203
settling because of the more solid nature of the feces. In the future, steps should be taken

to prevent differential settling of marker during subsampling.

108

APPENDIX B

Additional Original Data

Table 1. Effects of corn grain endosperm type and brown midrib 3 corn silage on
additional measures of feeding behavior and chewing behavior.

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI 62 S x G3
Eating rate, kg/min 0.36 0.39 0.40 0.39 0.02 0.10 0.38 0.19
Hunger ratio 0.16 0.18 0.18 0.14 0.02 0.59 0.56 0.07
Satiety ratio 0.18 0.20 0.22 0.19 0.02 0.32 0.63 0.07
Eating chews
/ kg NDF intake 3616 3314 3347 3342 169 0.18 0.10 0.11
/ kg starch intake 3196 2895 3010 2894 190 0.32 0.04 0.33
Ruminating chews
/ kg NDF intake 5625 4985 4800 4853 375 0.01 0.1 1 0.06
/ kg starch intake 4906 4319 4308 4176 290 0.02 0.02 0.13
Chew rate, chews/min
Eating 75.0 75.9 73.6 75.2 1.8 0.49 0.41 0.83
Ruminating 61.6 61.7 60.8 60.7 1.1 0.18 0.98 0.90

 

I S = Main effect of corn silage hybrid.

2 G = Main effect of ground corn grain endosperm type.

3 Interaction of corn silage hybrid and ground corn grain endosperm type.

109

Table 2. Effects of corn grain endosperm type and brown midrib 3 corn silage on
ruminal nutrient concentration and turnover time.

 

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI G2 S x G3

DM % 13.6 14.6 15.2 14.4 0.42 0.08 0.76 0.02

OM, % of DM 91.5 91.6 91.7 90.9 0.70 0.67 0.53 0.48

NDF, % of DM 53.5 52.1 51.3 49.5 1.31 0.02 0.11 0.86

pdNDF, % of DM4 27.1 25.8 32.6 31.3 1.14 <.0001 0.08 0.95

iNDF, % of DM5 26.4 26.4 19.2 18.2 0.80 <.0001 0.41 0.43

Starch, % of DM 6.76 7.60 7.48 8.88 0.52 0.05 0.03 0.57
Ruminal turnover time, h

DM 10.1 11.1 10.7 10.1 0.73 0.79 0.80 0.30

OM 10.6 11.5 11.2 10.5 0.79 0.78 0.88 0.29

NDF 23.0 24.8 23.9 21.5 2.0 0.48 0.85 0.26

pdNDFI 18.0 19.0 20.5 18.8 1.8 0.41 0.83 0.37

iNDFs 33.2 36.5 32.9 29.6 2.7 0.16 0.99 0.21

Starch 2.58 2.99 2.98 3.32 0.33 0.26 0.24 0.92
iNDF passage to

duodenum, kg/d 1.99 2.18 1.64 1.59 0.10 <.0001 0.23 0.07

 

I S = Main effect of corn silage hybrid.
2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.

4 Potentially digestible NDF.
5 Indigestible NDF.

110

Table 3. Effects of corn grain endosperm type and brown midrib 3 corn silage on
amount of ruminal VFA and ruminal pH during meal and rumination bouts.

Control bm3 P

 

 

Floury Vitreous Floury Vitreous SEM SI G2 S x G3

Fermentation acid, mmol

Acetate 73.8 76.4 75.7 75.8 1.1 0.37 0.06 0.08
Propionate 39.2 34.4 40.3 37.3 2.1 0.04 0.001 0.32
Butyrate 17.5 18.2 18.6 18.4 0.6 0.21 0.61 0.36
Isobutyrate 1.42 1.42 1.32 1.34 0.1 0.01 0.63 0.81
Valerate 3.03 2.80 3.11 2.94 0.2 0.36 0.11 0.78
Isovalerate 2.23 2.32 1.98 2.18 0.2 0.05 0.15 0.59
Formate 0.02 0.01 0.02 0.01 0.1 0.59 0.40 0.87
Branch chain VFA 3.65 3.74 3.31 3.52 0.3 0.03 0.21 0.63
Lactate 0.70 0.65 0.69 1.10 0.4 0.54 0.62 0.52
Meal initial pH 6.08 6.28 5.98 6.07 0.12 0.12 0.16 0.58
Meal final pH 5.95 6.10 5.89 5 .95 0.09 0.09 0.41 0.34
Rumination initial pH 6.00 6.20 5.86 5.97 0.11 0.05 0.09 0.59
Rumination final pH 6.06 6.26 5.99 6.11 0.11 0.13 0.03 0.61

 

G = Main effect of corn silage hybrid.
2 G = Main effect of ground corn grain endosperm type.
3 Interaction of corn silage hybrid and ground corn grain endosperm type.

Table 4. Effects of corn grain endosperm type and brown midrib 3 corn silage on
net energy for maintenance.

 

 

 

 

Control bm3 P
Floury Vitreous Floury Vitreous SEM SI 62 S x G3
NEM, Meal/d 10.2 10.3 10.2 10.3 0.22 0.93 0.31 0.80

 

IS= Main effect of corn silage hybrid.
2 G = Main effect of ground corn grain endosperm type.
I Interaction of corn silage hybrid and ground corn grain endosperm type.

111

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