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thesis entitled

EFFECTS OF ENHANCED l! VITRO NEUTRAL
DETERGENT FIBER DIGESTIBILITY OP
PORAGE OH FEED INTAKE AND
PERFORMANCE OF LACTATIRG COWS

presented by
HASAHITO 08A

has been accepted towards fulfillment
of the requirements for

ILS. degree in ANIMAL SCIENCE

M ' rprofessor

Date y

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

 

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EFFECTS OF ENHANCED IN VITRO NEUTRAL DETERGENT
FIBER DIGESTIBILITY OF FORAGE ON FEED INTAKE AND
PERFORMANCE OF LACTATING COWS
By

Masahito Oba

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1998

ABSTRACT

Effects of enhanced in vitro neutral detergent ﬁber digestibility of
forage on feed intake and performance of lactating cows

By

Masahito Oba

Effects of enhanced in vitro neutral detergent ﬁber (NDF) digestibility of forage on feed
intake, nutrient utilization and performance of high producing dairy cows were evaluated.
Brown midrib (bm3) mutation in corn silage increased in vitro NDF digestibility
compared to isogenic control. Experimental diets contained either bm3 or control corn
silage at similar dietary NDF contents. Cows fed bm3 corn silage increased feed intake,
milk production, passage rate for ruminal digesta, post-ruminal starch digestion, and
efﬁciency of microbial nitrogen production. Treatment had no effect on ruminal digesta
pool size, chewing activities, and NDF digestibility in the rumen. Bm3 treatment
decreased rumen pH, but did not increase ﬂuctuation in rumen pH. In vivo NDF
digestibility response to bm3 treatment was negatively related to response in dry matter
intake. Beneﬁcial effects of enhanced in vitro NDF digestibility of forage were greater

for high producing cows and cows fed high forage diets.

ACKNOWLEDGEMENTS

I would like to thank Dr. Allen for valuable advice throughout my Master’s program. His
enthusiasm for research kept me ambitious and encouraged me to think critically and
develop a variety of ideas. His open-door policy made me feel free to talk with him
whenever I had a question.

I would like to thank Dr. Ames, Dr. Beede, Dr. Bucholtz for serving as graduate
committee members. I am also grateful for Dr. Herdt, Dr. Hill, and Dr. Orth in support of
lab analysis.

I would like to express my thanks to Dave Main for all of his efforts: voluntary help in
sample collection, maintenance of feeding behavior monitoring equipment, and guidance
in lab work. Without his support, I could not have accomplished the experiments
successfully.

I also extend my thanks to Jackie Yun Ying. She gave me a lot of valuable advice
regarding the animal trial and lab analysis. I appreciate the voluntary help from Dewey
Longuski, Barry Choi, Frank Jing Xu, Elisabet Nadeau, Doug Mashek in sample
collection.

I would like to thank Cargill Hybrid Seeds for ﬁnancial support for animal trials.

Finally, I would like to thank my wife, Keiko, for sharing everything with me.

iii

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ viii
LIST OF FIGURES ................................................................................ xi
LIST OF ABBREVIATIONS ................................................................... xii
INTRODUCTION .................................................................................. 1
CHAPTER 1
A REVIEW OF LITERATURE ................................................................. 3
Regulation Mechanisms of Voluntary DMI ............................................. 3
Optimizing Rumen Fermentation ......................................................... 8
Effects on Brown Midrib Mutant Forages on Animal Performance ................. 11
Biochemistry of Brown Midrib Mutations ............................................. l6
Agronomic Aspects of Brown Midrib Forages ........................................ 21
Summary .................................................................................... 24
Tables ....................................................................................... 25
CHAPTER 2

Evaluation of the importance of forage NDF digestibility: effects on dry matter
intake and milk yield of dairy cows.

ABSTRACT ................................................................................ 27
INTRODUCTION ......................................................................... 28
MATERIAL AND METHODS ......................................................... 30
RESULTS AND DISCUSSION ......................................................... 33
DMI and Milk Production ....................................................... 33
Quantifying the effect of enhanced NDF digestibility ....................... 34

iv

CONCLUSION ............................................................................ 35
TABLES .................................................................................... 36
CHAPTER 3

Effect of enhanced in vitro NDF digestibility of corn silage by brown midrib 3
mutation on DMI and milk yield of high producing dairy cows.

ABSTRACT ................................................................................ 40
INTRODUCTION ......................................................................... 4 1
MATERIALS AND METHODS ........................................................ 44
Corn Silage ......................................................................... 44
Cows and Treatments ............................................................ 44
Sample Collection ................................................................ 45
Sample and Statistical Analysis ................................................. 46
RESULTS AND DISCUSSION ......................................................... 47
DMI and Milk Yield ............................................................. 47
Relationship Between Milk Yield and Milk Yield Response ............... 49
Nutrient Digestibility ............................................................. 50
CONCLUSION ............................................................................. 52
TABLES .................................................................................... 53
FIGURES ................................................................................... 59
CHAPTER 4

Effects of brown midrib 3 corn silage on feeding behavior and ruminal digesta
kinetics.

ABSTRACT ................................................................................ 65

INTRODUCTION ......................................................................... 66

MATERIAL AND METHODS .......................................................... 69

Corn Silage ......................................................................... 69
Cows and Treatments ............................................................. 69
Data and Sample Collection ..................................................... 70
Chemical Analysis ................................................................ 71
Statistical Analysis ............................................................... 72
RESULTS AND DISCUSSION ......................................................... 73
DMI and Meal Patterns .......................................................... 73
Ruminal Digesta Kinetics ....................................................... 75
Chewing Activities ................................................................ 77
CONCLUSION ............................................................................. 78
TABLES .................................................................................... 79
CHAPTER 5

Effects of brown midrib 3 corn silage on nutrient utilization by lactating cows

ABSTRACT ................................................................................ 88
INTRODUCTION ......................................................................... 89
MATERIAL AND METHODS .......................................................... 91
Experimental Designs ............................................................ 91
Data and Sample Collection .................................................... 93
Data and Sample Analysis ....................................................... 95
RESULTS ................................................................................... 97
DMI and Animal Performance .................................................. 97
Ruminal Fermentation ........................................................... 98

vi

Nutrient Digestibility ............................................................. 99

DISCUSSION ............................................................................. 100
Energy Intake and Utilization ................................................. 100
Microbial Efﬁciency ............................................................ 103
Milk Composition ............................................................... 103
CONCLUSION ........................................................................... 105
TABLES ................................................................................... 106
CHAPTER 6
General Discussion and Implications ........................................................ 113
Rate of Passage and bm3 Corn Silage ................................................. 113
Energy Utilization ........................................................................ 114
Homeostasis and DMI Depression ..................................................... 119
Figures ...................................................................................... 121
REFERENCE ..................................................................................... 123

vii

LIST OF TABLES

CHAPTER 1

Table 1. Effects of brown midrib 3 (bm3) corn silage on animal performance ............. 25
Table 2. Effects of brown midrib 3 (BMR) sorghum silage on animal performance .....26
Table 3. Effects of brown midrib 3 (BMR) pearl millet silage on animal

1)<:tfk)rrriziricze: ......................................................................................... 22(3
CHAPTER 2

Table 1. Summary of experiments used for statistical analysis ............................... 36
Table 2. Least square means for animal performance by different

levels of ﬁber digestibility (n=38) taking dietary NDF% as a covariate.

Calculated from 19 sets of treatment means from 8 articles of Journal

of Dairy Science ..................................................................................... 39
CHAPTER 3

Table 1. Nutrient composition of corn silage used to formulate experimental

diets ................................................................................................... 53
Table 2. Status of 32 cows at the beginning of experiment ................................... 54
Table 3. Ingredients and nutrient composition of experimental diets

(% of dietary DM) ................................................................................... 55
Table 4. Effects of enhanced NDF digestibility on feed intake .............................. 56
Table 5. Effects of enhanced NDF digestibility on milk production ........................ 57
Table 6. Effects of enhanced NDF digestibility on apparent total tract

digestibility (%) ..................................................................................... 58

viii

CHAPTER 4

Table 1. Nutrient composition of bm3 corn silage and normal control corn silage... ......79

Table 2. Ingredients and nutrient composition of experimental diets
(% of dietary DM) ................................................................................... 80

Table 3. Effects of bm3 and control corn silage at two dietary NDF contents on
nutrient intake during feeding behavior monitoring period (kg/d) ........................... 81

Table 4. Effects of bm3 and control corn silage at two dietary NDF contents on
meal patterns and eating activities ............................................................... 82

Table 5. Effects of bm3 and control corn silage at two dietary NDF contents on
digestion kinetics in the rumen ................................................................... 83

Table 6. Effects of bm3 and control corn silage at two dietary NDF contents on
ruminal digesta characteristics ..................................................................... 84

Table 7. Effects of bm3 and control corn silage at two dietary NDF contents on
rumination activities ................................................................................ 85

Table 8. Effects of bm3 and control corn silage at two dietary NDF contents on
total chewing activities ............................................................................ 86

Table 9. Pearson correlation coefﬁcient for chewing activity measurements among
32 observations ..................................................................................... 87

CHAPTER 5

Table 1. Effects of bm3 and control corn silage at two dietary NDF contents on
DMI and milk production ........................................................................ 106

Table 2. Effects of bm3 and control corn silage at two dietary NDF contents on
ruminal pH ......................................................................................... 107

Table 3. Effects of bm3 and control corn silage at two dietary NDF contents on
rumen fermentation ............................................................................... 108

Table 4. Effects of bm3 and control corn silage at two dietary NDF contents on
digestibility of DM and OM ..................................................................... 109

Table 5. Effects of bm3 and control corn silage at two dietary NDF contents on
digestibility of NDF .............................................................................. 110

ix

Table 6. Effects of bm3 and control corn silage at two dietary NDF contents on
digestibility of starch .............................................................................. 111

Table 7. Effects of bm3 and control corn silage at two dietary NDF contents on
nitrogen metabolism

........................................................................................................ 112

LIST OF FIGURES

CHAPTER 3

Figure 1. Relationship between average milk yield (kg/d) for 14 (1 prior to the
beginning of the experiment and the response in DMI (kg/d) to the bm3 treatment ...... 59

Figure 2. Relationship between average milk yield (kg/d) for 14 (1 prior to the
beginning of the experiment and the response in milk yield (kg/d) to the bm3
treatment. ................................................................ , ........................... 60

Figure 3. Relationship between responses to bm3 treatment: apparent total tract

DM digestibility (%) versus DMI (kg/d). ........................................................ 61
Figure 4. Relationship between responses to bm3 treatment: total tract NDF

digestibility (%) versus DMI (kg/d). ............................................................. 62
Figure 5. Relationship between responses to bm3 treatment: apparent total tract

starch digestibility (%) versus DMI (kg/d). ..................................................... 63
Figure 6. Relationship between responses to bm3 treatment: apparently digested

DMI (kg/d) versus DMI (kg/d). .................................................................. 64
CHAPTER 6

Figure 1. Glucose availability and utilization by the mammary gland over time:
model 1. ............................................................................................ 121

Figure 2. Glucose availability and utilization by the mammary gland over time:
model 2. Energy intake increases without increasing ﬂuctuation relative to Figure 1... 121

Figure 3. Glucose availability and utilization by the mammary gland over time:
model 3. Fluctuation increases without change in energy intake relative to Figure 1.... 122

Figure 4. Glucose availability and utilization by the mammary gland over time:
model 4. Energy intake increases with increased fluctuation relative to Figure 1 ....... 122

xi

ADF

BCS

BW

bm3

BMR

CP

DM

DMI

FCM

FC

NIEOP

NDF

NAN

NANNIN

OM

SCM

VF A

LIST OF ABBREVIATIONS

Acid detergent ﬁber

Body condition score

Body weight

Brown midrib 3 mutant for maize

Brown midrib mutant for sorghum and pearl millet
Crude protein

Dry matter

Dry matter intake

Fat-corrected milk

Forage to concentrate ratio.

Microbial nitrogen

Milk energy output as a percentage of NEL intake
Neutral detergent ﬁber

Non-ammonia nitrogen

Non-ammonia non-microbial nitrogen

Organic matter

Solid-corrected milk

Volatile fatty acid

xii

INTRODUCTION

Maximizing DMI is an important management goal for high producing dairy herds. As
genetic ability to produce milk increases, nutritional management to meet the energy
requirement of dairy cows becomes increasingly important. The amount of energy
needed for maintenance and milk production of high producing cows often exceeds the
amount of energy that they can consume. Milk yield of high producing dairy cows
usually exceeds 50 kg per day at peak lactation. Even though they cannot consume
enough feed to meet their energy requirements, cows in early lactation can sustain a high
level of milk yield by mobilizing body reserves. Therefore, they are more susceptible to
metabolic diseases such as ketosis or fatty liver. Even if high producing cows do not
experience a serious metabolic disorder, they may have subclinical disorders that might

compromise DMI and milk yield

Digestibility of forage NDF could be a key factor to maximize DMI of high producing
cows. Increasing feed intake with adequate ﬁber supply is a challenge. Dairy cows
require effective NDF in diets to maintain rumen function. NRC (1989) recommended
that dairy rations contain at least 25% NDF of dietary DM, and more than 75% of dietary

NDF come from forage source. However, if dietary NDF content is too high, maximum

DMI is limited by physical ﬁll in the rumen. When physical ﬁll limitation to maximum
DMI exists, enhanced case of NDF hydrolysis may result in rapid disappearance of the
NDF fraction from the rumen, reduce physical ﬁll in the rumen, and allow greater
voluntary feed intake. Although nutritional importance of NDF is accepted widely,
speciﬁc effects of NDF degradability are not clearly understood. Forage NDF varies
greatly in its digestibility (Nocek and Russell, 1988; Allen and Oba, 1996), which
indicates that forage NDF varies in its physical, chemical, and nutritional characteristics.

Nutritional importance of enhanced NDF digestibility needs to be evaluated.

A forage with brown midrib mutant consistently increases NDF digestibility (Cherney et
al., 1991). A comparison of brown midrib corn silage with isogenic control corn silage
may isolate the speciﬁc effect of NDF digestibility on animal performance because this
comparison minimizes other confounding nutritional and physical characteristics found in
forages to be compared. The objective of this series of studies is to evaluate the effects of
enhanced NDF digestibility on DMI and animal performance. Speciﬁcally, the effects of
brown midrib corn silage on DMI, ruminal fermentation, digestion characteristics, and

milk production of high producing dairy cows are focused.

CHAPTER 1

A REVIEW OF LITERATURE

Regulation Mechanisms of Voluntary DMI

Understanding regulation mechanisms of voluntary feed intake is essential before any
management action is taken to enhance DMI of high producing cows. The mechanisms
that regulate DMI have been studied extensively. In general, cows consume feeds to
meet their energy requirement. When cows are fed high energy diets which are palatable,
digestible and low in ﬁll, feed intake is regulated by the energy density of the ration
(Mertens, 1994). According to the theory of Conrad et a1. (1964), voluntary feed intake
is regulated by either energy density of a diet, or physical ﬁll depending on the
digestibility of the diet cows consume. Although this theory simpliﬁes the complex
mechanisms that limit DMI of dairy cows, feed intake is regulated by various factors
depending upon metabolic status of animals. Integration of several positive and negative

stimuli generally contributes to the regulation of voluntary feed intake (Forbes, 1977).

In general, taste, smell, texture and visual appearance influence feed intake. Less
palatable feeds often depress DMI. However, psychogenic factors may not enhance
voluntary feed intake of cows in early lactation. Nombekela et a1. (1994) found that dairy
cattle preferred sweet taste compared to the other primary tastes which are sour, salty and
bitter. They implied that a sucrose-sweetened diet had the potential to enhance feed
consumption. However, in their follow-up study, sucrose supplementation at 1.5% of
dietary DM did not enhance voluntary feed intake of dairy cows in early lactation
(Nombekela and Murphy, 1995). Although they observed a transient increase in DMI for
the sucrose-supplemented diet during ﬁrst two weeks after parturition, the effect of
sweetener did not have a long term (over 12 weeks of experimental period) effect on

DMI and milk yield.

The theory that dietary bulk and physical distention of the reticulorumen can limit feed
intake (Van Soest, 1994) has been accepted widely. DMI is more likely to be controlled
by physical ﬁll when cows are fed high forage diets (Johnson and Combs, 1991; Dado
and Allen, 1995). In the study of Dado and Allen, water-ﬁlled plastic containers, which
equaled 25% of pretrial rumen volume, were placed into the rumen of cows fed 25% of
NDF or 35% of NDF in the diet. The inert bulk decreased DMI for cows fed 35% NDF
in the ration, but did not affect DMI for cows fed 25% NDF in the ration, indicating
physical ﬁll regulates maximum DMI of dairy cows when they are fed high NDF diet.
Johnson and Combs (1991) studied the effect of rumen inert bulk on DMI by replacing

25% of reticulorumen contents with a water-ﬁlled bladder. They used cows in early

lactation which were in slightly positive energy balance. Both DMI and water intake

were observed to be depressed due to the rumen inert bulk.

NDF was proposed as a chemical indicator to predict DMI because of its ﬁlling
characteristics in the rumen (Mertens, 1987). Cell wall constituents ferment slowly and
stay in the rumen longer than other feed components. However, dietary NDF content
alone cannot predict the ﬁlling effect of a diet in the rumen. Filling effect of NDF is
often confounded by many other factors; for example, difference in rate of passage may
change the ﬁlling effects of NDF. Rate of passage is controlled by level of DMI, ambient
temperature, particle size, and particle density (NRC, 1987). Grinding and pelleting
feeds increases rate of passage (NRC, 1987). In addition, passage from the rumen
depends upon the quantity of particles in close proximity to the reticulo-omasal oriﬁce
(Allen and Mertens, 1988). Therefore, rate and potential extent of digestion for forage
NDF might also affect ruminal retention time of NDF because of their effect on particle
density or buoyancy (Allen, 1996). During fermentation, ﬁbrous particles are associated
with gases generated by ruminal microbes, which make particles buoyant longer and to
be selectively retained in the rumen. Passage from the rumen also depends upon the rate
of particle size reduction, which is stimulated by eating chews, ruminating chews as well
as microbial degradation (Allen and Mertens, 1988). In addition, NDF itself varies in its
degradability in the rumen. Ruminal digestibility of forage NDF has been reported to
range from less than 35% to over 75% for various forage types (Nocek and Russell,
1988). Although forage NDF digestibility in vivo varies by animals and feeding

conditions, much of the variation could be due to the composition and structural

differences of the forages; in vitro NDF digestibility after 30h incubation was reported to
vary from less than 30% to 60% for corn silage and alfalfa (Oba and Allen, 1996). Thus,
it is impossible that dietary NDF concentration alone measures all these factors that

might inﬂuence ﬁlling effects in the rumen.

Another study observed no effect of rumen inert bulk on DMI (Johnson and Combs,
1992). They inserted a water-ﬁlled bladder into the rumen of cows in early and mid-
lactation. Then, they offered two diets that differed in forage to concentrate ratio. There
was no signiﬁcant effect of rumen inert bulk on DMI regardless of energy density of
diets. It is probably because energy requirement of cows fed both diet groups was well
satisﬁed, and physical ﬁll did not limit feed intake of these cows. Dado and Allen (1995)
observed the effect of rumen inert bulk on DMI only for the cows fed a high forage diet.
Although physical ﬁll is one of the most important factors which regulate voluntary DMI
of high producing dairy cows, the relative effect of ﬁll on DMI greatly depends upon the

energy requirement of dairy cows.

Physiological satiety also regulates voluntary DMI of animals. Feeding rapidly
ferrnentable carbohydrates often depressed feed intake (McCarthy et al., 1989; Casper et
al., 1990; Oliveira et a1, 1993; Aldrich et al., 1993, Overton et al., 1995).
Chemoreceptors in the ruminal wall are sensitive to changes in pH, and may regulate
feeding behavior of animals (Harding and Leek, 1972). Edward and Poole (1983)
reported that the addition of sodium bicarbonate at 2% of dry matter signiﬁcantly

increased feed intake of dairy cows, indicating the possible role of rumen pH in feed

intake regulation. Increased serum glucose level and subsequent insulin secretion is the
major physiological satiety signals for non-ruminants. Similarly, Deetz and Wangsness
(1981) found that intrajugular insulin injection decreased feed intake of sheep.
Propionate as well as glucose possibly stimulates insulin secretion for ruminants (De
Jong, 1982; Harmon, 1992). Istasse et al. (1987) infused propionate into the rumen
approximately 50% of daily propionate supply in two 3-h pulses, and observed
concurrent sharp peaks in insulin concentration at the jugular vein. Lactating dairy cows
were also observed to respond to high starch diet by elevated plasma insulin
concentration (Lee et al., 1990). Propionate alone can directly affect feed intake
regulation. Anil and Forbes (1980) demonstrated that the liver has receptors which are
sensitive to propionate, and propionate infusion into the portal vein almost completely

prevented eating.

It is less likely that a single factor limits maximum feed intake although one factor may
sometimes become more dominant than others in regulation of maximum feed intake.
Depending upon feeding conditions, both physical ﬁll and physiological satiety can
synergistically interact with each other to regulate feed intake. Physical ﬁll limitation to
DMI does not necessarily mean that there is no additional capacity in the reticulorumen.
Dado and Allen (1995) found a reserve space of more than 16L in the reticulorumen of
the dairy cows challenged by both dietary ﬁber and rumen inert bulk. Mbanya et al.
(1993) suggested that DMI be affected by a combined effect of acid production and
distention, not by one of the effects alone. They infused acetate, propionate, or both, with

or without distention of the rumen by a balloon. Combination of VFA infusion and

reticulorumen distention signiﬁcantly depressed DMI while VFA infusion or distention
alone did not. The role of the osmo-receptor or chemo-receptor in the rumen may need to

be considered in the relation of distention of the reticulorumen.

Optimizing Rumen Fermentation

Increasing energy density of a diet is another management strategy to improve energy
intake of animals. Energy requirements of non-ruminant animals are satisﬁed relatively
easily by supplementing fat, but fat supplementation may lead to nutritional
complications for ruminant animals. Palmquist and Jenkins (1980) stated that physical
coating of forage particle, reduced cation availability, a toxicity to certain
microorganisms were associated with feeding excess fat in a dairy diet. It is generally
suggested that fat supplementation should be limited to 5% of DMI, to avoid negative
effects on animal performance (Palmquist and Jenkins, 1980; Cant et al., 1993). In
addition, fat supplementation may not be ﬁnancially feasible for some dairy producers.
Fat has 2.25 times as much energy as starch does, but fat supplementation can cost two to

three times more than cereal grains per calorie.

Non—structural carbohydrates are other major sources of energy for dairy cows.

However, starch cannot be fed beyond the buffering capacity of the rumen because starch
ferments very quickly in the rumen, and rapid fermentation acid production depresses
feed intake by lowering rumen pH and by destroying a stable rumen eco-system.
Previous research (McCarthy et al., 1989; Casper et al., 1990; Oliveira et a1, 1993;

Aldrich et al., 1993, Overton et al., 1995) indicated the negative relation between rapidly

fermentable non-structural carbohydrates and DMI (Knowlton et al., 1996). When excess
amount of non-structural carbohydrates are available in the rumen, lactate production
may increase due to rapid growth of starch fermenting microorganisms. In the rumen of
high producing dairy cows compared to that of steers, ruminal microorganisms grow
much more rapidly due to faster liquid dilution rate. Streptococcus bovis produces
lactate with a rapid turnover of ruminal digesta, although this organism ferments starch to
formate, acetate, and ethanol at slower growth rate (Russell et al., 1981; Russell and
Allen, 1983). Due to the activity of rapidly growing starch fermenters, lactate
accumulates in rumen ecosystem and lowers ruminal pH. Lactate producers are more
tolerant to lower rumen pH than cellulolytic microorganisms, and they dominate in the
rumen by producing more lactate and making ruminal pH even lower (Slyter, 1976).
Excess ruminal lactate concentration results in rumen acidosis and depression of DMI.
The diet of high producing dairy cows cannot largely rely on non-structural

carbohydrates as an energy source.

Optimum amount of starch fermentation in the rumen depends upon the buffering
capacity of the rumen because fermentation acid production needs to be balanced with its
removal to maintain stable rumen environment (Allen, 1997). Dairy cows require
physically effective NDF in diets to maintain rumen function. Physically effective ﬁber
should be distinguished from effective ﬁber. Effective ﬁber is determined by the
difference in milk fat content when a non-forage source NDF replaces forage NDF in a
dairy diet, whereas physically effective ﬁber is determined by its stimulatory effect on

chewing (Mertens, 1997). The contribution of dietary NDF to buffering capacity in the

rumen can be evaluated by chewing time because rate of saliva ﬂow is higher during
chewing than resting (Cassida and Stokes, 1986). Saliva contains bicarbonate and
phosphate ions which buffer the fermentation acids in the rumen (Bailey and Balch,
1961). Since the chemical composition of saliva is relatively constant (Erdman, 1988),
buffering capacity of the rumen is mainly determined by the quantity of saliva ﬂow or
chewing time. On the other hand, effective ﬁber does not directly measure the effect of
dietary NDF on rumen fermentation. Milk fat content may be confounded by metabolic
status of animals because a variable amount of milk fat is derived from mobilized body
fat, especially in early lactation (Mooney and Allen, 1997). Physically effective ﬁber
more accurately measures the contribution of dietary NDF to buffering capacity in the

rumen.

Dairy rations should contain at least 25% NDF, and more than 75% of the dietary NDF
should come from forage sources (NRC, 1989). Dietary forage NDF concentration is
more related to rumen pH than dietary NDF concentration (Allen, 1997). Particle length
of dietary ﬁber is closely related to chewing activities (Santini et al., 1983). Finely
ground forage may be less effective at stimulating saliva ﬂow into the rumen. The effect
of non-forage source NDF on total chewing time varies depending on the particle size of
a forage to be compared with (Mooney and Allen, 1997). However, physical
effectiveness of non-forage source N DF is generally less than that of forage NDF because
of smaller particle size and faster passage rate (Grant, 1997). Adequate amount of forage

NDF is needed to maintain rumen function.

10

The relationship between forage fermentability and physical effectiveness of fiber is not
clearly understood. Beauchemin (1991) and Nelson and Satter (1992) reported decreased
chewing activities for diets containing alfalfa harvested at early maturity than at late
maturity. Grant et al. (1995) found that total chewing time per kg NDF intake was lower
for brown midrib sorghum silage which is higher in NDF digestibility than normal
sorghum silage. On the other hand, Robinson and McQueen (1997) reported no

treatment effects of forage NDF fermentability on chewing activity.

Effects of Brown Midrib Mutant Forages on Animal Performance

Digestibility of forage NDF could be a key factor to maximize DMI of high producing
cows. Increasing feed intake with adequate ﬁber supply is a challenge. If dietary NDF is
not sufﬁcient, decreased chewing activity results in lower ruminal pH, leading to
decreased cellulolytic bacteria activities and increased risk of rumen acidosis. If dietary
NDF content is high, DMI may be limited by physical ﬁll in the rumen. Both situations
depress voluntary dry matter intake. When physical ﬁll limitation to maximum DMI
exists, enhanced ease of NDF hydrolysis may stimulate rapid disappearance of the NDF
fraction from the rumen, reduce physical ﬁll in the rumen, and allow greater voluntary
feed intake (Dado and Allen, 1995). Grant et al. (1995) and Dado and Allen (1996) fed
silages with similar NDF and CP content but different NDF digestibility to lactating dairy
cows, and found signiﬁcant increases in DMI and milk production for diets with higher

NDF digestibility.

11

Potential extent of NDF digestion and rate of digestion are partly determined by the
lignin content of a forage since lignin is indigestible (Van Soest, 1994). The effect of
NDF digestibility on voluntary DMI can be studied by using brown midrib mutant
forages. Brown midrib mutant forages are signiﬁcantly lower in lignin content and
higher in NDF digestibility than normal control forages (Chemey et al., 1991). The
effect of brown midrib 3 (bm3) corn silage on the livestock performance has been studied
extensively since the early 1970's. One experiment with outstanding performance
difference was reported by Muller et al. (1972). They compare an F2 population of
maize which segregated for bm3 with the F2 population of phenotypically normal maize
and a commercial hybrid. The ears were removed before ensiling so that different level
of grain yield would not affect the results. NDF concentration was about 60% for each
silage. Eighteen crossbred ram lambs were randomly assigned to the diets of bm3, the
normal counterpart, and another commercial hybrid. Lignin concentrations of silage
were 4.5%, 6.8%, and 6.9% for bmr3, normal, and another commercial hybrid,
respectively. They reported that 29% greater (P<.Ol) voluntary DMI for bm3 corn silage
than for normal silage, when fed to lambs ad libitum. The apparent digestibility of DM
(P<. 10) and NDF (P<.05) were higher for bm3 corn silage. They concluded that the less
ligniﬁed cell wall in the bm3 corn silage resulted in faster and higher extent of ruminal

digestion which is associated with greater dry mater intake.

The effects of bm3 corn silage on performance of lactating dairy cows were evaluated
(Table 1). Frenchick et al. (1976) reported that bm3 corn silage increased milk yield by

0.66 kg/d. Keith et al. (1979) found .9 kg and 1.0 kg more FCM yield when cows were

12

fed bm3 silage at 75:25 and 60:40 forage to concentrate ratio, respectively. Cows fed
bm3 corn silage increased BW gain. Rook et al. (1977) found that cows fed the diet with
bm3 corn silage gained 40 kg more BW than cows fed the control diet during the
experimental period from the 6th week to 13th week after calving. Absorbed nitrogen
and retained tissue nitrogen tended to be higher for cows fed bm3 corn silage. Similarly,
Sommerfeldt et al. (1979) reported that cows fed bm3 corn silage gained 58.4 g/d while

cows fed control corn silage lost 47.3 g/d.

Similar research has been conducted for sorghum and pearl millet with brown midrib
(BMR) mutant (Table 2, 3). The most pronounced BMR effect on lactating dairy cows
was recently observed by Grant et al (1995). They compared a BMR sorghum silage
with another normal sorghum hybrid, and reported a 24% greater dry matter intake and a
46% more FCM yield for cows fed BMR sorghum silage. Total volatile fatty acid
concentration in the rumen was higher, and the rate of forage passage was faster for cows
fed BMR sorghum than normal sorghum. Although a sorghum silage is generally
cosidered to be nutritionally inferior to alfalfa or corn silage (Lance et al., 1964), BMR
sorghum hybrids were shown to be as competitive as those forages (Grant et al., 1995;
Lusk et al., 1984). Cherney et al. (1990) reported BMR mutation in pearl millet also
improved digestibility, DMI, and ADG of castrated ram. The effect of BMR pearl millet
on dry matter intake and weight change was more speciﬁcally observed for the second
cuttings. They also observed that regrowth of pearl millet BMR was more palatable to

grazing lambs than the control; lambs spent 2.6 times more time on grazing BMR plots.

13

The effects of brown midrib forages on animal performance were not consistent in these
previous experiments although general animal responses were positive. Dry matter
intake was reported to be higher for animals fed brown midrib mutant plants (Muller et
al., 1972; Rook et al., 1977; Block et al., 1981; Chemey et al., 1990; Grant et al., 1995);
however, other research reported that DMI was not different between brown midrib
forages and control forages (Frenchick et al., 1976; Keith et al., 1979; Sommerfeldt et al.,
1979; Wedig et al., 1987). Rook et al. (1977) and Block et al. (1981) used early lactation
cows, and observed greater DMI for cows fed bm3 corn silage. High producing dairy
cows in early lactation usually experience negative energy balance, and physical ﬁll more
dominantly limits voluntary feed intake, especially when fed high NDF diet (Dado and
Allen, 1995). Enhanced N DF digestibility can stimulate rapid rumen turnover, allowing
greater feed intake. On the other hand, maximum DMI of heifers and steers (W edig et
al., 1987) or mid to late lactation cows (Kieth etal., 1979; Frenchick et al., 1976) may not
be limited by physical ﬁll, but more likely by chemostatic factors, therefore more
digestible ﬁber might not stimulate voluntary DMI. The most pronounced effects of
brown midrib forages were observed by Muller et al. (1972) and Grant et al. (1995).
Muller et a1 (1972) fed 60% NDF diet, removing ears from corn silage in order to detect
more accurate effect of bm3 treatment. Likewise, Grant et al. (1995) fed mid lactation

cows at relatively higher dietary NDF (40% of dietary DM).

When forage is supplemented with other feeds, the effect of brown midrib forages might
be masked. Frenchick et a1. (1976) did not observe signiﬁcant DMI difference between

bm3 corn silage and control, and attributed their results to the fact that forage made up

14

only half of the diet in their experiment. When heifers were offered bm3 corn silage with
corn grain at 0%, 1%, and 2% of body weight, DMI was signiﬁcantly higher only when
fed without corn grain (Kieth et al., 1981). Feeding more starch in diets might change
rumen environment and diminish the effect of enhanced NDF digestibility. Source of

starch and processing methods can affect ruminal ﬁber digestion.

Responses to brown midrib treatments in apparent total tract NDF digestibility also
varied among experiments. Greater total tract NDF digestibility was reported for brown
midrib treatments (Muller et al., 1972; Sommerfeldt et al., 1979; Stallings et al., 1982;
Weller and Phipps, 1986; Fritz et al., 1988; Chemey et al., 1990; Grant et al., 1995).
However, some experiments found that total tract digestibility was not improved by
brown midrib forages (Rook et al., 1977; Lusk et al., 1984; Wedig et al., 1988). Similar
total tract digestibility within comparisons was attributed to greater DMI for the bm3
treatment (Rook et al., 1977). Total tract digestibility declines as feed intake increases
(T yrrell and Moe, 1975). Greater DMI for brown midrib treatments can be associated
with faster rate of digesta passage, diminishing potential digestibility. Wedig et al.
(1986) implied that compensatory postruminal digestion might make it more difﬁcult to

see the positive effect of BMR treatment on apparent total tract digestibility.

Some researchers reported increased milk yield (Frenchick et al., 1976; Kieth et al., 1979;
Grant et al., 1995), while the others did not (Rook et al., 1977; Sommerfeldt et al., 1979;
Block et al., 1981; Stallings et al., 1982). Instead, Rook et al. (1977), Sommerfeldt et al.

(1979), and Block et al. (1981) observed that cows fed bm3 corn silage gained more BW.

15

Block et al. (1981) concluded that energy intake was not limiting milk production in their
experiment because extra energy was directed towards BW gain. Data of rumen pH and
volatile fatty acid proﬁle did not give clues to why the extra energy from bm3 corn silage
was utilized for BW gain (Frenchick et al., 1976; Rook et al., 1977; Sommerfeldt et al.,
1979). Milk fat depression was reported when cows were fed bm3 corn silage (Frenchick
et al., 1976; Rook et al., 1977; Block et al., 1981). Ruminal acetate to propionate ratio
(Block et al., 1981) and rumen pH (Frenchick et al., 1976; Block et al., 1981) were lower
for the cows fed bm3 corn silage. However, Grant et al. (1995) observed that milk fat
concentration is improved by feeding BMR sorghum silage. Inconsistent energy

utilization for bm3 treatments cannot be explained clearly.

Biochemistry of Brown Midrib Mutations

Lignin is amorphous bio-polymer. It plays an important role to maintain structural
integrity of plant tissues, but inhibits cell wall degradation in the rumen because of the
strong covalent bonds with structural carbohydrates. Lignin synthesis in plants begins
with deamination of phenylalanine and tryosine (Chemey et al., 1991). Phenylpropenoic
acids are metabolized via hydroxylation and methylation to p-coumaric, ferulic, and
synapic acids. Then, those phenolic acids are reduced to their respective aldehydes, and
subsequently to p-coumaryl, coniferyl and sinapyl alcohol. Those monomeric alcohols
are condensed to polyphenols which are more resistant to degradation by rumen-
microorganisms. The metabolized acids, aldehydes, and alcohol are involved in lignin
formation. p-Coumaric and ferulic acids are most commonly found in forage that

animals consume, and they inhibit the growth of aerobic and anaerobic bacteria (Jung and

16

Fahey, 1983). In addition, those phenolic monomers especially p-coumaric acid, are
cross-linked to structural carbohydrates and decrease the extent of digestion (Burn'tt et
al., 1984). Lignin content is closely related to the ambient temperature, and increases as

the physiological maturity of plant proceeds.

Brown midrib mutation modiﬁes the lignin content of plants. Jorgenson (1931) noticed
that brown midrib mutation reduces lignin content compared to near isogenic counterpart.
Phenotypically, reddish-brown pigmentation is associated with ligniﬁed tissues,
especially on the leaf midrib. Reduced lignin content possibly contributes to higher
forage quality because of more available energy and improvement in digestibility.
However, it had not been studied extensively until the 1970's because the trait reduced
grain yield, and economical value of corn silage was lower than corn grain. The brown

midrib trait had been considered to be less important by plant breeders.

Research conducted since early 1970‘s showed that the lignin content of a brown midrib
forage is signiﬁcantly lower than its normal counterpart (Chemey et al., 1991). Lignin
composition is also inﬂuenced by the brown midrib mutation. A brown midrib mutation
in sorghum stops aldehyde intermediates being incorporated into the polymer due to the
lack of enzymatic activities and results in their accumulation (Bucholtz et al., 1980).
Wedig et al. (1989) observed twice as much vanillin in a BMR sorghum as in normal
counterpart. Phenolic acid composition in bm3 is also different from normal, lower
concentration of p-coumaric acid and similar concentration of ferulic acid has been

observed (Fritz et al., 1990; Gerhardt et al., 1994). The lower ratio of p-coumaric acid to

17

ferulic acid in bm3 possibly explains the more extensive ruminal degradation of cell wall
components (Hartley and Jones, 1978). Lignin synthesis is associated with deamination
of phenylalanine and tyrosine, therefore brown midrib mutations may inﬂuence amino
acid composition of plants. Gordon and Neudoerffer (1973) reported higher
concentration of aspartic acid and lower concentration of phenylalanine and alanine in
young bm3 maize (74 days old). Lower concentration of phenylalanine and similar
concentration of tyrosine indicated that a brown midrib l mutation may block metabolism
between phenylalanine and prephenic acid which is a precursor of phenylalanine and

tyrosine. Subsequently ligniﬁcation may be blocked as well.

Cherney et al. ( 1988) studied the chemical composition of brown midrib mutant and
normal counterpart for pearl millet, sorghum, and corn. They used near-isogenic inbreds,
near-isogenic hybrids, and chemically induced BMR mutants for the comparisons within
sorghum lines, maize lines, and pearl millet lines, respectively. They found lower NDF
and lignin concentrations and higher crude protein concentration for brown midrib plants
than for normal counterparts of each plant. Fritz et al. (1988), Block et al. (1981), and
Keith et al. (1979) reported that the brown midrib forages were higher in crude protein
than normal types. Fritz et al. (1988) and Cherney et a1. (1990) observed lower NDF
concentration for BMR sorghum x sudangrass and pearl millet, respectively. Allen et al.
(1997) evaluated 14 different corn hybrids with and without bm3 mutation, and found
NDF content was not affected by bm3 mutant and CP content was slightly higher for bm3
corn silage. However, differences in chemical composition between brown midrib

mutations and isogenic counterparts have not been consistent for all experiments.

18

p-Coumaric acid is considered to be more important than ferulic acid in crosslinking of
lignin to hemicellulose (Burritt et al., 1984). p-Coumaric acid increased dramatically
with plant maturity, while ferulic acid in lignin changed little (Chaves et al., 1982). p-
Coumaric acid was found to be higher for normal than for a BMR genotype (W edig et al.,
1989b). The extent of hemicellulosic monosacharide digestion was negatively correlated
to the level of p—coumaric acid in plants (Wedig et al., 1989a, b). Ester-bonding occurs
between phenolic compounds and uronic acid of hemicellulose for grass (Harkin, 1973),

and it inhibits the ruminal degradation of hemicellulose (Van Soest, 1994).

Hemicellulose composition might be altered by brown midrib mutations. The high ratio
of xylose to arabinose indicates a more polymerized xylan and less branching with
hemicellulosic monosaccharides (Bittner, 1983). This ratio can be a function of forage
digestibility since it increases as plants mature (Wedig et al., 1989a). Normal genotypes
of sorghum were reported to have higher xylose to arabinose ratio (Cherney et al., 1986;
Wedig et al., 1989b). However, difference in hemicellulose modiﬁcation may be not
detectable for immature forages (W edig et al., 1989a). The difference in hemicellulose
composition between brown midrib and normal control has not been studied for maize
and pearl millet, and there is insufﬁcient data to say a brown midrib mutation changes

hemicellulose composition (Cherney et al., 1991).

Even though NDF digestibility is higher for brown midrib forages, a unit of brown midrib

lignin inhibits cell wall digestion more than normal lignin does. A higher degree of

19

cross—linking in the propane side chain of brown midrib lignin was observed (Gordon and
Grifﬁth, 1973). The ratio of indigestible residues to ADL was 37% higher for a brown
midrib mutant than for normal control, leading Thorstensson et al. (1992) to imply that a
unit of brown midrib lignin inhibits NDF digestion more than normal lignin does. Other
research reported this ratio was 55% greater for BMR than for normal (Grant et al.,
1995). A lower concentration of cinamic acids results in more condensed lignin (Wedig
et al., 1989a) and digestibility of brown midrib lignin was reported to be lower (Cherney

et al., 1986).

The effect of BMR sorghum on in vitro digestibility was observed as early as four weeks
after planting (Hana et al., 1981). Grand et al., (1985) reported that lower lignin content
of bm3 corn was detectable for 20 day old corn. However, corn silage is generally
harvested close to physiological maturity, so the bm3 effects on digestibility at late
maturity are more important. Ramadhan et al. (1976) observed that the effect of bm3 on
decreased lignin content and improved digestibility persisted in corn stalk residues even
after grain harvest. However, the effect of bm3 gene on improvement in nutritional
quality is speciﬁc for vegetative portion of a plant. Keith et al. (1980) reported no effect

of bm3 grain on performance of growing rats.

There are four distinct brown midrib loci in maize (Kuc' and Nelson, 1964), and they are
labeled bml , bm2, bm3, and bm4. Muller et al., (1971) found that acid detergent
permanganate lignin concentration of normal, bml, bm3, and bml/bm3 was 6.08%,

5.13%, 4.37%, and 4.60%, respectively. Lechtenberg et al. (1972) compared digestibility

20

of stover from mutant inbred corn plants harvested 35 days after silking. They reported
in vitro dry matter disappearance of F2 normal, bml , bm2, bm3, bml/bm3, and bm4 was
55.1%, 59.0%, 53.1%, 60.6%, 67.4% and 54.2%, respectively. They concluded that
genotype of bm3 was the most promising mutant allele, and most of the following
research has focused on bm3 locus of brown midrib maize mutant. On the other hand,
numbers associated with the gene symbol for sorghum brown midrib genotypes does not
mean that a brown midrib mutant allele is located on a particular locus. In the
experiment by Porter et al. (1978), brown midrib mutants of sorghum were chemically
induced. Nineteen independently occurring brown midrib mutants were identiﬁed and

were numerically designated BMR] to BMR19.

Brown midrib mutations have been identiﬁed for maize, sorghum, sudangrass, and pearl
millet. This trait can be induced chemically. Porter et al. (1978) soaked sorghum seeds
with diethyl sulfate, and induced BMR mutation. However, Cherney et a1. (1991)
reported that he could not develop a brown midrib mutation for other forage species, and
implied that brown midrib trait seems to be occurred only for C4 species. Since the
effects of brown midrib mutation is phenotypically expressed by the homozygous
recessive, it may not be found in the genomes of polyploid forage species (Vogel and

Sleper, 1994).

Agronomic Aspects of Brown Midrib Forages
Although nutritional advantages to animals exist, a brown midrib forage is agronomically

inferior to normal genotype due to a less ligniﬁed cell wall. Zuber et al. ( 1977) measured

21

crushing strength and stalk section weight of bm3 and normal corn. They suggested that
farmers harvest at near physiological maturity to prevent stalk lodging since their study
showed bm3 corn had 17 to 26% lower crushing strength and 8 to 14% lower stalk-
section weight than normal control. Brown midrib mutation affects early vigor and
growth rate, too. The average linear accumulation rate of total dry matter was 15%
higher for normal genotypes. Nicholson et al. (1976) implied that a brown midrib
genotype may be more susceptible to F. moniliforme and other fungi associated with stalk

and ear rots.

Silage yield may be lowered by brown midrib mutation. Brown midrib maize was
observed to delay silking by 3 days as average and decrease the ear proportion of total
dry matter by 4 to 6% (Weller et al., 1985). The average grain yield, stover yield, and
whole plant yield of brown midrib genotypes were 77%, 90%, and 84% of normal
genotypes, respectively (Miller et al., 1983). Another research group reported that the
bm3 maize yielded 20% lower grain and 17% lower stover than each isogenic normal
counterpart on average (Lee and Brebaker, 1984). Recently, Allen et a1. (1997) reported
bm3 decreased corn silage yield by 12% on average. Miller et al. (1983) as agronomists
concluded that normal genotypes offered more potential for silage breeding programs.
However, the response to a brown midrib mutant varies greatly by hybrid. Even though a
brown midrib mutant generally reduces yield compared to normal control genotype, some
hybrids yielded as much as normal counterpart did (Miller et al., 1983). Gentinetta
(1989) reported some bm3 hybrids, which contained signiﬁcantly lower lignin, produced

as much grain and stover as normal genotypes did. It might mean that forage breeders

22

can develop a hybrid that is more desirable as an animal feed while maintaining

agronomic strength.

Evaluation of speciﬁc effects of brown midrib mutation requires two isogenic plants that
differ only by the mutation. A brown midrib mutation is incorporated into hybrids by
backcross breeding (Allard, 1960; Stoskopf, 1993). Backcross breeding needs a recurrent
parent and a donor parent; only the brown midrib mutation should come from a donor
parent, and all the other genes must be from a recurrent parent (each hybrid). Therefore,
the completion of conversion depends upon number of backcross (BC). The donor parent
contributes less to the plant genetics as a backcross breeding program proceeds. The
proportion of genes from the donor parent is reduced by one-half following each

generation of backcrossing, and expressed by the formula:

(1 / 2)"n+l where n is the number of backcrosses.
In the absence of selection, proportion of the donor to the genome is (1/2)"6 = 0.0156 in
BCS and (1/2)"7 = 0.0078 in BC6 . After plants are backcrossed ﬁve or six times, they
still have 1.56% or 0.78% of genome from the donor parent, respectively. However, this
equation is a simpliﬁcation for convenience. Depending upon how genes are distributed

over the chromosomes, the number of genes from donors left in the plants varies.

Linkage needs to be considered because some genes are located on the same chromosome
where the brown midrib mutation is (Stoskopf, 1993). Linkage occurs when a gene is 50

map units or less from an adjacent gene, but genes more than 50 map units apart are not

23

linked and they segregate as if they were on different chromosomes. The probability of
eliminating undesirable genes is given by the formula:

1 - (1 - p )""+1 where p is the recombination fraction (p=1/map units)

n is the number of backcrosses.

If genes are 10 units away from bmr, the probability for eliminating those genes after ﬁve
backcrosses is only about 47% [ 1 - (1 - 0.1)A6 ]. Ten to twelve backcrosses may be
required to produce m isogenic line which is different from recurrent parent by only
one gene (Stoskopf, 1993). Although six backcross breeding with rigid selection is
considered to be sufﬁcient to get a near isogenic plant (Allard, 1960), the required
number of backcrosses varies depending on species, a trait which is transferred, breeding

methods, and how conservative you want to be.

Summary

Maximizing DMI is an important management goal for dairy producers and nutritionists.
Physical ﬁll is a factor limiting maximum DMI of high producing dairy cows. Enhanced
NDF digestibility may allow more feed intake by rapid turnover of rumen digesta. A
brown midrib forage is lower in lignin content and higher in NDF digestibility, and has

great a potential in dairy cattle diets despite agronomic defects.

24

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25

Table 2. Effects of brown midrib 3 (BMR) sorghum silage on animal performance

 

 

Animal DMI Milk Yield Milk Fat Body F :C
Wt. (NDF%)
Wedig (1) Steer + 0.35 kg --- --- --- (66%)
(2) Heifer + 0.20 kg --- --- --- (66%)
Fritz (R x G) H-non lact. - 0.20 kg --- --- --- (75%)
(R x P) H-non lact. + 2.10 kg ** --- --- --- (75%)
Grant et al. H-mid lact. + 21% * + 5.7 kg * + 0.54% * --- (40%)

 

* signiﬁcantly differ (P < 0.10)
** signiﬁcantly differ (P < 0.05)

Table 3. Effects of brown midn'b 3 (BMR) pearl millet silage on animal performance

 

 

Animal DMI Milk Yield Milk Fat Body Wt. F:C
(NDF%)
lst cutting Sheep + 0.3% --- --- + 2 g (66%)
2nd cutting Sheep + 37.8% *** --- --- + 172 g ** (66%)

 

** signiﬁcantly differ (P < 0.05)
*** signiﬁcantly differ (P < 0.01)

26

CHAPTER 2

EVALUATION OF THE IMPORTANCE OF FORAGE NDF
DIGESTIBILITY: EFFECTS ON DRY MATTER INTAKE AND

MILK YIELD OF DAIRY COWS

ABSTRACT
Effects of NDF digestibility on performance of dairy cows were evaluated statistically
using treatment means for nineteen sets of forage comparisons reported in the literature.
All comparisons reported signiﬁcant differences in NDF digestibility of forages in vitro,
in situ, or(and) in vivo. Treatment means were blocked by comparison within an
experiment. Statistical model included random effect of block, ﬁxed factorial effect of
NDF digestibility (high or low), and dietary NDF level as a covariate. Enhanced NDF
digestibility of forage signiﬁcantly increased DMI and milk production. One unit
increase in N DF digestibility in vitro or in situ was associated with 0.15 kg increase in
DMI and 0.24 kg increase in FCM(4%). Within this set of comparisons, DMI and FCM
(4%) were inﬂuenced more by NDF digestibility than by dietary NDF level. The

difference in NDF digestibility between the treatments was greater when measured in

27

vitro or in situ than in vivo. N DF digestibility in vitro or in situ might be a better
indicator of DMI than NDF digestibility in vivo because forages with high in vitro or in
situ NDF digestibility might have shorter rumen retention times, allowing greater DMI at
the expense of NDF digestibility in vivo. Digestibility of NDF is an important parameter
of forage quality.

(Key words: ﬁber digestibility, intake, fill limitations, forage quality)

Abbreviation key: FC = forage to concentrate ratio.

INTRODUCTION
Dairy cows require effective N DF in diets to maintain rumen function and maximize milk
production. Dairy rations should generally contain at least 25% NDF and a large
proportion of dietary NDF should come from forage sources (NRC, 1989). Because
dietary forage NDF concentration is closely related to chewing activity and is a
determinant of rumen pH of dairy cows (Allen, 1997), dairy rations are often formulated
to a speciﬁc N DF content and(or) forage NDF content. However, NDF content of forage
varies widely depending upon species, maturity, and growing environment. Forages are
routinely analyzed for NDF content because of its variation as well as its nutritional

importance.

Digestibility of NDF may be another important parameter of forage quality because
forage NDF varies widely in its degradability in the rumen (N ocek and Russell, 1988;
Allen and Oba, 1996) and NDF digestibility possibly inﬂuences animal performance.
Although dairy cows require forage NDF in diets, feeding excess dietary N DF often

28

limits voluntary feed intake by rumen ﬁll. Mertens (1987) suggested that DMI of dairy
cows can be predicted by dietary NDF because of positive relationship between NDF and
rumen ﬁll. Therefore, enhanced ease of NDF hydrolysis may stimulate rapid
disappearance of the NDF fraction from the rumen, reduce physical ﬁll in the rumen, and
allow greater voluntary feed intake (Allen and Oba, 1996). Grant et al. (1995) and Dado
and Allen (1996) fed silages with similar NDF and CP content but different NDF
digestibility to lactating dairy cows, and found signiﬁcant increase in DMI and milk
production. Several experiments comparing brown midrib mutant corn silage with its
isogenic normal control reported enhanced animal performance for cows fed brown
midrib corn silage (Frenchick et al., 1976; Rook et al., 1977; Kieth et al., 1979;
Sommerfeldt et al., 1979; Block et al., 1981; Stallings et al., 1982). Brown midrib
hybrids are consistently higher in in vitro NDF digestibility due to less ligniﬁcation of

plant cell wall (Cherney et al., 1991).

Isolating the speciﬁc effect of NDF digestibility on animal performance is difﬁcult.
Although many experiments have reported NDF digestibility data, interpretation of
results is difﬁcult because of a variety of confounding factors. Experiments comparing
forages that differ only in NDF digestibility are rare and it is difﬁcult to conﬁrm that
other factors which could potentially affect animal performance did not vary. If two
forages are compared at a different forage to concentrate ratio (FC), other dietary factors
such as ruminal starch digestion might affect DMI and(or) milk production. If the
forages are compared at similar PC but different dietary NDF levels, results are
confounded by differences in ﬁlling effects in the rumen from different NDF

concentrations.

Despite technical difﬁculties for evaluation of NDF digestibility, it is necessary to

quantify the effect of NDF digestibility on DMI and milk production because of its

29

potential economic importance. One approach to evaluate the effect of N DF digestibility
is a statistical analysis across forage studies. An appropriate statistical model enables
confounding factors found in individual experiments to be treated as covariates, then
allows development of relevant interpretations about the effect of NDF digestibility on
animal performance. The objectives of this analysis are to evaluate and quantify the
effect of NDF digestibility on DMI and milk yield of dairy cows based upon the

statistical analysis of treatment means reported in the literature.

MATERIALS AND METHODS

A database was developed using treatment means from experiments which reported
signiﬁcant differences in digestibility of NDF among forages either in vivo, in situ, or
(and) in vitro. Those experiments which compared legume with grass were excluded
from this statistical analysis because of much greater differences in NDF, forage NDF,
and (or) starch concentration for diets composed of these forages. In addition,
experiments which did not report dietary NDF% and N DF digestibility either in situ or in
vitro were excluded from the database. Nineteen sets of forage comparisons (n=38) taken
from eight articles in Journal of Dairy Science were utilized for this statistical analysis
(Table 1; Llamas-Lamas and Combs, 1990; Arieli and Adin, 1994; Orozco-Hemandez et
al., 1994; Ruis et al., 1995; Grant et al., 1995; Dado and Allen, 1996, Sheperd and Kung
Jr., 1996; Oba and Allen, 1998).

Llamas-lamas and Combs (1990) evaluated the effect of alfalfa maturity on ﬁber

utilization of mid-lactation (120 DIM) cows. Six cows were fed three diets based on

30

alfalfa hay harvested at early vegetative, late bud, and full bloom maturities. Diets were
formulated for 29.4% NDF and 19.3% CP and FC ratios among the treatments varied
from 45:55 to 68:32. Arieli and Adin (1994) utilized 168 cows under commercial dairy
operation to evaluate the effect of wheat crop maturity on lactational performance.
Experimental diets contained either early or late cut wheat silage and diets were
formulated to 33% NDF. Orozco-Hernandez (1994) fed 18 mid-lactation cows with two
timothy silages which were either wilted or direct-cut and treated with formic acid. The
silage treated with formic acid was higher in degradable NDF fraction in situ, and the
experimental diets contained approximately 51% NDF. Ruiz et al. (1995) utilized 48
mid-lactation (110 DIM) cows to compare four different forage species: corn silage,
dwarf elephant grass, forage sorghum, and berrnuda grass. They fed each forage at
dietary NDF levels of 31%, 35%, and 39% in a 4 x 3 factorial arrangement of treatments.
The dietary CP content and FC ratios were not constant within comparisons. Grant et a1.
(1995) fed 12 mid-lactation (90 DIM) cows with corn silage, normal sorghum silage,
brown midrib sorghum silage, and alfalfa silage at 65% of dietary DM. Except for the
alfalfa based diet, each diet contained approximately 40% NDF and 17% CP. Dado and
Allen (1996) prepared two alfalfa silages with similar NDF content (approximately 40%)
but different in NDF digestibility (38.3% vs. 40.2% after 24b in vitro fermentation).
They fed 12 early-lactation (13 DIM) cows with the silages at 83.3% of dietary DM in a
crossover design. Dietary NDF content and CP content were approximately 36% and
23.5%, respectively. In the study of Sheperd and Kung (1996), corn silage treated with
cellulase enzyme was reported to decrease in vitro NDF digestibility (48.1 vs. 54.0%)
after 196 day ensiling. They fed the enzyme treated corn silage and control silage to 19
mid-lactation (80 DIM) cows. The experimental diets contained approximately 36%
NDF and 16% CP, and FC ratio was 50:50. Oba and Allen (1998) compared brown

midrib corn silage with its isogenic normal control. They fed 32 mid-lactation (89 DIM)

31

cows in a crossover design. The experimental diets contained about 31% NDF and

19.6% CF at 56:44 FC ratio.

Reported treatment means of DMI, milk production, milk composition, BW change per
day, rumen pH were collected as continuous response variables, and the data were
blocked by comparison within a study to remove variation among the different types of
experiments. Digestibility of NDF was a ﬁxed class variable, classiﬁed as either high or
low, and the statistical model included forage to concentrate (FC) ratio, and the linear and
quadratic effects of dietary NDF as covariates. The ﬁt model procedure of JMP® (SAS

Inst. Inc., Cary, NC) was used for statistical analysis with the following model:

Yijk = U + Bi + F]. + Ncov + NNcov + FCcov+ eijk
where Y m, = response variable (DMI, milk yield, FCM, BW change, etc)

U = overall mean
Bi = random effect of block (i = l to 19)
Fj = ﬁxed factorial effect of ﬁber digestibility (j = high or low)
Ncov = effect of dietary NDF as a covariate
NNcov = quadratic effect of dietary NDF as a covariate
FCcov = effect of dietary forage to concentrate ratio as a covariate

e 1,1. = residual, assumed to be normally distributed

32

RESULTS AND DISCUSSION

DMI and Milk Production

A reduced model without the covariate effect of FC ratio will be discussed since FC ratio
was not signiﬁcant for all response variables. Cows fed diets with more digestible forage
NDF increased DMI (22.0 kg vs. 20.9 kg; P<0.001). However, for some comparisons in
this data set, forages with high NDF digestibility were fed at lower dietary NDF content
than their counterpart treatments, so diet NDF content was included as a covariate. The
effect of dietary NDF content as a covariate was signiﬁcant and negative probably
because of its effects on rumen ﬁll. However, inclusion of the effect of dietary NDF as a
covariate did not negate the effect of NDF digestibility of forage (Table 2); rumen ﬁll is
affected by both N DF content of the diet and by forage NDF digestibility.

Diets with more digestible NDF resulted in greater milk yield (28.9 kg vs. 27.3 kg;
P<0.001) and FCM(4%) yield (26.2 kg vs. 24.5 kg; P<0.006). Milk composition was not
different between the treatments although milk lactose content tended to be higher
(P<0.11) for high NDF digestibility treatments. The effect of dietary NDF content as a
covariate was signiﬁcant for both milk production and FCM(4%) production. Milk
production increased as dietary NDF content decreased. The quadratic effect of dietary
NDF content was signiﬁcant (P<0.04) for FCM(4%) yield, suggesting that either very
low or very high dietary NDF content reduced FCM(4%). Energy output in milk may be
limited by energy intake when dietary NDF content is high, but also affected by a
reduction in milk fat synthesis when dietary NDF level is low. However, inclusion of the
effect of dietary NDF as a covariate did not negate the effect of N DF digestibility of
forage. These results indicated that forages with high NDF digestibility improved milk

production by increasing energy intake.

33

Maximizing energy intake is essential for maximum milk production. Energy intake can
be increased either by increasing energy density of diets or by increasing DMI. Efforts to
increase energy density of diets by substituting grain for forage may not always result in
increased energy intake because excess fermentation in the rumen depresses voluntary
feed intake of animals. Forages with high NDF digestibility allow greater DMI when
physical ﬁll limitations to feed intake exist. More rapid hydrolysis of NDF fraction may
allow greater DMI by rapid disappearance of N DF fraction from the rumen by increased

rate of digestion or (and) passage.

Quantifying the Effect of Enhanced NDF Digestibility

Within this data set, differences in NDF digestibility among forage comparisons
determined in vitro or in situ averaged 7.2 units (63.3% vs. 56.1%). However, the
average difference in total tract NDF digestibility between high NDF digestibility and
low NDF digestibility was only 4.8 units (54.8% vs. 50.0%). Although differences in
total tract NDF digestibility in vivo were less than differences measured either in vitro or
in situ, they were positively correlated (P<0.0001 R2=0.72). Although the reason for
smaller differences between treatments for in vivo N DF digestibility is partly because
other sources of NDF diluted the forage NDF of interest in the diet, animal factors might
also be responsible. NDF digestibility is a function of the potentially digestible fraction
and its rate of digestion and rate of passage (Allen and Mertens, 1988). In this statistical
analysis, enhanced NDF digestibility was associated with greater DMI, which might
result in a higher rate of passage (NRC, 1987), reducing time for microbial degradation of
NDF in the rumen. In addition, exposure to acidic conditions in the small intestine and
fermentation in the large intestine in vivo might have reduced differences observed for
fermentation by rumen microbes in vitro or in situ. N DF digestibility measured in vitro
or in situ should more accurately reﬂect forage quality than in vivo NDF digestibility

because in vitro and in situ methods compare forages using a consistent retention time.

34

Because one of the possible beneﬁts of high NDF digestibility is to enhance DMI, forage
NDF digestibility should be evaluated by in vitro or in situ methods which exclude

animal factors.

The average difference of 7.2 units of NDF digestibility was associated with 1.1 kg
higher DMI resulting in 1.6 kg higher milk yield, and 1.7 kg higher FCM(4%). One unit
of enhanced NDF digestibility was positively associated with 0.15 kg of DMI, 0.22 kg
milk yield and 0.24 kg of FCM(4%). Theoretical energy balance calculations show that
this increase in DMI will support FCM(4%) in excess of this amount: One unit of
enhanced NDF digestibility increased DMI by 0.15 kg. Assuming a dietary energy
density of 1.70 Mcal NEL lkg, this increase in DMI is equivalent to 0.26 Meal. The 0.24
kg of increase in FCM(4%) requires 0.18 Mcal NEL Iday (NRC, 1989), which is about
70% of available extra energy. Although amount of energy from extra NDF digestion in
vivo varies depending on levels of DMI (Oba and Allen, 1998), it may contribute to
increasing energy intake even more. Energy not accounted for might have been utilized
for BW gain. In this statistical analysis, animals fed the diet with forages of higher NDF
digestibility tended to gain more BW (P<0.12).

CONCLUSION

Enhanced NDF digestibility of forage improves DMI and milk production of dairy cows.
NDF digestibility should be measured more routinely to assess forage quality. This
information is particularly important to accurately rank forages that might vary in yield
and different quality traits. NDF digestibility data from in situ or in vitro is more
valuable than in vivo data since it reﬂects forage quality more accurately by excluding

animal factors.

35

 

 

 

 

 

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38

Table 2. Least square means for animal performance by different levels of ﬁber
digestibility (n=38) taking dietary NDF% as a covariate. Calculated from 19 sets of
treatment means from 8 articles of Journal of Dairy Science.

 

 

LSM P—value R2

High FD Low FD FD NDF%
DMI 22.0 20.9 0.001 0.05 0.96
Milk 28.9 27.3 0.001 0.03 0.99
FCM(4%) 26.2 24.5 0.006 0.08 0.97
Fat% 3.40 3.34 0.33 0.52 0.88
Protein% 2.99 2.97 0.22 0.19 0.99
Lactose% 5.55 5.41 0.1 1 0.17 0.99
BW(kg/day) 0.36 0.20 0.12 0.16 0.55
Rumen-pH 6.33 6.36 0.71 0.65 0.89
in situ / in vitro NDF
digestibility, % 63.3 56.1 - - -
in vivo total tract NDF
digestibility, % 54.8 50.0 - - -

 

39

CHAPTER 3

EFFECT OF ENEHANCED IN VITRO NDF DIGESTIBILITY OF
CORN SILAGE BY BROWN MIDRIB 3 MUTATION ON DMI AND

MILK YIELD OF HIGH PRODUCING DAIRY COWS

ABSTRACT
Effect of enhanced in vitro neutral detergent ﬁber (NDF) digestibility of corn silage on
dry matter intake (DMI) and milk production was evaluated using 32 Holstein cows in a
crossover design with 28d periods. At the beginning of experiment, cows were 89 days
in milk, and produced 45.6 kg of milk per day. Experimental diets contained (44.6% of
dry matter) either brown midrib (bm3) corn silage or an isogenic normal (control) corn
silage. NDF digestibility estimated by 30b in vitro fermentation was higher for bm3 corn
silage by 9.7 units. NDF and lignin contents were lower for bm3 corn silage by 1.8 units
and 0.8 units, respectively. Diets were formulated for 19.6% crude protein, 31% NDF,
and 56:44 forage to concentrate ratio. Cows were blocked by days in milk, body weight,
milk yield, and body condition score (BCS), and cows within a block were randomly

assigned to each diet. DMI, milk production (3.5% fat-corrected milk), solids-corrected

40

milk, and BCS were 2.0 kg, 2.5 kg, 2.6 kg, and 0.09 units / 28d higher for the cows fed
bm3 corn silage, respectively. N 0 effect of treatment was observed for milk composition
or body weight change. Individual cow milk yield response to bm3 treatment was
positively related to pre-trial milk yield and DMI response tended to be positively related
to pre-tn'al milk yield. Enhanced in vitro NDF digestibility was associated with higher
energy intake, which resulted in increased milk production and body condition.

(Key words: bm3 corn silage, ﬁber digestibility, intake, ﬁll limitations)

Abbreviation key: BMR = brown midrib mutant for sorghum; bm3 = brown midrib

mutant for corn; BCS = body condition score

INTRODUCTION

Maximizing DMI is an important management goal for high producing dairy herds.

Cows in early lactation tend to be in negative energy balance, and energy status of
animals affects health and reproductive performance as well as milk production. Physical
ﬁll is considered to be one of the factors which regulate feed intake of dairy cattle (Allen,
1996). Fibrous fractions of feeds have a greater effect on rumen ﬁll than non-ﬁber
fractions because ﬁber ferments slowly and is retained in the rumen longer. Mertens
(1987) suggested that DMI of dairy cows can be predicted by dietary NDF because of
positive relationship between NDF content and bulk density of feeds. However NDF
varies in its degradability in the rumen. Ruminal digestibility of forage NDF has been
reported to range from less than 35% to over 75% for various forage types (Nocek and

Russell, 1988). Although forage NDF digestibility in vivo varies by animals and feeding

41

conditions, much of the variation could be due to the composition and structural
differences of the forages; in vitro NDF digestibility after 30h incubation was reported to
vary from less than 30% to 60% for corn silage and alfalfa (Allen and Oba, 1996).
Enhanced NDF digestibility is speculated to increase DMI when maximum DMI is
limited by rumen ﬁll. Faster disappearance of the NDF fraction from the rumen by
increased rate of digestion and(or) passage may reduce physical ﬁll in the rumen and

allow greater voluntary feed intake (Dado and Allen, 1995).

Evaluation of the effect of NDF digestibility on animal performance is complex because
it is difﬁcult to obtain two forages which differ only in NDF digestibility. Most
experiments that have had treatment differences for NDF digestibility have had these
effects confounded by differences in dietary NDF level, forage to concentrate ratio,
protein concentration, and(or) other dietary ingredients. Brown midrib mutants
consistently decrease lignin content of forages and increase NDF digestibility (Cherney et
al., 1991). The brown midrib 3 gene in corn hybrids was reported to decrease lignin
content by 1.1 units and increase 30b in vitro NDF digestibility by 8.4 units compared
with isogenic control hybrids with no effect on the NDF content of corn silage (Allen et
al., 1997). A comparison of corn silage from a hybrid converted to the brown midrib 3
mutation by backcrossing with its isogenic control hybrid might minimize the
confounding of differences in NDF content with a signiﬁcant difference in NDF
digestibility. In the 1970's, corn silage with a brown midrib mutation was extensively
studied (Block et al., 1981; Frenchick et al., 1976; Kieth et al., 1979; Rook et al., 1977;

Sommerfeldt, et al., 1979; Stallings et al., 1982). However, dietary NDF content or in

42

vitro NDF digestibility was not reported for most studies. Few studies used a total-
rnixed-ration and concentrates were fed according to milk production. This altered the
dietary NDF content and forage to concentrate ratio of experimental diets, making it
difﬁcult to draw conclusions about the effects of NDF digestibility on animal
performance. In addition, milk production of the cows used in previous experiments was
much lower than what is typical for high producing herds presently, which might have
affected the results because physical ﬁll might have been less of a limitation to feed

intake.

Recently, Grant et al. (1995) offered a sorghum hybrid with the low lignin brown midrib
(BMR) mutation and a different sorghum hybrid without the mutation in a total-mixed-
ration (65% forage, 40% dietary N DF) to mid-lactation dairy cows (90 - 202 DIM). The
BMR hybrid had higher NDF digestibility as measured by in situ and in vitro analyses.
The BMR sorghum with higher NDF digestibility resulted in a 24% increase in DMI and
a 46% increase in FCM. Although cows had relatively low milk yield, they were fed
diets with relatively high NDF content. Physical ﬁll might limit DMI of low producing
cows if dietary N DF content is high enough. However, the effects of enhanced NDF
digestibility on animal performance is of more interest for high producing cows. There
have not been any studies to evaluate the effects of enhanced NDF digestibility with high

producing cows fed diets with a typical NDF content.

43

The objective of this experiment was to evaluate the effects of enhanced NDF
digestibility on DMI and milk yield of high producing dairy cows consuming the diets

with normal NDF content.

MATERIALS AND METHODS

Com Silage

Two corn silage hybrids were planted in adjacent ﬁelds on May 12th, 1995 at the
Michigan State University campus farm. Row width was 76cm and plant density was
69,000 plants / ha for both hybrids. The two corn hybrids were Cargill 6727 and the
same hybrid that had been converted to the bm3 mutant. Corn was chopped at
approximately 30% DM on the 8th and 12th of September for the control hybrid and the
bm3 hybrid, respectively. Both hybrids were fermented and stored in adjacent 150 MT
concrete bunker silos. During harvest, each load was sub sampled, and the sub sample
was packed in 10 x 40 cm PVC experimental mini-silos, and allowed to ferment for 60 d.
Brown midrib corn silage was lower in lignin content by 0.8 units and higher in 30b in

vitro NDF digestibility by 8.4 units compared to the control corn silage (Table 1).

Cows and Treatments
Thirty-two multiparous Holstein cows (Table 2) in early to mid lactation (89 1:27 days in
milk; X :1: SD) from the MSU Dairy Cattle Teaching and Research Center were randomly

assigned to one of two diets after blocking by parity, BW, DIM, and average milk

production. Cows within a block were randomly assigned to adjacent stalls. The diet
contained either bm3 corn silage or control corn silage (80% of forage DM), alfalfa silage
(ensiled in a bag for this experiment, 20% of forage DM), dried ground corn, high
moisture corn, soybean meal, whole linted cottonseed, and premix of minerals and
vitamins (Table 3). Diets were formulated for 31% NDF and 19% CP (Table 3). Cows
were housed in tie-stalls throughout the experiment except for milking times. The
experiment was a cross-over design with two—28 day periods and the ﬁnal 7 days of each

period were used for collection of samples and data.

Sample Collection

Throughout the experiment, cows were fed once daily (1100 h) at 110% of expected
intake. Feed offered and refused were weighed for each cow daily during the collection
period. Samples of both rations (0.5 kg) and orts (10%) were collected daily during the
collection period and composited into one weekly sample per cow. All diet ingredients
were sampled separately daily and composited weekly. Cows were milked three times
per day in the milking parlor at 0600, 1400, and 2200 h. Milk yield was measured daily
during the collection period and averaged over the collection period. Milk was sampled
at every milking on days 1, 3, and 6 of each collection period and analyzed for fat,
protein, and lactose with infrared spectroscopy by Michigan DHIA (East Lansing, MI).
Body weight was measured twice on the two days immediately prior to the start of the
ﬁrst period and on the last two days of each period. Body condition score (BCS), on a
scale of 1 to 5, (1 = thin to 5 = fat) was determined by three trained investigators one day

prior to the start of the ﬁrst period, and on the last day of each period. Fecal samples

45

were collected from each cow at 0800 and 2000b of days 2 and 7 of each collection
period. Response variables were DMI, milk production, milk composition, body weight

change, body condition change, and apparent total tract digestibility.

Sample and Statistical Analysis

Ration, orts, silage, and fecal samples were dried in a 55°C forced air oven for 72 hours
and analyzed for DM content. All samples were ground with a Wiley mill (l-mm screen;
Authur H. Thomas, Philadelphia, PA). Samples were analyzed for ash, NDF, ADF,
lignin, indigestible NDF, CP, and starch. Ash content was determined after 5h oxidation
at 500° C in a mufﬂe furnace. NDF, ADF, and sulfuric acid lignin content were
determined sequentially (Van Soest et al., 1991; method A). Crude protein was analyzed
according to Hach et al. (1987). Starch was measured by an enzymatic method
(Karkalas, 1985) after a NaOH gelatinization step (O’Neil et al., 1993). Indigestible
NDF was estimated as NDF residue after 120h in vitro fermentation (Goering and Van
Soest, 1970) and used as an internal marker to calculate apparent total tract digestibility
(Cochran et al., 1986). Concentrations of all nutrients, except for DM were expressed as

percentages of DM determined from drying at 105° C in a forced—air oven.

All data was statistically analyzed using the ﬁt model procedure of JMP® using the

following model:

Yijk= u+Bi+Pj+Tk+eijk

46

where u = overall mean
Bi = effect of block (i = l to 16)
Pj = effect of period (j = 1 to 2)
Tk = effect of treatment (k = l to 2)

eijk = residual, assumed to be normally distributed

The data from four cows in the second period was excluded for the statistical analysis;
three cows had severe coliform mastitis and one cow lost function in two quarters after

teat injuries.

RESULTS AND DISCUSSION

DMI and Milk Yield

Dry matter intake was higher by 2 kg (P < 0.0001) for cows on the bm3 treatment (Table
4). Although both diets were originally formulated for the same NDF content, NDF
fraction of bm3 corn silage decreased over the experimental period (Table 2), and dietary
NDF level was 0.8 units lower for bm3 treatment diet. However, lower dietary NDF
content for the bm3 treatment was not the major factor allowing higher DMI of the cows
in this experiment because N DF intake for the bm3 treatment was 0.5 kg higher (P <
0.005) than that for normal control. Cows on the bm3 diet were able to consume more

NDF because NDF was more readily degraded in the rumen. Since the NDF fraction of

47

bm3 corn silage was more readily hydrolyzed, turnover time of ruminal digesta might

have decreased, allowing more space in the rumen and higher feed intake.

Milk yield, FCM(3.5%), and SCM were higher for the bm3 diet by 2.7 kg (P < 0.002),
2.5kg (P < 0.0005), and 2.6 kg (P < 0.0001), respectively. Milk composition was not
affected by treatment (Table 5). Production of fat (1.41 kg vs. 1.33; P < 0.01), protein
(1.23 kg vs. 1.14 kg; P < 0.0001), and lactose (2.03 kg vs. 1.87 kg; P < 0.001) were
signiﬁcantly higher for the cows fed bm3 silage than those fed control silage. Improved
milk production for the cows fed bm3 diet can be attributed to increased DMI because
total tract DM digestibility was not different (Table 6). Some researchers reported milk
fat depression with feeding bm3 corn silage (Block et al., 1981; Frenchick et al., 1976),
but others reported the opposite effect of an increase in milk fat with feeding bm3 corn
silage (Grant et al., 1995; Rook et al., 1977). This experiment and others (Kieth et al.,
1979; Sommerfeldt et al., 1979; Stallings et al., 1982) found that milk fat concentration
did not change signiﬁcantly. Inconsistent results may be explained by differences in diet
formulation or feeding conditions. Enhanced N DF degradability or (and) greater DMI
may optimize ruminal fermentation in some cases, but it also may lead to excess
fermentation acid production resulting in lower rumen pH and lower milk fat content
(Block et al., 1981) depending upon feeding conditions. The effects of enhanced NDF

degradability on milk composition should be investigated further.

Energy status of animals was improved by feeding bm3 corn silage (Table 5). Energy

balance was estimated as NEL intake minus NEL for maintenance and milk production.

48

Actual total tract DM digestibility was used to calculate N EL intake (Moe and Tyrrell,
1976) by ﬁrst converting to expected DM digestibility at 1X maintenance (dividing by
0.92) and converting this value to TDN by adding 4 to adjust for higher calories from
digestible dietary fat. This energy balance calculation indicated that cows were in
slightly positive energy balance during this experiment and that energy balance was
higher for the bm3 treatment. Change in body condition score was positive and 0.09 unit
/ 28d higher (P < 0.05) for the cows fed bm3 diets. Body weight gain was positive and
numerically higher for the bm3 treatment, but not statistically signiﬁcant. Although body
weight was measured on two consecutive days each period and averaged, variation was
large probably due to the variation of digesta weight in the rumen. BCS might be more
appropriate to monitor energy status of animals and to detect the energy balance change
in a relatively short period (28d) around a peak lactation. Assuming that one unit of BCS
is equivalent to 362 Mcal of NEL (Crooker and Weber, 1996), the difference in BCS
(0.09 unit / 28d) gain was calculated to be 1.2 Mcal NEL per day, which is equivalent to
the energy required to produce 1.7 kg of 3.5% FCM. Since the bm3 treatment improved
energy status of animals, evaluation of milk production response only might
underestimate the effects of enhanced in vitro NDF digestibility of corn silage on animal
productivity. Energy balance in the early lactation is likely to affect health of animals,

reproductive performance, and subsequent milk production.

Relationship Between Milk Yield and Milk Yield Response
Animal responses to enhanced in vitro N DF digestibility were generally positive, but

animals responded to the bm3 treatment in a different manner depending on production

49

level. The response in DMI (Figure 1) and milk production (Figure 2) to the bm3
treatment (the difference between the bm3 and control treatments within a cow) was
plotted against pre-trial milk production (the average milk production of 14 days
immediately prior to the beginning of experiment). Variation was relatively large
because some cows received the bm3 treatment in the ﬁrst period (earlier in lactation) and
received the control diet in the second period (later in lactation), overestimating the
response to the bm3 treatment. Others received the treatments in the opposite order,
underestimating the response to the bm3 treatment. However, high producing cows
beneﬁted more from enhanced in vitro NDF digestibility of corn silage; higher producing
cows had greater increases in DMI (P < 0.06), and milk yield (P < 0.03) for the bm3
treatment. It is more challenging for high producing cows to meet their energy
requirement, and this result indicates that DMI of higher producing cows is limited by
rumen ﬁll to a greater extent than lower producing cows. The degree of animal response
to the bm3 treatment in DMI and milk yield was highly correlated (r2=0.78), indicating

that improved DMI enhanced milk production.

Nutrient Digestibility

Apparent total tract digestibility of DM, OM, CP, and starch was not signiﬁcantly
different by treatment (Table 6) although total tract digestibility of NDF and ADF were
slightly higher for the bm3 treatment by 1.9 units (P < 0.02) and 2.9 units (P < 0.001),
respectively. Enhanced in vitro NDF digestibility contributed little to the increase in diet
energy density. Digestibility of NDF estimated by 30h in vitro fermentation was 9.7

units higher for bm3 corn silage (49.1% vs. 39.4%) while the bm3 treatment increased

50

the total tract NDF digestibility by only 1.9 units (32.8% vs. 30.9%). The difference in
NDF digestibility estimated by in vitro fermentation may overestimate the difference in
total tract NDF digestibility because the in vitro method evaluates NDF digestibility

using the same incubation time while actual residence time in the rumen is different (21)

A negative relationship was observed between response in DMI for the bm3 treatment
and total tract digestibility of DM (r2 = 0.31, P < 0.002; Figure 3) and NDF (r2 = 0.41; P
< 0.0002; Figure 4). A higher rate of passage associated with increased DMI might
reduce NDF digestibility. More rapid partial hydrolysis of bm3 NDF possibly
contributed to the reduction of rumen fill, allowing greater DMI. Since enhanced DMI
increases rate of passage (NRC, 1987), ruminal retention time of NDF for bm3 corn
silage might be shorter than that for control silage NDF. Digestibility of NDF is
determined by the potential extent of digestion, rate of digestion, and the time exposed to
microbial degradation (Allen and Mertens, 1988). The regression equation for this data
set (Figure 4) showed that the advantage of bm3 corn silage for apparent total tract NDF
digestibility was eliminated when the bm3 treatment increased DMI by approximately
3kg. Total tract starch digestibility was not affected by change in DMI (P > 0.36; Figure
5). In contrast to NDF digestion, starch can be digested extensively in the small intestine
as well as in the rumen, compensating for the possible reduction in ruminal digestion
from a higher rate of passage. Regardless of the inconsistent effects of enhanced in vitro
NDF digestibility on diet energy density, energy intake was increased by the bm3

treatment because of greater DMI (Figure 6).

51

CONCLUSION

Enhanced NDF digestibility with the brown midrib 3 mutation of corn silage improved
DMI, milk production, and BCS of high producing dairy cows. Milk composition was
not affected by treatment. Cows producing more milk beneﬁted to a greater extent from
high NDF digestibility of corn silage. Higher NDF digestibility in vitro does not
necessarily result in increased energy density of diets. The beneﬁts of enhanced NDF
digestibility should be investigated further in a whole lactation trial since improvement in
energy status at peak lactation might inﬂuence persistency of milk yield, animal health,

and reproductive performance.

52

Table 1. Nutrient composition of corn silage used to formulate experimental diets.

 

 

 

lBefore study 2During study
bm3 Control bm3 Control
DM, % 30.2 33.5 31.7 32.6
NDF (% of DM) 42.0 40.4 38.3 40.1
ADF (% OfDM) 21.1 21.0 19.9 21.2
Lignin (% of DM) 1.7 2.5 1.7 2.5
3NDFD, % 45.3 36.8 49.1 39.4
CF (% of DM) 8.7 8.4 9.7 9.5
Ash (% of DM) 4.2 3.8 4.5 4.0
Starch (% of DM) 4ND ND 33.1 33.3

 

' Samples were taken each load at harvest and fermented in PVC experimental mini silos

for 60 days (November/ 1995).

2 Samples were taken at feed out during the experiment. (May-July/ 1996)

3 NDF digestibility measured by in vitro fermentation for 30 h.

4 not determined

53

Table 2. Status of 32 cows at the beginning of experiment.

 

 

Average SD
Parity 2.66 i1.45
Body Wt. 636 kg i570 kg
BCS 2.30 i049
DIM 89 d :27.1 d
Milk Yield 45.6 kg $6.2 kg

 

54

Table 3. Ingredients and nutrient composition of experimental diets (% of dietary DM).

 

 

bm3 Control

bm3 corn silage 44.6 -
Control corn silage - 44.6
Alfalfa silage 11.2 11.2
Dry ground corn 5.6 5 .6
High moisture corn 9.3 8.9
Soybean meal 18.8 19.2
Whole linted cottonseed 5.6 5 .6
Vitamin & Mineral premix 5.0 5.0
Nutrient composition

NDF 30.8 31.6

ADF 16.8 17.4

CP 19.6 19.6

Starch 30.3 30.2

lNEL (Mcal / kg) 1.72 1.72

zeC ratio 56:44 56:44

 

' calculated assuming same book values for two corn silage

2 Forage to concentrate ratio.

55

Table 4. Effects of enhanced NDF digestibility on feed intake.

 

 

 

Treatment P - value
bm3 Control
Intake, kg / (1

DM 25.5 23.5 < 0.0001
NDF 7.7 7.2 < 0.01
Indigestible NDF 3.1 3.4 <0.0001
Starch 7.8 7.1 <0.0001
CP 5.1 4.7 <0.0001

 

56

Table 5. Effects of enhanced NDF digestibility on milk production.

 

 

 

Treatment
P - value
bm3 Control
Production, kg / (1
Milk 41.6 38.9 < 0.01
3.5% FCM 40.9 38.4 < 0.001
4.0% FCM 37.9 35.6 < 0.001
SCM 37.7 35.1 < 0.0001
Fat 1.41 1.33 < 0.01
Protein 1.23 1.14 < 0.0001
Lactose 2.03 1.87 < 0.001
Milk composition, %
Fat 3.43 3.46 0.89
Protein 2.99 2.95 0.35
Lactose 4.90 4.85 0.88
BW change, kg / d 0.17 0.08 0.45
BCS change /28 d 0.11 0.02 0.05
Energy Balance, Mcal NEL / d 2.93 1.00 0.02

57

Table 6. Effects of enhanced N DF digestibility on apparent total tract digestibility (%).

 

 

 

Treatment P - value
bm3 Control
DM 61.5 61.0 0.38
OM 63.0 62.6 0.48
NDF 32.8 30.9 0.02
ADF 34.7 31.8 <0.001
Starch 80.8 83.1 0.18
CF 66.8 67.4 0.45

 

58

 

 

 

8 ..
8° 6 -
8
t:
O
Q:
«E
”2"
C3
-2-
-4 I I I r r I
30 35 40 45 50 55 60

Initial milk yield (kg)

Figure 1. Relationship between average milk yield (kg/d) for 14 (1 prior to the beginning
of the experiment and the response in DMI (kg/d) to the bm3 treatment. DMI response

(kg/d) = 4.4 + 0.15 x pre-trial milk yield (kg/d); r2 = 0.13; P < 0.06.

59

Milk yield response (kg)

 

 

 

" 10 I l l T I I
30 35 40 45 50 55 60

Initial milk yield (kg)

Figure 2. Relationship between average milk yield (kg/d) for 14 d prior to the beginning
of the experiment and the response in milk yield (kg/d) to the bm3 treatment. Milk yield

response (kg/d) = -13.2 + 0.37 x pre-trial milk yield (kg/d); r2 = 0.17; P < 0.03.

60

 

Apparent total tract DM
digestibility response (%)

3..
C.”

 

,'_.
UI

 

'2 4 is
DMI response (kg)

I
.D

I
[\J
O
m'l

10

Figure 3. Relationship between responses to bm3 treatment: apparent total tract DM
digestibility (%) versus DMI (kg/d). Apparent total tract DM digestibility response (%)=

2.58 — 0.93 x DMI response (kg/d); 13 = 0.31; P < 0.01.

61

' I I—3 I—t N
5‘ U! C U‘ o Ur o
I I I L l I J

Total tract NDFD reponse (%)

 

 

I
H
M
—1
q

I
d

 

I
.h
l
N
O
N
A
O'\
00-1

10
DMI response (kg)

Figure 4. Relationship between responses to bm3 treatment: total tract NDF digestibility
(%) versus DMI (kg/d). Total tract NDF digestibility response (%) = 6.07 — 2.05 x DMI

response (kg/d); r2 = 0.41; P < 0.001.

62

l—L
O
I

M
1

O
l

I
M
1

Total tract starchD reponse (%)

 

 

.'_.
, O
.5
I
N—
o—
Nd
#-
o—
m-
H
o—

DMI response (kg)

Figure 5. Relationship between responses to bm3 treatment: apparent total tract starch

digestibility (%) versus DMI (kg/d). r2 = 0.03; P > 0.36.

63

Digested DMI response (kg)
[\3

 

 

 

-1 I l I I I I I

-4 -2 0 2 4 6 8 10
DMI response (kg)

Figure 6. Relationship between responses to bm3 treatment: apparently digested DMI

(kg/d) versus DMI (kg/d). Apparently digested DMI response (kg/d) = 0.64 + 0.38 x

DMI response (kg/d); r2 = 0.58; P < 0.0001.

CHAPTER 4

EFFECTS OF BROWN MIDRIB 3 CORN SILAGE ON FEEDING

BEHAVIOR AND RUMINAL DIGESTA KINETICS

ABSTRACT

Effects of brown midrib 3 (bm3) corn silage on meal patterns, chewing activities, and
ruminal kinetics of digesta were evaluated using eight multiparous ruminally and
duodenally cannulated dairy cows in a duplicated 4 x 4 Latin square design.
Experimental diets consisted of either bm3 corn silage or its isogenic normal control at
two concentrations (29% and 38%) of dietary neutral detergent ﬁber (NDF). In vitro
NDF digestibility at 30h was higher for bm3 corn silage by 9.4 percentage units. Feeding
behavior of animals was monitored continuously for 4 days each period by a
computerized data acquisition system. Ruminal digesta was evacuated twice per period
to determine the ruminal NDF pool size and its turnover time. Dry matter intake (DMI)
increased for bm3 treatments at- both dietary NDF contents. However, an interaction
between corn silage and dietary NDF content was observed for meal size and interval

between meals, indicating that greater DMI for bm3 treatments might be attributed to

65

different DMI regulation mechanisms at each dietary NDF content. There was no effect
of corn silage treatments on total chewing time either per day or per kg of NDF intake.
Although bm3 diets increased turnover rate of NDF in the rumen, ruminal NDF pool size
was maintained because of greater DMI. Enhanced in vitro N DF digestibility of corn
silage changed meal patterns and ruminal kinetics of NDF, but did not affect chewing
activities of high producing dairy cows.

(Key words: brown midrib 3 corn silage, ﬁber digestibility, feed intake regulation,
chewing activity)

Abbreviation key: bm3 = brown midrib mutant for maize

INTRODUCTION

Maximizing DMI is an important management goal for high producing dairy herds.
Cows in early lactation tend to be in negative energy balance, and energy status affects
productivity and health. Digestibility of NDF is an important quality parameter of forage
because it was shown to affect feed intake and milk yield (Oba and Allen, 1998a).
Physical ﬁll is one of the factors which limit maximum feed intake (Allen, 1996), and
more rapid hydrolysis of NDF fraction in the rumen may allow greater DMI when rumen
ﬁll limits maximum feed intake (Oba and Allen, 1998a). Converting a corn silage hybrid
to a bm3 mutant is one way to increase NDF digestibility of corn silage (Cherney et al.,
1991). Forages with brown midrib mutation increased DMI and milk yield compared
with control forages with similar NDF and CP contents (Grant et al., 1995; Oba and

Allen, 1998b).

66

Although it is logically hypothesized that reduced physical ﬁll in the rumen improves
DMI more when animals are fed a high NDF than a low NDF diet, research results have
not been consistent. Grant et a1. (1995) and Dado and Allen (1995) fed high NDF diets
(40% and 37%, respectively) and reported that enhanced in vitro NDF digestibility
increased DMI and milk yield. However, Robinson and McQueen (1997) reviewed
previous experiments which evaluated the effect of ruminal ﬁll on animal performance
(Miller et al., 1990; Robinson and McQueen, 1992; Varga et al., 1984) and summarized
that the positive effect of forage N DF fermentability might be higher for low NDF diets.
Robinson and McQueen (1997) fed forage ﬁber which differed in its fermentability at
three dietary NDF contents, and reported positive effect of enhanced ﬁber fermentability
on DMI and milk yield regardless dietary NDF content. These inconsistent animal
responses were explained partly by the difference in energy requirements of animals
across the studies; ruminal ﬁll from a high NDF diet may not limit maximum DMI if
energy requirement of animals is low, whereas a low NDF diet can limit DMI by ﬁll if
energy requirement of animals is not satisﬁed. However, the effects of forage NDF
degradability on the regulation mechanisms of voluntary DMI could be more complex
and needs further investigation. Because DMI per day is a function of meal size and
meal frequency, evaluation of meal patterns of cows fed forages which differed in in vitro
NDF digestibility at different dietary NDF concentration may give clues for relevant

interpretation of animal responses to NDF degradability.

67

Effect of forage NDF fermentability on physical effectiveness of ﬁber is another area of
interest. Dairy cows require a speciﬁc amount of effective NDF in diets to stimulate
chewing and maintain ruminal functions. Dietary forage NDF concentration and forage
NDF particles size are closely related with ruminal pH and chewing activities of dairy
cows, respectively (Allen, 1997). However, the relationship between fermentability of
forage NDF and chewing activities is not well understood. Beauchemin (1991) and
Nelson and Satter ( 1992) reported decreased chewing activities for diets containing
alfalfa harvested at early versus late maturity. Grant et a1. (1995) found that total
chewing time per kg NDF intake was lower for brown midrib sorghum silage than normal
sorghum silage. On the other hand, Robinson and McQueen (1997) reported no
treatment effects of forage ﬁber fermentability on chewing activity. It is generally
speculated that enhanced NDF degradation stimulates rapid clearance of NDF from the
rumen, resulting in reduced NDF mass in the rumen and reduced chewing activities.
However, little data are available to examine the relationships among chewing activities,

NDF intake, ruminal NDF pool size, and NDF retention time in the rumen.

The objectives of this experiment were to evaluate the effects of bm3 corn silage on meal

pattern and chewing activities of lactating dairy cows fed two concentrations of dietary

NDF, and to evaluate their relationship with digestion kinetics in the rumen.

68

MATERIALS AND METHODS

Corn Silage

Two corn silage hybrids were planted in adjacent fields on May 14th, 1996 at the
Michigan State University campus farm. The two corn hybrids were Cargill 6208FQ and
Cargill 657 which is the same hybrid that had been converted to the bm3 mutant. Corn
plant was chopped at approximately 30% DM on the 10th and 19th of September for the
control hybrid and the bm3 hybrid, respectively. Both hybrids were fermented and stored
in adjacent 150 MT concrete bunker silos. During harvest, each load was sub sampled,
and the sub sample was packed in 10 x 40 cm PVC experimental mini-silos, and allowed
to ferment for 30 (1. Brown midrib corn silage was lower in lignin content by 0.7
percentage units and higher in 30h in vitro NDF digestibility by 9.4 percentage units

compared with the control corn silage (Table 1).

Cows and Treatments

Eight multiparous Holstein cows were assigned to a duplicated 4 X 4 Latin square design
balanced for carry over effects. Treatment periods were 21 days with the ﬁnal 10 days
used for collection of samples and data. The experimental treatments were in a 2 x 2
factorial arrangement, and factors evaluated were dietary NDF content (29% and 38%)
and bm3 mutation (bm3 and normal control). Cows were cannulated ruminally and
duodenally prior to calving, and the experiment started approximately 70 days after
calving. The experimental diets contained either bm3 corn silage or normal control corn
silage (80% of forage DM) supplemented with alfalfa silage (20% of forage DM), dried

ground corn, whole cottonseed, premix of protein supplement (soybean meal, distiller’s

69

 

grains, and blood meal), and premix of minerals and vitamins (Table 2). A11 diets were
formulated for 18% of dietary CP content. Cows were housed and milked in tie-stalls

throughout the experiment.

Data and Sample Collection

Throughout the experiment, cows were fed once daily (1200 h) at 110% of expected
intake. Feed offered and refused were weighed for each cow daily during the collection
period; samples of all diet ingredients (0.5 kg) and orts (10%) from each cow were
collected daily and composited into one sample per period. Feeding behavior was
monitored from d 12 through d 16 (96 h) of each period by a computerized data
acquisition system (Dado and Allen, 1993). Data of chewing activities, feed
consumption, water consumption, and ruminal pH was recorded for each cow every 5
secs. Daily means were calculated for meal bouts, interval between meals, eating chew
rate, eating time, eating chews, meal size, ruminating bouts, interval between ruminating
bouts, ruminating chew rate, ruminating time, and ruminating chews. The daily means of
variables were averaged over the four days. The rumen was evacuated at 1400 h (2 h
after feeding) on d 19 and at 1000 h (2 h before feeding) on d 21. Total ruminal content
mass and volume were determined. During evacuation, a 10% aliquot of digesta was
separated for ease of sub sampling. The aliquot was squeezed through 4 layers of cheese
cloth to separate into solid and liquid. Samples were taken from both phases for

determination of nutrient pool size.

70

 

Chemical Analysis

Diet ingredients, orts, and ruminal digesta samples were dried in a 55°C forced air oven
and analyzed for DM content. DM content of ruminal digesta was also determined by
toluene distillation (AOAC, 1990). All samples were ground with a Wiley mill (1mm
screen; Authur H. Thomas, Philadelphia, PA). Samples were analyzed for ash, NDF,
ADF, lignin, indigestible NDF, CP, and starch. Ash content was determined after 5h
oxidation in a mufﬂe furnace at 500° C. Ash-free NDF, ADF, and sulfuric acid lignin
content were determined sequentially (Van Soesteta1., 1991; method A). Crude protein
was analyzed according to Hach et al. (1987). Starch was measured by an enzymatic
method (Karkalas, 1985) after a NaOH gelatinization step (O’Neil et al., 1993).
Indigestible NDF was estimated as NDF residue after 120h in vitro fermentation
(Goering and Van Soest, 1970). Concentrations of all nutrients, except for DM were

expressed as percentages of DM determined by drying at 105° C in a forced-air oven.

The ruminal pool sizes (kg) of DM, OM, NDF, indigestible NDF, and starch were
determined by multiplying the concentration of each nutrient by the ruminal digesta
weight (kg). Duodenal nutrient ﬂow was estimated by the method described in Chapter
5. Turnover rate in the rumen, passage rate from the rumen, and ruminal digestion rate of

each nutrients (%lh) were calculated by the following equations;

Turnover rate in the rumen (%/h) =

(intake of nutrient / ruminal pool of nutrient) / 24 x 100

71

Passage rate from the rumen (%lh) =
(duodenal ﬂow of nutrient / ruminal pool of nutrient) / 24 x 100
Digestion rate in the rumen (%/h) =

Turnover rate in the rumen (%/h) — Passage rate from the rumen (%lh)

Assuming that the indigestible NDF fraction disappears from the rumen only by passage,
indigestible NDF intake substituted for duodenal indigestible NDF ﬂow in this
calculation. Because of technical difﬁculty in ﬁltering indigestible NDF residues for
duodenal samples, duodenal indigestible NDF ﬂow had been overestimated; the

estimated duodenal ﬂow had exceeded intake.

Statistical Analysis
All data were analyzed using the fit model procedure of JMP® (Version 3.2, SAS

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

Yijkl = u + Si + ij + Pk + Tl + STil + eijkl
where
11 = overall mean,
Si = random effect of square ( i = 1 to 2),
CW, = random effect of cow within square ( j = 1 to 4),
Pk = random effect of period (k = 1 to 4),
TI = ﬁxed effect of treatment (1 = 1 to 4),

STil = interaction of square and treatment,

eijld = residual, assumed to be normally distributed.

72

A reduced model without square X treatment interaction was used when this effect was
not signiﬁcant (P > .10). Orthogonal contrasts were used to determine effect of dietary
NDF content, effect of com silage, and interaction of dietary NDF content and corn
silage. Treatment effects and their interaction were declared signiﬁcant at P < .05 and P

< .10, respectively.

RESULTS AND DISCUSSION

DMI and Meal Patterns

Cows fed bm3 corn silage had higher DMI at both dietary NDF contents (P < 0.05; Table
3). An interaction of dietary NDF content and corn silage was not detected (P > 0.54).
This agrees with the observation of Robinson and McQueen (1997) who reported a
positive effect of higher forage NDF fermentability on DMI regardless of dietary NDF
content. However, a signiﬁcant interaction between dietary NDF and corn silage was
observed for interval between meals (P < 0.08; Table 4) and meal size of DM (P < 0.08).
Observed differences in meal patterns indicate that greater DMI for bm3 treatments might
be a result of different DMI regulation mechanisms at each level of dietary NDF content.
Meal size was greater (2.2 kg vs. 2.0 kg) and interval between meals was longer (98.8
min vs. 87.8 min) for bm3 treatments compared with control at high NDF content,
whereas meal size was smaller (2.2 kg vs. 2.3 kg) and interval between meals was shorter

(90.4 min vs. 98.2 min) for bm3 treatments compared with control at low NDF diets.

73

When cows were fed the dietary treatment with higher NDF, higher DMI for the bm3
treatment was from greater meal size. It was previously noted that physical ﬁll limitation
to DMI changed meal patterns of animals (Baumont et al., 1990; Dado and Allen, 1995).
Baumont et a1. (1990) found that sheep increased frequency of eating bouts when bulk
was added to the rumen. Cows challenged by ruminal ﬁll from dietary NDF and ruminal
inert bulk increased the percentage of smaller meals and decreased the percentage of
larger meals (Dado and Allen, 1995). In these instances, physical ﬁll might have been
the dominant factor affecting satiety. When limitation of physical ﬁll to DMI exists,
animals might try to maintain DMI by increasing number of eating bouts (Baumont et al.,
1990). In our study, greater meal size and longer interval between meals for the bm3
treatments may indicate that bm3 corn silage is less ﬁlling in the rumen than the control

silage.

When cows were fed the dietary treatment with lower NDF, bm3 treatment increased
DMI not because of greater meal size, but because of shorter intervals between meals.
Physical ﬁll was less likely to limit their maximum DMI with low NDF diets because the
ruminal digesta mass and volume were lower for low NDF diets than high NDF diets
(Table 6), indicating that there is additional capacity available in the rumen. The
mechanisms, which explain shorter intervals between meals for the bm3 treatment at low
NDF diet, are not known and need further investigation. However, it can be attributed
partly to lower ruminal pH for the bm3 treatment (P < 0.01; Table 6); lower ruminal pH
might result in more rapid absorption of volatile fatty acids (VFA) because more VFA are

present in the associated form which is readily absorbed through the ruminal wall. It is

74

speculated that rapid absorption of VFA and subsequent metabolite uptake by peripheral
tissues makes the cows feel hunger sooner. Elucidating the interaction of ruminal

fermentation and physiology of lactating dairy cows may be a fertile area of research.

Ruminal Digesta Kinetics

Ruminal turnover rate of NDF tended to be higher for bm3 treatment (P < 0.07; Table 5),
indicating that NDF from bm3 corn silage was more readily degraded and passed from
the rumen faster, allowing additional capacity in the rumen. However, faster turnover
rate of bm3 NDF cannot be attributed to faster rate of digestion. In this experiment,
ruminal digestion rate for the potentially digestible NDF fraction was lower (P < 0.02)
for bm3 treatment possibly due to lower ruminal pH (P < 0.01; Table 6). The potential
advantage in NDF digestibility for the bm3 corn silage might be diminished because of

negative effects of low ruminal pH on ﬁber digesting bacteria (Hoover, 1986).

Faster rate of ruminal NDF turnover for bm3 treatment was a result of faster rate of
passage (P < 0.04). Rate of passage is inﬂuenced by rate of particle size reduction,
particle density, and buoyancy of digesta (Allen, 1996). Particle size reduction is not
totally dependent upon microbial fermentation, but occurs primarily as a result of
mastication and detrition (Mcleod and Minson, 1988). lllius and Gordon (1991)
proposed that rate of particle size reduction in the rumen can be estimated by indigestible
NDF content of forage because a ligniﬁed cell wall provides plant rigidity. Because in
vitro NDF digestibility was higher for bm3 corn silage than control, greater fragility for

bm3 NDF might contribute to faster passage rate for the ﬁbrous fraction of ruminal

75

digesta. Stensig and Robinson (1997) showed that passage rate for the digestible NDF
fraction and the indigestible NDF fraction can be different because each fraction is not
equally distributed in a plant and (or) a diet. In this experiment, passage rate for the
digestible NDF fraction was not affected by corn silage treatment possibly because the
passage from the rumen is inﬂuenced by other factors such as buoyancy of digesta and
(or) rate of digestion. However, passage rate for the indigestible NDF fraction was
higher for bm3 treatment (P < 0.0001), which indicates that fragility of plant cell wall
could enhance particle size reduction and subsequent passage from the rumen without
microbial degradation. Although diets with bm3 corn silage did not have faster digestion
rate of NDF in the rumen, enhanced ease of NDF degradation for bm3 corn silage could

contribute to the more rapid clearance of NDF from the rumen.

Cell wall fragility can be estimated by in vitro NDF digestibility to some extent, but the
effects of in vitro NDF digestibility on ruminal ﬁll and DMI need to be interpreted with
caution, especially for comparisons across different forage species. Hoffman et al.
(1998) compared alfalfa silage with perennial ryegrass silage at similar dietary NDF
content and forage to concentrate ratio, and found greater DMI for the diet based on
alfalfa which was lower in N DF digestibility. In general, legumes differ from grasses in
potential extent of digestion or (and) rate of digestion, which possibly affects buoyancy
of digesta in the rumen and ruminal ﬁll (Allen, 1996). Waghom et al. (1989) reported
that rate of particle size reduction by mastication is faster for alfalfa than ryegrass.

Although particle density and rate of particle size reduction partly account for NDF

76

turnover rate in the rumen, these factors are not estimated directly by in vitro digestion

methods.

Chewing Activities

There was no effect of corn silage on ruminating time either per day or per kg of NDF
intake (Table 7), or on total chewing time either per day or per kg of NDF intake (Table
8). Although the effectiveness of the more ruminally degradable bm3 NDF at stimulating
chewing was similar to control N DF, ruminating time and total chewing time per kg of
DMI tended to be lower for bm3 treatments (P < 0.07 and P < 0.11, respectively).
Turnover rate of NDF was higher for bm3 treatments, but the bm3 corn silage did not
affect chewing activities possibly because NDF mass in the rumen was maintained due to

greater DMI.

In contrast to our observations, it was reported that alfalfa harvested at an earlier maturity
decreased chewing activity per kg of alfalfa N DF intake compared with alfalfa harvested
at a later maturity (Bucheamin 1991). This implies that forages with higher NDF
digestibility might decrease the physical effectiveness at stimulating chewing because
NDF digestibility of alfalfa usually decreases with maturity. However, Bucheamin
(1991) reported that DMI was lower for cows fed earlier maturity alfalfa hay. Because
alfalfa DM disappearance in sacco was faster for the early maturity alfalfa, low DMI
could result in reduced NDF mass in the rumen and reduced chewing activity. Robinson
and McQueen (1997) found that forage NDF fermentability did not inﬂuence chewing
activities, and reported that DMI was higher for the diets with more fermentable forage

NDF. Nadeau and Allen (1998) showed that NDF digestibility of alfalfa does not affect

77

total chewing time or ruminating time per kg of alfalfa NDF intake, and reported no
effect on DMI and ruminal NDF pool size. Within current data set, total chewing time
per day were positively related to NDF intake (P < 0.001; r = 0.57) and ruminal NDF
pool size (P < 0.001; r = 0.55), but not related to ruminal turnover rate of NDF (Table 9).
These evidences may lead to the hypothesis that forage N DF degradability does not affect
chewing activities as long as enhanced DMI maintains a critical amount of NDF mass in

the rumen.

CONCLUSION

Feeding bm3 corn silage increased DMI regardless of dietary NDF content. Meal pattern
of animals fed bm3 corn silage was different at each level of dietary NDF, indicating that
greater DMI for bm3 treatments might be attributed to different DMI regulation
mechanisms at each level of dietary NDF content. Rate of NDF passage from the rumen
was faster for bm3 treatments. Chewing activities was not inﬂuenced by bm3 corn silage

probably because of greater DMI and maintained ruminal NDF pool.

78

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87

CHAPTER 5

EFFECTS OF BROWN MIDRIB 3 CORN SILAGE ON NUTRIENT

UTILIZATION BY LACTATING COWS

ABSTRACT

Effects of brown midrib 3 (bm3) corn silage on ruminal fermentation, site of digestion,
and energy utilization were evaluated using eight multiparous ruminally and duodenally
cannulated dairy cows (70 days in lactation) in a duplicated 4 x 4 Latin square design.
Experimental diets contained either bm3 corn silage or normal control at two
concentrations (29% and 38%) of dietary neutral detergent ﬁber (NDF). Digestibility of
NDF estimated by 30h in vitro fermentationwas higher for bm3 corn silage by 9.4
percentage units. Low NDF diets and bm3 treatments increased milk yield. The bm3
treatment depressed milk fat concentration when fed with the low NDF diet. Fat-
corrected milk yield (3.5%) tended to be higher for the cows fed bm3 corn silage,
whereas daily body weight gain was higher for the cows fed low NDF diets. Both bm3
treatments and low NDF diets lowered rumen pH, but bm3 treatments did not increase
ﬂuctuation of rumen pH. Feeding bm3 corn silage increased the energy utilized for milk
production, possibly because of consistent nutrient supply from the rumen. Passage rate

of ruminal digesta was higher for bm3 treatments, which might contribute to improved

88

microbial efﬁciency and to shifting site of digestion from the rumen to the intestine. The
beneﬁcial effects of bm3 corn silage on animal performance were greater when fed with
the high NDF diet.

(Key words: brown midrib 3 corn silage, ﬁber digestibility, rumen pH, energy utilization
efﬁciency)

Abbreviation key: bm3 = brown midrib mutant for maize; FCM = fat—corrected milk;
SCM = solid-corrected milk; BCS = body condition score; NAN = non-ammonia
nitrogen; MN = microbial nitrogen; NANMN = non-ammonia non—microbial nitrogen;

MEOP = milk energy output as a percentage of NEL intake

INTRODUCTION

As a genetic potential of dairy cows for milk production increases, it becomes more
challenging to meet the energy requirement of high producing cows. Cows in early
lactation tend to be in negative energy balance, and maximizing energy intake in this
period is essential for maximum productivity and farm proﬁtability. Enhanced
digestibility of forage NDF was reported to increase DMI and milk yield of lactating
dairy cows (Oba and Allen, 1998a). Because maximum feed intake of high producing
dairy cows is often limited by rumen ﬁll (Allen, 1996), feeding more degradable NDF
might allow greater feed intake by rapid turnover of the ﬁbrous fraction in ruminal
digesta by increasing rate of digestion, increasing rate of passage, or both (Oba and

Allen, 1998a).

89

Although animal responses to forages with higher NDF digestibility are usually positive,
their effects on farm proﬁtability are inﬂuenced by many other factors. In general, it is
more expensive to obtain a forage with higher NDF digestibility; harvesting a perenial
forage at earlier maturity usually sacriﬁces potential yields. Utilizing forages with a
brown midrib mutation is another alternative to obtain enhanced NDF digestibility
because it consistently lowers lignin content and enhances NDF digestibility (Cherney et
al., 1991; Allen et al., 1997). However, it is reported that the bm3 mutation decreased
corn silage yield an average of 12% across hybrids (Allen et al., 1997). Because forages
are a signiﬁcant portion of a dairy ration, their nutritional advantages and economical
potential depend upon an appropriate diet formulation to a great extent. Unfortunately,
little information is available regarding supplementation of forages with higher NDF

digestibility.

Comparison of a bm3 corn silage with its isogenic normal control might provide relevant
information about the effects of enhanced in vitro NDF digestibility on animal
performance because it minimizes confounding nutritional and physical characteristics of
forages with signiﬁcant difference in NDF digestibility. However, animal responses to
bm3 corn silage have not been consistent. Some experiments reported that feeding bm3
corn silage increased milk yield (Frenchick et al., 1976; Kieth et al., 1979; Oba and
Allen, 1998), whereas others reported that it increased body fat deposition (Rook et al.,
1977; Sommerfeldt et al., 1979; Block et al., 1981; Oba and Allen, 1998). Some studies

reported milk fat depression for cows fed a bm3 corn silage (Frenchick et al., 1976;

90

Block et al., 1981). Despite many production trials, the effects of bm3 corn silage on
energy utilization are not well understood. Some studies evaluated fermentation
characteristics in the rumen and speculated the relationships among rumen pH, acetate to
propionate ratio, and animal performance (Frenchick et al., 1976; Rook et al., 1977;
Sommerfeldt et al., 1979; Block et al., 1981). However, none of the previous
experiments evaluated the effects of bm3 corn silage on nutrient digestibility in the
rumen, microbial efﬁciency, and site of digestion. Understanding these digestion
characteristics is essential to supplement forages with enhanced NDF digestibility in the

most effective manner, and to maximize their utilization.

The objective of this experiment was to evaluate the effects of bm3 corn silage on
ruminal fermentation, microbial efﬁciency, site of digestion, and energy utilization by

lactating dairy cows fed diets with two dietary NDF concentrations.

MATERIALS AND METHODS

Experimental Designs

Two corn silage hybrids were grown at the Michigan State University campus farm, and
ensiled in adjacent 150 MT concrete bunker silos. The two corn hybrids were Cargill
6208FQ and Cargill 657 which is the same hybrid that had been converted to the bm3
mutation. Both hybrids were chopped at approximately 30% DM. Brown midrib corn
silage was lower in lignin content by 0.7 percentage units (1.3 vs. 2.0, % of DM) and

higher in 30b in vitro NDF digestibility by 9.4 percentage units (55.9 vs. 46.5, % of

91

NDF) compared with the control corn silage. The experimental treatments were in a 2 x
2 factorial arrangement, and factors evaluated were dietary NDF concentrations (29% and
38%) and bm3 mutation (bm3 and normal control). The experimental diets contained
either bm3 corn silage or control corn silage (80% of forage DM) supplemented with
alfalfa silage (20% of forage DM), dried ground corn, whole cottonseed, premix of
protein supplement (soybean meal, distiller’s grain, and blood meal), and premix of
minerals and vitamins. The diets were formulated for 18% of dietary CP content.

Chemical composition of corn silages and experimental diets were described in Chapter

4.

Eight multiparous Holstein cows were ruminally and duodenally cannulated prior to
calving. Duodenal cannulas were soft gutter type made of plastisol. The duodenum was
ﬁstulated proximal to the pylorus region and prior to the pancreatic duct and the cannulas
were placed between 10th and 11th ribs as described by Robinson et al. (1985). Both
ruminal and duodenal surgery were performed at the Department of Large Animal
Clinical Science, College of Veterinary Medicine, Michigan State University. Cows
were 70d i7.7d in lactation at the initiation of experiment, and assigned to a duplicated 4
X 4 Latin square balanced for carry over effects. Each period consisted of 21 days and
the ﬁnal 11 days were used for collection of samples and data. Cows were individually
housed in tie stalls, milked in their stalls twice daily (0600 h and 1800h), and fed once
daily (1200 h) at 110% of expected intake throughout the experiment.

Data and Sample Collection

92

Rumen pH was monitored from 12 d to 16 d of each period by a computerized data
acquisition system (Dado and Allen, 1993), and data was recorded for each cow every 5
seconds. Calibration of pH probes was checked once per day, and adjusted if necessary.
When pH differed from the calibration solutions at pH 4 and 7 by more than 0.05 units,
the data of last 24 hours was excluded from statistical analysis. Daily means and
variation of rumen pH, and the time spent below pH 6.0, 5.8, and 5.5 were determined.
The negative effects of low rumen pH were estimated by multiplying the time spent
below pH 6.0, 5.8, and 5.5 by the deviation from the pH 6.0, 5.8, and 5.5, respectively
(Mackie and Gilchrist, 1979). The rumen pH data were summarized for each day, and

the daily means were averaged over the 4 days.

The double marker method was utilized for estimating duodenal ﬂow of nutrients
(Faichney, 1980). Chromium was used as a solid phase marker, and closed as Cr-
mordanted wheat straw (30 g/d per cow; approximately 1.2 g Cr / d). Cobalt was used as
a liquid phase marker, and infused as Co-EDTA solution (400 ml/d; approximately 0.5 g
Co / (1). Both Cr-mordanted ﬁber and Co-EDTA solution were prepared by the method
described by Uden et al.(1980). Cr-mordanted ﬁber was ﬁlled in gelatin capsules (1.5 oz,
Tropac Inc., Airﬁeld, NJ) and dosed through the rumen cannula twice daily (0730 h and
1930 h). Co-EDTA solution was infused into the rumen for 1 minute out of every 10
minutes from d 11 through d 19 of each period. Both markers were prime-dosed (3x of
daily dose) on d 11 of each period. Duodenal digesta samples were collected every 3
hours from d 16 to d 19. The ﬁrst sample was collected at 1200 h on d 16 and collection

time was delayed one hour on each of the following days so that 24 samples were taken

93

for each cow which represented every hour of a 24-hour period to account for diurnal
variation. Duodenal digesta was separated to liquid and solid phases by ﬁltering through
a polypropylene screen with a 1000 um aperture size (Fisher Scientiﬁc, Pittsburgh, PA)
at each sampling time. The digesta retained on the screen and the digesta through the
screen were arbitrary deﬁned as solid phase digesta and liquid phase digesta,
respectively. Samples were immediately frozen at -20° C, and then lyophilized. Fecal
samples were collected every 6 hours concurrently with duodenal digesta sampling.
Rumen ﬂuid samples were collected every 3 hours, starting at 1200 h on d 19 and
continued for 24 hours for determination of VFA, lactate, and ammonia concentrations.
Samples were taken from 5 or 6 different sites within the rumen and strained through four

layers of cheesecloth, and frozen at -20° C until analysis.

Feed offered and refused were weighed for each cow daily during each data collection
period ((1 12 through d 21). During each collection period, samples of all diet ingredients
(0.5 kg) and orts (10% of refused feed) from each cow were collected daily and
composited into one sample per period. Milk yield and DMI were recorded daily during
each collection period and averaged. Milk was sampled at every milking on d 16, d 18,
and d 20 of each period and analyzed for fat, protein, and lactose contents with infrared
spectroscopy by Michigan DHIA (East Lansing, MI). Rumen empty body weight was
measured after evacuating rumen digesta on d -2 and d -1 of the ﬁrst period, and
measured twice on d 20 and d 21 of each period and averaged. Body condition score, on
a scale of 1 to 5 (l = thin, 5 = fat), was assessed by three trained scorers immediately

prior to the start of the ﬁrst period and on d 21 of each period.

94

Data and Sample Analysis

Diet ingredients, orts, and fecal samples were dried in a forced-air drying oven at 55° C
for 72 hours and analyzed for DM content. All samples were ground with a Wiley mill
(1mm screen; Authur H. Thomas, Philadelphia, PA). Samples were analyzed for ash,
NDF, ADF, lignin, indigestible NDF, CP, and starch. Ash content was determined after
5h oxidation at 500° C in a mufﬂe furnace. Ash-free NDF, ADF, and sulfuric acid lignin
content were determined sequentially (Van Soest et al., 1991; method A). Crude protein
was analyzed according to Hach et al. (1987). Starch was measured by an enzymatic
method (Karkalas, 1985) after a NaOH gelatinization step (O’Neil et al., 1993).
Indigestible NDF was estimated as N DF residue after 120h in vitro fermentation
(Goering and Van Soest, 1970). Concentrations of all nutrients, except DM were
expressed as percentages of DM determined from drying at 105° C in a forced-air drying

oven.

Feeds, solid phase duodenal digesta, liquid phase duodenal digesta, and fecal samples
were analyzed for concentrations of Co and Cr. Samples were digested with phosphoric
acid according to Williams et al. (1962), and read by ﬂame atomic absorption
spectrometry (Smith-Hieftje 4000, Thermo Jarrell Ash Co., Franklin, MA). Cobalt
concentration of feeds and solid phase duodenal digesta was analyzed by furnace atomic
absorption spectrometry by a commercial laboratory (VBSA Inc., Newton, IA).

Duodenal ﬂow of nutrients was calculated according to the method of Russell and

95

Armentano (1985). Passage rates of OM, NDF, and starch were estimated according to

the method described in Chapter 4.

Duodenal digesta were analyzed for purines and ammonia to estimate microbial nitrogen
ﬂow and non—ammonia non-microbial nitrogen ﬂow to the duodenum. Purines were used
as a microbial marker, and purine to microbial N ratio was estimated by analysis of
microbial pellets obtained after differential centrifugation of rumen ﬂuid (Overton et al.,
1995). Purines were measured by spectrophotometer (Beckman Instruments, Inc.,
Fullerton, CA) at 260 nm (Zinn and Owens, 1986). Dried duodenal digesta samples were
hydrolyzed for 20 minutes to extract ammonia retained in samples. Rumen ﬂuid samples
were centrifuged at 26,000 x g for 30 minutes and supematants were collected.

Ammonia concentration was determined for the duodenal samples and the centrifuged
rumen ﬂuid according to Broderick and Kang (1980). The supematants of centrifuged
rumen ﬂuid were analyzed for VFA and lactate by gas chromatography (Perkin Elmer,

Norwalk, CT), using pivalic acid as an internal marker.

All data were statistically analyzed using the ﬁt model procedure of JMP® (Version 3.2,

SAS Institute Inc., Cary, NC) according to the following model:

Yijkl = u + Si + Cm, + Pk + Tl + STil + eijkl
where

it = overall mean,

Si = random effect of square ( i = 1 to 2),

c.

J“, = random effect of cow within square ( j = 1 to 4),

Pk = random effect of period (k = 1 t0 4),

96

T1 = ﬁxed effected of treatment (1 = 1 to 4),

eijkl = residual, assumed to be normally distributed.

A reduced model without square x treatment interaction was used when this effect was
not signiﬁcant (P > .10). Orthogonal contrasts were made to determine effect of dietary
NDF content, effect of corn silage, and interaction of dietary NDF content and corn
silage. Treatment effects and their interaction were declared signiﬁcant at P < .05 and P

< .10, respectively.

RESULTS

DMI and Animal Performance

DMI was higher for low N DF treatments (P < 0.001) and bm3 treatments (P < 0.02;
Table 1). Energy intake, calculated from digested DMI, was higher for low NDF diets (P
< 0.01) but was not affected by corn silage treatments. Milk yield, FCM yield, solids-
corrected milk yield (SCM) were higher for bm3 treatments regardless of dietary NDF
concentration, whereas dietary NDF concentration did not affect milk production. A
signiﬁcant interaction of dietary NDF concentration and corn silage treatments was
observed (P < 0.06) for milk fat content, indicating that milk fat content was lower for the
bm3 treatment when fed with the low NDF diet (3.28% vs. 3.67%), whereas milk fat
content was not affected by corn silage treatments when dietary NDF content was high
(3.86% vs. 3.90%). Milk fat yield was not affected by either corn silage or dietary NDF

content. Brown midrib 3 treatments increased milk lactose content (P < 0.05) and did not

97

affect milk protein content, and resulted in significantly higher yield of each component
(P < 0.01). A signiﬁcant interaction (P < 0.10) for milk solids-non-fat (SNF) content
between dietary NDF content and corn silage indicated that the bm3 corn silage tended to
increase SNF when fed with the high N DF diet, but had no effect when fed with the low
NDF diet. Neither of BW gain or BCS change was affected by bm3 treatments, but low

NDF treatment increased BW gain compared to high NDF treatment (P < 0.01).

Ruminal Fermentation

Daily mean ruminal pH was lower for bm3 treatments (P < 0.01; Table 2) and tended to
be lower for low NDF diets (P < 0.06). Daily variance and daily range in rumen pH were
higher for low NDF diets, but not affected by corn silage treatments. The time spent
below pH 6.0 and 5.8 was greater for bm3 treatments, but was not affected by dietary
NDF content. Signiﬁcant interactions between dietary NDF content and corn silage
treatments were observed for ruminal concentration of total VFA (P < 0.07), lactate (P <
0.07), and butyric acid (P < 0.05; Table 3). For high NDF diets, total VFA
concentrations were 92.6 and 80.4 mM for bm3 and control, respectively. For low NDF
diets, total VFA concentrations were 92.8 and 98.3 mM for bm3 and control,
respectively. Low NDF diets increased propionate concentration, and decreased acetate
concentration and acetate to propionate ratio compared to high NDF diets. Ammonia

concentration tended to be lower for bm3 treatments (P < 0.09).

98

Nutrient digestibility

Apparent total tract DM digestibility was not affected by corn silage treatment, but
numerically lower for cows fed bm3 corn silage (Table 5). Therefore, amounts of
apparently digested DM were not affected by corn silage treatments even though dry
matter intake was higher for bm3 treatments (P < 0.02). A signiﬁcant interaction of
dietary NDF content by corn silage treatments was observed for true ruminal OM
digestion (kg/d; P < 0.01). Truly digested OM in the rumen was higher for the bm3
treatment with the high NDF diet, but lower for the bm3 treatment with the low NDF diet
compared to the control. However, more OM was digested post-ruminally for bm3
treatments than the control regardless of dietary N DF concentration. Corn silage
treatments did not affect NDF digestibility in the rumen, post-rumen, or total tract (Table
5). Ruminal starch digestibility was higher for control treatments (P < 0.01), whereas
post-ruminal starch digestibility was higher for bm3 treatments (P < 0.01; Table 6).
Passage rate from the rumen for NDF (P < 0.04), indigestible NDF (P < 0.0001), and

starch (P < 0.02) was higher for bm3 treatments (Table 5, 6).

Nitrogen intake and duodenal ﬂow of non-ammonia nitrogen (NAN) were higher (P <
0.01; Table 7) for bm3 treatments. Although non-ammonia non-microbial nitrogen
(NANMN) was not affected by corn silage treatments, bm3 treatment increased microbial
nitrogen (MN) ﬂow at the duodenum (P < 0.04). Microbial efﬁciency estimated by
duodenal ﬂow of MN as a percentage of truly digested OM in the rumen was higher for
bm3 treatments (P < 0.01). Duodenal ﬂow of NAN was also higher for low NDF diets (P

< 0.001); low NDF diets increased NANMN ﬂow (P < 0.0001), but did not increase MN.

99

Postruminal digestibility of duodenal NAN was higher for low NDF diets and bm3
treatments. Apparent total tract digestibility of nitrogen was not affected by corn silage

treatments.

DISCUSSION

Energy Intake and Utilization

Cows fed bm3 corn silage increased yields of milk, FCM, and SCM, which are partly
attributed to greater DMI for bm3 treatments. However, bm3 treatments did not
signiﬁcantly increase digested DMI (Table 1). Therefore, enhanced milk production for
bm3 treatments might be attributable to factors other than greater digestible energy
intake. In this experiment, bm3 treatments lowered rumen pH without increasing its
daily variation compared to control treatments (Table 2). Low rumen pH usually
decreases methane production in the rumen, which might increase metabolizable energy
available for animals. In addition, VFA are more readily absorbed through the rumen
wall as rumen pH declines. Because rate of VFA absorption in the rumen is a function of
rumen pH and concentration gradient, low rumen pH with less ﬂuctuation might result in
greater and more consistent energy supply from the rumen to animals. In contrast to
adipose tissue and skeletal muscle, metabolism in the mammary gland is not regulated by

insulin (Zhao et al., 1996), and nutrient uptake should occur more consistently. It is

100

speculated that consistent energy supply to animals results in enhanced milk production

for bm3 treatments.

Milk energy output as a percentage of NEL intake (MEOP) was higher for bm3 treatments
(P < 0.05; Table 1). Energy intake (NEl) was calculated from digested DMI.

N umerically higher energy intake for bm3 treatments may raise a question whether
mathematical dilution of maintenance energy output leads to a greater MEOP. However,
signiﬁcant MEOP difference between corn silage treatments was not negated (P < 0.05)
when MEOP was analyzed using the statistical model with energy intake as a covariate,
suggesting that more energy was utilized for milk production when cows were fed bm3

corn silage regardless of amounts of energy intake.

In contrast, low NDF diets increased energy intake (P < 0.01) and BW gain (P < 0.01),
but did not increase SCM yield (P > 0.26) compared to high NDF diets (Table 1). It is
observed that low NDF diets lowered rumen pH with higher daily variation and greater
range in variation compared to high NDF diets (Table 2). Greater ﬂuctuation in rumen
pH might indicate inconsistent energy supply to animals, resulting in an increase in
energy metabolite utilization by adipose and skeletal muscle tissue and relatively less
substrate available for milk production because the mammary gland is not sensitive to
insulin (Zhao et al., 1996). The relationship between ruminal fermentation pattern and

energy utilization warrants further investigation.

101

Feeding bm3 corn silage shifted the site of starch digestion from the rumen to post-
ruminal digestion (Table 6). Ruminal starch digestion was lower for cows fed bm3 corn
silage than control, probably because of faster rate of passage (P < 0.02), whereas post-
ruminal starch digestion was higher for cows fed bm3 corn silage probably due to
compensatory digestion from reduced ruminal digestion. It has been suggested that
metabolism of starch through glucose absorbed from the small intestine may be
energetically more efﬁcient than metabolism through fermentation to propionic acid in
the rumen and subsequent gluconeogenesis in the liver (Black, 1971). Owens et al.
(1986) reported ruminal starch digestion was energetically about 70% as efﬁcient as
starch digestion in the small intestine for steers. Although the total tract starch
digestibility tended to be lower for bm3 treatment in the current study, more starch was
digested post-ruminally for bm3 treatments, which might contribute to enhanced milk
production (Nocek and Tamminga, 1991). However, greater starch digestion in the small
intestine does not necessarily mean greater glucose absorption. Kreikemeier et al. (1991)
infused corn starch at 60 g/h into the abomasum of steers, and found that only 38% of
corn starch disappearance resulted in net portal glucose absorption, indicating that starch
can be fermented to VFA or lactate in the intestines. In addition, ruminants may have
limited capacity of starch digestion and absorption in the small intestine (Owens et al.,
1986; Nocek and Tamminga, 1991; Harmon, 1992). Although enhanced milk production
for bm3 treatments could be attributed partly to increased post-ruminal starch digestion,

the relationship between site of digestion and milk production is not understood clearly.

102

Microbial Efficiency

Duodenal flow of NAN and post-ruminal NAN digestion were higher for low NDF diets
and bm3 treatments (Table 7), possibly contributing to greater milk and milk protein
yield (Table 1). Low NDF diets increased duodenal NANMN ﬂow compared with high
NDF diets (Table 7). Lana et al. (1997) reported that low rumen pH increased amounts
of escape protein by decreasing ruminal deamination and ammonia accumulation.
However, bm3 treatments increased duodenal MN ﬂow and microbial efﬁciency, which
might be attributed to faster passage rate of ruminal digesta for cows fed bm3 corn silage
compared with those fed control corn silage. Greater rate of passage of ruminal digesta
might increase ﬂow of attached microorganisms. Ammonia concentration in the rumen
tended to be lower for cows fed bm3 corn silage (P < 0.09; Table 3), indicating that more
ruminal ammonia was incorporated into microbial protein. Although low NDF diets
increased passage rate of ruminal digesta, they did not increase duodenal MN ﬂow and
microbial efﬁciency, possibly because lower pH and greater ﬂuctuation in rumen pH

inhibited microbial growth.

Milk Composition

Milk fat content was lower when bm3 corn silage was fed with the low NDF diet,
compared with the other treatments (Table 1). However, fat yield was not affected by
either dietary NDF content or corn silage treatment. The observed lower milk fat content
might be explained by relative difference between the rate of milk fat synthesis and the
rate of milk lactose synthesis. Milk lactose yield and milk volume were increased for

bm3 treatments possibly because of consistent energy supply from the rumen and

103

increased post-ruminal starch digestion. Low milk fat content is commonly associated
with reduction in the acetate to propionate ratio in the rumen, but bm3 treatments did not
affect acetate to propionate ratio in this experiment. Although milk fat yield was not
different among treatments, milk fat was diluted by increased milk yield when cows were
fed bm3 corn silage with low dietary NDF. Cows fed bm3 corn silage at low dietary
NDF gained more BW, and improved energy status might have limited mobilization of
body fat for milk fat synthesis. Milk fat is derived from mobilized body fat, dietary fat,
and fatty acids synthesized in the mammary gland. Milk fatty acid composition needs to
be evaluated to understand why milk fat content is often depressed when a bm3 corn

silage is fed.

Milk lactose content was increased by bm3 treatments, but its mechanism is not well
understood. Lactose is synthesized in Golgi vesicles of secretory cells and draws water
into the vesicles by osmotic pressure. Therefore, milk lactose content generally does not
vary because osmolarity of milk tends to be maintained constant (Holt, 1983). However,
mineral elements also exert osmotic pressure in milk. There is negative correlation
between lactose content and those of Na and K, and total osmotic pressure of milk is kept
constant and equal to that of blood (Holt, 1983). When milk lactose content is increased,
it may mean either that mineral content in milk decreases (Linzell, 1967) or that both

milk osmolarity and blood osmolarity increase (W heelock et al., 1965).

104

CONCLUSION

Brown midrib 3 corn silage increased milk production without a signiﬁcant increase in
digested DMI. Enhanced performance for cows fed bm3 corn silage might be attributed
to consistent energy supply from the rumen, faster rate of passage, increased post-ruminal
starch digestion, and greater microbial protein production. Microbial efﬁciency was
greater for bm3 treatment. NDF digestion either in the rumen or in total tract was not
affected by corn silage treatments. Milk fat content was lowest when cows were fed bm3
corn silage with the low NDF diet. The beneﬁcial effects of bm3 corn silage on animal

performance might be greater when fed with a high NDF diet.

105

 

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CHAPTER 6

GENERAL DISCUSSION AND IMPLICATIONS

Rate of Passage and bm3 Corn Silage

Brown midrib 3 corn silage increased DMI and milk yield, which were partly attributed
to increased rate of passage for bm3 treatments. Enhanced DMI and animal performance
cannot be attributed to the difference in NDF digestibility in vivo. The second study
(Chapter 5) showed that N DF digestibility either in the rumen or in the total tract was not
affected by corn silage treatments. Although the ﬁrst experiment (Chapter 3) showed that
total tract NDF digestibility was slightly higher for cows fed bm3 corn silage, cows that
responded more to the bm3 treatment with greater DMI decreased total tract NDF
digestibility. Enhanced in vitro NDF digestibility does not necessarily increase NDF
digestibility in the rumen or in the total tract. Digestibility of NDF is a function of
potential extent of digestion, rate of digestion, and rate of passage (Allen and Mertens,
1988). Faster rate of passage is associated with enhanced DMI, and reduces NDF
digestibility (NRC, 1987) because ruminal retention time for NDF is shortened.

However, differences in ease of NDF hydrolysis and cell wall fragility within a forage

113

species might be estimated by in vitro N DF digestibility. These chemical and physical
characteristics of bm3 NDF could contribute to faster rate of passage. Nutritional
advantages associated with bm3 treatments were enhanced DMI, improved microbial
efficiency, and shifting site of starch digestion from the rumen to the intestines, and all of

these could be a result of a faster rate of passage.

Energy Utilization

Although maximum milk production of high producing dairy cows is generally limited by
energy intake, enhanced energy intake does not necessarily lead to greater milk energy
output. In the second experiment (Chapter S), cows fed low NDF diets increased energy
intake and milk yield, but did not increase SCM yield compared to those fed high NDF
diets. On the other hand, digested DMI was not signiﬁcantly higher for bm3 treatments,
but bm3 treatments increased milk yield and SCM yield. It is speculated that ﬂuctuation
of rumen pH inﬂuences energy utilization of lactating cows. Both low NDF diets and
bm3 treatments lowered rumen pH, but daily variation and range of rumen pH were
increased by low NDF diets, not by bm3 treatments. Greater daily variation in rumen pH
reﬂects greater variation in fermentable energy supply to the rumen and is likely to result

in ﬂuctuation of supply of energy metabolites from the rumen to animals.

An objective of the model discussed below is to demonstrate the importance of
ﬂuctuation in serum metabolite ﬂow to determine energy utilization of lactating dairy
cows. To simplify the model, it is assumed that milk volume is solely determined by

lactose synthesis in the mammary gland due to the osmotic effect of lactose. Rate of milk

114

lactose synthesis (MLS) is a function of rate of glucose uptake (GU) and glucose
metabolism efﬁciency (GME).

MLS (g/min) = GU (g/min) x GME (1)
GME probably varies diumally. However, incorporating GME in the model is beyond
the scope of this discussion, and it is generally agreed that glucose uptake is the limiting
step for milk lactose synthesis (Kronfeld, 1982). Therefore, assuming constant GME (=

1.0) for convenience of calculation, equation (1) is modiﬁed as:

MLS (g/min) = GU (g/min) (2)

Because glucose uptake in the mammary gland is not regulated by insulin, glucose uptake
should occur more consistently (Zhao et al., 1996). It is reported that rate of glucose
uptake in the mammary gland increases linearly when serum glucose concentration
increases from 2.2 mM to 3 mM, and becomes constant when the serum glucose
concentration exceeds 3.0 mM (Kronfeld, 1982). Because glucose ﬂow to the mammary
gland per min (GF) is determined by blood ﬂow rate (BF) and blood glucose
concentration (GC), serum glucose content gives only partial information about total ﬂow
of glucose to the mammary gland. However, this study (Kronfeld, 1982) showed that a
limitation to rate of glucose uptake (GU) exists at a certain point of serum glucose ﬂow.
This critical point of GU is arbitrarily set to 1.0 for this discussion. Glucose potentially

available (GA) to the mammary gland is a function of GP and uptake efﬁciency (K).

GF (g/min) = GC (g/L) x BF (L/min) (3)
GA (g/min) = GF (g/min) x K (4)

GU (g/min) = 1.0 if GA 2 1.0 (5)

115

GU (g/min) = min {GA, 1.0} (6)
In contrast to the mammary gland, glucose transport in adipose tissues is stimulated by
insulin at higher serum glucose concentrations, then limitation to glucose transport in

adipose tissues is less likely.

Although serum glucose concentration is regulated by a homeostatic mechanism, some
diurnal variation exists. Four graphs demonstrate several patterns of glucose supply and
glucose uptake in the mammary gland for 420 minutes (Figure 1-4). The undulant curve
indicates GA, and the area below the GA curve shows total glucose ﬂow that is
potentially available to the mammary gland (GT). The line (y = 1.0) indicates the
potential capacity of mammary gland to transport serum glucose into the cells. The line
GU consists of either the GA curve or the line (y = 1.0), whichever is lower, indicates the
rate of glucose uptake in the mammary gland, and area below this line shows the amount
of glucose taken into the mammary gland (GM). Maximum GM occurs by minimizing
the period for which GA value is below 1. The area, above the line (y = 1.0) but below
the line GA, indicates the amount of serum glucose needed to shift towards adipose

tissues, and denoted as GF.

GT (g) = sum {GA x min} (7)
GM (g) = sum {GU x min} (8)
GF (g) = GT - GM (9)

The comparison between Figure 1 and Figure 2 demonstrates how greater energy intake

inﬂuences glucose utilization when ﬂuctuation in energy supply is maintained. As total

116

glucose ﬂow (GT) increases from 420g to 450g, both areas GM and GF increases from
407g to 420g and 13g to 30g, respectively. This implies increased milk yield and BW
gain. The comparison between Figure 1 and Figure 3 demonstrates how a greater
ﬂuctuation in energy supply inﬂuences glucose utilization when energy intake is
maintained. Total glucose ﬂow (GT) does not change. However, the area GM decreases
from 407g to 393g and the area GF increases from 13g to 27g. This implies decreased
milk yield and increased BW gain. Comparison between Figure 1 and Figure 4
demonstrates how greater energy intake inﬂuences glucose utilization when a ﬂuctuation
in energy supply increases. As total glucose ﬂow (GT) increases from 420g to 450g, the
area GF increases from 13 g to 43g. This implies increased BW gain. In this example,
the area GM does not change, implying that greater energy intake can result in no change
in milk yield. Although actual regulation mechanisms for glucose utilization could be
more complex, this modeling approach shows the importance of ﬂuctuation in the serum

glucose ﬂow.

Volatile fatty acids are other energy metabolites for ruminants. Utilization of serum VFA
by lactating dairy cows might be explained by the model discussed above. The
ﬂuctuation in the blood VFA ﬂow possibly inﬂuences their utilization more dramatically
because serum concentration of VFA ﬂuctuates greatly. It has been reported that acetate
and butyrate can ﬂuctuate diumally more than 10-fold (Quigley and Heitmann, 1991).
Although the mammary gland is insulin resistant, adipose tissue increases acetate
utilization with action of insulin (Vernon, 1988). More ﬂuctuation in the serum ﬂow of

acetate and butyrate may allow the adipose tissues to uptake more of these metabolites.

117

Low rumen pH without increased daily variation for bm3 treatment might indicate high
and consistent VFA supply from the rumen into the blood circulation. This observation
implies that serum energy metabolite level was maintained high without increased
ﬂuctuation, which possibly accounts for increased FCM yield for bm3 treatments
regardless energy intake level. Low NDF diets in the second experiment increased
energy intake and milk yield, but did not improve FCM yield. The low NDF diets
lowered rumen pH with greater daily variation and range of rumen pH. Enhanced energy
intake increased milk yield; total glucose ﬂow might be high enough to offset the
possible loss of mammary glucose uptake resulting from ﬂuctuation of serum glucose
ﬂow. However, ﬂuctuation of serum acetate or butyrate concentration is generally
greater than that of serum glucose concentration, and it is speculated that a total ﬂow of
acetate or butyrate might not compensate the loss of mammary uptake resulting from the
ﬂuctuation in ﬂow of these substrates. Accordingly, cows fed the low N DF diets
increased BW gain instead of increasing milk energy output. This theory does not negate
other possible factors that account for milk fat depression, but indicates that milk fat
depression can be partially attributed to the ﬂuctuation of serum ﬂow of substrates. At
this point, we do not know that milk fat synthesis is limited more by enzyme activities in
the mammary gland or by rate of uptake for milk fat precursors. However, if substrate
uptake limits milk fat synthesis to some extent, the potential exists to alter energy

utilization of lactating cows by manipulating ruminal fermentation pattern.

118

Homeostasis and DMI Depression

It has already been discussed that the energy metabolites that exceed uptake capacity of
the mammary gland might be shifted towards adipose tissue under the inﬂuence of
insulin. However, insulin action is sometimes inhibited; in early lactation, all peripheral
tissues become insulin resistant so that more nutrients are available for the mammary
gland. Insulin resistance in early lactation might be related to the regulation mechanism
of DMI. Animals consume feeds to meet their energy requirement. However, it is
physiologically more accurate to say that animals consume feeds to maintain homeostasis
of the blood or to make up for metabolites used for maintenance, milk production, and
body deposition (M. S. Allen, personal communication). Although milk yield generally
sets the energy requirement of cows, cows in the early stage of lactation typically
consume less than when they produce the same amount of milk later in lactation. Low
DMI in early lactation is attributed partially to ﬁll limitation, but metabolic mechanisms
might be involved also. The hypothesis that animals consume feeds to maintain
homeostasis would explain low DMI in early lactation. Animals can consume more feeds
in later stage of lactation probably because rate of serum metabolite uptake is enhanced
by insulin. On the other hand, insulin resistance in early lactation suppresses the capacity
of peripheral tissues to remove metabolites from the blood pool, and animals need to
limit DMI to maintain homeostasis. In other words, animals more seriously need to

regulate metabolite inﬂow to the blood because they have less control on efﬂux from the

blood.

119

Feeding rapidly fermentable carbohydrate often depresses DMI. The satiety signals such
as lower rumen pH, greater serum propionate concentration, or insulin secretion may act
to prevent excessive accumulation of serum metabolites and to maintain homeostasis of
blood. Fill limitation to maximum DMI demonstrates the concept that physical capacity
of the rumen is limited and that restricted digesta ﬂow limits feed intake. Similarly,
capacity of the metabolite pool in the blood is physiologically limited, and extremely
rapid metabolite inﬂow or restricted metabolite efﬂux to peripheral tissue might limit
voluntary feed intake. This leads to the theory that ruminal fermentation pattern possibly
inﬂuences the DMI regulation mechanism of lactating dairy cows (M. S. Allen, personal

communication).

Fluctuation of rumen pH could be an important factor to inﬂuence DMI and energy
utilization of lactating dairy cows. However, it is difﬁcult to isolate speciﬁc effects of
ﬂuctuation in rumen pH from the effects of low rumen pH; a low NDF diet typically
increases fermentation acid production and decreases buffering capacity of rumen digesta
simultaneously. It is often observed that low NDF diets decrease DMI, NDF digestion in
the rumen, milk fat content, and FCM yield, and these observations have been attributed
to low rumen pH. However, they might directly result from greater ﬂuctuation of rumen
pH. In the second experiment (Chapter 5), bm3 corn silage treatments lowered rumen pH
without negative effects on animal performance. Although daily means of rumen pH and
serum metabolite concentration for cows fed a variety of diets are routinely reported in
the literature, little data are available for their diurnal variation and variation range.

Elucidating the relationship among fermentation pattern in the rumen, serum energy

120

metabolite ﬂow, DMI regulation mechanisms, and energy utilization of lactating dairy

cows warrant further investigation.

121

 

— - - Capacity
----GAl
—GU1

 

glucose (g/min)

 

 

0 so 160 1E0 230 2E0 350 330 450

minutes
Figure 1. Glucose availability (GA) and utilization by the mammary gland (GU) over
time: model 1. Total glucose ﬂow potentially available to the mammary gland (GT) =
420g; Amount of glucose taken into the mammary gland (GM) = 407g; Amount of serum
glucose needed to shift towards adipose tissue (GF) = 13g.

 

"—- GAZ
——-GU2

 

- - - Capacity]

 

1.2- ..

1. A A

glucose (g/min)

 

 

0 20 1:10 1&0 260 2&0 350 3;) 450

minutes
Figure 2. Glucose availability (GA) and utilization by the mammary gland (GU) over
time: model 2. Energy intake increases without increasing ﬂuctuation relative to Figure
1. Total glucose ﬂow potentially available to the mammary gland (GT) = 450g; Amount
of glucose taken into the mammary gland (GM) = 420g; Amount of serum glucose
needed to shift towards adipose tissue (GF) = 30g.

122

 

- — - Capacity
----- GA4
—GU4

 

 

 

glucose (g/min)

 

 

0 E0 100 lso 200 2E0 300 3&0 400
minutes

Figure 3. Glucose availability (GA) and utilization by the mammary gland (GU) over

time: model 3. Fluctuation increases without change in energy intake relative to Figure 1.

Total glucose ﬂow potentially available to the mammary gland (GT) = 420g; Amount of

glucose taken into the mammary gland (GM) = 393g; Amount of serum glucose needed

to shift towards adipose tissue (GF) = 27g.

 

- - - Capacity
“-— GA3
—-GU3

 

 

glucose (g/min)

 

 

0 so 100 1;!) 200 2so 300 3;0 400

minutes
Figure 4. Glucose availability (GA) and utilization by the mammary gland (GU) over
time: model 4. Energy intake increases with increased ﬂuctuation relative to Figure 1.
Total glucose ﬂow potentially available to the mammary gland (GT) = 450g; Amount of
glucose taken into the mammary gland (GM) = 407g; Amount of serum glucose needed
to shift towards adipose tissue (GF) = 43g.

123

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