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This is to certify that the
thesis entitled

Effect of rumen-protected fatty acid saturation on milk yield,
intake, chewing behavior and ruminal fermentation in lactating
dairy cows

presented by

Kevin J. Harvatine

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Animal Science

 

 

 

 

 

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6/01 c/CIRC/DatomopGS-DJ 5

 

EFFECTS OF RUMEN-PROTECTED FATTY ACID SATURATION ON MILK
YIELD, INTAKE, CHEWING BEHAVIOR AND RUMINAL F ERMEN TATION
IN LACTATING DAIRY COWS

By

Kevin J. Harvatine

A THESIS

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

MASTERS OF SCIENCE

Department of Animal Science

2003

ABSTRACT

Effects of rumen-protected fatty acid saturation on milk yield, intake, chewing
behavior and ruminal fermentation in lactating dairy cows

By

Kevin J. Harvatine
Two experiments were conducted to investigate the effects of rumen-protected
unsaturated (UNS) and saturated (SAT) fatty acids (FA) on milk yield, intake, chewing
behavior, and ruminal fermentation of lactating dairy cows. In the ﬁrst experiment, UNS
(2% calcium salts of palm FA) decreased dry matter intake (DMI, 0.8 kg/d), rumination
time (25 min/d), plasma insulin and milk protein concentrations compared to SAT (2%
prilled FA) in a 32 cow crossover design. In the second experiment, UNS (2.5 % calcium
salts of blended FA) decreased milk fat percent and increased body weight gain
compared to SAT (2.5% prilled FA) in 8 duodenally and ruminally cannulated cows.
Calcium salts only provided partial rumen-protection as UNS FA were highly
biohydrogenated. A simpliﬁed model of rumen biohydrogenation was developed and
used to determine that UNS decreased fractional biohydrogenation rate of trans-C18: 1.
Saturated FA decreased rumen organic matter digestibility possibly because of
modiﬁcation of particle passage rate related to increased rumination. UNS also
decreased dry matter intake and meal size compared to SAT (1.6 kg), and SAT increased
rumination over 50 min per day compared to control and UNS. Duodenal FA proﬁle is
important for prediction and manipulation of animal response because of physiological

and metabolic effects of individual FA.

Dedicated to the Harvatine and Foster Families of

Northeastern Pennsylvania

iii

ACIG‘JOWLEDGEMENTS

I would like to ﬁrst thank Dr. Allen for his guidance and patience throughout my
graduate program. Under his mentorship I have developed a strong science background,
learning the rules of science and experimental research. Dr. Allen also challenged me to
apply integrative thinking within my research and learn the process of critically
understanding biology and scientiﬁc theory. Most importantly he has highlighted the fun
and rewards of working in science. I would also like to thank Dr. Allen, the MSU
Graduate School, the MSU Department of Animal Science and the Milk Specialties
Company for providing ﬁnancial support and freedom to think and explore during my
research program. I would also like to recognize the Thomas Fellowship for allowing me
to attend the International Animal Functional Genomics Conference.

I would like to thank Dr. Herdt, Dr. Romsos, and Dr. VandeHaar for their
guidance, advice, and feedback as members of my graduate committee. I would also like
to acknowledge Dr. Beede and Dr. Bucholtz, unofﬁcial members of my guidance
committee, for their interest, advice and constant interaction. They and the other
professors of the dairy nutrition group have provided an invaluable service as
professional and personal role models.

One person alone does not conduct research projects of the size and scale
accomplished for this thesis. I would like to thank the Dr. Allen’s research technicians,
Dave Main, Dewey Longuski, and Jackie Yun Ying for their help and support during my
experiment and lab work. They are each experts in their individual roles. I would also

like to thank Justin Zyskowski for his time and knowledge in setting up and running fatty

iv

acid analysis, and Dr. Herdt for use of his GC for fatty acid analysis. The help of Dr.
Palmquist of The Ohio State University for fatty acid analysis and Dr. Boisclair of
Cornell University analysis for plasma leptin was appreciated. I would like to extend my
appreciation to Christy Taylor, Steve Mooney, Jenn Voelker, Barry Bradford, Jill
Davidson, Jim Liesman, Larry Chase, Bob Kreﬁ and the MSU Dairy Barn staff for their
excellent technical assistance and academic discussion. I would also like to recognize the
dedicated group of undergraduate students that provide help in our lab, especially Amy
Nash, Heather Wieczocek and Allison Rober.

Finally, I would like to thank my parents Paul and Susan, my brothers Josh and
Will, my sister-in-law Julie, and my close extended family for their continual

encouragement, advise, and understanding.

TABLE OF CONTENTS

 

 

 

LIST OF TABLES -- - - - -- - -- - -- xi
LIST OF FIGURES - - - - - - - - xv
LIST OF ABBREVIATIONS - - - ...... . -- -_ xvi
INTRODUCTION ............................................................................................... 1
CHAPTER 1
LITERATURE REVIEW
Dietary Fat ................................................................................................. 4
Fat and the Rumen ..................................................................................... 5
Sources of Dietary Fat ............................................................................... 11
Metabolic Utilization of Absorbed Fatty Acids .......................................... 17
Fatty Acid Requirements ........................................................................... 18
Fatty Acid Digestibility .............................................................................. 24
Dietary Fatty Acid Effects on Intake .......................................................... 27
Regulation of Intake ................................................................................... 28
Satiety and the Gastrointestinal Tract ......................................................... 3O
Satiety and Absorbed Nutrients .................................................................. 31
Satiety and Energy Balance ........................................................................ 32
Other Factors Stimulating Satiety ............................................................... 33
CHAPTER 2

The effect of production level on feed intake, milk yield and plasma metabolite
response to rumen protected fatty acid saturation in lactating cows.

ABSTRACT .............................................................................................. 35

vi

INTRODUCTION ..................................................................................... 36

MATERIALS AND METHODS ............................................................... 38
Cows and Treatments ..................................................................... 38
Data and Sample Collection ........................................................... 39
Sample Analysis ............................................................................. 40
Statistical Analysis ......................................................................... 41
RESULTS AND DISCUSSION ................................................................. 42
Intake and Chewing Behavior ......................................................... 43
Production ...................................................................................... 48
Energy Balance and Efﬁciency ....................................................... 51
Plasma Metabolites and Hormones ................................................. 53
CONCLUSIONS AND IMPLICATIONS .................................................. 54
CHAPTER 3

Effect of rumen-protected fatty acid saturation on milk yield, milk fatty acid profile,

energy balance and plasma metabolites and hormones of lactating dairy cows

ABSTRACT .............................................................................................. 68
INTRODUCTION ..................................................................................... 69
MATERIALS AND METHODS ............................................................... 70
Cows and Treatments ..................................................................... 71
Data and Sample Collection ........................................................... 72
Sample and Statistical Analysis ...................................................... 73
RESULTS AND DISCUSSION ................................................................. 75

vii

Milk and Milk Component Yield .................................................... 76

Milk Fatty Acid Proﬁle .................................................................. 77

Energy Intake and Balance ............................................................. 78

CONCLUSION ......................................................................................... 82
CHAPTER 4

Kinetic model of rumen biohydrogenation: effects of rumen protected fatty acid
saturation on fractional rate of biohydrogenation and duodenal fatty acid ﬂow in

lactating dairy cows.

ABSTRACT .............................................................................................. 93
INTRODUCTION ..................................................................................... 94
MATERIALS AND METHODS ............................................................... 96
Cows and Treatments ..................................................................... 96
Data and Sample Collection ........................................................... 97
Sample and Statistical Analysis ...................................................... 100
RESULTS AND DISCUSSION ................................................................. 102
Fatty Acid Intake and Duodenal Flow ............................................ 102
Rumen Pool and Turnover .............................................................. 104
Kinetics of Biohydrogenation ......................................................... 107
Extent of Biohydrogenation ............................................................ 110
Model Simpliﬁcations and Assumptions ......................................... 111
CONCLUSION ......................................................................................... 1 13

viii

CHAPTER 5
Effects of rumen-protected fatty acid saturation on ruminal and total tract nutrient

digestion in lactating dairy cows.

ABSTRACT .............................................................................................. 123
INTRODUCTION ..................................................................................... 124
MATERIALS AND METHODS ............................................................... 125
Cows and Treatments ..................................................................... 125
Data and Sample Collection ........................................................... 127
Sample and Statistical Analysis ...................................................... 127
RESULTS AND DISCUSSION ................................................................. 130
Intake ............................................................................................. 13l
Flow Marker .................................................................................. 131
Ruminal Carbohydrate Digestion .................................................... 132
Ruminal Digestion Kinetics ............................................................ 133
Post-Ruminal Digestion .................................................................. 134
Total Tract Digestion ...................................................................... 135
Fatty Acid Digestion ...................................................................... 135
CONCLUSION ......................................................................................... 141
CHAPTER 6

Effect of rumen-protected fatty acid saturation on feed intake, and feeding and
chewing behavior of lactating dairy cows

ABSTRACT .............................................................................................. 152

ix

INTRODUCTION ..................................................................................... 153

MATERIALS AND METHODS ............................................................... 154

Cows and Treatments ..................................................................... 154

Data and Sample Collection ........................................................... 155

Sample and Statistical Analysis ...................................................... 157

RESULTS AND DISCUSSION ................................................................. 159

Intake ............................................................................................. 159

Ruminal Pools ................................................................................ 162

Feeding and Chewing Behavior ...................................................... 162

Nutrient Selection ........................................................................... 165

Ruminal pH .................................................................................... 166

CONCLUSION ......................................................................................... 166
CHAPTER 7

CONCLUSIONS AND IMPLICATIONS .......................................................... 173

APPENDIX ......................................................................................................... 177

REFERENCES .................................................................................................... 179

LIST OF TABLES

CHAPTER 2
Table 1. Status of 31 cows at the beginning of experiment ..................................... 55
Table 2. Ingredients and nutrient composition of treatments .................................. 55

Table 3. Ingredient and nutrient composition of experimental

diets ................................................................................................................. 56
Table 4. Effects of fatty acid saturation on intake and

feeding behavior .................................................................................................... 5 7
Table 5. Effects of fatty acid saturation on production ........................................... 58

Table 6. Responses (saturated — unsaturated) of intake and
production by pretrial 3.5% fat-corrected milk yield .............................................. 59

Table 7. Responses (saturated — unsaturated) of production
by dry matter intake response (saturated - unsaturated) .......................................... 60

Table 8. Effects of fatty acid saturation on plasma hormones
and metabolites .................................................................................................... 61

Table 9. Effects of fatty acid saturation on energy intake
and partitioning ..................................................................................................... 62

Table 10. Responses (saturated - unsaturated) of energy

balance and plasma metabolites and hormones ...................................................... 63
CHAPTER 3

Table 1. Paramater means for cannulated and non-cannulated

cows used in the experiment .................................................................................. 83
Table 2. Composition of treatment mixes .............................................................. 83
Table 3. Ingredient and nutrient composition of experimental diets ....................... 84

Table 4. Effects of dietary rumen-protected fatty acids on milk
production of cannulated cows .............................................................................. 85

xi

Table 5. Effects of dietary rumen-protected fatty acids
on milk production of non-cannulated cows .......................................................... 86

Table 6. Effects of dietary rumen-protected fat on
milk fatty acid proﬁle for cannulated cows ............................................................ 87

Table 7. Effects of dietary rumen-protected fatty acids
on milk fatty acid proﬁle for non-cannulated cows ................................................ 88

Table 8. Effects of dietary rumen-protected fatty acids
on energy balance and efﬁciency for cannulated cows ........................................... 89

Table 9. Effects of dietary rumen-protected fatty acids
on intake and total tract digestion for non-cannulated cows ................................... 90

Table 10. Effects of dietary rumen-protected fatty acids
on energy balance and efﬁciency for non-cannulated cows .................................... 91

Table 11. Effects of dietary rumen protected fatty acids

on plasma metabolites and hormones ..................................................................... 92
CHAPTER 4
Table 1. Fatty acid composition of diets ................................................................ 118

Table 2. Effects of rumen-protected fatty acids varying in
saturation on FA intake, duodenal ﬂow, and biohydrogenation .............................. 119

Table 3. Effects of rumen-protected fatty acids varying in
saturation on ruminal pools and turnover rates ....................................................... 120

Table 4. Effects of rumen-protected fatty acids varying in

saturation on rates of passage from the rumen and rates

of biohydrogenation .............................................................................................. 121
Table 5. Effects of rumen-protected fatty acids varying in

saturation on extent of biohydrogenation and

biohydrogenation index ......................................................................................... 122
CHAPTER 5

Table 1. Ingredient and nutrient composition of
experimental diets ................................................................................................. 143

xii

Table 2. Effects of rumen protected fatty acids varying in
saturation on digestion of DM and OM .................................................................. 144

Table 3. Effects of rumen protected fatty acids varying in
saturation on digestion of total NDF and potentially
digestible NDF (pdNDF) ....................................................................................... 145

Table 4. Effects of rumen protected fatty acids varying in
saturation on digestion of starch ............................................................................ 146

Table 5. Effects of rumen protected fatty acids varying in
saturation on ruminal fermentation ........................................................................ 147

Table 6. Effects of rumen protected fatty acids varying in
saturation on ruminal digestion kinetics ................................................................. 148

Table 7. Effects of rumen protected fatty acids varying in
saturation on digestion of energy ........................................................................... 149

Table 8. Effects of rumen protected fatty acids varying in
saturation on digestion of FA ................................................................................. 150

Table 9. Effects of rumen protected fatty acids varying in
saturation on digestion of fatty acids ...................................................................... 151
CHAPTER 6

Table 1. Effects of dietary rumen-protected fatty acids
on duodenal fatty acid flow ................................................................................... 168

Table 2. Effects of dietary rumen-protected fatty acids
on intake of nutrients ............................................................................................. 169

Table 3. Effects dietary rumen-protected fatty acids
on ruminal nutrient pool. ....................................................................................... 169

Table 4. Effects of dietary rumen-protected fatty acids
on meal patterns and water consumption .............................................................. 170

Table 5. Effects of dietary rumen-protected fatty acids
on chewing behavior ............................................................................................ 171

Table 6. Effects of dietary rumen-protected fatty acids on
nutrient selection .................................................................................................. 172

xiii

Table 7. Effects of dietary rumen-protected fatty acids
on ruminal pH ....................................................................................................... 172
CHAPTER 7

Table 1. Effects of dietary rumen-protected fatty acids on
fatty acid intake of noncannulated cows ................................................................ 177

Table 2. Effects dietary rumen protected fatty acids on
ruminal pool variance ............................................................................................ 178

Table 3. Effects dietary rumen-protected fatty acids on ﬂux
of ruminal fatty acid disappearance ....................................................................... 178

xiv

LIST OF FIGURES

CHAPTER 2

Figure 1. Milk protein percent response by pretrial fat

corrected milk (FCM) yield ................................................................................... 64
Figure 2. Milk protein yield response by pretrial fat corrected

milk (FCM) yield .................................................................................................. 65
Figure 3. Milk protein percent response by insulin response .................................. 66

Figure 4. Relationship between leptin and bST concentration across
animal periods. ...................................................................................................... 67

CHAPTER 4

Figure 1. Simpliﬁed model of rumen biohydrogenation for calculation of fractional
passage and biohydrogenation rates ....................................................................... 114

Figure 2. Model recognizing different fatty acid (FA) pools describing rumen availability
of FA for bacterial uptake ...................................................................................... 117

XV

BCS
BHBA
BW
CCK
CLA
CON
CP
DE
DIM
DM
DMI
FA
FCM
GE
GI
GLP-l
INDF

INT

MUN
NDF

NEFA

LIST OF ABBREVIATIONS
Body Condition Score
B-hydroxybuturate
Body Weight
Cholecystokinin
Conjugated Linoleic Acid
Control
Crude Protein
Digestible Energy
Days in Milk
Dry Matter
Dry Matter Intake
Fatty Acid
Fat Corrected Milk
Gross Energy
Gastro-intestinal
Glucagon-like-peptide- 1
Indigestible Neutral Detergent Fiber
Intermediate
Linear
Milk Urea Nitrogen
Neutral Detergent Fiber

Non-esteriﬁed Fatty Acid

xvi

NEL
NEm
OM
pdNDF
pFCMY

PUFA

SAT
TRT
UNS

VFA

Net Energy of Lactation

Net Energy of Maintence
Organic Matter

Potentially Digestible NDF
Pretrial Fat Corrected Milk Yield
Poly unsaturated FA
Quadratic

Rumen Protected Fatty acids
Saturated

Treatment

Unsaturated

Volatile Fatty Acid

xvii

INTRODUCTION

Early lactation, high producing dairy cows experience negative energy balance due to
limitation of energy intake, while late lactation, low producing cows gain excessive body
weight due to failure to control energy intake. Although, lactation and maintenance of
body weight are under homeorhetic control, milk production and tissue gain are not
perfectly coordinated with intake causing asynchrony of nutrient intake and energy
expenditure. Negative energy balance places the cow under metabolic stress, decreasing
milk yield and persistency and increasing susceptibility to metabolic disorders.

Excessive body weight gain wastes feed resources and decreases whole farm efﬁciency,
and increases the incidence of dystocia, ketosis, and fatty liver during the next lactation.
Dairy nutritionists attempt to regulate energy balance through dietary
intervention. Concentrates are commonly fed to increase the energy density of a diet,
which increases fermentation acid production, and decreases dietary ﬁber and daily
rumination time (Allen, 1997). A reduction in ruminal pH with increased concentrate
feeding can decrease rumen ﬁber digestion, decrease microbial protein production, and
increase systemic acidosis. In contrast, fat supplementation increases dietary energy
density without increasing diet fermentability. Fat sources can be protected in the rumen;
decreasing FA biohydrogenation and inhibition of fermentation (Wu et al., 1991).
Research in other animal models demonstrates the bioactivity of FA, including
modulation of intake and body weight gain. Although increased dietary energy density is
not required in late lactation, FA supplementation may be used to decrease intake and

body weight gain, or decrease digesta passage rate and increase diet digestibility. In late

lactation, decreasing intake and partitioning nutrients towards milk synthesis and away
from fat storage would allow continued feeding of a high concentrate diet that provides
more propionate to help maintain higher levels of milk yield (Leaver, 1988; and Hansen
et al., 1991).

Intake and diet nutrient density and digestibility determines total nutrients
absorbed. Fat supplementation increases the energy density of the diet, but intake and
digestibility must be maintained to increase daily energy intake. The intake response to
dietary fat depends on the FA proﬁle reaching the duodenum; with unsaturated FA more
hypophagic than saturated FA (Drackley et al., 1992; Christensen et al., 1994; Bremmer
et al., 1998; and Allen, 2000). Saturated FA digestibility is lower than unsaturated FA
fed in triglyceride form, but saturation may not affect digestibility when fed as
unesteriﬁed FA (Pantoja et al., 1995; Pantoja et al., 1996; and Elliot et al., 1999).

Fatty acid protection methods and effects of FA supplements on rumen
fermentation have not been tested with high producing cows fed fermentable diets and
high concentrations of rumen available FA. Few studies in lactating cows have reported
digestibility of unesteriﬁed FA differing in saturation directly in the same experiment
(Eastridge and Firkins, 1991; Palmquist, 1991; and Schauff and Clark, 1992), and there
are no reports of FA digestibility in high producing cows with high passage rates.

Cow response to energy supplementation may depend on metabolic state. High
and low producing cows are considerably different in energy metabolism and intake
regulation. Two experiments were conducted to evaluate cow response to rumen-
protected FA saturation. A crossover experiment using 2% calcium salts of palm oil and

prilled, hydrogenated FA was ﬁrst used to observe difference in response to FA

saturation by low and high milk yield cows. A second intensive experiment used 2.5% of
a more unsaturated FA calcium soap than palm oil and prilled, hydrogenated FA to
observe effects on ruminal digestion and feeding behavior. An additional block of non-
cannulated cows was used in the second experiment to increase the number of intake and
milk production observations.

It may be possible to select FA supplements to modify metabolism based on
production goals such as increased energy intake and efﬁciency in early lactation and
decreased body weight gain and intake in late lactation. The objective of this research
was to evaluate the effects of supplemental FA saturation on milk yield, energy balance,
ruminal fermentation, feed intake, and chewing behavior. The hypothesis of the ﬁrst
experiment was that more highly unsaturated FA would decrease intake relative to
saturated FA at equal FA concentrations, and individual cow response would depend on
production level. The hypothesis of the second experiment was that more unsaturated FA
would decrease intake by decreasing meal size, and increase ruminal digestion by

decreasing passage rate, but would not affect FA digestibility.

CHAPTER 1

A REVIEW OF LITERATURE

Dietary Fat

Dietary fatty acids (FA) serve a number of functions in lactating dairy cows.
Traditionally, fat has been considered an energy source, providing energy required for
maintenance and production of tissue and product. Dietary FA also serve as integral
structural components of cellular membranes, and regulatory molecules. More recently,
FA are appreciated as biological modiﬁers of physiology and metabolism, making them
bioactive compounds (Drackley, 2000). Dairy cows experience vastly different metabolic
states during a lactation cycle and dietary FA serve different roles during these states. It
is reasonable to speculate that cow response will depend on FA proﬁle, metabolic state,
and their interaction.

Fat supplementation in ruminants is not a new area of investigation. Palmquist
and Jenkins (1980) provided a short history of fat research, discussing a 1907 review of
data from 10 European experiment stations showing little beneﬁt of fat on milk and milk
fat yield (Kellner, 1907). However, research in the late 1920’s to early 1940’s
consistently observed a 2 to 10% milk production response to increased dietary lipids. In
a 1960 review, Warner (1960) discussed reduced ﬁber digestion and milk production
with fat supplementation, leading to the conclusion that fat was rarely superior to cereal
grain. Palmquist and Jenkins (1980) focused their review on the renewed interest in

using fat supplementation to increase dietary energy density, without increasing dietary

starch content, to support energy requirements of high producing cows. Recently, dietary
fat has also gained interest for increasing reproductive efﬁciency (Staples et al., 1998),
and changing the FA proﬁle of animal products (Grummer, 1991; Pahnquist and
Beaulieu, 1993; and Mansbridge and Blake, 1997). Consumers are increasingly
concerned about the intake of saturated FA with their link to health problems including
heart disease and diabetes (Mansbridge and Blake, 1997). Increasing CLA intake may
decrease the incidence of cancer and obesity (Kelly, 2001). Dietary manipulation may
allow designing FA proﬁles of meat and milk products to meet consumer demands for
low saturated FA concentration (Grummer, 1991). Lastly, FA type has a profound effect
on animal physiology including metabolic signaling and gene transcription that may have

application to increase production and efﬁciency (Drackley, 2000).

Fat and the Rumen

Dietary FA must ﬁrst pass through the rumen before absorption in the intestine,
making rumen lipid metabolism an important starting point for discussion of fat
supplementation. Rumen activity and fate of FA is well studied and the subject of many
reviews including those by Harfoot (1981), Harfoot and Hazlewood (1988), and Jenkins
(1993). Extensive lipolysis of triglycerides and hydrogenation of unsaturated FA occur
in the rumen by bacteria and protozoa. Theoretically, digestion of long-chain FA in the
rumen should be low, with minimal absorption across the ruminal epithelium and
minimal catabolism to VFA and C02 (Jenkins, 1993). Microbial de novo FA synthesis for

incorporation into phospholipids should produce a net positive ﬂow of lipids to the

duodenum (Drackley, 2000). In contrast to the expected positive rumen FA flow,
digestion studies commonly observe a net loss of FA in the rumen (Jenkins, 1993).

Lipid complexes (triglycerides, galactolipids and phospholipids) are hydrolyzed
to their individual components in the rumen, releasing long-chain FA. The rapid
hydrolysis of esteriﬁed FA, especially diglycerides, was recognized with identiﬁcation of
the lipolytic capacity of many bacteria (Harfoot, 1981). Although considered a rapid
process, lipolysis may be rate limiting, as different biohydrogenation end products are
observed in esteriﬁed and non-esteriﬁed treatments (Palmquist and Jenkins, 1980). In
addition, the rate of lipolysis may be affected by the saturation of FA in lipid complexes
(Elliot et al., 1997).

Free unsaturated FA are biohydrogenated by rumen bacteria and protozoa. The
importance of protozoal biohydrogenation was disregarded after observing that
defaunation had little effect on rumen FA biohydrogenation (Harfoot, 1981). Protozoa
may play a secondary role to bacteria, or bacterial hydrogenation capacity may be very
high. Hydrogenation of unsaturated FA may be a protective mechanism, as unsaturated
FA are toxic to rumen bacteria (Harfoot, 1981). However, in the rumen, unsaturated FA
are normally present as a triglyceride or are associated with feed particles that would
have little interaction with bacteria (Harfoot, 1981). Harfoot (1981) proposed that
because bacteria require more highly saturated FA for formation of phospholipid
membranes they may hydrolyze and biohydrogenate FA for incorporation into their
membranes, eliminating the metabolic burden of synthesizing saturated FA.

Microbial biohydrogenation is a multi-step process of which the rate and control

are not well understood. A free carboxyl group is required for hydrogenation, limiting

the availability of FA in triglycerides or associated with metal cations. The requirement
for a free carboxyl group was ﬁrst concluded after observing a lower plasma appearance
rate with ruminal infusion of linoleic acid in free compared to esteriﬁed form, and was
later demonstrated in vitro (Harfoot, 1981). Biohydrogenation of linoleic and linolenic
acid are multistep pathways that include trans-diene intermediates (Harfoot and
Hazelwood, 1988). The ﬁrst step of hydrogenation of linoleic acid is isomerization of the
cis-l2 bond to trans-11, forming cis-9, trans-11 C18:2, known as conjugated linoleic acid
(CLA) (Drackley, 2000). A hydrogenation reaction then removes the cis-9 double bond
forming vaccenic acid. In the ﬁnal step, the trans-11 bond is removed producing stearic
acid. Biohydrogenation of oleic acid also includes formation of a number of trans-F A
intermediates (Mosley et al., 2002). The competition between FA biohydrogenation and
passage rates determines duodenal FA ﬂow. Considerable levels of vaccenic acid and
other trans-C1821 isomers reach the duodenum, but very little CLA escapes from the
rumen (Piperova et al., 2002).

Allen (2000) proposed that the extent of biohydrogenation is a result of the
characteristics of the fat source, retention time in the rumen, and characteristics of the
microbial population. Using simple enzyme kinetic theory, total biohydrogenation is
determined by the pool size of available FA, rumen retention time, and bacterial
hydrogenation capacity that is a function of bacteria concentration, microbial population,
and rumen environment. Beam et a1. (2000) observed decreased rates of lipolysis and
biohydrogenation with increased concentrations of polyunsaturated triglycerides. Even at
high concentrations of FA the rate of lipolysis was over three times the rate of

biohydrogenation, thus lipolysis was not rate limiting (Beam et al., 2000). However,

Van Nevel and Demeyer (1996) observed decreased lipolysis with lower pH, making
lipolysis the rate—limiting step. In vitro experiments showed severe inhibition of
fermentation with addition of free unsaturated FA, but no effect of esteriﬁed unsaturated
FA or free saturated FA (Chalupa et al., 1984). Traditionally, hydrolysis of triglycerides
is considered rate limiting, but it appears that numerous factors affect hydrolysis. In
some situations unsaturated FA availability is limited or slowed by esteriﬁcation, but not
in all. Factors determining the rate of hydrolysis are not well understood, limiting
nutritionist’s ability to predict the level of rumen FA protection provided by
esteriﬁcation.

Loss of dietary FA from the rumen through absorption across the rumen wall and
oxidative metabolism is often considered minimal, and bacterial synthesis of PA is ‘
commonly expected to produce a net positive ﬂow of FA through the rumen. Low
absorption and metabolism of FA from the rumen was ﬁrst concluded with minimal
plasma recovery of radioisotope labeled linoleic acid infused into the rumen while
diverting nutrients with a reentrant cannula (Jenkins, 1993). Ruminal bacteria contain
10-15% lipid on a DM basis. These fats originate from preformed FA uptake and de
novo synthesis (Jenkins, 1993). Rumen bacteria and protozoa readily incorporate dietary
FA into their cellular membranes, and increased availability of exogenous FA decreases
endogenous synthesis (Palmquist and Jenkins, 1980). Wu and Palmquist (1991) reported
synthesis of 6.6 mg of FA per g of non-lipid diet during 24 h in vitro incubations; lipid
synthesis was not affected by source of fat (calcium salts and T6) or addition of acetate
or isoacids. Duodenal FA ﬂow cannot be partitioned into dietary and microbial

synthesized origins in simple digestion studies, and bacterial synthesis may hide possible

oxidation and absorption of FA from the rumen. In contrast to the net positive FA ﬂow
expected, Jenkins (1993) observed that 15 out of 47 published treatment means reported a
loss of FA from the rumen. Regression analysis predicted an 8 percent loss of lipid
intake and showed up to 30 percent lipid loss in the dataset (Jenkins, 1993). Ferlay et a1.
(1993) reported a 14% increase in FA ﬂow with control diet and 36.7 and 21.3% rumen
FA loss with rapeseed FA fed as calcium salts and triglycerides, respectively. Loss of FA
in the rumen may be due to ﬂow marker error causing under-prediction of duodenal ﬂow.
However, Doreau and Chillard (1997) proposed that negative FA ﬂux observed through
the rumen is not because of ﬂow marker bias, but caused by absorption and oxidation of
FA especially with higher fat diets. Fatty acid oxidation was observed by rumen
epithelium in vitro, and bacteria adhering to the rumen wall can absorb oxygen from the
epithelial cells and are capable of oxidative metabolism (Doreau and Chilliard, 1997).
The authors proposed that higher fat diets experience greater loss of FA and hypothesized
that FA are less adsorbed to feed particles in high fat diets leading to increased contact
with the rumen wall and increased opportunity for absorption and oxidation. The
increasing occurrence of rumen FA loss reported in digestion studies merits investigation
of rumen FA metabolism that has been ignored as technical bias.

Dietary fat can alter microbial grth and have profound associative effects on
ruminal nutrient digestibility. Chalupa et al. (1984) observed that unsaturated FA
inhibited fermentation, but saturated FA had no effect. Fat supplementation has variable
effects on ruminal digestion, but normally ﬁber digestion is decreased, and nonstructural

carbohydrate digestion is not changed (Jenkins, 1993). Total tract digestibility is

normally not affected by fat supplementation because of compensatory digestion in the
lower tract (Merchen et al., 1997).

Devendra and Lewis (1974) reviewed four theories for fat mediated depression of
ﬁber digestion including: 1. physical coating of ﬁber preventing microbial attachment, 2.
modiﬁcation of rumen microbial population due to toxic effects, 3. inhibition of
microbial activity due to coating of bacterial cell surface, and 4. reduction in cation
availability for microbes from formation of salts with long-chain FA. The authors
preferred the physical coating of ﬁber theory. In contrast, Palmquist and Jenkins (1980)
concluded that most data support inhibitory effects on microbial activity, which changes
bacterial competitiveness and shifts the microbial population, especially causing a
decrease in protozoa and cellulytic bacteria. Jenkins ( 1993) attributed the variable effects
of fat on ﬁber digestion to the structure of the lipid including degree of saturation and
presence of a free carboxyl group. Free FA can directly inhibit microbial growth through
disruption of membrane function (Jenkins, 1993). Unsaturated FA are more toxic than
saturated FA, possibly because of increased FA absorption, detrimental effects of
biohydrogenation, or disruption of cellular membrane function (Jenkins, 1993).

Inhibitory effects of FA on ﬁber digestion can be partially alleviated. Addition of
metal cations (ex. Ca) increases formation of insoluble salts, blocking FA absorption and
inhibition of microbial growth (Palmquist and Jenkins, 1980). Increasing saturation and
chain length of the FA increases the amount and strength of salt formed (Jenkins and
Palmquist, 1982). The formation of the metal salts is determined by the binding afﬁnity
of the cation and the dissociation constant of the FA. Fatty acid binding to metal cations

is partially dependent on pH of the rumen and the pK, of the FA. Sukhija and Palmquist

10

(1990) determined the pK., for calcium salts of stearate, tallow, palm FA and soy oil to be
4.5, 4.5, 4.6 and 5.6 respectively. These pK, values are misleading because they are
determined for a FA mixture. Soy oil contains a much higher concentration of
unsaturated FA than the other treatments and demonstrates the high pKa of unsaturated
FA. Finally, addition of other feed particles, particularly ﬁber, decreases detrimental
effects presumably through increased competition in FA absorption (Doreau and
Chilliard, 1997).

Ruminal lipid metabolism changes FA proﬁle reaching the duodenum. The
proﬁle of long-chain FA absorbed in the small intestine is the combined result of the FA
fed and ruminal biohydrogenation. Duodenal FA are more saturated than dietary FA, and
include many FA isomers from incomplete biohydrogenation, and odd-carbon and

branch-chain FA from microbial synthesis.

Sources of Dietary Fat

Diets of dairy cows contain four appreciable sources of FA including forages,
grains, oilseeds, and fat supplements. The sources vary in FA form and type and have
different effects in the rumen (Allen, 2000).

The large dietary proportion of forage fed makes it a signiﬁcant source of lipid,
even though forage contains a small concentration of FA (l-4%). This is especially true
in low total fat diets. Forage lipids are found predominantly in the plant leaf, mostly in
the form of glycolipids and some as phospholipids (Harfoot, 1981). The high
concentration of glycolipids may cause an overestimation of the energy value because

glycolipids have less energy than estimated by the 2.25 factor used in calculation of total

11

digestible nutrients (TDN; Van Soest, 1996). Vegetative FA composition is highly
unsaturated, normally containing over 70% linoleic and linolenic acid. Doreau and
Chilliard (1997) found it interesting that plant FA are only slightly less hydrogenated in
the rumen than free oils. High efﬁciency of forage lipid hydrogenation may not be
surprising since forage lipids are mostly associated with the leaf which is rapidly broken
down in the rumen, especially with legumes. The FA are in complex lipids but are in
close association with bacteria digesting the leaves. Furthermore, high concentrations of
FA are present in the leaf chloroplast (Harfoot, 1981) that would be spilled when plant
cells are ruptured.

Grain supplements vary in their FA concentration, proﬁle and availability. Corn
grain FA content varies with variety including specially bred high-oil corn. Although
corn FA content is low, it can contribute considerably to FA intake when fed at high
inclusion rates. Grain byproducts can also provide a considerable amount of lipid.
Soybean meal retains residual FA after extraction and many corn grain byproducts
contain considerable concentrations of FA including com distillers grain (10% EE, NRC,
2001). Fat availability is expected to depend on association of FA with grain
components, particle size and processing methods. The saturation, esteriﬁcation, and rate
of rumen availability of grain FA is expected to vary considerably.

Oilseeds contain a high concentration of lipid in the form of triglycerides and can
increase the dietary lipid concentration even at low inclusion rates (2-12% of DM).
Cottonseed, soybean, canola, and rapeseed are common oilseeds fed to dairy cows
depending on location, availability, and price. Oilseed lipid contains high concentrations

of unsaturated fat (50—90% of FA) mostly as oleic and linoleic acid with low

12

concentrations of linolenic, although there are large differences in the oleic to linoleic
acid ratios between oilseeds (Van Soest, 1996). Most oilseed FA are in the form of
triglycerides and must be released by lipolysis before biohydrogenation, as previously
discussed. The triglycerides are contained within the seed coat and are adsorbed to the
seed components. The seed must be mechanically broken down by chewing and
microbial digestion to release the triglycerides. Triglyceride release is predicted from the
digestion rate (inherent feed characteristics and fermentation) and passage rate from the
rumen.

Although oilseeds contain very high concentrations of unsaturated FA, processing
method determines the extent of FA biohydrogenation and interference with
fermentation. Grinding, extruding, roasting, ﬂaking, ferrnaldehyde coating, and
hydroxide treatment change FA availability in the rumen. The processing challenge is to
slow the rate of unsaturated FA availability in the rumen while not impeding FA
digestion in the small intestine. In vitro fermentation observed decreased lipolysis and
increased ﬁber digestion with whole and roasted oilseed compared to free oil (Reddy et
al., 1994). Roasting decreased FA availability linearly with increasing roasting
temperature and extrusion had little protective effect presumably caused by the release of
oil from cells during processing (Reddy et al., 1994). In agreement, in viva comparison
of extruded, ground, and roasted soybeans concluded that FA were protected the least
with extrusion and the greatest with roasting (Chouinard et al., 1997). Tice et al. (1994)
observed no difference in reducing particle size of roasted soybeans on rumen
biohydrogenation; biohydrogenation of roasted soybean C18 unsaturated FA ranged from

52.1 to 60.1% and was not different from calcium salts of palm oil control (57.6%).

13

Whole rapeseed and canola seed provide excellent rumen protection of FA but are also
indigestible in the small intestine. Grinding rapeseed and canola seed increases intestinal
digestibility but dramatically increases rumen FA availability, while chemical treatment
to weaken the seed coat increases intestinal digestibility while maintaining rumen
protection (Hussein et al., 1996). Whole cottonseed was moderately biohydrogenated
(566-67. 1% of unsaturated C18 FA), maintained total tract FA digestibility (62.5-
70.9%), and had no detrimental effect on ﬁber digestion and microbial fermentation when
fed at different concentrations in diets differing in ferrnentability (Harvatine et al., 2002).
Animal and vegetable fat by-products provide an economical source of FA for
dairy rations. These fat sources vary in their rumen activity and can be processed to
increase saturation or decrease esteriﬁcation. Fatty acids that do not interfere with rumen
fermentation are considered rumen inert, protected, or bypass. Protected fat sources do
not interfere with rumen fermentation as their characteristics make FA unavailable to
microorganisms and prevent coating of feed particles. Highly saturated FA are naturally
protected in the rumen considering their low interference with fermentation (Chalupa et
al., 1984), presumably because of their high melting point, hydrophobia, and limited
disturbance of cellular membranes. Esteriﬁcation may partially decrease FA availability
and protect microbial fermentation when lipolysis limits FA availability. In vitro,
fermentation is not inhibited by esteriﬁed FA (Chalupa et al., 1984), but results may
differ depending on hydrolysis rate, which may be affected by bacterial population,
rumen environment and triglyceride saturation. Vegetable oil and animal fat are mostly
esteriﬁed FA with varying FA proﬁle depending on source. The degree of saturation of

esteriﬁed fat has variable effects on rumen fermentation and digestibility. Saturation of

14

tallow normally does not change rumen fermentation in dairy cows (Pantoja et al., 1995;
Pantoja et al., 1996), although decreased DM and ﬁber digestibility have been observed
in steers (Elliott et al., 1997)

Dietary rumen available FA concentrations above 5% DM are generally
considered detrimental to rumen fermentation. Protected products have been developed
including encapsulated, calcium soaps, prilled and hydrogenated, and amide bonded FA
(Jenkins, 1993). Rumen protected fat serves the dual purpose of protecting rumen
fermentation against the detrimental effects of FA, and protecting unsaturated FA from
biohydrogenation.

The ﬁrst protection method was developed over 30 years ago and included
encapsulation of oil by formaldehyde-treated protein that was indigestible in the rumen,
but was digestible in the abomasum (Doreau and Chilliard, 1997). The method was only
partially effective with challenges in manufacturing and breakdown during mastication
(Doreau and Chilliard, 1997).

Calcium soaps of long-chain FA are a common, commercially available product
used as a source of protected unsaturated FA. Calcium ions are bound to the free carboxyl
groups of FA rendering them unavailable for biohydrogenation. Calcium salts of palm
oil seem to have no effect on rumen ﬁber digestion although biohydrogenation of the FA
may be extensive (Doreau and Chilliard, 1997). Wu et al. (1991) and Wu and Palmquist
(1991) reported in vivo and in vitro hydrogenation of calcium salts of palm oil at 57 and
47% of unsaturated FA respectively. As previously presented, pH has a large effect on
the dissociation of the metal salts, and increasing FA saturation increases cation binding

(Sukhija and Palmquist, 1990). The large postprandial drop in pH is expected to result in

15

dissociation of the calcium complex, especially from highly unsaturated FA as
demonstrated in vitro (Doreau and Chilliard, 1997). Sakhija and Palmquist (1990)
determined 10% dissociation of calcium salts of palm oil and tallow at pH 5.5. They also
determined that soy oil, which contains a high concentration of unsaturated fat, had a pKa
of 5.5, much higher than the 4.5 pKa of the more highly saturated palm FA (Sukhija and
Palmquist, 1990). In a mixed blend, unsaturated FA will dissociate more than saturated
FA as pH decreases because unsaturated FA have a higher pK, Fatty acids may reattach
to calcium when rumen pH increases after the postprandial drop, but can be
biohydrogenated while dissociated.

Saturated FA are considered inert in the rumen as their chemical characteristics
limit their dispersion in the rumen and decrease their direct contact with bacteria.
Saturated FA also have limited physiological inﬂuence on bacteria because they do not
interfere with membrane integrity and are not easily absorbed because of their
hydrophobic nature. Prilling and hydrogenating fat takes advantage of this characteristic
by simply saturating fat sources. The process results in very saturated free FA that do not
interfere with rumen fermentation and digestion of DM and ﬁber (Elliott et al., 1994;
Elliott et al., 1997).

The most recently developed technique to create rumen protected fat binds FA to
primary amines producing fatty acyl amides (Fotouhi and Jenkins, 1992; and Jenkins et
al, 1996). This process protects FA by blocking the carboxyl group much the same as
metal cations. Fatty acyl amides provide exceptional rumen protection with a 0.4%/h rate
of biohydrogenation compared to 4.7%/h for free FA (Fotouhi and Jenkins, 1992), and

either do not affect or increase total tract digestion of DM and ﬁber (Jenkins, 1999; and

16

DeLuca and Jenkins, 2000). The goal of manufactured fat products is to protect the FA
in the rumen to prevent interference with fermentation, but still deliver highly digestible

FA to the small intestine.

Metabolic Utilization of Absorbed Fatty Acids

Dietary FA are used as a concentrated source of energy. Early nutritionists
recognized the increased energy value of fat, assigning it a physiologic fuel value 2.25
times that of protein and carbohydrates (Stipanuk, 2000). This is the result of increased
efﬁciency during digestion, oxidation, and tissue deposition. Although some rumen loss
of FA has been reported, FA are not extensively destroyed by fermentation in the rumen.
Whereas, fermentation of dietary carbohydrates results in a large loss of energy from
maintenance and growth of bacteria and methane production. Fatty acid digestion in the
small intestine results in roughly 80% absorption of available FA (Drackley, 2000).
Biochemically, FA contain a large amount of free energy stored in carbon bonds. The
metabolism of FA yields energy for maintenance and production through complete
oxidation or partial oxidation and ketogenesis. Finally, transferring dietary fat to product
is very efﬁcient as preformed FA can be directly deposited in adipose or milk and do not
have to enter synthesis pathways that result in energy loss.

Biological systems are engineered to use fat for insulation, cushioning, cellular
structure, long-term storage of energy, and production of second messengers. Animals
can synthesize FA de novo from nutrients such as protein and glucose. However, the
ability to produce unsaturated FA are limited. Mammals can desaturate FA but cannot

place a double bond within nine carbons from the methyl end of the FA. This prohibits

17

the synthesis of FA with double bonds in the 00-6 and 00-3 positions that are required for
normal formation of cellular membranes and synthesis of key regulatory molecules such
as prostaglandins (Sardesai, 1992). Many of the required FA can be synthesized by
elongation and desaturation of linoleic and linolenic acid. In addition, unsaturated fat and
more speciﬁcally poly-unsaturated FA (PUFA) help maintain the ﬂuidity of cellular
membranes (Sardesai, 1992). Increasing unsaturated FA increases membrane ﬂuidity
because of their lower melting point. Changes in ﬂuidity can affect membrane
integration and movement of proteins such as receptors and transporters (Drackley,
2000). These changes affect the activity and efﬁciency of membrane transporters,

enzymes and receptors.

Fatty Acid Requirements

The many roles of FA and their bioactivity complicate the determination of
dietary FA requirements. This highlights the ambiguous nature of deﬁning nutrient and
animal requirements. The terms dispensable and indispensable are used to categorize
amino acids. Reeds (2000) discussed application of these categorizes in protein
metabolism and highlighted dependence on their deﬁnition that may change from a
nutritional, metabolic or functional perspectives. Likewise, the same concept has been
applied to FA, categorizing each as essential or nonessential based on the animals
capacity to synthesize or conserve the required amounts (Cunnane, 2000). Cunnane
(2000) proposed that the essential FA should be renamed “conditionally dispensable” due
to adequate capacity to synthesize or conserve the essential long-chain polyunsaturates

and their parent molecules.

18

Animal requirements are difﬁcult to quantify as they may be deﬁned as the
substrate required for maintenance and sustained production, or nutrient concentrations
that stimulate maximum production through changing physiology and metabolism. The
ﬁrst deﬁnition employs simple accounting and a factorial approach to ﬁrst calculate
expenditure in maintenance and production activities, and then determines required
intake based on biochemical assumptions of efﬁciency and metabolic conversion. A FA
requirement is thus the amount of the FA secreted in milk plus that retained in tissue and
oxidized for energy. The second deﬁnition recognizes that absorbed nutrients change
physiology and metabolism that determine animal response. Through this deﬁnition, FA
requirement depends on the amount and proﬁle of FA that directs nutrients to lactation
and increases efﬁciency through gene regulation and endocrine stimulation. Recognizing
the second level of complexity demands research into not only the energy value of dietary
nutrients consumed, but also the physiological and metabolic effect of individual FA.

Linoleic and linolenic acid are required as sources of (.0-6 and (o-3 FA that the
cow cannot synthesize. These FA are normally considered essential and must be supplied
in the diet (Drackley, 2000). Although it is easy to label these FA as essential, a
requirement is much harder to deﬁne. The metabolic requirement for the essential FA
has not been determined in the ruminant, but would be expected to be a function of FA
oxidation and turnover, grth and production of message.

Essential FA have been a subject of conversation in ruminant nutrition for many
years. The discussion has centered on the ruminant’s ability to absorb PUFA when their
diet is low in fat and the rumen actively biohydrogenates PUFA. Although essential FA

ﬂow to the duodenum is severely limited, there are no reports of FA deﬁciency in adult

19

ruminants. Mattos and Palmquist (1977) measured linoleic acid biohydrogenation and
transfer to milk fat in cows fed a high grain diet, and observed linoleic acid available at
twice the requirement for female weanling rats on a metabolic body weight basis. In
addition, ruminants may be adapted to sparing PUFA, preserving them for required
purposes. Essential FA are less available for oxidation since they are highly incorporated
into phospholipids and cholesteryl esters (Drackley, 2000). The slow turnover of
phospholipids and cholesteryl esters pools ensure retention of the essential FA. Dietary
FA are also incorporated into milk and tissue, however the efﬁciency of conversion of
dietary unsaturated FA to milk is lower than saturated FA (Chilliard, 1993). A ﬁnal
conservation method for PUFA is lower oxidation. Reid and Husbands (1985) observed
lower linoleic acid oxidation in cultured hepatocytes, and Linsay and Valerio (1975)
showed a 25-40% lower oxidation rate for linoleic acid than stearic and palmitic acid.
The essential FA may be more correctly labeled conditionally dispensable since the cow
appears to have adapted adequate conservation methods and does not experience clinical
deﬁciency.

Using the factorial approach it appears that essential FA are normally available in
adequate concentrations, especially since there are no reported cases of deﬁciency. Adult
ruminants have adapted their metabolism to work with limited PUFA, although there may
be beneﬁts to FA supplementation including improving reproductive efﬁciency,
increasing energy balance, and modulating physiology.

Some nutritionists have proposed that essential FA limit maximum reproductive
efﬁciency. Supplemental FA may increase reproductive efﬁciency through increased

energy balance, increased progesterone synthesis, and increased or decreased

20

prostoglandin F 2,, secretion, although results are inconsistent as reviewed by Grummer
and Carroll (1991), Staples et a1. (1998), and Williams and Stanko (1999). Dietary FA
supplementation to increase reproductive efﬁciency is an example of an attempt to
maximize production by increasing nutrient intake above the pure substrate requirement.
Energy balance has long been recognized as a major cause of anestrous and
infertility as the cow directs nutrients to lactation. Anestrous and infertility in low energy
balance may be mediated through low plasma leptin, insulin, or IGF-I concentration.
Insulin and IGF-1 are associated with increased follicular growth (Williams and Stanko,
2000), and leptin may mediate GnRH secretion through neuropeptide Y in the
hypothalamus (Harris, 2000; and Spicer, 2001). The effect of dietary FA on energy
intake and balance will be discussed in detail later. Plasma cholesterol is consistently
increased with dietary fat supplementation, being linked to increased demand for lipid
transport. Cholesterol is a precursor for progesterone synthesis in the corpus luteum and
fat supplementation has been shown to increase blood plasma progesterone concentration
(Staples et al., 1998). Progesterone synthesis may not be controlled by substrate
availability as maximum synthesis of progesterone was reached in vitro at a much lower
cholesterol concentration than normally observed in blood (Carroll et al., 1992). In
addition, Rabiee et a1. (1999) observed no relationship between ovarian uptake of
cholesterol and progesterone release; failing to implicate cholesterol availability as a
limitation of the steroids production. Increased plasma progesterone concentration is
expected to increase maintenance of pregnancy especially during early gestation.
Increasing unsaturated FA is proposed to increase prostoglandin-F2Cl secretion through

substrate availability, or decrease secretion through direct synthesis inhibition. As

21

previously mentioned, the essential FA are used to produce the precursors of PGan and
the availability of the FA may limit synthesis during early lactation when PGqu is
required for termination of anestrous and maintaining normal estrus cycles. Linoleic acid
has also been shown to inhibit PGan secretion both in vitro and in viva possibly through
competitive inhibition of arachidonic acid at a key synthesis enzyme (Staples et al.,
1998). Inhibition of PGan is expected to protect implanted embryos that are not
recognized by the dam and decrease fetal mortality in the ﬁrst 28 d of gestation. It has
been proposed that the concentration of the FA reaching the target tissues dictates the
stimulatory or inhibitory response of PGF2a(Staples et al., 1998),

The reproductive beneﬁts of FA supplementation are normally associated with
unsaturated FA, although reproductive beneﬁts are observed across all fat sources
(Staples et al., 1998). Biohydrogenation is normally not measured in reproductive trials,
and the oilseed, tallow, and palm oil treatments used are expected to provide limited
polyunsaturated FA to the duodenum. The metabolic hormone, energy balance and
cholesterol mechanisms proposed to increase reproduction are not speciﬁc for PUFA.
Energy balance is not consistently increased with fat supplementation because of
decreased dry matter intake, especially with higher unsaturated FA concentrations (Allen,
2000), and increased milk yield in some experiments (Chilliard, 1993). Cholesterol was
increased with duodenal FA infusion with no effect of FA type (Drackley et al.,1992;
Christensen et al., 1994; and Bremmer et al., 1998). Increased reproductive efﬁciency
through stimulating or attenuating PGan has a number of practical limitations. First
PUFA are highly biohydrogenated in the rumen, even when initially complexed as

calcium soaps. Secondly, if unsaturated FA was capable of both increasing and

22

decreasing PGan secretion dependent on plasma concentration we would expect to
observe variable effects (increased or decreased) on reproductive efﬁciency with essential
FA supplementation. Logically, additional essential FA supplementation cannot be
utilized to both increase PGan secretion to minimize anestrous and maintain normal
estrus cycles, and decrease PGan secretion to protect newly implanted embryos. All
animals have an indispensable requirement for energy to maintain homeostasis, repair
tissue, and grow, lactate and reproduce. The energy requirement of the high producing
dairy cow is extreme with the large demand of lactation. In many cases cows cannot
consume enough dietary carbohydrate to meet their energy requirement, and increasing
diet energy density through increasing fermentability leads to rumen acidosis (Allen,
1997). Dietary FA are over twice as energy dense as other dietary components.
Substitution of dietary fat for starch may make room in the diet for other nutrients such as
protein and ﬁber. Decreasing starch and increasing ﬁber is expected to increase
rumination and stabilize rumen fermentation (Allen, 1997). In this situation, dietary fat
might allow greater production if DMI is maintained

Fatty acids can regulate physiology and metabolism through modiﬁcation of
metabolic hormones, tissue sensitivity, and gene expression (Drackley, 2000). Dietary
FA stimulate secretion of a number of gut peptides including gastric inhibitory peptide
(GIP), glucagon-like-peptide-l (GLP-l), and Cholecystokinin [CCK (Dawson et al.,
1999; Meier et al., 2002; and Reidelberger, 1994)]. These hormones have large impacts
on insulin secretion and glucose disposal after a meal (van der Burg et al., 1995). Fatty
acids also directly stimulate pancreatic insulin secretion, with longer chain and more

saturated FA having greater affects (Gravena et al., 2002). At the tissue level,

23

unsaturated FA increases insulin sensitivity in rats (Clarke, 2000). Palmquist and Moser
(1981) observed decreased insulin sensitivity in cows fed calcium salts of palm oil
compared to no fat control. Fatty acid saturation may have differential effects on insulin
signaling between cows and rodents, or palm oil may not contain a high enough
concentration of unsaturated FA to increase insulin sensitivity. Polyunsaturated FA have
a clear impact on glycolytic, lipolytic, and lipid oxidation enzyme gene regulation in
rodent models (Sessler and Ntambi, 1998; and Raclot and Oudart, 1999). A high PUFA
diet results in preferential partitioning of ingested energy towards oxidation at the
expense of storage (Raclot and Oudart, 1999). The interaction of metabolic regulation
and FA metabolism is demonstrated in leptin repression of stearoyl-CoA desaturase-l in
the liver that is important in leptin mediated weight loss (Cohen et al., 2002). Effect of
fat type an endocrine response and gene expression in cattle has not been investigated.

Dietary FA concentration and proﬁle are quite different from the rat to the
ruminant, but FA appear to have an important role in mammalian metabolic regulation.
Drackley (2000) identiﬁed F A regulation of physiology as “one of the most exciting
current areas of research in lipid metabolism.” In the future we may select FA

supplementation to manage metabolic states and increase product yield or efﬁciency.

Fatty Acid Digestibility

Fat supplements must be efﬁciently digested and absorbed to beneﬁt the cow.
Fatty acid digestibility and the associative effects of FA on ruminal nutrient digestion are
very important considerations in energy intake. Lipid complexes are hydrolyzed in the

rumen delivering nonesteriﬁed FA to the small intestine, in contrast to the esteriﬁed FA

24

ﬂow in non-ruminants. Abomasal acid secretion decreases digesta pH to less than 2.0,
and greatly reduces the concentration of FA associated with metal cations. Pancreatic
juice and bile entering in the duodenum are essential for digestion of any remaining
triglycerides, formation of miscelles, and absorption of FA (Drackley, 2000). Bile salts
and lysolecithins aid in absorption of FA from feed particles and the formation of
miscelles that diffuse across the intestinal cell membrane in the jejunum (Doreau and
Chilliard, 1997). Lipases are present in pancreatic secretion but have an optimal pH of
7.5 with little activity below 5.0 (Noble, 1981). The low pH of the duodenum prohibits
lipase function and conditions for appreciable lipase activity are not expected until well
into the jejunum, delaying digestion of triglycerides and decreasing the opportunity for
FA absorption (Noble, 1981). Total tract digestibility of esteriﬁed FA is lower than
unesteriﬁed FA (Elloitt et al., 1994; and Elloitt et al., 1999), and triglyceride digestibility
decreases with increasing saturation (Pantoja et al., 1996; and Pantoja et al. 1995). Elliot
et a1. (1999) observed that highly saturated TG are more resistant to ruminal and
intestinal lipolysis than unsaturated TG, leading to low digestibility of highly saturated
TG. Finally, low intestinal pH is also expected to decrease the solubility of FA and bile
salts, but may solubilize calcium soaps allowing their absorption.

Lower tract and total tract digestibility can be determined for total FA, but such
measures for individual FA are meaningless due to the hydrogenation of unsaturated FA,
and bacterial synthesis of FA (Merchen et al., 1997). Biohydrogenation and microbial
FA synthesis, as discussed in reference to rumen digestion, also occur in the large
intestine, but to a lesser extent (Bock et al., 1991; Ferlay et al., 1993; and Elliot et al.,

1999). Individual FA digestibility can be measured through experimental treatments

25

differing in FA proﬁle and with duodenally and illeally cannulated cows. Nutritionists
have attempted to assign digestibility values to individual FA in a mixed FA diet (Moate
et al., 2000). This is a misrepresentation of the data as the recovered saturated FA pool is
inﬂated and unsaturated FA pool is decreased by hydrogenation in the rumen and hindgut
and oxidation in sample preparation and handling (Palmquist, personal communication).
There are very few direct comparisons of rumen protected saturated and unsaturated FA
digestibility, because of the cost and complexity of multiple cannulation experiments
necessary to make such comparisions. Christensen et al. (1994) and Bremmer et a1.
(1998) measured digestibility of abomasally infused free FA and observed no difference
between saturated and unsaturated fat treatments. Also, Schauff and Clark (1989),
Grummer et a1. (1988) and Palmquist (1991) directly compared calcium salts of palm oil
and prilled FA, ﬁnding no difference in total tract digestibility of energy, lipid and FA.
Doreau and Chilliard (1997) summarized 64 treatment groups reporting FA digestibility
in the small intestine or the lower tract, ﬁnding signiﬁcant differences across chain
lengths (although C16 and C18 did not differ), and observed only slight differences
between lower tract saturated and unsaturated C18 FA digestibility (77, 85, 83 and 76%
for 0, l, 2 and 3 double bonds respectively). The small differences in C18 FA digestion
are at least partially attributed to biohydrogenation of unsaturated FA in the large
intestine because the majority of the data set measured FA disappearance across both the

small and large intestine.

26

Dietary Fatty Acid Effects on Intake

Fatty acid supplements increase the energy density of the diet, but daily energy
intake depends on energy concentration and dry matter intake. Intake is highly regulated
by animal nutrient requirements and metabolic state, and also by the type and temporal
pattern of fuels absorbed (Allen, 2000). Fatty acid supplementation can cause
hypophagia, and fat source, form and type are signiﬁcant predictors of intake response
(Allen, 2000). Within commonly fed rumen protected FA sources, calcium salts of palm
oil linearly decreased intake with increasing dietary concentration while saturated FA had
no effect an intake (Allen, 2000). Benson et a1. (2001) summarized 11 infusion studies
representing 26 treatment groups showing intake depression with all but two treatments;
regression analysis revealed a negative relationship between infused C18:1 and C18:2 FA
concentration and intake, with C18:2 creating greater intake depression. Abomasal
infusions of unsaturated FA with a lower C16:C18 FA ratio decreased DM1 and
digestible energy intake (Drackley et al., 1992), and DM1 and gross energy intake
(Christensen et al. 1994). Bremmer et al. (1998) demonstrated a negative relationship
between intake and unsaturated FA with the same C16:C18 FA ratio. Experiments
feeding free oil versus protected unsaturated FA provide comparison of saturated and
unsaturated FA treatments because free oil is highly biohydrogenated in the rumen.
Oleamide consistently decreased intake compared to free oil and linearly decreased
intake with increasing inclusion rate (Jenkins, 2000; Jenkins et al., 2000; and DeLuca and
Jenkins 2000). Finally, four-day continuous intravenous infusion of both palmitic and
oleic acid signiﬁcantly decreased intake, while stearic acid only numerically decreased

intake (Vandermeerschen-Doize and Paquay, 1984).

27

Regulation of Intake

Fatty acid proﬁle reaching the duodenum determines hypophagia, and FA
saturation may affect intake through a number of modes including: metabolic fate of
nutrients, metabolic hormones, and gut peptides. The goal of intake regulation is
balancing short-term and long-term nutrient supply and demand. Numerous tissues are
proposed to be involved in intake regulation including splanchnic, hepatic, and adipose
tissue. The gut and liver are well positioned to ﬁrst detect absorbed nutrients, and
adipose tissue is well positioned to detect long-term energy balance.

Koopmans (1995) proposed that intake is regulated by internal signals arising
from the small intestine and the metabolic effect of absorbed nutrients, with differences
between nutrients and metabolic states. In the rodent model, intake compensation for
intravenously infused energy depends on the nutrient, with a 55% compensatory
reduction in energy intake for infused glucose energy, 103% for amino acid energy, and
41% for lipid (Walls and Koopmans, 1992). In addition, intake recovered rapidly
following glucose and amino acid treatments, returning to baseline within 2 d after
infusion, while DMI recovery following lipid treatment and incomplete (Walls and
Koopmans, 1992). Intravenous and intragastric infusions were used to explore the
interaction of the gut in intake regulation. Intragatric infusion decreased energy intake
15% more than intravenous nutrient infusion (Burggraf et al., 1997 ). The interaction of
absorbed nutrients and gut signals was shown with intravenous nutrient infusion during
fed and fasted states. Energy intake decreased to compensate for intravenous nutrient

infusion during the dark cycle while rats actively ate (99% energy intake compensation),

28

but daily intake failed to decrease to compenstate for energy infused during the light
period when rats were fasted [56% energy intake compensation (Walls et al., 1991)]. The
authors concluded that intake response to absorbed nutrients depends on a threshold level
of gut hormones (Walls et al., 1991). Nutrient infusion during a fed state may decrease
intake because endogenous gut peptide secretion has primed the regulatory system to
respond to absorbed nutrients. Without gut peptide secretion, intake regulatory
mechanisms might fail to respond to absorbed nutrients.

Parabiotic cross-intestinal rats are created by sewing together the skin and muscle
along the ﬂank, creating one peritoneal cavity (Koopmans et al., 1997). Connection of
skin and muscle creates a slow exchange of blood (1%/min), with full blood exchange
expected in 3-4 h (Koopmans et al., 1997). Intestinal crossing was achieved by splicing a
30 cm segment of the lower duodenum and upper jejunum of one rat’s intestine into the
intestine of its partner without severing nerves or blood supply. The rat losing nutrients
through absorption in its partner’s intestine increased intake by 50% and the rat gaining
nutrients decreased intake by 50% (Koopmans, 1991). Intake adjustment was quick and
repeatable, and body weight of rats was not different at the end of the experiments
(Koopmans, 1991). Plasma glucose, insulin, and glucagon did not vary with food intake
across rats, but plasma lactate was increased in the low intake rat and decreased in the
high intake rat (Koopmans, 1997). With a slow turnover of blood, transfer of metabolites
is expected to create long-term energy balance in the pair, but temporal signals of nutrient
absorption and short half-life hormones such as the gut peptides (CCK, GIP, and GLP-1)
would be blocked. The large difference in intake between the paired rats with the same

energy balance emphasizes the importance of temporal variation in nutrient absorption.

29

Further research using different dietary treatments for each rat in the pair may provide

more insight into interaction of the gut and intake.

Satiety and the Gastrointestinal Tract

The gastrointestinal tract serves as an endocrine gland to change gut motility,
stimulate digestive secretions, and regulate intake. The gastrointestinal tract has the ﬁrst
opportunity to sense the energy density and amount of food ingested. Four important
hormones released in response to a meal are CCK, GLP-l, glucagon-like-peptide-2
(GLP-2), and gastric inhibitory peptide (GIP). CCK is a well-researched peptide that
decreases gut motility and stimulates satiety in other species (Reidelberger, 1994). GLP-
1, GLP-2, GIP are also involved in prandial endocrine response and stimulation of satiety
(Dawson et al., 1999; Meier et al., 2002; and Burrin et al., 2003)

In ruminants, dry matter intake was decreased and postprandial CCK was
increased when diets were supplemented with calcium salts of palm oil (Choi and
Palmquist, 1996). Direct intravenous infusions of CCK depressed reticular-rumen
motility and intake in sheep (Grovum, 1981). Finally, Nicholson and Omer (1983)
showed that intestinal infusion of unsaturated FA decreased rumen motility of sheep.
These observations are consistent with CCK decreasing gut motility and centrally
signaling satiety (Reidelberger, 1994). Recently, Benson and Reynolds (2001) tested the
effect of unsaturated fat (rapeseed oil) infusion on gut peptide secretion, observing
increased plasma GLP-l without an increase in CCK, although CCK was pooled over
time eliminating observation of temporal variation. Rapeseed infusion had no effect on

feeding or chewing behavior, although a large reduction in time spent ruminating was

30

noted for some cows (Benson and Reynolds, 2001). Experiments testing gut peptide
response to FA inﬁrsion have only tested unsaturated FA and low FA controls, little data
is available reporting the effect of saturated FA an endocrine signaling and rumen
motility. Gut peptides may act centrally to stimulate satiety or may change rumen

motility and passage rate, increasing gut ﬁll and stimulating tension receptors.

Satiety and Absorbed Nutrients

Metabolic fate of nutrients can regulate intake through the site, rate and timing of
oxidation. Hepatic oxidation of FA has been demonstrated to decrease intake in the rat
(Langhans and Scharrer, 1987; Scharrer and Langans, 1989; and Friedman et al., 1999).
The location and function of the liver as a central site of fuel storage and metabolic
conversion makes it an ideal site to assess fuel utilization, and communicate this signal to
the brain via afferent nerves (Langhans, 1995). The hepatic oxidation theory of satiety
proposes that oxidation of nutrients increases hepatocyte intracellular ATP that affects
the ﬁring rate of the hepatic vagus; the exact mechanism by which hepatocytes interact
with the hepatic vagus has not been elucidated (Langhans, 1995). Emery et a1. (1992)
proposed that uptake and oxidation of FA by the liver might depress intake particularly in
early lactation. Pahnquist (1994) and Allen (2000) found hepatic oxidation regulation of
intake appealing and defendable, however it has not been proven in the ruminant.

The rate of FA oxidation would depend on FA uptake by the liver, and enzyme
concentration and activity. Emery et a1. (1992) reported hepatic NEFA uptake as a
constant proportion to blood concentration, and reviewed malonyl CoA and propionic

acid inhibition of oxidation and ketogenesis. In nonruminants, PUFA are preferentially

31

oxidized and saturated FA appear to be directed towards fat storage (Storlein, 2000). The
essential PUFA are conserved in the ruminant since they are highly associated with
phospholipid pools and have low reported oxidation rates. Substantial intakes of PUFA
are not expected even with protected sources, but high levels of mono-unsaturated FA
including trans- FA are absorbed. The oxidation rates of these FA have not been studied.
Dietary unsaturated FA may change liver oxidation and phosphorylation potential by
changing rate of gluconeogenesis, PUFA affect gene regulation of metabolic enzymes,
and FA have a large effect on insulin signaling through gut peptides and directly in the

pancreas.

Satiety and Energy Balance

Fatty acids may change energy balance through increased digestibility and
metabolic efﬁciency, or decreased milk fat production. Chemostatic regulation of intake
may feedback to decrease intake through leptin stimulation of satiety in the paraventrical
nucleus (Ingvartsen and Bosclair, 2001). Energy balance is expected to be an important
factor in intake response to fat supplementation, as long-term intake of fat should
increase energy balance and thus decrease intake. Fatty acid type may change
partitioning of FA by preferential oxidation or storage of FA causing changes in leptin
secretion. Increased oxidation of FA in the liver could also increase function of the
somatotropic axis, increasing IGF-l production, and changing nutrient partitioning
(Renaville et al., 2002). Recent research has identiﬁed trans-FA intermediates of rumen
PUFA biohydrogenation as key regulators of milk fat synthesis (Bauman and Griinari,

2003). Infusion of trans-10,cis-12 CLA reduces milk fat by decreasing lipogenic enzyme

32

gene expression (Baumgard et al., 2002a). Tyrell and Moe (1972) observed decreased
efﬁciency of ME utilization for milk synthesis during milk fat depression. Milk fat
depression normally occurs with little effect on dry matter intake leading to increased
body weight gain in proportion to milk fat depression (Baumgard et al., 2002b).
Increased body weight gain in response to trans-10,ci512 CLA in lactating cows is
contrary to decreased body fat gain observed in growing animals (Mersmann, 2001).
Milk fat depression of 25 to 50% resulted in no change in plasma glucose, insulin and
leptin concentration or insulin stimulated glucose clearance, but did result in 24 to 33%
reduced lipolytic response to an epinephrine challenge consistent with slightly increased
energy balance (Baumgard et al., 2002a). Gaynor et al. (1996) observed no effect of
abomasal infusion of cis or trans-C18:1 on disappearance rates of glucose, secretion of
insulin after glucose challenge, and appearance rates of NEF A and triglycerides after
norepinephrine challenge. Romo et a1. (1996) measured energy metabolism during cis
and trans-C18:1 FA infusion, observing increased production of milk energy for cis
compared to trans-FA but failed to ﬁnd differences in energy expenditure or tissue
retention. Incomplete compensatory adjustment in energy intake resulting in increased
body weight gain during milk fat depression represents a failure or shift in regulation of
energy balance that cannot be attributed to homeostatic signaling or regulation of lipid

and glucose metabolism.

Other Factors Stimulating Satiety
The rate of feed intake and rate of digesta passage out of the rumen create an

important balance to maximize rumen digestion but not limit intake by physical ﬁll.

33

Dietary lipids can have profound effects on rumen fermentation as previously presented.
Changing microbial activity may decrease digestion rate of ﬁber and other feed
components. Decreased digestion rate results in slower passage since feed particles must
be reduced in size and buoyancy before they may pass out of the rumen (Allen, 1996). If
dietary fat has a large impact on digestion and passage rate of ﬁber, ruminal ﬁll will
increase and stimulate tension receptors (Allen, 2000).

Isomers of unsaturated FA have profound biological activity as observed with
CLA and milk fat depression (Bauman and Griinari, 2003). Feeding dietary fat yields a
number of isomers from incomplete rumen biohydrogenation. The type and amount of
isomer is related to the type of fat fed, ruminal fermentation and residence time. The
effect of unsaturated FA type on animal physiology and metabolism may be mediated
through very small quantities of certain FA isomers. Which may explain the variation
observed in feeding experiments. Abomasal infusion of trans—C18:1 isomers numerically
decreased intake compared to cis-C18:1 FA although there are a large number of
candidate isomers that could depress intake including the trans-C18:1 isomers (Romo et
al., 1996; and Romo et al., 2000). Trans-FA isomers may be involved in intake
regulation, but satiety is consistently demonstrated with duodenal infusion of vegetable
oils low in trans-FA isomers (Benson and Reynolds, 2001).

Forbes proposed that intake is not regulated by one mechanism, but is determined
by the central integration of many signals (Forbes, 1996). The animal likely integrates
these signals and adjusts intake to minimize physical and metabolic discomfort (Forbes,
2000). The possible mechanisms discussed included both short and long-term regulators,

both of which probably play a role in FA induced intake depression.

34

CHAPTER 2

The effect of production level on feed intake, milk yield and plasma metabolite

response to rumen protected fatty acid saturation in lactating cows.

ABSTRACT

Animal response to dietary treatment may interact with metabolic state, which
differs for cows across a wide range of milk yield. Response to dietary saturated versus
unsaturated rumen-protected fatty acids (FA) was evaluated using 31 multiparous
Holstein cows arranged in a crossover design with 14 (1 periods. Cows averaged 43.7 kg
milk (range 34.0-57.5 kg) for the 4 (1 immediately prior to initiation of the experiment
when fed a diet intermediate in composition to the treatment diets. Treatments were
2.5% FA rumen-protected fat sources from unsaturated FA (UNS, calcium soaps of palm
FA) or saturated FA (SAT, prilled hydrogenated free FA). UNS had decreased DMI (0.8
kg/d) and time spent ruminating (25 min/d) with no effect on time spent eating compared
to SAT. No difference was observed between treatments for milk or 3.5% fat corrected
milk (F CM) yield. Intake and milk yield responses were not related to milk yield across
cows. SAT signiﬁcantly increased milk protein and lactose concentration, but treatment
did not affect yield of milk components. SAT increased insulin over 25% and decreased
non-esteriﬁed FA nearly 20% with no effect on plasma bST, leptin, glucose or [3-
hydroxybuturate. Milk protein concentration and yield responses to treatment were

positively correlated with pretrial fat-corrected milk yield. Milk protein response was

35

negatively correlated to insulin response, signifying the importance of insulin sensitivity
in control of milk protein synthesis. UNS decreased DM intake and rumination time
compared to SAT, consistent with reports of unsaturated fat increasing satiety and
decreasing gut motility. Depression of milk protein synthesis by fat supplementation

may be related to FA saturation and milk yield of cows.

INTRODUCTION

Energy required for milk yield is often greater than the cow’s ability to consume
dietary energy, resulting in a negative or low energy balance. Addition of fat to the diet
increases energy density without increasing rumen acid production, thus stabilizing
rumen function relative to addition of grain. Prilled, hydrogenated free fatty acids (FA)
and calcium salts of FA are two commercially available fat sources that have been
designed to reduce adverse effects of rumen active FA on rumen microbial fermentation.

The ability of the cow to increase daily energy intake depends on the digestible
energy density of the diet and daily dry matter intake (DMI). Intake is highly regulated
by animal nutrient requirements and metabolic states, and also by the type and temporal
absorption of fuels. Allen (2000) observed in a meta-analysis that fat supplements
differing in FA source, form, and type have different hypophagic effects. Within
commonly fed rumen protected FA sources, calcium salts of palm oil linearly decreased
intake with increasing dietary concentration while hydrogenated FA had no effect an
intake (Allen, 2000). The concept that FA of varying saturation reaching the duodenum

have different hypophagic effects was demonstrated through a series of abomasal

36

infusion studies that showed decreasing FA saturation decreased feed intake (Drackley et
al., 1992; Christensen et al., 1994; and Bremmer et al., 1998).

Dietary fat also has demonstrable effects on milk protein production (DePeters
and Cant, 1992). Decreased milk protein yield and concentration by increased dietary fat
might be caused by changes in rumen fermentation, endocrine signaling, milk yield or
mammary nutrient metabolism (DePeters and Cant, 1992). Drackley et al. (1992)
observed a linear decrease in milk crude protein yield with abomasal unsaturated FA
infusion, and Christensen et a1. (1994) observed decreased milk true protein and casein
yield with C18 unsaturated FA inﬁrsion compared to saturated FA, indicating that FA
proﬁle has an important role in fat-stimulated milk protein depression.

Dietary energy density is often increased by addition of fat in an attempt to
improve the energy balance of high producing cows that are unable to consume the
required amount of forage and grain to meet energy requirements. It is expected that
cows response to energy supplementation will depend on cow metabolic state or milk
yield. Crossover design experiments with a pretrial covariant period have been used to
study cow responses across production levels to evaluate low lignin corn silage (Oba and
Allen, 1999) and forage to concentrate ratio (Voelker et al., 2002). Similarly, response to
fat supplementation was expected to differ for cows varying in milk yield.

The objective of this experiment was to determine the relationship between
production level and responses for feed intake, milk yield, and plasma hormones and
metabolites to diets supplemented with saturated or unsaturated rumen-protected FA. We

hypothesized that more highly unsaturated FA would decrease intake relative to saturated

37

FA at equal FA concentrations, and individual cow response would depend on milk

yield.

MATERIALS AND METHODS

Cows and Treatments

Thirty-two multiparous Holstein cows (Table l) in mid- to late lactation (14425 70;
Mean :1: SD) at the Michigan State University Dairy Cattle Teaching and Research Center
were randomly assigned to sequence in a crossover design with a pretrial covariant
period. Treatments were 2.5% added dietary FA from saturated FA (SAT) or unsaturated
FA (UNS) sources (SAT- prilled hydrogenated free FA, Energy Booster 100®, Milk
Specialties Company Inc., Dundee, IL; UNS- calcium soaps of palm FA, Megalac®,
Church and Dwight Company, Inc., Princeton, NJ). Treatments were fed as a mix using
ground corn as a carrier, and were balanced for calcium and FA concentration using
limestone and rice hulls (Table 2). Covariant and treatment periods were 14 d in length
with the ﬁrst 10 d for diet adaptation followed by 4 d of sample collection. Diets
contained alfalfa silage (~50% of forage DM), corn silage (~50% of forage DM), dry
ground corn, whole linted cottonseed (12.5% of ration DM), protein mix (soybean meal,
corn gluten meal, and blood meal), and mineral and vitamin premix (Table 3). The base
diet contained ~5.0% FA with 2.0% FA from cottonseed. Milk yield averaged 43.7 kg/d
and ranged from 34.0 to 57.5 kg/d during the 4 (1 immediately prior to the experiment,
when cows were fed a diet intermediate to both treatments. Cows were housed in tie stalls
throughout the experiment, except for a 1.5-hour exercise period twice daily prior to

milking in a parlor. Samples and data were collected during the last 4 d of each period.

38

Experimental procedures were approved by the All University Committee on Animal Use

and Care at Michigan State University.

Data and Sample Collection

Throughout the experiment, cows were fed once daily (1100 h) at 110% of
expected intake. The amount of feed offered and arts were weighed for each cow daily
during the collection period. Samples of all dietary ingredients (0.5 kg) and treatment
diets were collected daily during the collection period and composited into one sample
per period. Samples of arts (12.5%) were collected daily during the collection period and
composited into one sample per cow period. Blood was collected from the coccygeal
vessel into a tube containing sodium heparin. Six samples were collected over two days
((1 10 and 11) represent 4-h intervals of a 24-h period to account for diurnal variation.
Blood was centrifuged at 2,000 x g for 15 min immediately after sample collection, and
plasma was harvested and frozen at -20°C until analysis. Cows were milked twice daily
in the milking parlor throughout the experiment. Milk yield was measured and sampled
at each milking from d 11 to d 14 and averaged over the period. Feeding behavior was
recorded manually every ﬁve minutes for 24 h on d 14. Activity was classiﬁed as eating,
ruminating, drinking, or idle. Cows were fed and milked as normal during feeding
behavior observation. Body weight was recorded on the day prior to the start of the ﬁrst
period and on d 14 of each period to determine body weight change. On the same days,
three trained investigators determined body condition score using a ﬁve-point scale (1 =

thin, 5 = fat; Wildman et al., 1982).

39

Sample Analyses

Milk samples were analyzed for fat, true protein, and lactose by infrared
spectroscopy at Michigan DHIA (East Lansing). Diet ingredients and arts were dried in a
55°C forced-air oven for 72 h. All samples were ground with a Wiley mill (l-mm screen;
Arthur H. Thomas, Philadelphia, PA). Diet ingredients were analyzed for DM, NDF,
ADF, lignin, starch, crude protein, ash, and FA concentration and proﬁle. NDF
concentrations were determined with the addition of heat-stable amylase (Van Soest et
al., 1991; method A). Starch was measured by an enzymatic method (Karkalas, 1985)
after samples were gelatinized with sodium hydroxide. Crude protein was analyzed
according to Hach et al. (1987). Ash content was determined after 6 h oxidation at 500
degree C in a mufﬂe furnace. Total FA concentration and FA proﬁle were analyzed by
GLC (Sukhija and Palmquist, 1988). Concentrations of all nutrients except DM were
expressed as percentages of DM determined from drying at 105 degree C in a forced-air
oven.

Blood samples were analyzed for insulin, glucagon, somatotropin (bST),
Cholecystokinin (CCK), leptin, non-esteriﬁed fatty acids (N EFA), glucose and beta-
hydroxybutyrate (BHBA). Commercial radioimmunoassay kits were used to determine
plasma concentration of insulin (Coat-A-Count; Diagnostic Products Corporation, Los
Angeles, CA), glucagon (Glucagon kit GL-32K; Linco Research, St. Charles, MO), and
CCK (Euria-CCK kit RB30; ALPCO, Windham, NH). Plasma somatotropin and leptin
concentration was determined by radioimmunoassays (Gaynor et al. 1995; and Ehrhardt
et al. 2000). Enzymatic kits were used for determination of glucose (Glucose kit #510;

Sigma Chemical Co., St. Louis, MO), NEFA (NEFA C-kit; Wako Chemicals USA,

40

Richmond, VA), and B-hydroxybutyrate (BHBA kit #310-A; Sigma Chemical Co., St.
Louis, MO).

Net energy of body weight change and milk production was calculated according to
NRC 2001.
Milk NEL (Meal/d) = MY (kg) x [0.0929 x (Fat %) + 0.0563 x (True Protein%) + 0.0395

x (Lactose%)]

Statistical Analyses

For treatment effects, all data were analyzed by the ﬁt model procedure of J MP
Version 5.0 (JMP, 2000) according to the following model:

Yijkl = it + Si + Cj(Si) + Pk + T1 + eijkl

where

u: overall mean,

S; = effect of sequence (1 = 1 to 2),

Cj(Si) = effect of cow nested in sequence (j = l to 16),

Pk = effect of period (k = 1 to 2),

T. = effect of treatment (1 = 1 to 2),

eiJ-kl = residual error.

41

Pretrial fat—corrected milk yield (pFCMY) was calculated as the average daily
production over eight milkings during the 4 (1 immediately prior to the initiation of the
experiment. Relationships between response to treatment and pFCMY were analyzed
according to the following model:

Y,=p+S,+pM+pM2+e,

Where

Yi = ySAT — YUNs

ySAT = response for the saturated FA treatment
yUNg = response for the unsaturated FA treatment

it = overall mean,

S, = effect of sequence (i = 1 to 2),

pM = pFCMY

pM2 = pFCMY2

e; = residual error

Data points with Studentized Residuals greater than 3 were considered outliers and
excluded from the data set. One cow was diagnosed with clinical mastitis in the ﬁrst

treatment period and was excluded from statistical analysis.

RESULTS AND DISCUSSION
Thirty-one cows completed the experiment and are proﬁled in Table 1. Cows
ranged from 34.0 to 57.5 kg milk during the covariant period. Fat treatments differed in
FA proﬁle with UNS containing nearly 2.5 times more unsaturated FA than SAT,

primarily as C18:1 and C18:2 (Table 2). Treatments also differed in C16:C18 FA ratio

42

because the UNS treatment contained high levels of palmitic acid. Diets contained nearly
equal concentrations of starch, CP, and FA, but differed slightly in NDF concentration
because of the addition of rice hulls in the UNS mix to equalize calcium concentrations

across treatments (Table 3).

Intake and Chewing Behavior

UNS decreased DMI 0.8 kg/d relative to SAT (P<0.01). UNS decreased intake of
starch, crude protein and total FA (P<0.01). In previous direct comparisons of saturated
and unsaturated rumen protected FA, no differences were observed in intake when
supplemented at 0.68 kg of FA (Grummer, 1988; and Schauff and Clark, 1989), and at 2
and 5% of the diet (Eastridge and Firkins, 1991). However, both of these studies had
fewer observations, lower producing cows and lower basal dietary FA concentration
compared to the present study. Allen (2000) reported that in 11 out of 24 studies calcium
salts of palm oil caused a linear decrease in feed intake, while 22 of the 24 trials resulted
in a numerical decrease in feed intake. In contrast, hydrogenated TG or FA resulted in
decreased feed intake in only one study and increased feed intake in two out of 21 studies
reported.

Heinrichs et al. (1982) observed that supplemented fat decreased the length and
size of the initial meal but tended to increase the number of spontaneous meals. De
Visser et a1. (1982) reported smaller meals and a slower eating rate with concentrates
containing added fat. Manual observation of feeding behavior, as used in this study, lacks
sensitivity for accurate determination of meal length and time between meals compared

to automated methods (Dado and Allen, 1993). Meals were identiﬁed and data was

43

analyzed for meal length and intermeal interval; no response variables were signiﬁcantly
different and the data is not presented in this paper.

Abomasal infusion of unsaturated fat consistently decreases feed intake relative to
no fat and saturated fat infusions (Benson and Reynolds, 2001). Drackley et al. (1992)
and Christensen et al. (1994) both showed abomasal infusions of 450g of unsaturated FA
with a lower C 16:C18 FA ratio decreased DM intake compared to an equal amount of
more saturated FA, while Drackley et al. (1992) also reported decreased digestible energy
intake. The experimental treatments in the current study differed in both unsaturated fat
concentration and C16:C18 FA ratio. Bremmer et al. (1998) demonstrated the negative
relationship between intake and diet unsaturated FA concentration at the same C 16:C18
FA ratio in abomasal infusions. In addition, protected Oleamide FA consistently linearly
decreases intake compared to free oil that is readily biohydrogenated in the rumen
(Jenkins, 1998; Jenkins, 2000; and DeLuca and Jenkins, 2000). The above experiments
provide strong evidence that FA-mediated intake depression is a ﬁrnction of FA
saturation, not chain length.

Rumen biohydrogenation of unsaturated FA can be extensive and may explain
differences in the magnitude and consistency of response when unsaturated fat is fed
versus directly infused into the abomasum. Klusemeyer and Clark (1990 and 1991) and
Wu et al. (1991) reported rumen biohydrogenation of unsaturated C18 FA fed as calcium
salts of palm oil at 34, 33 and 48% respectively. Unsaturated fat in the form of calcium
salts must ﬁrst dissociate from the calcium ion and then be absorbed by rumen bacteria
and biohydrogenated. The rate of this process is expected to be a ﬁrnction of the pK, for

the calcium salt, rumen pH, and the microbial population, which affects absorption of FA

and enzyme activity for biohydrogenation. The extent of biohydrogenation of the UNS
treatment in the current study is unknown. However, we expect that less unsaturated FA
reached the duodenum than was fed, which demonstrates the powerful hypophagic effects
of unsaturated FA.

There was no effect of treatment on time spent eating but UNS decreased time
spent ruminating 25 min/d (P<0.01) and increased time spent idle 25 min/d (P<0.01).
Total chewing time and time spent ruminating per kg of DM1 was not different between
treatments. This might be expected since cows consumed less feed for UNS treatment
and would have less digesta to ruminate, which is supported by the lack of treatment
effect for measures of time spent chewing per kg of DM consumed (Table 4). Although
its regulation is poorly understood, time spent chewing is primarily related to dietary
intake and concentration of ﬁber and forage ﬁber, and is poorly related to DMI (Allen,
1997). Total chewing time and time spent ruminating per kg of NDF intake was
decreased by UNS (P < 0.05 and P < 0.01, respectively). Therefore, differences in
feeding behavior observed in the current experiment cannot be attributed to differences in
feed intake. Treatment diets also contained the same base ration and are not expected to
differ in effectiveness of stimulating rumination, although associative effects on rumen
ﬁber digestion and passage could have affected rumen digesta pool size, leading to
changes in rumination.

Deswysen et al. (1987) reported a strong positive relationship between the number
of rumen contractions and rumination time, decreased time spent ruminating may be
indicative of less reticular-rumen motility. Nicholson and Omer (1983) showed that

intestinal infusion of unsaturated FA decreased rumen motility of sheep. Grovum (1984)

45

reported almost total cessation of rumen motility after 13 h of intragastric infusion of
unsaturated fat while intravenous infusion had little effect. Decreased intake and
frequency of biphasic and triphasic rumen contractions were observed within 3 h of
intragastric infusion of unsaturated FA (Grovum, 1984). Differences in gastric and
venous infusions implicate involvement of the gut in FA depression of reticular-rumen
motility. Dry matter intake was decreased and postprandial CCK was increased when
diets were supplemented with calcium salts of palm oil (Choi and Palmquist, 1996), and
direct intravenous infusions of CCK depressed reticular-rumen motility and intake in
sheep (Grovum, 1981). These observations are consistent with CCK decreasing gut
motility and centrally signaling satiety (Reidelberger, 1994). Benson and Reynolds
(2001) observed increased plasma concentrations of glucagon-like-peptide-l (GLP—l)
with infusion of unsaturated fat (rapeseed oil). GLP-1 is a gut peptide with similar
actions and secretion patterns of CCK (Hellstom and Naslund, 2001). Benson and
Reynolds did not observe a change in CCK secretion, although samples taken overtime
were pooled, and temporal variation could not be evaluated (Benson and Reynolds,
2001). These observations are consistent with gut peptide secretion responding to FA
ingestion with subsequent effects an intake and gut motility (Reidelberger, 1994; and
Hellstom and Naslund, 2001).

Experiments testing the effect of FA on rumen motility and gut peptide secretion have
used no fat controls. The effect of FA saturation on endocrine signaling and rumen
motility has not been explored. Decreased rumination with UNS in the current
experiment is consistent with FA saturation changing gut peptide secretion. Unsaturated

FA may directly stimulate increased gut peptide secretion or change temporal release of

46

gut peptides relative to a meal, causing a decrease in gut motility. Absorption of FA and
stimulation of gut peptide secretion likely coincide with rumination bouts between meals
as the rumen creates a lag between intake of FA and their ﬂow to the duodenum.
Decreased reticulorumen motility may then decrease time spent ruminating. Decreased
rumination and reticular motility may lead to increased distension from physical ﬁll
because of slower digestion and passage of digesta.

Dry matter intake response (SAT-UNS) was not related to pFCMY or pretrial fat
yield. In addition, there was no relationship between pretrial parameters and chewing
behavior responses (Table 6). Failure to detect a relationship of DM1 with pFCMY
discounts physical ﬁll and absorbed energy as mechanisms of unsaturated FA induced
hypophagia. Intake of high producing cows in a less positive energy balance is expected
to be limited by physical ﬁll, while intake of lower producing cows in a more positive
energy balance is expected to be limited by absorbed fuels. Observed intake responses in
the current experiment cannot be attributed entirely to either mechanism. Digestibility of
saturated FA is commonly thought to be lower than unsaturated fat. However, FA
digestibility cannot be determined by measuring digestibility of individual FA fed in a
mixture because hindgut biohydrogenation inﬂates recovery of saturated FA at the
expense of unsaturated fat, causing overestimation of unsaturated FA and
underestimation of saturated free FA digestibility. Other experiments have concluded that
the intake depression of unsaturated FA is not mediated by differences in digestibility
when supplemented in FA form. The abomasal infusions previously discussed (Drackley
et al.1992; Christensen et al. 1994; and Bremmer et al. 1998) did not show differences in

FA or energy digestibility when directly comparing saturated and unsaturated FA

47

treatments. Finally, Schauff and Clark (1989), Grummer (1988), and Palmquist (1991)
directly compared calcium salts of pahn FA and saturated free FA (same treatments as
this experiment) and found no difference in apparent total tract digestibility of energy,

lipid and FA, respectively.

Production

There were no treatment effects of FA type on yield of milk or milk components.
Production response to supplemental fat is inconsistent across experiments. Chilliard
(1993) reviewed the effect of fat supplementation and noted little difference in FCM in
short-term experiments, presumably because of a 2-3 wk production lag observed in
long-term fat studies. Experimental periods of 14 d, as used in the current experiment,
may be too short to establish effects on milk yield.

Unsaturated FA treatment decreased milk protein and lactose concentration
relative to SAT (protein 3.07% and 3.02%, and lactose 4.80% and 4.75% for SAT and
UNS respectively). Response (SAT — UNS) of milk protein yield and concentration were
positively related to milk yield (Figure 1, P = 0.02, R = 0.18; and Figure 2, P < 0.05, R =
0.46, respectively). High producing cows had larger milk protein yield responses when
fed SAT compared to UNS than cows with lower milk yield. Response in individual cow
FCM, milk fat yield, and fat percent were not related to pretrial production. The
production level or metabolic state by response interaction that we observed for milk
protein may explain the inconsistent reports of FA effects on milk protein synthesis.
Non-responding cows in some experiments, but not in others, may simply dilute

treatment effects or add unexplainable variation.

48

Decreased milk protein concentration is commonly attributed to the diluting effect
of increased milk yield in some experiments. In the current experiment, there was no
relationship between the response for milk protein percent (SAT — UNS) and the
response for milk yield (SAT — UNS, R2 = 0.41, P = 0.23). There was no dilution effect
of milk protein observed because milk protein yield response linearly increased with
increased milk yield response (R2 = 0.55, P < 0.001). Although not observed in the
current experiment, dilution of milk protein by increased milk production merits further
investigation. Protein and lactose concentration of milk are both very stable and expected
to be highly correlated; Wu and Huber (1994) reported a linear relationship between milk
and milk protein yield with an R2 of 0.90. Dilution of milk protein by increased milk
yield would represent a deviation from normal and should not be ignored.

Emery (1972) reported that milk protein concentration decreased 0.1 to 0.3
percentage points with added fat, and DePeters and Cant ( 1992) reviewed the effect of fat
on milk protein showing variation in published responses. Chilliard (1993) reviewed fat
effect on production and noted that milk protein concentration decreased in response to
fat supplementation to a greater extent in early lactation compared to peak lactation (0.8
vs. 0.5 g/kg) and in short-term experiments compared to long-term experiments (1.0 vs.
0.5 g/kg). In direct comparisons of dietary protected saturated and unsaturated FA,
Grummer (1988) showed unsaturated FA decreased milk protein 0.13% compared to no
fat control, and saturated fat treatment maintained milk protein. Schauff and Clark (1989)
observed no effect of fat type on milk protein. Dietary FA saturation appears to be an

important factor affecting milk protein response to FA treatment. Possible mechanisms

49

include inhibition of microbial protein production, modiﬁcation of insulin signaling, and
changes in the somatotropic axis.

Unsaturated FA fed as calcium salts of palm oil are partially available for
biohydrogenation, and may interfere with microbial growth rate or efﬁciency. Fatty acids
may decrease milk protein because of decreased microbial protein yields and less protein
absorbed and available for milk protein synthesis. However, feeding protected oleic acid
in the form of Oleamide decreased milk protein concentration and yield (Jenkins, 2000)
compared to raw canola oil (high oleic acid). Free oil interferes with ruminal
fermentation more than Oleamide but had less of an effect on milk protein synthesis than
the physiological effects of absorbed unsaturated FA.

Hyperinsulinemic-euglycemic clamp studies have identiﬁed insulin, or its
stimulation of IGF, as a regulator of milk protein synthesis (McGuire et al., 1995; and
Griinari et al., 1997). Increased milk protein synthesis in the clamp procedure is not
solely the effect of infused glucose sparing amino acids because cows supported
increased protein synthesis by increasing the extraction efﬁciency of essential amino
acids, mammary blood ﬂow, and glucose uptake (Mackle et al., 2000). In the present
study, saturated fat increased insulin 25% as well as milk protein concetration and yield
consistent with the insulin clamp model. Surprisingly, although milk protein response
was related to pFCMY, insulin response (SAT - UNS) was not. More puzzling is that
there was a signiﬁcant negative linear relationship of milk protein response and insulin
response (Figure 3). Cows with the greatest milk protein yield response (SAT - UNS)
had the lowest insulin response between treatments. If insulin regulates milk protein

synthesis, it is reasonable to expect that not just the plasma insulin concentration, but also

50

tissue sensitivity to insulin stimulation is important. Palmquist and Moser (1981) studied
the relationship of dietary unsaturated fat, plasma glucose and insulin, and milk protein
production. Glucose tolerance tests were used to measure insulin responsiveness and
sensitivity. Cows fed calcium salts of palm oil responded to glucose infusion with more
insulin secretion and had slower clearance of glucose, suggesting increased insulin
resistance. The authors proposed that fat-stimulated insulin resistance may reduce amino
acid transport into the mammary gland. The negative relationship between insulin and
milk protein response observed in the current experiment supports the hypothesis that
tissue insulin sensitivity is important to milk protein production.

Insulin stimulation of the somatotropic axis cannot be ruled out as a possible
mechanism for increasing milk protein synthesis. Molento et al. (2002) showed insulin
stimulated IGF-l production in early to mid lactation cows. They proposed that the bST
to insulin ratio was an important predictor of IGF -1 production, with higher ratios
correlating to higher IGF-l concentrations. In the present study, there was a signiﬁcant
positive relationship between the bST/insulin response and milk protein response (Table
10), with increasing bST/insulin ratios (higher expected IGF-l concentrations) linearly

related to higher milk protein responses.

Energy Balance and Efficiency

Treatments did not affect body weight gain or body condition score changes (P =
0.37, P = 0.74, respectively). There was also no relationship between measures of energy
balance and pretrial milk yield. Experimental periods were only 14 d in length, making

measurement of body tissue changes difﬁcult. The lack of effect on body weight, body

51

condition score, leptin, or bST indicates that cows did not change energy balance or that
experimental periods were too short to observe differences.

Treatment diets contained nearly equal nutrient compositions and were considered
to contain the same gross energy density. Efﬁciency calculated as FCM yield per kg
DMI was greater for UNS than SAT cows (P < 0.001). This calculation does not account
for changes in body energy, and treatment effects were not signiﬁcant for either milk
yield or body weight change. Efﬁciency calculated as net energy (NE) of body weight
change plus NEL milk production over DMI was not different between treatments. Body
weight energy gain, and milk energy yield were not different between treatments,
attributing any efﬁciency difference to changes in dry matter intake.

Calculation of marginal return or efﬁciency involves analysis of the response
stimulated by marginal inputs. The return provided by increased dry matter intake is a
more responsive and informative variable than absolute efﬁciency. Milk yield and milk
protein yield responses (SAT — UNS) were linearly increased with increased DMI
response (P < 0.01 and P < 0.001, respectively). Fat corrected milk, milk fat percent, and
milk fat yield responses (SAT - UNS) were affected quadratically by increasing DMI
response (P < 0.01). Marginal milk and milk protein yield were linearly increased and
marginal milk fat yield was affected quadratically with increasing DMI response (Table
7). Increasing DMI increased production of milk and milk components. The cost of the
additional production is merely the increased DMI when using marginal return. Marginal
return and efﬁciency is gaining prominence over the absolute efﬁciency calculation

presented and provides a more responsive decision making tool.

52

Plasma Metabolites and Hormones

Metabolic hormones analyzed included insulin, growth hormone, leptin, and
CCK, and metabolites assayed were glucose, NEFA, and BHBA. Saturated FA increased
insulin over 25% compared to UNS (12.8 vs. 10.1 uIU/mL, P < 0.001). Type of FA
determines insulin secretion in vitro with saturated and longer chain FA being more
insulinotropic (Stein et al., 1997). Plasma leptin and growth hormone concentrations were
not affected by treatment. Saturated FA treatment decreased NEFA over 20% compared
to UNS (89.3 vs. 115.5 ueq/l respectively, P<0.001), but plasma glucose and BHBA were
not affected by treatment. Decreased NEFA with no change in glucose may show
different responsiveness or regulation in fat and glucose metabolism to insulin signaling.
A quadratic effect of pFCM was observed on plasma NEF A. Interestingly, all cows
decreased plasma NEFA when fed SAT compared to UNS. No other plasma hormone or
metabolite was related to pFCMY.

A possible change in insulin sensitivity is noted using glucose to insulin ratio as a
proxy for insulin stimulation of glucose uptake. Unsaturated FA had a higher glucose to
insulin ratio than SAT (P < 0.02), but had no effect on the ratio of bST to insulin (data
not shown). Plasma BHBA did not change, but it is not possible to conclude difference in
FA oxidation between treatments because BHBA clearance is not known.

A quadratic effect of pFCM was observed on plasma NEFA. Interestingly, all
cows decreased plasma NEF A when fed SAT compared to UNS. No other plasma
hormone or metabolite was related to pFCMY.

Leptin is commonly correlated to fat cell size and body fatness. There is interest

in the interaction of insulin and leptin and the ability of insulin to directly stimulate leptin

53

secretion. The large increase in insulin with SAT treatment provides an interesting
opportunity to observe the relationship of insulin and leptin response in high producing
cows. Saturated fat treatment produced a large increase in insulin but no difference in
plasma leptin, and leptin response (SAT -UNS) was not related to insulin response.
Plasma concentrations of insulin and leptin were not related. Plasma bST was negatively
related to leptin as expected; higher bST is associated with a lower or negative energy

balance and higher leptin is associated with positive energy balance (Figure 4).

CONCLUSIONS AND IMPLICATIONS
Fatty acid proﬁle reaching the duodenum is important for predicting intake

response to fat supplementation. Increasing unsaturated FA concentration of the diet
decreased intake with no relationship to milk yield across cows. Unsaturated FA
decreased time spent ruminating, which may be the result of reduced gut motility as
previously observed in abomasal infusions. Dietary FA saturation affects insulin
secretion and plasma NEFA concentration. Saturated FA increased milk protein and the
magnitude of the response appears to be related to production level, insulin signaling, or

IGF-l stimulation.

54

Table 1. Status of 31 cows at the beginning of experiment. 1

 

 

Mean SD
BW, kg 655 45
BCS 2.35 0.38
DIM 130 70
Milk yield, kg 43.7 6.3

 

1 Data were collected from 32 cows for this experiment, but data from one cow was
excluded due to illness.

Table 2. Ingredients and nutrient composition of treatments’.

 

 

 

 

 

 

 

Nutrient SAT UNS
DM, % as fed
% of DM
Ingredients
Ca Soaps FA - 57.7
Prilled FA 48.9 -
Rice Hulls - 10.9
Limestone 19.2 -
Ground Corn 31.9 31.4
Composition
Total FA 49.7 46.9
Calcium 6.8 7.1
FA Proﬁle % of FA
C16 34.0 46.9
C18:0 46.2 4.3
C18:1 9.9 36.3
C18:2 2.54 9.31
C18:3 0.04 0.30
Unsaturated FA 13.2 46.1
Cl6:18 Ratio 0.58 0.94

 

1 SAT- saturated fatty acids treatment of prilled fatty acids, UNS- unsaturated fatty acids
treatment of calcium salts of palm oil

55

Table 3. Ingredient and nutrient composition of experimental diets].

 

Pretrial SAT UNS
Ingredients --------------- % of DM -------------
Corn silage2 22.5 22.4 22.5
Alfalfa silage3 18.9 20.0 20.1
Ground Corn 23.8 23.1 23.1
Whole Cottonseed 10.4 10.3 10.3
Protein mix4 14.1 13.8 13.9
SAT mixs 2.5 5.0 -
UNS mixs 2.4 - 4.6
Mineral vitamin mix6 4.4 4.5 4.5
Molasses mix7 1.0 0.9 0.9
Nutrient
DM 52.6 52.2 52.1
Total FA 7.1 7.0 6.8
% Unsaturated FA 3.8 3.3 4.0
Starch 26.6 25.5 25.7
NDF 26.0 26.4 26.9
Forage NDF 16.1 17.0 17.1
CF 18.6 17.3 17.4
Ash 5.3 5.4 5.1
Rumen-undegraded CP8
(% CP) 35.7 35.6 35.6

 

’ Pretrial- covariant period; SAT- saturated fatty acids treatment of prilled fatty acids,
UNS- unsaturated fatty acids treatment of calcium salts of palm oil
2 Corn silage contained 37.2% DM (as fed) and 39.6% NDF, 7.4% CP, 27.9% starch, and
3.0% ash on a DM basis
3 Alfalfa silage contained 30.9% DM (as fed) and 40.5% NDF, 17.0% CP, 4.2% starch,
and 7.2% ash on a DM basis.
4 Protein mix contained 74.1% soybean meal (44% CP), 20.1% corn gluten meal, and
5.8% blood meal.
5 Mix composition listed in Table 2.
6 Mineral vitamin mix contained 16.5% vitamin E, 41.0% vitamin D, 44.4% vitamin A,
1.9% trace mineral premix, 4.1% urea, 4.6% salt, 8.4% limestone,10.5% dicalcium
phosphate, 11.7% sodium bicarbonate, and 57.9% dry ground corn as a carrier

‘ Molasses mix was 66% DM and contained 30.3% CP.
8' Rumen-degraded protein estimated using values from NRC (2001).

56

Table 4. Effects of fatty acid saturation1 an intake and feeding behavior.

 

 

 

 

 

SAT UNS SE P

Intake --------- (kg/d) --------

DM 27.4 26.7 0.4 <0.01
NDF 6.93 6.88 0.09 0.39
Starch 6.79 6.62 0.10 <0.01
CP 4.57 4.46 0.06 <0.01
Total FA 4.08 3.86 0.05 <0.001
Eating time min

/d 210 205 6 0.33

/kg DMI 7.8 7.8 0.3 0.91

/kg NDF intake 31 30 1.0 0.40
Ruminating time

/d 535 510 10 <0.01

/kg DMI 20 19 0.5 0.26

/kg NDF intake 77 74 1.8 0.03
Total chewing time

/d 745 715 13 <0.0l

fkg DMI 27 27 0.6 0.26

/kg NDF intake 108 104 2.3 0.02
Time spent idle

/d 670 695 10 <0.01

 

' SAT- saturated fatty acids treatment of prilled fatty acids, UNS- unsaturated fatty acids
treatment of calcium salts of palm oil

57

Table 5. Effects of faty acid saturation onproduction‘.

 

 

 

SAT UNS SE P

Yield ----------- (kg/d) ----------

Milk 41.8 42.3 1.1 0.12
3.5% FCM" 41.8 42.4 1.2 0.11
SCM3 39.0 39.3 1.1 0.33
Fat 1.47 1.49 0.05 0.25
Protein 1.27 1.27 0.03 0.79
Lactose 2.00 2.01 0.06 0.23
Milk Composition %

Fat 3.54 3.54 0.09 0.92
Protein 3.06 3.02 0.05 0.04
Lactose 4.80 4.74 0.03 <0.001
Tissue Gain

BW change, kg/d 0.50 0.29 0.16 0.37

BCS change4 0.11 0.10 0.04 0.74

 

 

1 SAT- saturated fatty acids treatment of prilled fatty acids, UNS- unsaturated fatty acids

treatment of calcium salts of palm oil
2 3.5% fat-corrected milk yield (kg/d)

3 Solids-corrected milk yield (kg/d)
4 Change in body condition score (BCS; ﬁve-point scale where 1 = thin to 5 = fat) over a

14-d period.

58

Table 6. Responses (saturated — unsaturated) of intake and production by pretrial 3.5%
fat-corrected milk yield.

 

 

 

 

 

 

Response R2 Predictorl P2 Coefﬁcient3 Intercept4
DMI 0.19 pFCMY 5 0.14 -- --
MY (kg/d) 0.39 pFCMY 0.24 -- --
3.5% FCM (kg/d) 0.19 pFCMY 0.48 -- --
Fat, % 0.06 pFCMY 0.84 -- --
Fat yield, kg 0.02 pFCMY 0.75 -- --
Protein, % 0.46 Sequence6 <0.001 0.174 -0204
pFCMY <0.05 0.049
Protein yield, kg 0.18 Sequence 0.48 -0.008 -0. 173
pFCMY 0.02 0.004
Lactose, % 0.34 Sequence <0.01 0.006 1 .05
pFCMY 0.01 0.01 1
(pFCMY)2 <0.01 0.009
Lactose yield, kg 0.66 Sequence <0.001 -0.095 0.965
pFCMY <0.01 -0.049
(pFCMY)2 <0.01 0.0006

 

' Regression term

2 P- signiﬁcance of regression term

3 Coefﬁcient of the regression term

4 Intercept of the regression equation

5 Pretrial 3.5% fat-corrected milk yield (kg/d).
6 Sequence parameter estimate

59

Table 7. Milk yield and component responses (saturated — unsaturated) by dry matter
intake response (saturated — unsaturated)

 

 

 

 

 

 

Response R2 PredictorI P2 Coefficient3 Intercept"
MY (kg/d) 0.53 Sequence <0.01 -0.98 -l.10
DMI Response 5 <0.01 0.82
3.5% FCM (kg/d) 0.43 Sequence 0.09 -0.69 -l .26
DMl Response 0.67 0.17
(DMI Response)2 0.01 0.36
Milk Fat, % 0.32 Sequence 0.17 0.06 0.015
DMI Response 0.03 -0.05
(DMI Response)2 <0.01 0.01
Milk Fat Yield, Kg 0.35 Sequence 0.63 -0.01 -0.040
DMI Response 0.67 -0.01
(DMI Response)2 <0.01 0.02
Milk Protein, % 0.41 Sequence <0.001 0.09 0.034
DMI Response 0.24 0.01
Protein yield, kg 0.39 Sequence 0.23 0.01 -0.023
DMl Response <0.001 0.04

 

' Predictor— regression term

2 P - signiﬁcance of regression term
3 Coefﬁcient of the regression term

4 Intercept of the regression equation
5 DMI Response = (saturated DMI — unsaturated DMI)

Table 8. Effects of fatty acid saturation1 on plasma hormones

and metabolites.

 

CCK, pmol/L
Insulin, uIU/mL

BST, ng/mL

Leptin, ng/mL
Glucose, mg/dL
NEFA, ueq/L
BHBA, mg/dL

Glucose/insulin
ratio, mg/uIU

Insulin Std. Dev.

NEFA Std. Dev.

 

SAT UNS SE P
12.5 14.1 0.91 0.08
12.8 10.1 0.6 <0.001
1.94 1.98 0.14 0.76
2.17 2.56 0.09 0.18
62.0 61.4 0.5 0.19
89.3 115.5 3.3 <0.001
5.25 5.28 0.16 0.86
5.50 6.50 0.28 0.15
6.53 4.76 0.48 0.01
16.9 23.8 2.0 <0.01

 

1 SAT- saturated fatty acids treatment of prilled fatty acids, UNS-
unsaturated fatty acids treatment of calcium salts of palm oil

61

Table 9. Effects of fagy acid saturationl on energy intake and partitioning.

 

 

SAT UNS SE P

Milk Energy, Mcal

NE,2 28.7 29.9 0.77 0.33
Tissue energy gain},

Mcal NEL 2.90 2.36 1.11 0.73
Efﬁciency,

FCM/DMI 1.53 1.59 0.04 <0.001
Energy efﬁciency4 0.54 0.55 0.02 0.76

 

‘ SAT- saturated fatty acids treatment of prilled fatty acids, UNS- unsaturated fatty acids
treatment of calcium salts of palm oil

2 NEumnk) (Mcal/d) = MY (kg) x (0.0929 x fat% + 0.0563 x true protein% + 0.0395 x
lactose%) (NRC 2001).

3 NEL gain calculated from body weight gain (NRC 2001)

4 (Milk energy, Mcal NEL + Tissue energy gain, Mcal NEL) / kg DM

62

Table 10. Responses (saturated — unsaturated) of energy balance and plasma metabolites
and hormones

 

 

 

 

 

 

Response R2 PredictorI P2 Coefﬁcient3 Intercept4
Body wt gain 0.01 pFCMY 5 0.64 -- --
Time spent
Eating 0.49 pFCMY 0.29 -- --
Ruminating 0 pFCMY 0.95 -- --
ldle 0.38 pFCMY 0.72 -_ --
Plasma
Insulin, ulU/mL 0.65 pFCMY 0.84 -- --
Leptin, 0.1 1 pFCMY 0.54 -- --
Glucose, mg/dL 0.04 pFCMY 0.75 -- --
BHBA, mg/dL 0.05 pFCMY 0.26 -- --
Glucose/Insulin
bST, ng/mL 0.20 Sequence 0.25 -0.1 17 -5.054
pFCMY 0.07 0.251
(pFCMY)2 0.06 -0003
NEFA 0.28 Sequence 0.46 -l .90 - 190.5
pFCMY 0.02 8.27
(pFCMY)2 0.01 -010
bST / Insulin 0.36 Sequence <0.01 0.073 -1 .287
pFCMY 0.03 0.063
(pFCMY)2 0.02 -0.00078

 

' Predictor- regression term

2 P -— signiﬁcance of regression term

3 Coefﬁcient of the regression term

4 Intercept of the regression equation

5 Pretrial 3.5% fat-corrected milk yield (kg/d).

63

0.4 ..

 

: Y=-0.204+0.006x

03- P=0.048 .

' ” R2=0.46
e\° " 0
a,“ . O
m ..
C 0.2+-
O .
D.
in .
0’ .
D! 0.1-
.E ’
O .
‘6 i
L
o. 0 ‘
:5
E t

'0.1:- . . .

O
-0_2hr..41....lerrlririlirriliriil.1111

 

 

25 30 35 40 45 50 55 60

Pretrial FCM, kg/d

Figure 1: Milk protein percent response by pretrial fat corrected milk (FCM) yield.
Relationship between milk yield over the 14 (1 prior to the beginning of the experiment
and the response (saturated —- unsaturated) in milk protein concentration to the
unsaturated FA treatment.

 

03: Y=-0.38+0.0092x
. P=002 . ,

E ; R2=0.18 0
3 0.2- o
a; .
ID .
g r
a 0.1:.
in
o F
a: .
2 0—
.2
>.
c r-
i; 4L1 -
u f
O 1 o
h
a, t o o o
:5 -02_— 9
s E '-

-o_3brirrl”4.1....1...rnirn.1....1....l

 

 

25 30 35 40 45 50 55 60

Pretrial FCM, kg/d

Figure 2: Milk protein yield response by pretrial fat corrected milk (FCM) yield.
Relationship between milk yield over the 14 (1 prior to the beginning of the experiment
and the response (saturated — unsaturated) in milk protein yield to the unsaturated FA
treatment.

65

0.4

o v = 0.07 - 0.014 x
o P = <0.001
0'3 r12 = 0.375
00

0.2

0.1

 

Milk Protein Response, %

  

IITIﬁIUﬁTIIITIIT‘TI'TIU IFTIII]

 

 

0
to
-0.1 o o o.
-0.2 I I I I I I 1 L I l I I I J—l I I PI 1 I I I I l I I I I l
-10 -5 0 5 10 15 20

Insulin Response, plU

Figure 3: Milk protein percent response by insulin response. The relationship between
milk protein concentration response (saturated — unsaturated) and insulin response to
fatty acid saturation.

66

 

4.5 _
- o Y=3.67+-0.616x+0.262 x2
4:. L<0.001
- Q<0.001
: R2=0.51
_l 3.5 1-
E .
\
U!
C
.. 3 __
C 1-
: 1-
% ..
.1 2.5 -
C
2’.
O. O
1.5 hrerliirrlrrrrlrrrrlirnrJenn.Llirrrlrrrrl

 

 

bST, ngImL

Figure 4: Relationship between mean leptin and bST concentration across animal periods.

67

CHAPTER 3

Effect of rumen-protected fatty acid saturation on milk yield, milk fatty acid profile,

energy balance and plasma metabolites and hormones of lactating dairy cows.

ABSTRACT

Saturated and unsaturated rumen protected fat sources were evaluated for effects on yield
of milk and milk components, concentration of milk components including milk fatty
acid (FA) proﬁle, and energy balance. Eight ruminally and duodenally cannulated cows
and eight non-cannulated cows were used in a replicated 4x4 Latin square design with 21
(1 periods. Treatments were control (CON) and a linear titration of 2.5% added rumen
protected FA varying in unsaturation including saturated (SAT; prilled hydrogenated free
FA), 50:50 ratio of SAT and unsaturated (UNS; calcium soaps of long-chain FA), and
UNS. SAT did not change milk fat concentration, but UNS linearly decreased milk fat in
cannulated cows and tended to decease milk fat in non-cannulated cows. Milk fat
depression with UNS corresponded to increased milk trans-10, cis-12 conjugated linoleic
acid. Milk fat proﬁle did not change with SAT compared to CON, but UNS decreased
concentration of short and medium-chain FA and increased concentration of long-chain
FA. Digestible energy intake tended to decrease linearly with increasing UNS in
cannulated and non-cannulated cows. Increasing UNS linearly increased empty body
weight and net energy gain in cannulated cows, while plasma insulin and beta-

hydroxybutyrate concentrations were decreased linearly. Efﬁciency of conversion of

68

digestible energy to milk tended to decrease linearly with UNS for cannulated cows only.
Addition of rumen-protected fat did not change energy balance of cannulated cows, but
decreased energy balance of non-cannulated cows. Addition of SAT provided little

beneﬁt to production and energy balance, while UNS decreased milk energy yield.

INTRODUCTION

High producing dairy cows have large energy requirements that may exceed their
ability to consume dietary energy, resulting in less than maximum milk yield. Addition of
fat to the diet increases energy density without increasing rumen acid production, or
maintains energy density while increasing ﬁber for stabilization of rumen fermentation
(Allen, 1997). Prilled saturated free fatty acids (FA) and calcium salts of FA are two
manufactured products marketed to minimize effects of fat on ruminal fermentation.
However, calcium salts of FA are not entirely protected in the rumen and dissociation of
the calcium ion allows rumen biohydrogenation of unsaturated FA (Wu et al. 1991).

Traditionally, FA are considered a source of energy, but are now appreciated as
biological modiﬁers of physiology and metabolism. Incomplete biohydrogenation of
polyunsaturated FA increases duodenal ﬂow of trans-C18:1 and conjugated linoleic acids
(CLA), which have been implicated in milk fat depression through decreased lipogenic
gene expression (Bauman and Griinari, 2003; Peterson et al., 2003). Milk fat depression
normally occurs with little effect on dry matter intake leading to increased body weight

gain in proportion to milk fat depression (Baumgard et al., 2002b). Increased body

69

weight gain in response to trans-10, cis-12 CLA in lactating cows is contrary to
decreased body fat gain observed in growing animals (Mersmann, 2001).

The proﬁle of FA absorbed in the duodenum can alter the FA proﬁle of animal
products, especially modifying FA saturation and CLA concentration (Grummer, 1991;
and Mansbridge and Blake, 1997). Consumers are increasingly concerned about FA
intake. Decreasing saturated FA intake may decrease heart disease and diabetes
(Mansbridge and Blake 1997), and increasing CLA intake may decrease the incidence of
cancer and obesity (Kelly, 2001). Strategies for dietary FA supplementation can be
developed to alter the FA proﬁle of meat and milk products to meet consumer demands.

The objective of this experiment was to determine effects of rumen-protected FA
differing in FA saturation on milk and milk component yield, milk fatty acid proﬁle and

energy partitioning.

MATERIALS AND METHODS
This paper is the ﬁrst of four papers in a series from one experiment that
evaluated effects of rumen-protected FA (RPF) differing in FA saturation. This paper
discusses treatment effects on milk yield, milk FA proﬁle and energy balance, and the
companion papers discuss ruminal kinetics and extent of biohydrogenation (Chapter 4),
ruminal digestion kinetics and site of digestion (Chapter 5), and DM1 and feeding and
chewing behavior (Chapter 6). Experimental procedures were approved by the All

University Committee on Animal Use and Care at Michigan State University.

70

Cows and Treatments

Eight ruminally and duodenally cannulated (77: 8.7 DIM; mean :1: SD) and eight
non-cannulated (106:1: 15 DIM; mean :1: SD) multiparous Holstein cows from the
Michigan State University Dairy Cattle Teaching and Research Center were blocked by
cannulation and assigned randomly to replicated 4 x 4 Latin squares in a dose-response
arrangement of treatments plus a control. Non-cannulated cows were included in the
experiment to increase the number of observations for intake and milk yield. Cows were
blocked by cannulation because they were selected at different times and differed in
DIM, body condition score and surgical preparation. Treatments were a control diet
(CON) containing no added RPF or 2.5% added RPF from saturated (SAT - prilled
hydrogenated FA, Energy Booster 100®, Milk Specialties Company Inc., Dundee, IL),
intermediate mixture of saturated and unsaturated (INT), or unsaturated (UN S) FA (Ca
Soaps of LCFA, Megalac-R®, Church and Dwight Company, Inc., Princeton, NJ).
Treatment periods were 21 d with the ﬁnal 11 (I used for sample and data collection.
Surgery was performed at the Department of Large Animal Clinical Science, College of
Veterinary Medicine, Michigan State University. Immediately prior to initiation of the
experiment, mean empty BW (ruminal digesta removed) of cannulated cows was 516 z
33 kg (mean :1: SD) and mean BW of non-cannulated cows was 638 z 51 kg (mean :t SD).

Treatment mix composition is shown in Table 2. Treatment mixes included
limestone and rice hulls to balance for calcium and FA concentration and 50% ground
corn as a carrier. The base ration was formulated to provide 2.5% rumen available FA
from cottonseed, as would be expected in commercial diets, and treatments were

formulated to provide 2.5% rumen-protected FA as commonly recommended.

71

Experimental diets contained 40% forage (66:33 corn silage: alfalfa silage), 13.5% whole
cottonseed, dry ground corn, premixed protein supplement (soybean meal, corn gluten
meal, and blood meal), a mineral and vitamin mix, and 2.5% added rice hulls (CON),
saturated FA (SAT), 50:50 mix of saturated and unsaturated fat (INT) or unsaturated FA
(UNS) treatment (Table 3). All diets were fed as a total mixed rations. Final diet FA

concentration and composition is shown in Table 3.

Data and Sample Collection

Throughout the experiment, cows were housed in tie-stalls and fed once daily
(0900 h) at 115% of expected intake. Amounts of feed offered and orts were weighed for
each cow daily. Samples of all diet ingredients (0.5 kg) and arts from each cow (12.5%)
were collected daily on d 11 to 14 and combined into one sample to represent four days
for digestibility determination ((1 11-14). Cows were milked twice daily in their stalls
during the feeding behavior monitoring phase ((1 16 to 19) and in a milking parlor during
the rest of each period. Milk yield was measured at each milking on (1 11-19, and milk
was sampled at each milking on d 16 to 19.

Methods for determining fecal output and digestibility for cannulated cows are
described in Chapter 5. Indigestible NDF (iNDF) was used as a marker to calculate total
tract fecal ﬂow for non-cannulated cows. Fecal samples (1,000 g) were collected every 9
h from d 12 to d 14 yielding eight samples representing every 3 h of a 24-hour period to

account for diurnal variation.

72

Sample and Statistical Analysis

Feed and fecal samples were processed and analyzed as described in Chapter 5.
Milk samples were composited based on milk fat yield and centrifuged at 17,800 x g for
30 min at 8°C. Fat cake (300-400 mg) was extracted according to Hara and Radin (1978)
and methyl esters were formed according to Christie (1982) as modiﬁed by Chouinard et
al. (1999). FA were quantiﬁed by GC (Model 8500, Perkins-Elmer Corp, Norwalk, CT),
using a SP-2560 capillary column (100m X 0.20 mm id with 0.02-pm ﬁlm thickness;
Supelco, Bellefonte, PA). Oven temperature was 140°C for 5 min, then ramped 4°C/min
to 240°C and held for 15 min. Helium ﬂow was 20 cm/sec. Milk samples were analyzed
for fat, true protein, and lactose with infrared spectroscopy by Michigan DHIA (East
Lansing).

Commercial radioimmunoassay kits were used to determine plasma concentration
of insulin (Coat-A-Count, Diagnostic Products Corporation, Los Angeles, CA), and
glucagon (Glucagon kit GL-32K, St. Charles, MO). Commercial enzyme kits were used
for analysis of glucose (Glucose kit #510; Sigma Chemical Co., St. Louis, MO), NEF A
(N EF A C-kit; Wako Chemicals USA, Richmond, VA), and B-hydroxybutyrate (BHBA;
kit #310-A; Sigma Chemical Co., St. Louis, MO).

Energy values were calculated as follows:

DE intake = GEI x GE digestibility [GE digestibility as reported in Chapter 4]

NEL intake- calculated from DE through ME according to NRC 2001.

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

NEM = 0.080 x 13w 0'75 (NRC, 2001); and

73

NEL balance = NEL intake — NEM - Milk NEL

All data were analyzed using the ﬁt model procedure of JMP® (Version 5, SAS
Institute, Cary, NC) according to the following model:

Yijk=ll+Ci+Pj+Tk+6ijk
where

u = overall mean,

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

Pj = ﬁxed effect of period (j = 1 to 4),

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

eijk = residual error.

There was a signiﬁcant block by treatment effect for milk production and other
variables of primary interest (P<0.10) so cannulated and non-cannulated cow data was
separated for presentation. Period by treatment interaction was evaluated, but was
removed from the statistical model when not signiﬁcant (P > 0.10). Period by treatment
interaction was not signiﬁcant for any variable of primary interest; variables with
signiﬁcant interactions are noted in the tables. Data points with Studentized Residuals
greater than three were considered outliers and excluded from analysis. Few points were
excluded in analysis and rarely more than one per response variable. Preplanned
contrasts included the effect of addition of RPF (CON vs. SAT, INT and UNS), linear
effect of increasing concentration of unsaturated fat [L (SAT vs. UNS)], and quadratic
effect of increasing concentration of unsaturated fat [Q (INT vs. SAT and UNS)]. The
preplanned contrasts do not allow individual comparison of each fat treatment to the

control. Protected LSD was used for mean separation in the discussion when the model

74

treatment effect was signiﬁcant. Pearson correlation coefﬁcients were determined
between cow-period observations for some parameters. Average parameters for each
block presented in Table 1 were determined by including black in the above model.
Treatment effects, linear and quadratic responses, and correlations were declared
signiﬁcant at P < 0.05, and tendencies were declared at P < 0.10.

Data from two cow-periods were excluded from statistical analysis. One
cannulated cow developed clinical mastitis on d 19 of period 3, rumen samples, body
weight and body condition score was not collected for this period. Data previously
collected in this period was included in our analysis. The cow did not fully recover and

data from period 4 was not used.

RESULTS AND DISCUSSION

Characteristics of cows within each block are presented in Table 1. Cannulated
cows were 30d earlier in lactation at the start of the experiment, with lower BW and
greater milk yield than non-cannulated cows. Although blocks only slightly differed in
DIM, there was a large difference in BCS indicating differences in metabolic state.

Diets were based on the same forage and concentrates and differed only in
addition of FA treatment mix (Table 2). The CP concentration of the diets averaged
16.1%, which was lower than the target CP formulated for 17.8% because a number of
dietary ingredients contained lower concentrations of CP during the experiment than
measured before the experiment began. Treatments were formulated to contain the same
calcium concentrations using limestone, and rice hulls were used to take the place of FA

in the CON to maintain approximately the same fermentable and digestible carbohydrate

75

concentration (Table 2). Treatments differed in FA concentration and proﬁle. Control
diet contained 5.5 % FA and RPF diets contained 8.3, 8.1 and 7.8 % FA for SAT, INT
and UNS. Treatment mixes (Table 2) were formulated based on manufacturer’s product
speciﬁcations. Calcium salts of long-chain FA contained much lower FA than expected.
To compensate for the lower FA concentration of UNS, a greater concentration of UNS
mix was included in the UNS and INT treatments and CON mix (rice hulls only) was
used to compensate for the increased inclusion rate in CON, SAT and INT. The small
FA concentration difference among the ﬁnal RPF treatments is attributed to variation
during the experiment. Dietary unsaturated FA density increased from SAT to UNS
treatment (3.9, 4.4 and 4.9 % for SAT, INT, and UNS). Increased unsaturated FA from
SAT to UNS included increased C18:1, C18:2 and C18:3 and decreased Cl8:0 (Chapter
4). Addition of RPF increased the C16:C18 ratio, but the ratio was not changed within

RPF.

Milk and Milk Component Yield

Rumen-protected FA did not affect milk yield of cannulated cows (Table 4) or
non-cannulated cows (Table 5). However, within RPF treatments, milk yield was
reduced linearly with increasing UNS for cannulated cows only. Increasing UNS linearly
decreased milk fat concentration of cannulated cows and tended to decrease milk fat
concentration of non-cannulated cows. The linear reduction in milk yield and milk fat
concentration with increasing UNS linearly decreased fat-corrected milk and milk fat
yield for cannulated cows. Yields of milk and fat-corrected milk as well as

concentrations of milk fat and lactose were not different for SAT compared to CON for

76

either black of cows. Yield and concentration of milk protein was not affected by
treatment for cannulated or non-cannulated cows and milk urea nitrogen was linearly

decreased with UNS for cannulated cows only.

Milk Fatty Acid Profile

Milk FA proﬁle of cannulated and non-cannulated cows was consistent with
previous reports during milk fat depression (Baumgard, 2002a; and Peterson, 2003).
Addition of SAT did not increase cis-9, trans-11 CLA or trans-C18:1 FA concentration,
but UNS increased their concentration in cannulated and non-cannulated cows. In the
cannulated cows, trans-10, cis—12 CLA was increased by SAT and increasing UNS
linearly increased trans-10, cis-12 CLA. Non-cannulated cows did not increase trans-10,
cis-12 CLA with SAT, but UNS increased the CLA compared to control. Milk fat
concentration had a strong quadratic relationship with trans-10, cis-12 CLA [Y = 3.50 -
17.41 + 131.6(x - 0.03)2; R2= 0.60,], cis-9, trans-11 CLA [Y = 3.58 — 27.99 + 214.65(X
- 0.0204)2; R2: 0.66], and trans-C18:1 [Y = 4.03 — 0.27 + 0.02(x — 3.74)2;R2= 0.56] in
agreement with others (Peterson et al. 2002; Bauman and Griinari, 2003), although only
tran-10, cis-12 CLA has been demonstrated to induce milk fat depression (Baumgard et
al. 2000). Addition of SAT had very little effect on milk FA proﬁle, but increasing UNS
decreased concentration of short and medium chain FA and increased concentration of
long-chain FA. Milk fat depression, induced by increased duodenal ﬂow of trans-10, cis-
12 CLA, is mediated through decreased gene expression of lipogenic enzymes, leading to
decreased mammary de nova FA synthesis (Baumgard et al. 2002a; and Peterson et al.

2003). The observed FA proﬁle changes are consistent with decreased FA synthesis

77

causing a decrease in concentration of short and medium chain FA. Addition of UNS FA
increased the unsaturated proportion of C18 FA, but also increased trans-C18:1
concentration. Increased unsaturated FA and CLA with UNS should increase consumer
appeal, but increased total trans-C18:1 is expected to negatively affect consumer appeal
because of the association of trans-C18:1 and heart disease. Although we recognize the
importance of separation of trans-C18:1 isomers they could not be separated with the FA

analysis procedure used.

Energy Intake and Balance
Cannulated cows

Rumen-protected FA decreased DE intake and increasing UNS FA tended (P =
0.06) to linearly decrease DE intake, but NEL intake was not affected by treatment (Table
8). Calculation of NEL intake accounts for increased efﬁciency of converting DE from
FA to NE. Saturated FA treatment did not affect milk yield, but INT and UNS decreased
NEL milk because of lower milk yield and milk fat concentration with increasing UNS.
Empty BW gain and calculated net energy of tissue gain were increased with increasing
UNS but BCS was not affected by treatment. Energy balance, NEL milk, and NE
maintenance as a fraction of energy intake were not affected by treatment. Simple
efﬁciency calculated as NEL milk as a fraction of DE intake tended to decrease with
increasing UNS FA (P = 0.10). But NEL for production (tissue gain plus milk) as a
fraction of DE intake was not changed by treatment.

Fatty acids may change energy balance through modiﬁcation of multiple

physiological processes that affect intake, digestibility, metabolic efﬁciency, or

78

production, complicating prediction of energy balance. Treatment changed nutrient
partitioning; UNS decreased milk and milk fat yield compared to CON and SAT, and
increasing UNS linearly increased BW gain resulting in no difference in energy output.
Tyrrell and Moe (1972) observed decreased efﬁciency of ME utilization for milk
synthesis during milk fat depression, consistent with the tendency for decreased
efﬁciency of converting DE to NEL milk, because of increased energy use for tissue gain.
Milk production and BW gain are both homeorhetically controlled. Feed intake is
normally expected to decrease with increasing BW gain through Chemostatic regulation.
Increased BW gain may be physiologically directed to regain body condition lost in early
lactation, and increased BW gain may not feedback on energy intake depending on
metabolic state. Milk fat depression induced by biohydrogenation intermediates
normally does not result in decreased intake (Baumgard et al., 2002a). Increased tissue
energy gain as a result of decreased milk energy output, instead of decreased energy
intake, is unexpected because it demonstrates a disconnect of production and intake
leading to an imbalance in energy homeostasis (Chapter 6). Although it is possible that
biohydrogenation intermediates that induce milk fat depression may also increase BW
gain in dairy cows, this is opposite of the effect of CLA in monogastrics (Mersmann,
2001). Milk fat depression increases availability of acetate that is either directed or
demanded by adipose tissue to increase tissue gain. Increased BW gain may represent
directed growth or it may represent disposal of a metabolite to correct an imbalance in
metabolites.

Metabolic control of the increased body weight gain is not well understood. Milk

fat depression of 25 to 50% resulted in no change in plasma glucose, insulin and leptin

79

concentration or insulin stimulated glucose clearance, but did result in 24 to 33% reduced
lipolytic response to an epinephrine challenge (Baumgard et al., 2002a). Gaynor et al.
(1996) observed no effect of abomasal infusion of cis or trans-C18:1 on disappearance
rates of glucose, insulin secretion following a glucose challenge, and appearance rates of
NEFA and triglycerides alter a norepinephrine challenge. In the current study, SAT
increased plasma insulin concentration compared to CON, but UNS had no effect.
Furthermore, increasing SAT linearly increased insulin concentration. We have
previously reported increased plasma insulin with saturated FA compared to calcium salts
of palm oil (Chapter 2), consistent with in vitro insulinotropic effects of saturated FA
(Stein et al., 1997). There was no difference among treatments for plasma glucagon, and
B-HBA was linearly decreased with increasing UNS concentration presumably because
of lower FA intake. A period by treatment interaction was observed for plasma glucose
and NEFA concentration so treatment effects cannot be determined. Romo et al. (1996)
measured energy metabolism during cis and trans-C18:1 FA infusion, observing
increased production of milk energy with cis-C18:1 FA, but failed to detect differences
in energy expenditure or tissue retention. Increased body weight gain and little change in
intake during milk fat depression represents a failure in energy balance regulation that
cannot be attributed to homeostatic signaling or regulation of lipid and glucose
metabolism.
Nan-cannulated cows

Observation of nutrient intake, ruminal digestion kinetics and site of digestion of
cannulated cows is presented in companion papers (Chapter 4-6). Non-cannulated cow

DM and OM intakes were not affected by treatment. Intakes of NDF and starch were

80

decreased and intake of total FA was increased by addition of RPF. Greater NDF and
lower FA intake for CON compared to RPF was because rice hulls were included in place
of FA for the CON diet. Addition of RPF increased total tract digestibility of NDF and
pdNDF, and tended to increase OM digestibility (Table 9). Increasing UNS tended to
quadratically increase total tract NDF digestibility and tended to quadratically decrease
total tract starch digestibility. Changes in nutrient digestibility with UNS tended to
increase the amount of NDF and pdNDF digested in the total tract with a quadratic
response across RPF treatments.

Rumen-protected FA did not increase DE or NE intake in non—cannulated cows,
and increasing UNS tended to linearly decrease DE and NEL intake (Table 10). There
were no effects of treatment on NEL milk production, BW gain, or NEL tissue gain,
although RPF tended to increase BCS. Observing BW as opposed to rumen empty BW
increases error and bias due to variation and treatment effect on rumen digesta weight.
Although BCS tended to increase, calculated energy balance for cows decreased with
RPF. Rumen-protected FA also increased milk energy and maintenance energy as a
percent of energy intake. Treatment did not change efﬁciency of milk production or
energy utilization. Large differences in energy balance are not expected because plasma
insulin, glucagon, glucose, and B-HBA were not affected by treatment.

Cannulated and non-cannulated cows responded differently to treatment. Milk fat
concentration was signiﬁcantly depressed by UNS for cannulated cows but only tended to
be decreased by UNS for non-cannulated cows. Less signiﬁcant milk fat depression and
milk CLA concentration indicates less duodenal trans-FA ﬂow or decreased sensitivity to

trans-FA isomers in the mammary gland. Duodenal trans-FA ﬂow may be decreased by

81

more effective protection of PUFA or more complete biohydrogenation of trans-F A.
Fatty acid protection and biohydrogenation may differ between the blocks because
differences in intake or passage rate. Non-cannulated cows were had much more adipose
tissue (1.2 BCS greater) and had lower milk yield (5.7 kg) and were expected to be in a
different metabolic state. Metabolic state may interact with metabolic and physiologic

response to FA biohydrogenation intermediates.

CONCLUSION
Increasing unsaturated FA treatment decreased milk fat yield and intake of
digestible energy. Milk fat depression was consistent with the biohydrogenation theory
of milk fat depression with decreased milk fat concentration associated with increased
trans-10, cis-12 conjugated linoleic acid and lower concentrations of short and medium
chain fatty acids. Cows experiencing milk fat depression increased body weight gain.
Increased body weight gain may be because of the type of fuels available and incomplete

intake compensation to maintain energy homeostasis.

82

Table 1. Parameter means1 for cannulated and non-cannulated cows used in the

 

 

experiment.

Parameter Cannulated Non-cannulated P2
Pretrial DIM 77 106 <0.0012
13w 614 677 <0.013
BCS 1.9 3.1 <0.0015
Milk 45.6 39.9 0.083
FCM 40.7 38.7 0.543

 

1 All means except pretrial DIM are the mean over the entire experiment.
2 Linear contrast of cannulated vs. non-cannulated

Table 2. Composition of treatment mixes].

 

 

 

 

Nutrient CON SAT UNS
% of DM

Ingredients
Ca Soaps FA - - 57.5
Prilled FA - 50.5 -
Rice Hulls 50.5 - 10.9
Limestone 16.7 16.7 -
Ground Corn 32.7 32.7 31.6

Nutrient
Total FA 1.6 58.5 43.4
Unsaturated FA 1.0 6.1 23.6

 

1 Treatments were CON- control with supplemental rumen-protected fatty acids (FA),
SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

83

Table 3. Ingredient and nutrient composition of experimental diets'.

 

 

CON SAT INT UNS
Ingredients ...... % of DM2 .......
Corn silage3 24.6 24.7 24.7 24.6
Alfalfa silage“ 12.6 12.6 12.6 12.6
Ground Corn 28.7 28.8 28.8 28.7
Whole Cottonseed 13.5 13.5 13.5 13.5
Protein mix5 10.5 10.6 10.5 10.5
Mineral vitamin mix° 4.3 4.3 4.3 4.3
CON Mix7 5.7 0.5 0.2 -
SAT Mix7 - 5.0 2.5 -
UNS Mix7 - - 2.9 5.7
Nutrient

DM 55.6 55.7 55.7 55.7
OM 926° 92.9b 931° 93.1“
Total FA 5.5d 8.3a 8.1b 7.8°
Unsaturated FA 3.6d 39° 4.4b 49°
Starch 30.8“ 30.3“b 30.5bc 307°
NDF 29.1a 27.3d 27.5° 27.7b
Indigestible NDF 11.2a 9.7b 10.0b 99°
Forage NDF 169° 17.0a 16.9bc 17.0b
CP 16.2a 16.1" 16.1b 16.1"
Rumen-undegraded CP8 5.1a 4.8d 5.1b 49°
% NDF from forage 57.4d 61.4’l 60.9b 60.5c
GE MCal/Kg 4.55b 472° 4.72' 4.71'

 

' Treatments were CON- control with supplemental rumen-protected fatty acids (FA), SAT- saturated fat
from prilled FA, INT- intermediate saturation as a mix of prilled FA and calcium soaps of LCFA, and
UNS- unsaturated fat fed as Ca soaps of LCFA.

2 Means with different superscripts differ by P<0.05

3 Corn silage contained 34.7% DM (as fed), and 43.4% NDF, 8.4% CP, 10.7% indigestible NDF, 24.1%
starch, and 4.8% ash on a DM basis.

4 Alfalfa silage contained 36.3% DM (as fed) and 48.1% NDF, 16.2% CP, 25.7% indigestible NDF, 2.6%
starch, and 9.7% ash on a DM basis.

5 Protein mix contained 74.1% soybean meal, 20.1% corn gluten meal, and 5.8% blood meal.

6 Mineral vitamin mix contained 12.7% sodium bicarbonate, 11.5% limestone, 5.5% salt, 2.2% trace
mineral premix, 2.0% urea, 2.0% dicalcium phosphate, 0.6% vitamin D, 0.48% vitamin A, 0.12% vitamin
E, and 62.9% dry ground earn as a carrier

7 Mix composition listed in Table 2

8Rumen-degraded protein estimated using values from NRC (2001).

84

Table 4. Effects of dietary rumen-protected fatty acids on milk production of cannulated
cows.

 

 

 

Treatment LS Meansl P 2
CON SAT INT UNS SE Tlt RPF L Q
Yield, kg/d
Milk 47.0 46.6 45.2 43.7 2.7 0.06 0.10 0.02 0.95
3.5% FCM2 43.8 42.3 40.2 37.0 2.7 0.01 0.01 0.01 0.71
Milk fat 1.45 1.37 1.26 1.10 0.10 0.00 0.01 <0.001 0.68
Milk protein 1.33 1.34 1.30 1.30 0.06 0.62 0.61 0.26 0.66
Milk lactose 2.33 2.27 2.20 2.14 0.14 0.10 0.07 0.08 0.95
Milk solids 4.11 4.06 3.94 3.86 0.22 0.19 0.14 0.11 0.85
Milk composition,
%
Fat 3.06 2.93 2.78 2.43 0.19 <0.001 0.01 <0.001 0.40
Protein 2.84 2.88 2.89 2.96 0.08 0.20 0.14 0.15 0.47
Lactose 4.95 4.9 4.83 4.83 0.05 0.00 <0.001 0.01 0.16
Solids 8.77 8.75 8.67 8.74 0.09 0.42 0.36 0.88 0.17
Milk Urea N,
ﬂg/dL 16.1 16.4 15.6 14.2 0.81 0.04 0.29 0.01 0.63

 

1 Treatments were CON- control with supplemental rumen—protected fatty acids (FA),
SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 Trt: treatment effect, RPF: effect of rumen-protected FA, L: linear effect of saturation,
and Q: quadratic effect of saturation.

3 3.5% fat-corrected milk.

85

Table 5. Effects of dietary rumen-protected fatty acids on milk production of non-

cannulated cows.

 

 

 

Treatment LS Meansl P 2
CON SAT INT UNS SE Trt RPF L Q

Yield, kg/d

Milk 39.4 39.8 39.5 40.8 2.1 0.54 0.46 0.38 0.37

3.5% FCM2 38.9 39.1 38.0 39.1 2.2 0.81 0.86 0.99 0.35

Milk fat 1.34 1.35 1.30 1.32 0.10 0.81 0.67 0.69 0.44

Milk protein 1.25 1.25 1.21 1.26 0.05 0.58 0.88 0.83 0.18

Milk lactose 1.91 1.91 1.85 1.95 0.10 0.61 0.93 0.56 0.23

Milk Solids 3.53 3.54 3.43 3.59 0.18 0.59 0.91 0.64 0.21
Milk composition, %

Fat 3.45 3.43 3.31 3.25 0.23 0.17 0.15 0.08 0.84

Protein 3.18 3.16 3.11 3.11 0.07 0.21 0.12 0.19 0.49

Lactose 4.82 4.79 4.75 4.79 0.06 0.1 1 0.07 0.93 0.08

Solids 8.95 8.90 8.80 8.85 0.11 0.01 0.01 0.25 0.05
Milk Urea N, mg/dL 16.7 16.8 16.8 16.5 0.61 0.92 0.95 0.56 0.72

 

I Treatments were CON- control with supplemental rumen—protected fatty acids (FA),

SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.
2 Trt: treatment effect, RPF: effect of rumen-protected FA, L: linear effect of saturation,

and Q: quadratic effect of saturation.

3 3.5% fat-corrected milk.

86

Table 6. Effects of dietary rumen-protected fat on milk fatty acid proﬁle for cannulated

 

 

 

 

COWS.
Treatment LS Meansl P 2
Fatty Acid CON SAT INT UNS SE Trt RPF L Q
g/ 100 g fatty acids

6:0 2.21 2.29 1.59 1.61 0.16 0.001 0.02 <0.001 0.05
8:0 1.33 1.28 0.89 0.83 0.09 <0.001 <0.001 <0.001 0.03
10:0 3.09 2.85 1.99 1.84 0.19 <0.001 <0.001 <0.001 0.01
12:0 3.43 3.15 2.35 2.26 0.18 <0.001 <0.001 <0.001 <0.01
14:0 1 1.0 10.7 8.9 8.6 0.36 <0.001 <0.001 <0.001 0.02
15:0 1.12 1.15 0.85 0.86 0.08 0.003 0.03 0.002 0.09
16:0 28.3 30.1 29.2 28.4 0.82 0.18 0.26 0.06 0.94
16:1, cis 1.53 1.62 1.62 1.72 0.18 0.63 0.33 0.47 0.76
17:0 0.67 0.74 0.59 0.51 0.02 <0.001 0.003 <0.001 0.06
18:0 10.8 10.9 11.0 9.15 0.65 0.02 0.41 0.006 0.13
18:1, trans 3.16 2.96 4.14 6.27 0.39 <0.001 0.004 <0.001 0.29
18: 1, cis-9 18.5 18.9 21.0 19.6 0.72 0.007 0.01 0.23 0.006
18:2, cis-9,cis-12 2.71 2.66 3.10 3.65 0.11 <0.001 0.002 <0.001 0.67
18:3 0.22 0.19 0.21 0.23 0.009 <0.001 0.30 <0.001 0.70
20:0 0.12 0.13 0.13 0.1 1 0.006 0.03 0.56 0.008 0.19
cis-9,tranS-11 CLA 0.01 0.02 0.03 0.05 0.005 <0.001 <0.001 <0.001 0.13
trans-10, cis-12 CLA 0.02 0.03 0.04 0.08 0.006 <0.001 <0.001 <0.001 0.006
Total

<C16 23.7 22.8 17.7 17.2 0.91 <0.001 <0.001 <0.001 0.003

C 18 36.3 36.2 40.3 40.1 1.16 0.004 0.02 0.003 0.07

Uns/Sat C18 0.70 0.70 0.73 0.77 0.01 <0.001 0.004 <0.001 0.52
Yield g/d

<C16 34.1 31.7 24.1 19.4 3.2 <0.001 <0.001 <0.001 0.48

C16 43.8 44.0 41.1 33.5 3.6 0.003 0.08 <0.001 0.38

>C16 54.8 51.7 56.2 46.3 4.3 0.05 0.27 0.11 <0.05

 

1 Treatments were CON- control with supplemental rumen-protected fatty acids (FA),

SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCF A.
2 Trt: treatment effect, RPF: effect of rumen-protected FA, L: linear effect of saturation,

and Q: quadratic effect of saturation.

87

Table 7. Effects of dietary rumen-protected fatty acids on milk fatty acid proﬁle for non-

cannulated cows.

 

Fatty Acid

6:0

8:0

10:0

12:0

14:0

15:0

16:0

16:1, cis

17:0

18:0

18:1, trans

18:1, cis-9

18:2, cis-9,ciS-12
18:3

20:0

cis-9,trans-ll CLA
trans-10, cis-12 CLA

Total
<C l 6
C18
Uns/Sat C18

Yield
<Cl6
C16
>C16

 

 

Treatment LS Meansl P 2
CON SAT INT UNS SE Trt RPF L Q
g/ 100 g fatty acids
2.35 2.05 1.69 1.89 0.22 0.08 0.03 0.52 0.19
1.46 1.25 1.03 1.10 0.13 0.01 0.004 0.23 0.18
3 .49 3.04 2.47 2.51 0.30 <0.001 <0.001 0.03 0.15
3.89 3.49 2.89 2.83 0.29 <0.001 <0.001 0.007 0.17
11.5 11.0 10.0 9.3 0.34 <0.001 <0.001 <0.001 0.49
1.21 1.27 1.05 0.88 0.10 <0.001 0.009 <0.001 0.62
29.2 30.4 29.9 28.8 0.69 0.02 0.28 0.004 0.53
1.49 1.45 1.41 1.38 0.14 0.59 0.27 0.43 0.97
0.70 0.80 0.65 0.46 0.03 <0.001 0.03 <0.001 0.51
10.5 10.9 10.9 10.0 0.47 0.06 0.78 0.02 0.14
2.61 2.88 3.43 4.52 0.60 <0.001 0.005 <0.001 0.44
17.4 18.0 19.4 19.2 0.82 0.02 0.01 0.07 0.18
2.72 2.58 2.85 3.69 0.12 <0.001 0.005 <0.001 0.02
0.21 0.17 0.21 0.24 0.009 <0.001 0.34 <0.001 0.76
0.1 1 0.14 0.13 0.12 0.006 0.001 0.001 0.005 0.37
<0.01 0.01 0.02 0.02 0.008 0.10 0.06 <0.10 0.70
0.01 0.02 0.02 0.03 0.01 <0.10 0.03 0.28 0.61
25.5 23.8 20.4 19.7 1.3 <0.001 <0.001 0.001 0.15
34.2 35.1 37.6 38.9 1.2 <0.001 <0.001 <0.001 0.41
0.69 0.69 0.71 0.74 0.01 <0.001 0.005 <0.001 0.51
34.3 32.4 27.0 27.0 3.2 0.002 0.003 0.01 0.13
41.6 43.6 41.0 40.3 3.3 0.36 0.99 <0.10 0.57
48.8 49.7 51.2 53.6 3.5 0.12 0.11 0.07 0.81

 

I Treatments were CON- control with supplemental rumen-protected fatty acids (FA),

SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCF A.
2 Trt: treatment effect, RPF: effect of rumen-protected FA, L: linear effect of saturation,
and Q: quadratic effect of saturation.

88

Table 8. Effects of dietary rumen-protected fatty acids on energy balance and efﬁciency
for cannulated cows.

 

Treatment LS Meansl P 2

 

CON SAT INT UNS SE Trt RPF L Q

 

Intake

DE5,Mca1/d 78.0 75.3 74.0 70.1 3.1 0.07 0.04 0.06 0.59
NEL",Mcal/d 42.3 41.7 41.3 39.2 1.8 0.38 0.32 0.20 0.61
Production

MilkNEL5,Mcal/d 29.8 28.6 27.5 25.8 1.67 <0.01 <0.01 <0.01 0.68

Empty BWchange,kg/d 0.21 0.11 0.49 0.94 0.24 0.08 0.28 0.02 0.89
NEL6 Empty BW gain,/d 1.06 0.43 2.14 4.16 1.03 0.07 0.32 0.02 0.90

BCS change, /21d 0.05 0.04 0.13 0.08 0.04 0.42 0.51 0.43 0.17
Balance
N15L balance’, Meal/d -55 -5.6 4.2 47 1.4 0.85 0.68 0.63 0.52
Milk NEL, %Nl~:L intake 43.0 45.3 43.8 46.8 2.0 0.29 0.20 0.48 0.21
NEM,,,,, %NF.L intake 69.8 68.9 66.8 65.4 2.9 0.61 0.37 0.34 0.91
Efficiency
NF:L Milk/DE Intake 0.40 0.41 0.38 0.38 0.02 0.31 0.54 <0.10 0.53
NE], Prod / DE Intake 0.42 0.42 0.41 0.44 0.02 0.86 0.70 0.69 0.53

 

’ Treatments were CON- control with supplemental rumen-protected fatty acids (FA),
SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 Trt: treatment effect, RPF : effect of rumen-protected FA, L: linear effect of saturation,
and Q: quadratic effect of saturation.

2 Digestible energy intake as reported (Chapter 5)

4 NEW...) = DMI (kg) x (0.0245 x TDN(%)) (NRC 1989).

5 NEumilk) (Meal/d) = MY (kg) x (0.0929 x fat% + 0.0563 x true protein% + 0.0395 x
lactose%) (NRC, 2001).

6 NEuempty body weight change) calculated according to NRC 2001

7 NEMintake) - NEL(maintenance)“ NEL(milk), Where NEL(maintenance) = 0-080 X BWOJS (NRC
2001

2 NE, Milk Yield + NEL Body Weight Gain) / DE Intake

89

Table 9. Effects of dietary rumen-protected fatty acids an intake and total tract digestion
for non-cannulated cows.

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE Trt RPF L Q
Intake, kg/d
DM 26.7 26.3 25.4 25.4 0.97 0.26 0.12 0.27 0.54
OM 24.7 24.4 23.6 23.6 0.91 0.35 0.18 0.30 0.54
NDF 7.7 7.2 7.1 7.1 0.24 0.02 0.003 0.52 0.68
pdNDF 4.7 4.7 4.5 4.6 0.16 0.62 0.46 0.50 0.39
Starch 8.4 7.8 7.7 7.8 0.33 0.08 0.01 0.88 0.53
Total FA 1.4 2.2 2.0 2.0 0.07 <0.001 <0.001 0.004 0.54
C16 FA 0.28 0.51 0.48 0.46 0.02 <0.001 <0.001 0.01 0.71
C18 FA 0.99 1.40 1.31 1.28 0.05 <0.001 <0.001 0.06 0.004
TT Digested, kg/d
OM 16.5 17.1 16.3 16.0 0.68 0.31 0.97 0.08 0.61
NDF 3.2 3.5 3.3 3.5 0.15 0.02 0.01 0.078 0.05
pdNDF 3.2 3.5 3.3 3.5 0.15 0.02 0.01 0.78 0.05
Starch 7.8 7.4 7.3 7.2 0.30 0.09 0.02 0.43 0.76
Total FA 1.0 1.6 1.4 1.4 0.06 <0.001 <0.001 0.09 0.35
C16 FA 0.21 0.39 0.36 0.35 0.01 <0.001 <0.001 0.03 0.37
C18 FA 0.71 0.98 0.92 0.94 0.04 <0.001 <0.001 0.38 0.34
TT Digested, % Intake
OM 66.6 70.1 69.0 68.1 1.1 0.21 0.08 0.23 0.95
NDF 42.0 48.7 46.9 50.3 1.2 <0.001 <0.001 0.31 0.07
pdNDF 68.8 75.0 73.3 77.4 1.8 0.01 0.003 0.32 0.16
Starch 93.6 94.6 94.7 92.9 0.39 0.007 0.27 0.003 0.06
Total FA 71.3 70.8 70.6 73.7 1.3 0.29 0.80 0.11 0.29
C16 FA 75.7 76.0 74.5 76.4 1.1 0.54 0.99 0.77 0.16
C18 FA 71.6 69.7 70.3 73.9 1.4 0.15 0.86 0.04 0.35

 

’ Treatments were CON- control with supplemental rumen-protected fatty acids (FA),
SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCF A, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 Trt: treatment effect, RPF: effect of rumen-protected FA, L: linear effect of saturation,
and Q: quadratic effect of saturation.

90

Table 10. Effects of dietary rumen-protected fatty acids on energy balance and efﬁciency
for non-cannulated cows.

 

 

 

Treatment LS MeansI P 2
CON SAT INT UNS SE Trt RPF L Q

Intake

DE2, Meal/d 77.4 83.8 78.9 78.2 3.4 0.17 0.25 0.07 0.44

NEL", Meal/d 42.2 47.5 44.4 43.8 2.0 0.07 0.06 0.06 0.45
Production

Milk NELS, Meal/d 26.8 26.9 26.0 26.9 1.4 0.70 0.76 0.99 0.26

Empty BW change, kg / d 1.6 0.6 2.0 1.6 0.7 0.46 0.58 0.83 0.14

NEL‘5 BW change, / d 11.8 4.5 15.3 6.3 4.9 0.41 0.59 0.80 0.12

BCS change, /21d 0.05 0.14 0.17 0.17 0.05 0.27 0.06 0.63 0.78
Balance

NEL balance7, Meal/d 3.52 1.76 -0.50 -1.53 1.6 0.07 0.04 0.09 0.72

Milk NEL, %NEL intake 64.2 57.3 58.8 61.4 2.7 0.10 0.04 0.15 0.82

NEM,,,,,, %NEL intake 45.2 40.4 42.0 42.4 2.0 0.12 0.03 0.30 0.73
Efficiency

NEL Milk / DE Intake 0.35 0.32 0.33 0.34 0.01 0.27 0.17 0.16 0.73
NELProd / DE Intake 0.50 0.38 0.52 0.43 0.06 0.39 0.43 0.59 0.15

 

' Treatments were CON- control with supplemental rumen-protected fatty acids (FA),

SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCF A, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 Trt: treatment effect, RPF: effect of rumen-protected FA, L: linear effect of saturation,
and Q: quadratic effect of saturation.

2 Digestible energy intake as reported (Chapter 5)

4 NEuinmke) = calculated from DE through ME according to NRC (2001)

5 NEW.) (Meal/d) = MY (kg) x (0.0929 x fat% + 0.0563 x true protein% + 0.0395 x
lactose%) (NRC, 2001).

6 NEuempty body weigh, change) calculated according to NRC 2001

7 NEUbalance) " I\IEMmaintenance) - NEL(milk), Where NEL(maintenance) : 0-080 X BWOJS (NRC
2001)

8(N13, Milk Yield + NEL Body Weight Gain) / DE Intake

91

Table 11. Effects of dietary rumen protected fatty acids on plasma metabolites and
hormones.

 

 

 

Treatment LS Meansl P2
CON SAT INT UNS s13 Trt RPF L Q

Cannulated Cows

1nsu1in5, pIU/ml 6.9 8.3 6.8 5.8 0.86 0.04 0.92 0.006 0.78

Glucagon, pg/ml 114.3 100.8 109.5 107.8 7.1 0.30 0.17 0.31 0.38

Glucose, 4'5 mg/dl 58.2 57.6 58.3 58.3 0.95 -5 — — -

NEFA, 5'5 mM 74.7 64.1 76.0 68.0 3.0 - - - -

B-HBA, 5 (mg/d1) 4.9 5.1 4.7 4.5 0.22 0.19 0.58 0.04 0.79
Non-cannulated cows

lnsulin2, ulU/ml 9.2 8.6 9.0 9.2 1.2 0.95 0.78 0.61 0.93

Glucagon, pg/ml 124.4 125.6 130.6 128.7 9.9 0.85 0.54 0.69 0.62

Glucose, 4 mg/dl 56.0 57.7 57.0 57.7 1.2 0.49 0.16 0.98 0.53

NEFA, 5'5 mM 62.6 60.0 62.9 59.7 4.6 - - - -

B-HBA, 5 (mg/d1) 5.4 4.8 5.0 5.1 0.25 0.34 0.12 0.39 0.85

 

' Treatments were CON- control with supplemental rumen-protected fatty acids (FA),
SAT- saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 Trt: treatment effect, RPF: effect of rumen-protected FA, L: linear effect of saturation,
and Q: quadratic effect of saturation.

2 Measured for a composite of 8 samples collected over 3 (1, representing 3-h intervals of
a 24-h day.

4 Calculated using 8 samples collected over 3 (1, representing 3-h intervals of a 24-h day.
5 Signiﬁcant Period by Treatment interaction.

92

CHAPTER 4

Kinetic model of rumen biohydrogenation: effects of rumen-protected fatty acid
saturation on fractional rate of biohydrogenation and duodenal fatty acid flow in

lactating dairy cows.

ABSTRACT

Saturated and unsaturated rumen protected fatty acid sources were evaluated for effects
on fractional rate and extent of rumen biohydrogenation and duodenal fatty acid (FA)
ﬂow. Eight ruminally and duodenally cannulated multiparous Holstein cows (77i 12
DIM, meant SD) were used in a replicated 4x4 Latin square design with 21 d periods.
Treatments were control and a linear titration of 2.5% added rumen-protected FA (RPF)
varying in saturation: saturated (SAT; prilled hydrogenated free FA), intermediate mix of
SAT and unsaturated (UNS; calcium soaps of long-chain FA), and UNS FA. A simple
model of rumen FA metabolism is proposed that allows calculation of ﬁrst order
fractional rate of FA biohydrogenation and FA passage after determination of ruminal FA
pool Size and duodenal ﬂux. Rumen protected FA increased rumen FA turnover rate.
Passage rates of C16:0, C18:0 and total C18 were linearly decreased and trans-C18:1
fractional passage rate was quadratically affected with increasing UNS. Increasing UNS
increased extent of C18:2 and C18:3 biohydrogenation, and decreased extent of 18:1 and
trans-18:1 biohydrogenation. Calcium salts failed to protect polyunsaturated FA from

rumen biohydrogenation despite a mean ruminal pH of 6.0, and UNS decreased rumen

93

biohydrogenation of trans-C18:1 leading to increased duodenal ﬂow. This model allows
a mechanistic description of rumen biohydrogenation and determination of extent of

C18:1 biohydrogenation.

INTRODUCTION

Dietary fat serves a number of functions in lactating dairy cows including
substrate for energy, components for cellular structure, and second messengers. Some
fatty acids (FA) are biologically active with the ability to modify reproductive efﬁciency
(Staples et al., 1998), milk fat synthesis (Bauman and Griinari, 2003), and metabolism
(Drackley, 2000). Prilled, hydrogenated free fatty acids (FA) and calcium salts of FA are
two commercially developed products marketed to minimize effects of fat on ruminal
fermentation. Fatty acids bound to calcium ions are unavailable for bacterial uptake, but
FA become available by dissociation of the calcium ion. Dissociation of the calcium ion
is affected by pH and the binding afﬁnity of the FA, with greater dissociation at lower
pH, especially with more highly unsaturated FA (Sukhija and Palmquist, 1990).

Unsaturated FA available for uptake are partially biohydrogenated in the rumen
leading to the production of trans-FA isomers and saturated FA (Harfoot and Hazlewood,
1988). Increasing FA saturation decreases the toxic effects of unsaturated FA on
bacteria. Harfoot (1982) proposed that the goal of biohydrogenation by ruminal microbes
is the production of saturated FA for incorporation into their plasma membranes. The
high lipolytic capacity of ruminal microbes increases the pool of unsaturated free FA; if

bacteria did not hydrolyze esteriﬁed FA there would be very low levels of available FA

94

in the rumen. Recognizing this goal of biohydrogenation may provide insight into
biohydrogenation capacity and control.

Allen (2000) proposed that the extent of biohydrogenation is determined by the
characteristics of the fat source, retention time in the rumen, and characteristics of the
microbial population. Using Simple enzyme kinetic theory, total biohydrogenation is
determined by pool Size of available FA, rumen retention time, and bacterial
hydrogenation capacity that is a function of bacteria concentration, microbial population,
and rumen environment. Microbial biohydrogenation is a multistep process for which the
kinetics are not well documented. Beam et al. (2000) presented a schematic of lipid
metabolism in the rumen that included lipolysis, isomerization and hydrogenation,
resulting in formation of saturated FA. Hydrogenation of linolenic and linoleic acid
results in the formation of trans monounsaturated FA after the formation of trans-diene
intermediates that are rapidly metabolized (Harfoot and Hazelwood, 1988).
Biohydrogenation of oleic acid also includes formation a number of trans-C18:1
intermediates (Mosley et al. 2002). Trans-C18:1 can be hydrogenated to stearic acid or
passed to the duodenum. Numerous in vitro studies have demonstrated efﬁcient
biohydrogenation of C18:2 and C18:3, but have noted decreased capacity for trans-C18:1
biohydrogenation, especially with increased polyunsaturated FA (PUF A; Beam et al.
2000), decreased pH (Martin and Jenkins, 2002) and decreased dietary ﬁber (Harfoot and
Hazlewood, 1988).

Fatty acid proﬁle reaching the duodenum is determined by FA metabolism in the
rumen. The proﬁle of absorbed FA can be altered to maximize animal efﬁciency and

increase value of animal products by manipulating FA metabolism in the rumen if the

95

kinetics of rumen FA metabolism are understood. The objective of this experiment was
to determine effects of protected FA differing in FA saturation on ruminal
biohydrogenation and duodenal FA ﬂow and more mechanistically describe the process

of biohydrogenation.

MATERIALS AND METHODS

This is the second of four papers in a series from one experiment that evaluated
effects of rumen-protected fat sources differing in fat saturation. This paper discusses
treatment effects on rumen FA biohydrogenation and duodenal ﬂow, and the companion
papers focus on milk yield, milk FA proﬁle and energy balance (Chapter 3), ruminal and
post-ruminal nutrient digestion (Chapter 5), and intake, and feeding and chewing
behavior (Chapter 6). Experimental procedures were approved by the All University
Committee on Animal Use and Care at Michigan State University.
Cows and Treatments

Eight early lactation (77: 8.7 DIM; mean :1: SD) ruminally and duodenally
cannulated multiparous Holstein cows from the Michigan State University Dairy Cattle
Teaching and Research Center were assigned randomly to replicated 4 x 4 Latin squares
in a dose-response arrangement of treatments plus a control. Treatments were diets
containing no added protected fat or 2.5% rumen protected FA (RPF) from saturated
(SAT- prilled hydrogenated free FA, Energy Booster 1002, Milk Specialties Company
Inc.), an intermediate mixture of saturated and unsaturated (INT), or unsaturated FA
(UNS- Ca Soaps of LCF A, Megalac-R®, Church and Dwight Company, Inc., Princeton,

NJ). Treatment periods were 21 d with the ﬁnal 11 (1 used for sample and data collection.

96

Cows were ruminally and duodenally cannulated prior to calving and assigned randomly
to treatment sequence. Surgery was performed at the Department of Large Animal
Clinical Science, College of Veterinary Medicine, Michigan State University.
Immediately prior to initiation of the experiment, empty BW (ruminal digesta removed)
was 516 :1: 33 kg (mean 2 SD).

Treatment mix and diet composition are reported in Chapter 3. Experimental
diets contained 40% forage (66:33 corn silage: alfalfa silage), 13.5% whole cottonseed,
dry ground corn, premixed protein supplement (soybean meal, corn gluten meal, and
blood meal), a mineral and vitamin mix, and 2.5% added rice hulls (CON), saturated FA
(SAT), 50:50 mix of saturated and unsaturated fat (INT) or unsaturated FA (UNS). All

diets were fed as a total mixed ration.

Data and Sample Collection

Throughout the experiment, cows were housed in tie-stalls and fed once daily
(0900 h) at 115% of expected intake. Amounts of feed offered and orts were weighed for
each cow daily. Samples of all diet ingredients (0.5 kg) and arts from each cow (12.5%)
were collected daily on d 11 to 14 and combined into one sample to represent four days
for digestibility determination ((1 11-14). Indigestible NDF was used as a marker to
calculate duodenal ﬂow for the cannulated cows. Duodenal samples (1,000 g) were
collected every 9 h from d 12 to d 14 yielding eight samples representing every 3 h of a
24-hour period to account for diurnal variation.

Ruminal contents were evacuated manually through the ruminal cannula at 1350 h

(4.5 h after feeding) on d 20, and at 0700 h (2 h before feeding) on d 21 of each period.

97

Total ruminal content mass and volume were determined. During evacuation, 10%
aliquots of digesta were separated to allow accurate sampling. Aliquots were squeezed
through a nylon screen (1 mm pore size) to separate into primarily solid and liquid

phases. All samples were frozen immediately at -—20°C.

Sample and Statistical Analysis

Forages and arts were ground with dry ice in a Wiley® mill (6mm screen; Authur
H. Thomas, Philadelphia, PA), sub-sampled and lyophilized (Tri-Philizer’“ MP, F TS
Systems, Stone Ridge, NY) for analysis of DM concentration. Dried forage, arts, and
RPF were reground in a Wiley® mill with a 1mm screen, dry ice was ground with the
RPF to prevent fat from melting in the grinder. Concentrates were ground in a cyclone
mill with a 2 mm screen (Udy Mill, Seedburo Equipment Co, Chicago, IL). Rumen
liquid and solid subsamples were lyophilized, ground in a Wiley® mill with a 1mm
screen, and recombined according to the original ratio of solid and liquid DM. Duodenal
samples were thawed, combined, and ﬁltered into primarily solid and liquid phases using
nylon mesh (1 mm pore size) to minimize sampling errors due to segregation of samples
into solid and liquid phases. Both phases were weighed, and sub-samples were taken
from each phase. Liquid and solid sub-samples were lyophilized, ground in a Wiley®
mill with a 1mm screen, and recombined by weight according to the original ratio of solid
and liquid DM. A portion of all samples was placed in a Whirl Pac bag (NASCO, Fort
Atkinson, WI) ﬂushed with nitrogen gas and frozen for FA analysis.
Concentrations of all nutrients except DM were expressed as percentages of DM

determined by drying at 105° C in a forced-air oven for more than 8 h. Indigestible NDF

98

was estimated as NDF residue after 240-h in vitro fermentation (Goering and Van Soest,
1970). Rumen ﬂuid for the in vitro incubations was collected from a non-pregnant dry
cow fed only alfalfa hay. Feeds and rumen and duodenal digesta FA were extracted
according to Sukhija and Palmquist (1988). Ruminal pool sizes (kg) of nutrients were
determined by multiplying the concentration of each component by the ruminal digesta
DM mass (kg).

A simple model was developed to calculate biohydrogenation rate of unsaturated
FA in the rumen (Figure 1). The model assumes that unsaturated FA are not oxidized,
but are hydrogenated and appear in a less saturated pool, pool sizes are representative of
steady state conditions and biohydrogenation follows ﬁrst-order kinetics. Ruminal
turnover, fractional passage rate (kp) and fractional biohydrogenation rate (k1,) for each

FA (FAi) pool were calculated using the following equations:

Turnover rate in the rumen (% h'l) =

(FA; ﬂow into pool from intake and biohydrogenation, g/h / Ruminal FA, pool, g)
x 100
Fractional passage rate from the rumen (% h") =

(Duodenal FA, ﬂow, g/h / Ruminal FA; pool, g) x 100

Fractional disappearance rate from the rumen (% h'l) =

(Rumen FA, turnover rate, g/h/ Rumen FA, Pool, g) x 100

Fractional biohydrogenation rate from the rumen ((% h")=

99

Fractional FA, disappearance rate (% h'l) - Fractional FA; passage rate (% h'l)

Extent of biohydrogenation (% biohydrogenated) =
= Fractional FA, biohydrogenation rate (% h'l) / [Fractional F A, biohydrogenation

rate (% h'1)+ Fractional FA, passage rate (% h“)]

Total biohydrogenation index (Proportion of double bonds, %) was calculated according
to Tice et al. (1994) =
100 — [100 * (Duodenal ((C18:1 + (C18:2 * 2) + (C18:3 * 3)) / Total C18)/

Intake (C18:1 + (C18:2 * 2) + (C18:3 * 3)) / Total C18))]

C18:3 biohydrogenation index (Proportion of C18:3, %) =

100 — [100 * (Duodenal (C18:3 /Total C18) /Intake (C18:3 / Total C18))]

C18:2 biohydrogenation index (Proportion of C18:2, %) =

100 — [100 * (Duodenal (C18:2 / Total C18) / Intake (C18:2 / Total C18))]

All data were analyzed using the ﬁt model procedure of JMP“ (Version 5, SAS
Institute, Cary, NC) according to the following model:

Yijk=lk+Ci+Pj+Tk+eijk
where

u = overall mean,

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

100

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

eijk = residual error.

Period by treatment interaction was evaluated, but was removed from the
statistical model when not declared signiﬁcant (P > 0.10). Period by treatment was not
signiﬁcant for any variable of primary interest; variables with signiﬁcant interactions are
noted in the tables. Data points with Studentized Residuals greater than 3.0 were
considered outliers and excluded from analysis. Very few observations were excluded
and rarely more than one per response variable. Preplanned contrasts include the effect
of addition of RPF (CON vs. SAT, INT and UNS), linear effect of increasing
concentration of unsaturated fat [L (SAT vs. UNS)], and quadratic effect of increasing
concentration of unsaturated fat [Q (INT vs. SAT and UNS)]. The preplanned contrasts
do not allow individual comparison of each fat treatment to the control. Protected LSD
was used for mean separation when treatment was signiﬁcant. Pearson correlation
coefﬁcients were determined between cow-period observations for some parameters.
Treatment effects, linear and quadratic responses, and correlations were declared
signiﬁcant at P < 0.05, and tendencies were declared at P < 0.10.

One cow developed clinical mastitis on d 19 of period 3, and her rumen was not
evacuated. Data collected in this period before d19 for this cows was included in
statistical analysis. The cow did not fully recover and data from period 4 was not

included in the dataset.

101

RESULTS AND DISCUSSION
Fatty Acid Intake and Duodenal Flow

Fatty acid intake and duodenal ﬂow are reported in Table 1. Fatty acid intake is a
function of dietary FA concentration and daily DMI. Rumen protected FA treatment
increased total FA intake. Increasing UNS linearly decreased total FA intake primarily
because of a large decrease in DMI (Chapter 5) but also from slightly lower FA
concentration. Saturated FA increased intake of C16:0 and C18:0, while UNS increased
intake ofcis-18:1, 18:2 and 18:3.

Dietary FA are biohydrogenated by bacteria in the rumen, and animal response is
highly dependent on the resulting FA proﬁle reaching the duodenum. Dietary treatments
were selected to maximize the difference in duodenal unsaturated FA ﬂow, especially
polyunsaturated FA (PUFA), using commercially available products. Saturated FA
treatment increased FA ﬂow by increasing dietary FA density with little effect on DMI,
while UNS failed to increase duodenal FA ﬂow compared to control because DM intake
was depressed. Within RPF, increasing UNS linearly decreased duodenal FA ﬂow.
Duodenal ﬂow of C16:0 and C18:0 was increased by SAT compared to CON, but was
not affected by UNS. Increasing SAT linearly increased C18:0 ﬂow, with SAT
delivering nearly twice the ﬂux of C18:0 to the duodenum as UNS. In contrast, duodenal
flow of cis-C 1 8:1 was not different between SAT and CON, but ﬂows were increased by
INT and UNS. Increasing UNS quadratically affected duodenal ﬂow oftrans-18:1 FA
and linearly increased cis-18:1 FA. Although treatments were expected to drastically

change duodenal PUFA ﬂow, treatment did not affect ﬂow of C18:2 FA reaching the

102

duodenum, and C18:3 FA was linearly decreased by UNS. UNS treatment failed to
increase duodenal ﬂow of C18:2 and C 18:3 FA because of intake depression and
incomplete protection of unsaturated FA. Addition of RPF increased duodenal ﬂow of
saturated FA for SAT treatment and monounsaturated FA for UNS treatment in this
experiment.

Rumen protected FA increased duodenal C 16:0 concentration, and UNS linearly
increased C 16:0. Increasing UNS linearly decreased duodenal C1820 concentration and
linearly increased cis- and trans-C18:1, C18:2 and C18:3 concentration. Using mean
separation, SAT did not change, but UNS increased trans-C18:1 and C18:2 concentration
relative to CON. Saturated FA decreased concentration ofC18z3, but UNS did not differ
from control.

Duodenal ﬂow of CLA isomers could not be detected with the analytical
procedures used. The FA extraction and methylation procedure of Sukhija and Palmquist
(1988) causes partial transformation of CLA with only 57.4 and 54.9% methylated
recovery of cis-9, trans-11 and trans-10, cis-12 CLA respectively (Duckett et al. 2002).
However, duodenal ﬂow of CLA is extremely low relative to total VFA; Piperova et al.
(2002) reported 1.0 to 1.8 g/d ﬂow of total CLA with 0.24 to 0.53 g/d ﬂow of trans-9, cis-
11 and 0.05 to 0.26 g/d ﬂow of trans-10,cis12 CLA. Similar CLA ﬂow results were
reported in steers (0.63 to 1.2 g/d CLA; Duckett et al., 2002), and in sheep (0.12 to 0.20
g/d cis-9,trans-ll CLA; Kucuk et al, 2001).

The large synthesis of cis-9,trans-11 in the mammary gland is a major contributor
to total milk CLA secretion (7.2-9.1 g/d CLA), making rumen contribution of total CLA

small compared to de nova synthesis (Piperova et al., 2002). However, some CLA

103

isomers are secreted in smaller amounts than duodenal ﬂow suggesting the importance of
rumen synthesis for some isomers.

Indigestible NDF was used as the ﬂow marker for calculation of duodenal ﬂow.
Chromic oxide was originally intended for use as an external marker but resulted in
unrealistically high duodenal ﬂow possibly because of inadequate subsampling
procedures for duodenal liquid. Indigestible NDF is prone to treatment bias as duodenal
FA might affect in vitro fermentation for determination of indigestible residue. To insure
that this was not the case, we also calculated duodenal and fecal ﬂow using acid
detergent-sulfuric acid lignin and 120 hr indigestible ADF after ether extraction. Results

were similar for all markers and iNDF was chosen for calculation of duodenal ﬂows.

Rumen Pool and Turnover

Rumen FA pool size is the mean of the pool size before and after feeding, and is
assumed to represent steady state pool Size (Table 3). Rumen protected FA increased
rumen pool Size of total and C16:0 FA. Saturated FA linearly increased C18:0 pool
relative to UNS, while UNS was not different from CON. The rumen trans-C18:1 pool
was linearly increased by UNS, but treatment did not affect cis-18:1, 18:2 or 18:3 pool
sizes. We were unable to ﬁnd previous research reporting FA pool sizes in the rumen.
However, Abughazaleh et al. (2002, 2003) reported 2.6-8.7% trans-11 C18:1, 0.09-0.26%
cis-9,trans-11 CLA, and up to 0.13 % trans-10,cis-1 l CLA as a percentage of total FA in
rumen grab samples. Fatty acid proﬁle of grab samples from the rumen may not
represent true FA proﬁle of rumen contents because FA may associate differently with

liquid and solid fractions of the rumen, biasing the sample. In the current study, trans-

104

C18:1 FA ranged from 5.4 to 8.4% of total FA, and CLA was not detected, possibly
because of limitations of our FA analysis method. Conjugated linoleic acid rumen pools
may be of minimal importance in discussion of rumen FA biohydrogenation, but trans-
C18:1 is an important pool for modeling ruminal biohydrogenation because of its much

greater size.

Kinetics of Biohydrogenation

Biohydrogenation is traditionally reported as the proportion of unsaturated FA or
double bonds removed in the rumen (Wu et al., 1991, and Tice et al. 1994). Although
this is an index of rumen metabolism it fails to mechanistically describe FA
biohydrogenation. Also, this method is inadequate for calculation of biohydrogenation of
monounsaturated FA because appearance from biohydrogenation of more PUFA is not
determined. Rate of biohydrogenation may be determined by in vitro batch and
continuous culture systems (Wu and Palmquist, 1991, and Beam et al. 2000), although
both may have limited application to normal rumen fermentation. In situ methods have
also been employed (Enjalbert et al. 2003), but have limited application because of FA
entrance and exit from the in situ bags.

Methods for modeling rumen carbohydrate digestion (Allen and Mertens, 1998)
can be applied to rumen FA metabolism. The pool and ﬂux method determines ﬁrst
order rumen passage and digestion rates using duodenal ﬂow and rumen pool size
(F irkins et al., 1998). Measuring FA pool Size and duodenal passage allows calculation
of rumen retention time and determination of the fractional rate of FA biohydrogenation,

assuming all disappearance of FA is because of biohydrogenation and not from oxidation

105

or absorption. The model also allows calculation of the extent of biohydrogenation of
individual FA calculated from passage and biohydrogenation rates.

Determination of fractional passage and biohydrogenation rates are complicated
by heterogeneous pools (ex. free in rumen, adsorbed to feed, associated with metal ion,
etc), and determination of ﬁrst order kinetics rely on the assumption that enzyme
concentration is not limiting. In addition, FA entry rate for some pools is from
biohydrogenation in addition to intake. Two versions of a Simpliﬁed model were
developed to calculate rumen FA biohydrogenation rates while accounting for appearance
of FA from biohydrogenation (Figure 1). Model A (Figure la) assumes
biohydrogenation of C18:3 and C18:2 directly to C18:1, and biohydrogenation of C18:1
to C18:0. Model B (Figure 1b) partitions C18:1 to cis- and trans-C18:1 FA pools and
assumes biohydrogenation of C18:3, C18:2 and cis-C18:1 to trans-18:1, and
biohydrogenation of trans-C18:1 to C18:0. The C18:3 and C18:2 pools are cis isomer
pools only, representing cis-9,cis-12 C18:2 and cis-9,cis-12,cis-15 C18:3, reference to
C18:3 and C18:2 simply refers to these FA in the remainder of the paper.

Harfoot and Hazlewood (1988) provide detailed description of FA
biohydrogenation, including the production of trans-diene intermediates during
biohydrogenation of C18:3 and C18:2. Ruminal pools of these intermediates were not
detectable in the current experiment, and others have reported low rumen concentration
of trans—diene FA (Aquhazeleh et al. 2002 and 2003). Biohydrogenation intermediates
have very high rates of biohydrogenation as indicated by high rates of C18:2 and C18:3

biohyrogenation compared to very small pool sizes of biohydrogenation intermediates.

106

Production of trans-diene intermediates and more complex pathways are recognized, but
were not expected to improve model utility.

Biohydrogenation of cis-C18:1 to C18:0 was traditionally believed to occur by
direct biohydrogenation without formation of intermediates (Harfoot and Hazlewood,
1988). However, increasing rumen available oleic acid has been shown to increase trans-
C18:1 FA concentration in rumen digesta (Aquhazaleh et al., 2003), concentration in
rumen digesta and duodenal ﬂow (Kalscheur et al. 1997) and concentration in milk
(DeLuca and Jenkins, 2000; Jenkins, 2000; Kalscheur et al. 1997, 2003; and
Aquhazaleh et al. 2003). Mosely et al. (2002) observed production of a number of
trans-C18:1 intermediates during in vitro biohydrogenation of cis-9 C18:1. In viva
observation of increased trans-C18:1 with cis-18:1 treatments may be from cis-C18:1
inhibition of biohydrogenation. Direct inhibition by cis-18:1 has not been explored, but
increasing C18:2 concentration decreases biohydrogenation in vitro (Beam et al. 2000).
In vitro production of trans-C18:1 from cis-C18:1 provides strong evidence for formation
of the trans-C18:1 intermediate. Direct formation of C18:0 from cis-C18:1 requires a
kinetic approach to verify as both paths result in the formation of C18:0. The presence of
the direct path and partitioning of biohydrogenation between the direct and indirect route
is not known and may vary with bacterial population and rumen environment. In
addition, Proell et al. (2002) observed 15% formation of cis-1 8:1 from labeled trans-9
18:1 in in vitro batch culture, although it appears to be a slow reaction resulting in only a
slight enrichment of the cis-18:1 pool aﬁer 48 h of incubation. In addition, trans-9 C18:1
is a very small pool in the rumen, which combined with the slow rate, limits appreciable

formation of cis-C1 8:1 in the rumen. Data concerning formation of cis-C18:1 from trans-

107

C18:1 FA present in the rumen in higher concentrations is not available. Reverse
reactions expected to result in very small ﬂuxes are expected to be of little consequence
because of dilution by the large forward ﬂux and are ignored in these simple models.
Therefore, model B assumes all biohydrogenated cis-18:1 enters the trans-18:1 pool.

The models assume that disappearance of FA is because of biohydrogenation and
not absorption or oxidation. Loss of FA from the rumen through absorption and
oxidation are often overlooked or considered minimal. Ruminal infusion of radioactive
labeled linoleic acid and diversion of duodenal ﬂow showed minimal plasma recovery of
label (Jenkins, 1993). However, ruminal loss of FA is commonly observed in digestion
studies; Jenkins (1993) reported FA loss in 15 of 47 published studies. Although this
could be attributed to ﬂow marker bias, Doreau and Chillard (1997) discussed evidence
supporting FA oxidation and absorption in the rumen, citing in vitro oxidative capacity of
rumen epithelium and possible oxygen transfer across the epithelium to bacteria capable
of oxidative metabolism. Rumen digestion or absorption of C18:3, C18:2 and cis-C18:1
will inﬂate the biohydrogenation rate of trans-C18:1 because of an overestimation of
inﬂux to the trans-C18:1 pool. Wu et al. (1991) proposed calculation of
biohydrogenation as a proportion of total C18 to correct for rumen loss of FA. The same
could be done for the kinetic models by setting C18 intake equal to duodenal C18 ﬂow by
calculating individual C18 FA intake as duodenal total C18 ﬂow multiplied by the C18
proportion of the FA fed. However, actual observed intake and duodenal ﬂow values
were used in model parameterization in the present study. Fractional rumen
disappearance of total and C16 FA was not changed by treatment, but disappearance rate

of C18 tended to increase with increasing UNS. Apparent rumen digestion of C16 FA

108

was increased with RPF, and increasing UNS RPF tended to increase total and C18 FA
(Chapter 5). Rumen turnover of total, C18, and C16 FA was increased by RPF (Table 3).
Dietary FA in the control treatment were fed as whole cottonseed that would be expected
to be retained in the rumen ﬁber mat increasing ruminal retention time. Rumen protected
FA fed in granular form with smaller particle size are expected to ﬂow from the rumen
faster than FA associated with cottonseed because they are less likely to be retained in the
ﬁbrous mat. Increasing UNS had no effect on rumen FA turnover.

Rumen protected FA did not affect fractional passage rate of total FA, C18:2 or
C 18:3 from the rumen (Table 4). Fractional passage rate of total C18 FA was linearly
increased by SAT, but was not different for UNS compared to CON. Increasing UNS
linearly decreased C16:0, C18:0 and total C18 fractional passage rate, did not affect
fractional passage rate of cis-C18: l, and affected fractional passage of trans-C1 8:1
quadratically with the highest value for INT. Differences in FA passage rates within RPF
may represent different FA pool types, association with different ﬁactions, or associative
effects on rumen passage of other nutrient pools; fractional passage rate of indigestible
NDF from the rumen increased linearly with increasing SAT (Chapter 5).

Fractional rate of biohydrogenation of individual FA in the rumen is an important
mechanistic measure as it reports FA biohydrogenation as a proportion of the available
FA. Increasing UNS had no effect on C18:2 biohydrogenation but linearly increased
fractional biohydrogenation rate of C18:3 (Table 4). Increased fractional rate of C18:3
biohydrogenation with UNS identiﬁed poor rumen protection by calcium salts. More
highly unsaturated FA are expected to have a higher pKa (Sukhija and Palmquist, 1990)

increasing dissociation of the calcium FA complex as pH decreases. However, ruminal

109

pH in this experiment was moderate and not affected by treatment with a mean of 6.0
(Chapter 6).

Addition of SAT did not affect rate of biohydrogenation of total C18:1, trans-
C18:1 and cis—C18:1 compared to CON. However, UNS linearly decreased rate of
biohydrogenation of C18:1 and trans-C18:1, and tended (P = 0.08) to increase rate of cis-
C18:1 biohydrogenation. A potential bias of increased rumen FA loss with UNS may
have increased the observed fractional rate of C18:1 biohydrogenation.
Biohydrogenation oftrans-18zl is considered the rate limiting step of rumen
biohydrogenation (Harfoot and Hazelwood 1988), allowing trans-C18:1 to accumulate in
the rumen. Biohydrogenation of trans-C18:1 cannot be determined by previous
measurements of rumen FA metabolism, but the proposed models allow determination of
C18:1 biohydrogenation. Increased duodenal ﬂow oftrans-C18:1 for UNS is from
greater intake of unsaturated FA (particularly cis-C18:1), increasing inﬂow to the trans-
C18:1 pool by biohydrogenation, and a decreased rate of biohydrogenation and increased

passage rate of trans-C18:1 from the rumen.

Extent of Biohydrogenation

Extent of biohydrogenation of C18:1 and more speciﬁcally trans-C18:1 was
linearly decreased by increasing concentration of unsaturated FA (Table 5). There was
no difference in extent of biohydrogenation ofcis-C18:1, but biohydrogenation of C18:2
and C18:3 were increased by increasing unsaturated FA. In agreement, biohydrogenation
index calculated according to Tice et al. (1994) shows a linear increase in the number of

double bonds destroyed with increasing UNS. Modiﬁcation of the index to calculate

110

biohydrogenation of individual PUFA showed that biohydrogenation of C18:3 tended to

increase linearly and C18:2 tended to be affected quadradically with increasing UNS.

Model Simpliﬁcations and Assumptions

Determination of ﬁrst order kinetics by the pool and ﬂux method relies on the
assumption of homogenous pools and that the entire pool is available for
biohydrogenation. This could cause errors in the parameters because not all FA are in the
same form, and FA form affects availability for bacterial uptake. Fatty acids that are
esteriﬁed, associated with a metal ion, or simply adsorbed to feed particle are not
available for bacterial uptake and subsequent biohydrogenation. In the current study, RPF
were non-esteriﬁed but differed in association with metal cations. A more realistic
representation of available FA pool is presented in Figure 2, and represents a submodel of
biohydrogenation for each individual FA. Each FA pool can be subdivided ﬁrst into
esteriﬁed and free FA, and secondly into available and unavailable pools within esteriﬁed
and unesteriﬁed pools. Esteriﬁed FA undergo lipolysis by bacteria to yield free FA,
however not all esteriﬁed FA are physically available to bacteria. The availability of
esteriﬁed FA depends on their location within plant cellular structure, seed coats, within
pellets, or adsorbed to particles. The lag for availability represents hydration and
breakdown of feed particle making esteriﬁed FA available for bacterial hydrolysis. Fatty
acids produced from hydrolysis are expected to enter the available free FA pool. Free or
unesteriﬁed FA also enter the rumen directly by feed intake. Unavailable free FA
represents FA adsorbed to or protected by feed particles and FA associated with metal

cations. Nutritionists have recognized that the addition of metal cations such as calcium

111

can partially alleviate FA inhibition of ﬁber digestion through formation of an insoluble
salt that blocks FA uptake by microbes (Palmquist and Jenkins, 1980). Increasing
saturation and chain length of the FA increases the amount and strength of salt formed
(Jenkins and Palmquist, 1982). The formation of the metal salts is determined by the
binding afﬁnity of the cation and the dissociation constant of the fatty acid. Fatty acid
binding to metal cations is partially dependent on pH of the rumen and the pK, of the FA.
Sukhija and Palmquist (1990) determined the pK, for calcium salts of stearate, tallow,
palm FA and soy oil to be 4.5, 4.5, 4.6 and 5.6 respectively. These pKa values are
somewhat misleading since they are determined for a mixture of FA. Soy oil contains a
much higher concentration of unsaturated FA than the other treatments, and demonstrates
the high pK, of unsaturated FA. Variation in rumen pH is expected to change the amount
of F A available for bacterial uptake and biohydrogenation as FA salts can be formed and
destroyed in the rumen. The pool size of FA available for biohydrogenation by the model
represented in Figure 2 is a function of the rate of esteriﬁed FA availability (ktag), the rate
of lipolysis (knp), rate of free FA availability (km), rate of complexing as a salt (kc), rate
of passage (Kp) and rate of biohydrogenation (kb). Although total esteriﬁed and free FA
pools can be determined and may represent a signiﬁcant improvement in representing
biohydrogenation, the available and unavailable pools of each fraction cannot be
realistically evaluated.

Differences in the fractional biohydrogenation rate calculated using the simpliﬁed
models (Figure 1) indicates that the pools are not homogeneous and differ in availability,

or that rates are affected by the microbial population and are not ﬁrst-order. Future

112

 

research Should resolve the relative inﬂuence of each on rate of FA biohydrogenation in

the rumen.

CONCLUSION

Calculation of individual FA biohydrogenation with a simpliﬁed model allows a
more mechanistic description of rumen FA biohydrogenation. The model described
increased duodenal trans-C18:1 FA ﬂow as the result of increased ruminal production of
trans-C18:1 and decreased trans-C18:1 biohydrogenation. Future ability to predict
duodenal FA proﬁle depends an observation and analysis of individual steps of FA
metabolism. Modeling biological systems requires assumptions and Simpliﬁcation Of
unknown of undeterminable events. Assumptions for this model must be tested in the
future. It is our hope that this model will be challenged and improved with new
experimental data. More complex models may be developed in the future to model
production of individual trans isomers, although ﬂux through such pathways may require

isotope labeling.

113

Figure 1

 

 

 

 

 

 

 

 

 

 

 

 

Panel A
FA Intake
C18:3 C18:2
kb‘l kb2
§ i g kp2
k“ C18:1 kbs C1820

 

 

 

 

 

 

 

k at

Duodenal Flow

 

 

 

 

Figure 1: Panel A. Simple model of rumen biohydrogenation for calculation of fractional

passage and biohydrogenation rates

114

 

 

Figure 1 (Cont)

Panel B FA Intake

 

 

 

 

 

01833 cis-C18:1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1...] [1.

Duodenal Flow

 

 

 

 

Figure 1. Simpliﬁed model of rumen biohydrogenation for calculation of fractional
passage and biohydrogenation rates. The model allows calculation of rumen FA
fractional biohydrogenation and passage rates while accounting for appearance of FA
from biohydrogenation. The simple model (Panel A) assumes biohydrogenation of C18:3
(kbl) and C18:2 (kbz) directly to C18:1, and biohydrogenation of C18:1 (kb3) to C1820.
The second model partitions C18:1 to cis- and trans-C18:1 FA pools. The more complex
model in Panel B assumes biohydrogenation of C18:3 (km), and C18:2 (khz) to trans-
C18:1, and isomerization ofcis-C18:1 (kg) to trans-C18:1. Finally, trans-C18:1 (km) is
biohydrogenated to C18:0. The C18:3 and C18:2 pools represent cis-9,ciS-12 C18:2 and

115

cis-9,ciS-12,ciS-15 C18:3 and do not contain trans isomers. Production of trans-diene
intermediates is ignored due to their small pools relative to their large ﬂux. Each FA
pool is available for passage and each passes at its own rate signiﬁed by different

subscript (kp1-5).

116

 

 

 

klag

 

 

 

 

 

  
 

 

 

 

 

 

 

 

 

 

 

 

 

f Unavailable I Available
9‘3 Esteriﬁed l Esteriﬁed
FA kli
Intake k‘” p
fnefa
\ Unavailable Available
FA FA
kp2 kb
Y
Duodenal Flow Transfomed

FA

 

 

Figure 2: Model describing fatty acids (FA) available for bacterial uptake in the rumen
recognizing heterozygous FA pools. Fatty acid intake is partitioned into an esteriﬁed
FA fraction (fcfa) and a non-esteriﬁed FA fraction (fnefa). Unavailable FA include FA in
cellular structures, adsorbed to feed particles, protected in seedcoats, and bound to metal
cations. A lag function (king) represents the rate that esteriﬁed FA become available for
bacterial lipolysis, and kup is the rate that available esteriﬁed FA are hydrolyzed to free
FA. Hydrolyzed FA enter the available FA pool. Unavailable FA become available by
breakdown of feed particle in digestion and dissociation of metal cations due to pH, and
available FA can become unavailable by complexing with a metal cation (kc). Available
FA are taken up by microbes and biohydrogenated, transforming them to different FA or
FA isomer. The rate of biohydrogenation is represented as k1,. All FA pools are

available for passage to the duodenum and pass at different rates signiﬁed by different

subscripts (kp1-4).

117

Table 1. Fatty acid composition of diets

 

CON SAT INT UNS
Fatty Acid ----------- % of Total FA2 -----------
C1420 0.5d 1.4° 1.0b 0.6°
C16:0 188° 22.6b 22.9ab 23.1b
C18:0 2.6° l7.6° 10.5b 3.0°
C18:1 trans-l 1 005° 040° 0.27b 012°
C18:1 cis-9 15.6b 120° 15.61) 194°
C18:1 cis-11 0.69b 060° 0.69b 0.78°
C18:2 466° 31.3d 351° 39.3b
C18:3 28° 1.8d 2.l° 2.3b
> C20 1.4° 1.3b 1.3° 1.2d
Total FA, % DM 5.5d 83° 8.1b 7.8°
Unsaturated FA, % DM 3.6d 39° 4.4b 49°
C16:C18 ratio 0.285 036° 036° 036°

 

1 Treatments were CON- control with no supplemental rumen-protected fatty acids (FA),
SAT- saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCF A.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.

2 Means with different superscripts differ by P<0.05

118

Table 2. Effects of rumen-protected fatty acids varying in saturation on FA intake,
duodenal ﬂow, and biohydrogenation.

 

 

Treatment LS Means’ P2
CON SAT INT UNS SE TRT RPF L Q
Intake, g/d
Total FA 1500 2100 2000 1800 70 <0.001 <0.001 <0.001 0.68
C16:0 280 490 460 430 20 <0.001 <0.001 0.003 0.97
C1810 40 380 210 50 10 <0.001 <0.001 <0.001 0.19
C18:1 trans 1.2 12 6.9 2.7 0.4 <0.001 <0.001 <0.001 0.39
C18:1 cis 240 270 320 360 12 <0.001 <0.001 <0.001 0.21
C18:2 690 670 700 710 30 0.09 0.77 0.02 0.28
C18:3 41 40 44 47 1 <0.001 0.04 <0.001 0.50
Duodenal Flow, g/d
Total FA 1400 2000 1900 1500 120 <0.001 0.003 <0.001 0.25
C16:0 290 440 430 350 30 <0.001 <0.001 0.007 0.29
C18:0 580 880 680 470 53 <0.001 0.07 <0.001 0.89
C18:1 trans 160 170 280 260 24 <0.001 0.003 0.001 0.02
C18:1 cis 75 84 100 100 6.3 <0.001 <0.001 0.003 0.15
C18:2 94 100 99 97 6.6 0.57 0.28 0.37 0.99
C18:3 7.4 7.8 7.6 6.6 0.4 0.16 0.87 0.04 0.41
Duodenal Composition, % Total FA
Cl6:0 20.4 22.3 22.8 23.4 0.33 <0.001 <0.001 0.03 0.88
C18:0 41.1 44.6 36.7 30.9 1.4 <0.001 0.03 <0.001 0.53
C18:1 trans 11.1 8.5 15.0 18.2 1.4 <0.001 0.07 <0.001 0.31
C18:1 cis 5.2 4.2 5.4 6.9 0.16 <0.001 0.11 <0.001 0.47
C 18:2 6.7 5.1 5.3 6.4 0.28 <0.001 0.003 0.002 0.15
C18:3 0.53 0.39 0.41 0.47 0.02 0.001 <0.001 0.02 0.39

 

l Treatments were CON- control with no supplemental rumen-protected fatty acids (FA),
SAT- saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

119

 

Table 3. Effects of rumen-protected fatty acids varying in saturation on ruminal pools and

turnover rates

 

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q

Ruminal Pool, g

Total FA 920 1040 1040 960 40 0.04 0.02 0.10 0.29

C16:0 200 230 240 230 10 0.001 <0.001 0.82 0.24

C1820 280 390 330 260 20 <0.001 <0.05 <0.001 0.99

C18:1 trans 56 56 75 80 4.7 <0.001 0.001 <0.001 0.06

C18:1 cis 75 79 85 90 6.6 0.24 0.15 0.15 0.99

C18:2 160 170 170 150 15 0.82 0.86 0.52 0.61

C18:3 4.5 4.8 4.8 4.3 0.4 0.64 0.70 0.29 0.57
Rumen Turnover, % h.l

Total FA 6.8 8.1 7.9 7.8 0.32 0.03 0.005 0.60 0.93

C16 6.0 8.1 7.9 7.7 0.32 0.001 <0.001 0.44 0.94

C18 7.2 8.1 8.1 8.1 0.34 0.13 0.02 0.90 0.98

 

l Treatments were CON- control with no supplemental rumen-protected fatty acids (FA),
SAT- saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

120

 

 

Table 4. Effects of rumen-protected fatty acids varying in saturation on rates of passage
from the rumen and rates of biohydrogenation.

 

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q
Passage Rate (Kp), % h-l
Total PA 6.4 7.3 7.4 6.5 0.36 0.14 0.16 0.11 0.26
C1620 6.3 7.5 7.2 6.2 0.40 0.06 0.12 0.03 0.47
C1810 8.4 9.6 8.7 7.6 0.62 0.11 0.78 0.02 0.86
C18:1-trans 5.0 5.2 7.1 6.3 0.56 0.04 0.07 0.16 0.04
C18:1-cis 4.2 4.7 4.9 4.8 0.31 0.37 0.11 0.85 0.57
C18:2 2.4 2.6 2.5 2.6 0.19 0.67 0.28 0.89 0.62
C18:3 7.1 6.9 6.9 7.1 0.65 0.99 0.90 0.88 0.91
Total C18 6.5 7.5 7.4 6.6 0.33 <0.05 0.07 0.04 0.36
Digestion Rate, % h.l
TFA 0.31 0.60 0.74 1.43 0.37 0.17 0.15 0.11 0.54
C16 -0.20 0.81 0.77 1.6 0.46 0.05 0.02 0.19 0.41
C18 0.72 0.69 0.97 1.64 0.35 0.20 0.35 0.07 0.66
Biohydrogenation Rate (kb), % h"l
Model A
C1823 31.1 28.2 34.2 38.9 2.3 0.02 0.31 0.003 0.81
C18:2 15.0 14.6 16.1 16.7 1.08 0.44 0.50 0.15 0.76
C18:1 20.6 20.0 15.8 15.7 1.44 0.01 0.02 0.02 0.18
Model B
C18:1 trans 48.4 47.5 34.1 33.4 3.4 <0.001 0.004 0.001 0.07
C18:1 cis 9.4 10.1 11.3 12.0 0.79 0.09 0.06 0.08 0.78

 

I Treatments were CON- control with no supplemental rumen-protected fatty acids (FA),
SAT- saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCF A, and UNS- unsaturated FA fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

121

 

 

Table 5. Effects of rumen-protected fatty acids varying in saturation on extent of
biohydrogenation and biohydrogenation index.

 

 

 

 

 

Treatment LS Means' P2
CON SAT INT UNS SE TRT RPF L Q

Biohydrogenation Extent (% Intake)

C18:1 73.4 71.5 61.9 63.6 2.8 0.01 0.02 0.04 0.09

C18:1 trans 90.5 90.1 82.0 83.0 2.1 0.006 0.02 0.01 0.06

C18:1 cis 69.0 68.0 69.1 71.6 1.8 0.41 0.74 0.12 0.74

C18:2 86.4 84.5 86.1 86.6 0.77 0.07 0.33 0.02 0.42

C18:3 81.8 80.2 82.9 84.9 1.1 0.03 0.47 0.005 0.78
Biohydrogenation Index, (% intake)3

Total C18 72.0 69.6 64.7 61.8 1.5 0.001 0.001 <0.001 0.55

C18:3 79.6 78.4 80.8 81.1 1.1 0.24 0.68 0.07 0.42

C18:2 84.9 83.1 84.6 83.1 0.70 0.16 0.12 0.85 <0.10

 

1 Treatments were CON- control with no supplemental rumen-protected fatty acids (FA),
SAT- saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.

2 Calculated according to Tice et al. 1994.

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CHAPTER 5

Effects of rumen-protected fatty acid saturation on ruminal and total tract nutrient

digestion in lactating dairy cows

ABSTRACT

Saturated and unsaturated rumen-protected fat sources were evaluated for effects
on ruminal digestion kinetics, and ruminal and post-ruminal nutrient digestion. Eight
early lactation ruminally and duodenally cannulated cows (77:h 12 DIM, meanzl: SD) were
used in a replicated 4x4 Latin square design with 21 d periods. Treatments were control
(CON) and a linear titration of 2.5% added rumen-protected fatty acids varying in
unsaturation; saturated (SAT; prilled hydrogenated free FA), 50:50 mix of SAT and
unsaturated (UNS; calcium soaps of long-chain FA), and UNS. The base ration included
37.2% forage and 13.5% cottonseed. SAT linearly decreased ruminal digestibility of DM
and OM because of a linear reduction in ruminal neutral detergent ﬁber (N DF)

dige stibility. The reduction in ruminal NDF digestibility was because of a linear decrease
in digestion rate and a linear increase in passage rate of potentially digestible NDF for
SAT treatment. Digestibility was not different between treatments because of
compensatory post-ruminal digestion. Ruminal FA and C18 FA digestibility tended to
increase linearly with UNS, and post-ruminal C18 FA digestibility increased with UNS.

SAT linearly decreased ruminal OM digestibility and decreased intestinal long-chain FA

123

 

 

digestibility, although differences in FA digestibility may be partially explained by FA

intake.

INTRODUCTION

Fat is commonly included in diets of dairy cows to increase energy density.
Rumen available fatty acids (FA), from oilseeds, byproducts and tallow, can be used to
increase dietary FA concentration up to 3 percentage units with minimal negative effects
on microbial growth and rumen function (NRC, 2001). However, unprotected
unsaturated FA are absorbed by rumen bacteria and can be toxic to the microbes unless
saturated by biohydrogenation (Harfoot, 1981).

Prilled FA and calcium salts of FA are two commercially available rumen-
protected fat (RPF) sources commonly used to increase dietary FA concentration. Prilled
FA are highly saturated FA developed to decrease interference with microbial
fermentation. Calcium salts of long-chain FA are FA complexed with a calcium ion
making them insoluble. Microbes cannot absorb FA as calcium salts and FA salts have
little effect on microbial fermentation. However, the complex dissociates as ruminal pH
decreases allowing microbial uptake and biohydrogenation (Wu et al., 1991).

Rumen-protected FA are often prescribed to increase daily energy intake,
although the ability of the cow to increase daily energy intake depends on the energy
density of the diet, diet digestibility and site of nutrient digestion. Associative effects of
FA may shift site of nutrient digestion from the rumen to the intestine possibly reducing

diet digestibility. We previously reported that UNS increased trans-F A duodenal ﬂow

124

 

through increased biohydrogenation of polyunsaturated FA and decreased rates of
biohydrogenation and passage of trans-18:1 (Chapter 4). Saturated FA treatment
increased C16:0 and C1820 ﬂow to the duodenum and had little effect on ruminal
metabolism of FA compared to control.

The objective of this experiment was to determine the effects of RPF saturation
on ruminal digestion kinetics and site and extent of nutrient digestion. We hypothesized
that the more unsaturated RPF source would decrease feed intake, increasing ruminal

retention time and ruminal OM digestibility compared to the saturated RPF treatment.

MATERIALS AND METHODS
This paper is the third of a series of four papers from one experiment that
evaluated effects of RPF differing in FA saturation. This paper discusses treatment
effects on ruminal digestion kinetics and site of digestion, and the companion papers
focus on milk production, milk FA proﬁle and energy balance (Chapter 3), ruminal
kinetics and extent of biohydrogenation (Chapter 4), and feed intake and feeding and
chewing behavior (Chapter 6). Experimental procedures were approved by the All

University Committee on Animal Use and Care at Michigan State University.

Treatments and Cows

Eight ruminally and duodenally cannulated multiparous Holstein cows (77 1 8.7
DIM; mean 1 SD) from the Michigan State University Dairy Cattle Teaching and
Research Center were used in a replicated 4 x 4 Latin square design experiment. Cows

were randomly assigned to treatment sequence. Treatments were a control diet (CON)

125

 

containing no added RPF or 2.5% RPF from saturated (SAT, prilled hydrogenated free
FA, Energy Booster 100®, Milk Specialties Company Inc., Dundee, IL), intermediate
mixture (50:50) of saturated and unsaturated (INT), or unsaturated (UN S, Ca Soaps of
LCF A, Megalac-R®, Church and Dwight Company, Inc., Princeton, NJ) FA. Treatment

periods were 21 d with the ﬁnal 11 d used for sample and data collection. Cows were

 

surgically prepared prior to calving and duodenal cannulas were soft gutter type made of
tygon and vinyl tubing (Crocker et al., 1998). The duodenum was ﬁstulated proximal to
the pylorus region and prior to the pancreatic duct and cannulas were placed between the
10th and 11th ribs as described by Robinson et al. (1985). Both ruminal and duodenal
surgeries were performed at the Department of Large Animal Clinical Science, College
of Veterinary Medicine, Michigan State University. Immediately prior to initiation of the
experiment, empty BW (ruminal digesta removed) of cows was 516 :1: 33 kg (mean 2
SD).

Diet components and composition are presented in a companion paper (Chapter

 

3). Brieﬂy, experimental diets contained 40% forage (66:33, corn silage: alfalfa silage),
13.5% whole cottonseed, dry ground corn, premixed protein supplement (soybean meal,
corn gluten meal, and blood meal), a mineral and vitamin mix, and 2.5% added rice hulls
(CON), saturated FA (SAT), 50:50 mix of saturated and unsaturated FA (INT) or
unsaturated FA (UNS). The base diet contained 5.5% FA with 2.5% FA from whole

cottonseed. All diets were fed as a total mixed rations.

126

Data and Sample Collection

Cows were housed and fed as previously described (Chapter 2). Indigestible NDF
was used as a marker to calculate duodenal ﬂow and ruminal digestibility and Chromic
oxide was used as a marker to calculate digestibility in the total tract. Gelatin capsules
(1.5 oz., Tropac Inc., Airﬁeld, NJ) containing 5 g of Chromic oxide and ground spelt hulls
(Wiley® mill, 2 mm screen; Authur H. Thomas, Philadelphia, PA) were dosed through
the ruminal cannula at 0700, 1500, and 2300 h (total of 15 g Cr203 /d) from 7 to 14 d with
a priming dose of 3X on d 7. Duodenal samples (1,000 g), fecal samples (500 g), and
rumen ﬂuid samples (100 ml) were collected every 9 h from d 12 to d 14 so that 8
samples were taken for each cow each period, representing every 3 h of a 24-hour period
to account for diurnal variation. Rumen ﬂuid samples were obtained by combining and
straining digesta from 5 different sites in the rumen. All samples were immediately
frozen at -20° C.

Ruminal contents were evacuated manually through the ruminal cannula at 1350 h
(4.5 h after feeding) on d 20 and at 0700 h (2 h before feeding) on d 21 of each period.
Total ruminal content mass and volume were determined. During evacuation, 10%
aliquots of digesta were separated to allow accurate sampling. Aliquots were squeezed
through a nylon screen (1 mm pore size) to separate into primarily solid and liquid

phases. Samples were taken from both phases for determination of nutrient pool size.

Sample and Statistical Analysis
Diet ingredients, orts, rumen contents and duodenal digesta were processed as

previously described (Chapter 3). Brieﬂy, forages and orts were coarse ground with dry

127

 

ice, lyophilized (Tri-Philizer'" MP, FTS Systems, Stone Ridge, NY) and then ﬁnely
ground. Rumen solid and liquid fractions were lyophilized and recombined based on
original DM ratio of solid and liquid fractions. Duodenal digesta was similarly split into
solid and liquid fractions, subsampled, lyophilized and recombined based on the DM
ratio of the fractions. Fecal samples were lyophilized, ground using a Willey mill® (1mm
screen; Authur H. Thomas, Philadelphia, PA) and combined on an equal DM basis into
one sample per cow per period. A portion of all samples were placed in a Whirl PacTM
bag (N asco, Fort Atkinson, WI) ﬂushed with nitrogen gas and frozen for FA analysis to
minimize FA oxidation.
All dried samples were analyzed for DM, ash, NDF, 240-hour in vitro indigestible

NDF (iNDF), potentially digestible NDF (pdNDF), CP, starch, gross energy (GE), and
FA concentration and proﬁles. Ash concentration was determined after 5 h oxidation at
500° C in a mufﬂe fumace. Concentration of NDF was according to Van Soest et al.
(1991, method A). Indigestible NDF was estimated as NDF residue after 240-h in vitro
fermentation (Goering and Van Soest, 1970). Rumen ﬂuid for in vitro incubations was
collected from a non-pregnant dry cow fed only alfalfa hay. Fraction of pdNDF was
calculated by difference (1.00 —-iNDF). Crude protein was analyzed according to Hach et
al. (1987). Starch was measured by an enzymatic method (Karkalas, 1985) after samples
were gelatinized with sodium hydroxide. Glucose concentration was measured using a
glucose oxidase method (Glucose kit #510; Sigma Chemical Co., St. Louis, MO), and
absorbance was determined with a micro-plate reader (SpectraMax 190, Molecular
Devices Corp., Sunnyvale, CA). Gross energy was assayed by bomb calorimeter (Parr

Instrument Inc., Moline, IL). Rumen ﬂuid was analyzed for concentration of major VFA

128

 

 

and lactate by HPLC (Waters Corp., Milford, MA). Fatty acids were extracted according
to Sukhija and Palmquist (1988), and quantiﬁed by GC (Model 8500, Perkins-Elmer
Corp, Norwalk, CT), using a SP-2560 capillary column (100m X 0.20 mm id with 0.02-
pm ﬁlm thickness; Supelco, Bellefonte, PA). Oven temperature was 140°C for 5 min,
then ramped 4°C/min to 240°C and held for 15 min. Helium ﬂow was 20 cm/sec.
Concentrations of all nutrients except DM were expressed as percentages of DM
determined by drying at 1050 C in a forced-air oven for more than 8 h.

Fecal samples were analyzed for concentration of chromium. Samples were
digested with phosphoric acid (Williams et al., 1962), and chromium was quantiﬁed by
ﬂame atomic absorption spectrometry (SpectraAA 220, Varian, Victoria, Australia)
according to manufacturer's recommendation. Nutrient intake was calculated using the
composition of feed offered and refused on CI 11-14. Ruminal pool sizes of nutrients
were determined by multiplying the concentration of each component by the ruminal
digesta DM mass. Turnover rate in the rumen, passage rate from the rumen, and ruminal
digestion rate of each component were calculated according to Oba and Allen (2003).

To determine differences between treatments, all data were analyzed using the ﬁt
model procedure of JMP® (Version 5, SAS Institute, Cary, NC) according to the
following model:

Yijk=u+Ci+Pj+Tk+eijk
where

u = overall mean,

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

Pj = ﬁxed effect of period (j = 1 to 4),

129

Tk = ﬁxed effect of treatment (k = l to 4), and

Cijk = residual error.

Period by treatment interaction was evaluated, but was removed from the
statistical model when not signiﬁcant (P > 0.10). Period by treatment interaction was not
signiﬁcant for any variable of primary interest; variables with signiﬁcant interactions are
noted in the tables. Data points with Studentized Residuals greater than three were
considered outliers and excluded from analysis. Few points were excluded in analysis
and rarely more than one per response variable. Preplanned contrasts included the effect
of addition of RPF (CON vs. SAT, INT and UNS), linear effect of increasing
concentration of unsaturated fat [L (SAT vs. UNS)], and quadratic effect of increasing
concentration of unsaturated fat [Q (INT vs. SAT and UNS)]. The preplanned contrasts
do not allow individual comparison of each fat treatment to the control. Protected LSD
was used for mean separation in the discussion when the model treatment effect was
signiﬁcant. Pearson correlation coefﬁcients were determined between cow-period
observations for some parameters. Treatment effects, linear and quadratic responses, and
correlations were declared signiﬁcant at P < 0.05, and tendencies were declared at P <
0.10. For reasons previously described (Chapter 4), rumen pool data from one period and

all data from one cow-period was excluded from statistical analysis.

RESULTS AND DISCUSSION
Treatment diet content and composition were previously presented (Chapter 3).
Diets contained the same base ration and differed in 2.5% RPF or rice hulls. Control diet

contained 5.5% FA and RPF diets contained 8.3, 8.1 and 7.8 % FA for SAT, INT and

130

 

 

UNS. Dietary unsaturated FA density increased from SAT to UNS treatment (3.9, 4.4 and

4.9 % for SAT, INT, and UNS).

Intake

Addition of RPF decreased DMI, and increasing UNS linearly decreased intake
within RPF. Intake of other nutrients including NDF, pdNDF, starch and CP followed
the same response pattern. Intake and feeding behavior are discussed in a companion

paper (Chapter 5).

Flow Marker

Indigestible NDF was used as the ﬂow marker for calculation of duodenal ﬂow
and chromic oxide was used for determination of fecal ﬂow. Chromic oxide was also
intended for prediction of duodenal ﬂow, but resulted in unrealistically high duodenal
ﬂow possibly because of inadequate subsampling procedures for duodenal liquid. Use of
indigestible NDF has the potential to cause treatment bias because of effects of duodenal
FA on in vitro fermentation for determination of indigestible residue. To insure that this
was not the case, we also calculated duodenal and fecal ﬂow for all cow periods using
acid detergent-sulfuric acid lignin as well as 120 hr indigestible ADF after ether
extraction. These markers provided results similar to ﬂow and digestibility calculated
with iNDF. Chromic oxide was used as the total tract fecal marker because it was not

affected by subsampling.

131

 

Ruminal Carbohydrate Digestion

SAT linearly decreased apparent ruminal DM and OM digestibility (Table 2)
because of a reduction in ruminal NDF digestibility (Table 3). Apparent ruminal
digestibility of starch was not affected by FA saturation. Increasing SAT concentration

reduced the amount of DM and tended to reduce the amount of OM and NDF digested in

 

the rumen, but did not affect the amount of starch digested. Addition of RPF did not
affect (P>0.58) apparent ruminal digestibility of DM, OM, NDF, or starch but decreased

the amounts of OM and starch apparently digested in the rumen (Tables 4, 5, and 6).

Rumen-protected FA treatments decreased ruminal VFA concentration and

changed VFA proﬁle by decreasing acetate and increasing propionate concentrations

 

(Table 5). Total ruminal VFA concentration tended to decrease linearly with increasing
SAT, and the molar proportion of acetate tended to decrease and branch chain VFA
proportion increased with increasing SAT. Decreased total VFA and acetate
concentration is consistent with the reduction in apparent ruminal OM digestibility.
The reduction in ruminal OM digestibility with SAT was not expected, because
saturated FA are not expected to interfere with microbial fermentation. In vitro
ferrnentations with addition of free stearic acid at 10% of DM was not different from
esteriﬁed stearic acid, although fermentation was decreased with free palmitic and oleic
FA (Chalupa etal., 1984). Although higher concentrations of free stearic acid, ranging
from 10-20% of DM, did resulted in a linear decrease of VFA production (Chalupa et al.,
1984), these are much higher FA concentrations than observed in the rumen in this
experiment (Chapter 3), and the authors noted low metal cation concentration in the

buffer that were expected to limit formation of FA salts. Most reports of prilled,

132

hydrogenated free FA have shown no effect on total tract nutrient digestion in lactating
dairy cows (Schauff and Clark, 1989; Palmquist, 1991; Elliott et al. 1996). However,
there is some evidence of a reduction in ruminal or total tract digestibility by prilled,
hydrogenated FA. Grummer (1988) observed decreased total tract NDF digestibility with
a low inclusion rate (0.68 kg/d) of prilled, hydrogenated FA compared to a low fat
control, although ruminal VFA concentration and in situ DM and NDF digestion rates
were not affected, and there were no effects on digestibility with a high FA inclusion rate
(0.91 kg/d). Saturated free FA compared to low fat and esteriﬁed saturated FA treatment
decreased ruminal OM digestibility in steers (Elliott et al., 1997), and total tract DM
digestibility of lactating cows (Eastridge and F irkins, 1991). In the current study, it

appears that SAT decreased ruminal digestibility via alterations of rumen passage rates.

Ruminal Digestion Kinetics

Saturated RPF decreased the fractional digestion rate and linearly increased the
fractional passage rate of pdNDF (Table 6). Increasing passage rate and decreasing
digestion rate resulted in a linear decrease of up to 24% for ruminal digestibility of
pdNDF by SAT (Table 3). Contrary to its effects on pdNDF, SAT linearly decreased
iNDF passage rate. Opposite effects on pdNDF and iNDF passage rates might be related
to SAT effects on chewing behavior. SAT linearly increased rumination time per day
and rumination time per kg DMI, and SAT cows ruminated over 50 min more per day
than CON and UNS (Chapter 5). Size and density of digesta particles are constraints to
passage from the rumen, and are affected by chewing and fermentation (Allen, 1996).

Increased rumination is expected to increase particle size reduction rate as well as

133

 

increase rumen movements and sorting of small particles entrapped in the ﬁbrous mat
increasing their rate of escape from the rumen (Allen, 1996). Increased rumination for
SAT may have increased escape of particles containing more rapidly digested NDF,
explaining the greater passage rate of pdNDF and a slower digestion rate for the pdNDF
remaining in the rumen. The slower rate of fermentation may increase iNDF buoyancy in
the rumen and decrease iNDF passage rate (Allen, 1996).

Ruminal FA turnover was increased by RPF treatment, no differences were
detected for fractional rates of passage and digestion of total FA. Ruminal turnover of
DM, OM, NDF, pdNDF, and starch were not affected by RPF or FA saturation. Ruminal
FA pool size, and FA biohydrogenation and passage kinetics are reported in companion

papers (Chapter 3).

Post-Ruminal Digestion

Treatment did not affect post-ruminal digestibility of OM, NDF, pdNDF and
starch as a percent of duodenal ﬂow (Tables 2, 3, 4). Differences in the amount of
duodenal ﬂow decreased the amount of OM and tended to decrease the amount of starch
digested post-ruminally with increasing UNS. SAT linearly decreased post-ruminal
digestibility of DM and OM as a percent of intake, representing a shift in site of digestion
away from the rumen and towards the intestine. The changes in post-ruminal digestion

are consistent with compensatory nutrient digestion in the intestine and hindgut.

134

 

Total Tract Digestion

Rumen-protected FA and FA saturation had no effect on total tract DM, OM,
NDF or starch digestibility because of compensatory post-ruminal digestion. Saturated
FA numerically decreased DM, OM, NDF and pdNDF total tract digestibility compared
to INT and UNS providing support for decreased ruminal digestibility, especially because
total tract and ruminal digestibilities were determined using independent markers.
Although FA digestibility linearly decreased with increasing SAT (Table 8), the effect

was too small to affect the digestible energy concentration of the diets (Table 7).

Fatty Acid Digestion

Rumen-protected FA increased and UNS linearly decreased total, C16, and C18
FA intake (Tables 8 and 9). Intake and duodenal ﬂow of individual FA were described in
Chapter 4, it was reported that UNS linearly decreased duodenal C 1 8:0 concentration and
linearly increased duodenal C18: 1, C18:2 and C18:3 concentration. Rumen-protected FA
did not affect total or C18 ruminal FA digestibility, but increased C16 FA ruminal
digestibility. Increasing UNS tended to linearly increase ruminal digestibility of total and
C18 FA.

Loss of dietary FA from the rumen through absorption across the rumen wall and
oxidative metabolism is often considered minimal, and bacterial synthesis of FA is
commonly expected to produce a net positive ﬂow of FA through the rumen. Low
ruminal absorption and metabolism of FA was ﬁrst concluded with minimal plasma
recovery of labeled carbon after ruminal infusion of radioisotope labeled linoleic acid

while diverting nutrients with a reentrant cannula (Jenkins, 1993). In addition, microbial

135

 

F A synthesis was reported during in vitro fermentation (Wu and Palmquist, 1991). In
contrast to the net positive FA ﬂow expected, Jenkins (1993) observed that ruminal FA
loss was reported for 15 out of 47 published treatment means. Regression analysis
predicted an 8 percent loss of FA intake, and up to a 30 percent FA loss was reported in
the dataset (Jenkins, 1993). F erlay et al. (1993) reported a 14% increase in FA ﬂow with
control diet and 36.7 and 21 .3% ruminal FA loss with rapeseed FA fed as calcium salts
and triglycerides respectively. Ruminal FA loss may be because of ﬂow marker bias
under-predicting duodenal ﬂow. However, Doreau and Chillard (1997) proposed that
negative ruminal FA ﬂux is not due to ﬂow marker bias, but caused by absorption and
oxidation of FA, especially with higher fat diets. Rumen epithelium and bacteria adhering
to the rumen wall may be capable of oxidative metabolism of FA (Doreau and Chilliard,
1997). Doreau and Chillard (1997) noted that higher FA concentration diets experience
greater FA loss and hypothesize that FA are less adsorbed to feed particles leading to
increased contact with the rumen wall and increased opportunity for absorption and
oxidation. In addition, increased dietary FA concentration decreases microbial FA
synthesis (Jenkins, 1993), which may allow detection of ruminal FA digestion. Variation
in reported rumen FA loss may be because of bacterial FA synthesis, especially in low
FA diets. Bacteria incorporate dietary FA into their plasma membranes and bacterial FA
cannot be considered entirely from microbial production, so true FA digestibility
occasionally reported in the literature has little meaning. Simple digestion studies cannot
partition duodenal FA ﬂow into dietary and microbial synthesized fractions limiting the

ability to determine the extent of rumen FA synthesis and digestion.

136

Investigations of ruminal FA loss have not considered effects of FA saturation.
Increased ruminal disappearance of unsaturated FA observed in the current study may
represent increased metabolism of unsaturated FA, and many reports of ruminal FA loss
were unsaturated FA treatments (F erlay et al., 1993; and Wu et al., 1991). It is logical
that unsaturated FA may be more highly oxidized in the rumen because they are less
hydrophobic than saturated FA and are more dispersed in the rumen allowing increased
contact with ruminal bacteria. In addition, ruminal bacteria absorb unsaturated FA during
biohydrogenation. Unsaturated FA that are absorbed in excess of bacterial requirements
for cellular membranes might be oxidized to eliminate their toxic effect. Fatty acid
saturation and concentration in the diet might explain the inconsistent ruminal FA loss
noted by Jenkins (1993). The increasing occurrence of ruminal FA loss reported in
digestion studies merits investigation of ruminal FA metabolism.

Digestibility of total FA as a percent of duodenal FA ﬂow tended to decrease and
C16 FA digestibility decreased with RPF treatments. Increasing SAT tended to decrease
total FA digestibility and decreased C18 FA digestibility as a proportion of FA ﬂowing to
the duodenum. Total tract digestibility of total FA was not affected by RPF treatments
but increasing SAT linearly reduced total, C16 and C18 FA digestibility. SAT increased
duodenal ﬂow of FA but UNS did not change F A ﬂow compared to CON because of
decreased intake and increased ruminal FA loss. Within RPF, increasing SAT linearly
increased duodenal FA ﬂow because of increased intake, less ruminal FA disappearance
and unintentionally higher dietary FA concentration (see discussion of treatments in
Chapter 3). Amount of total and C16 FA digested post-ruminally tended to increase with

increasing SAT. Post-ruminal digestion of total and C18 FA as a percent of intake was

137

 

 

 

not different across treatment, as a result of differences in ruminal digestion. This shows
equal efﬁciency of capturing dietary FA energy between SAT and UNS. Treatment did
not affect total tract total and C16 FA digestibility but decreased total tract digestibility of
C18 FA for non-cannulated cows on the same diets (Chapter 3). Saturated FA are

considered less digestible than unsaturated FA. Differences in FA digestibility reported

 

in the literature are biased because of ruminal FA oxidation, confoundation of
esteriﬁcation, biohydrogenation in the large intestine and oxidation of unsaturated FA
prior to analysis. Total tract digestibility of esteriﬁed FA is lower than unesteriﬁed FA
(Elloitt et al., 1994; Elloitt et al., 1999), and triglyceride digestibility decreases with
increasing saturation (Pantoja et al., 1996; Pantoja et al., 1995). Elliot et al. (1999)
observed that highly saturated triglycerides are more resistant to ruminal and intestinal
lipolysis, resulting in lower digestibility. The low pH of the duodenum prohibits lipase
function, and esteriﬁed FA are not hydrolyzed until the jejunum, decreasing the
Opportunity for FA absorption (Noble, 1981). Decreased ruminal lipolysis of saturated
FA increases duodenal ﬂow of esteriﬁed FA. The belief that saturated FA are less
digestible may be an erroneous conclusion based on decreased total tract digestibility of
saturated esteriﬁed FA causing decreased ruminal lipolysis and increased duodenal ﬂow

of less digestible esteriﬁed FA.

 

Sample handing and preparation may bias digestibility calculation because of
partial oxidation of FA(Palmquist, personal communication). In the current study,
samples were ﬂushed with nitrogen gas and frozen to minimize sample oxidation.
However, the methylation procedure of Sukhija and Palmquist (1988) may also cause

partial loss of unsaturated FA. Oxidation of unsaturated FA from improper storage and

138

sample preparation decreases unsaturated FA concentration, leading to an over-prediction
of unsaturated FA digestibility compared to saturated FA, which are much less prone to
oxidation.

Data for total tract and intestinal digestibility of individual FA supports decreased
digestibility of saturated FA compared to unsaturated F A. However, measures for
individual FA are meaningless because of FA biohydrogenation and synthesis in the large
intestine (Merchen et al., 1997). Digestibility of individual FA only be determined with
duodenally and illeally cannulated cows, although the cost and complexity of multiple
intestinal cannulation has prevented such measures. However, digestibility of unsaturated
FA can be compared to saturated FA by observing total, C16 or C18 FA digestibility
between treatments differing in duodenal FA proﬁle. Christensen et al. (1994) and
Bremmer et al. (1998) measured digestibility of total FA for abomasally infused free FA
and observed no difference between saturated and unsaturated FA treatments. Schauff
and Clark (1989), Grummer et al. (1988) and Palmquist (1991) directly compared
calcium salts of palm oil and prilled, saturated free FA, ﬁnding no difference in total tract
digestibility of energy, lipid and FA. Elliott et al. (1996) observed 8-percentage units
lower total tract FA digestibility with prilled FA compared to calcium salts of palm oil,
although treatments were not compared in the statistical contrasts. Doreau and Chilliard
(1997) summarized 64 treatment groups reporting FA digestibility in the small intestine
or the lower tract, ﬁnding no difference between C16 and C18 FA, and observed only
slight differences between lower tract saturated and unsaturated C18 FA digestibility (77,

85, 83 and 76% for 0, 1, 2 and 3 double bonds respectively).

139

 

 

 

Lastly, true loss of ruminal FA discounts the value of measurement of total tract
digestibility. Increased ruminal digestion may increase total tract FA digestibility
without increasing energy available to the animal. If unsaturated FA are more highly
degraded in the rumen, increased unsaturated FA digestion in the total tract might not
increase intestinal FA absorption. The energy efﬁciency of ruminally digested FA is not
known, but considerable energy loss is expected because of bacterial maintenance energy
requirements and loss of energy in chemical transformation. Digestion studies have
reported substantial ruminal FA digestion (Wu et al. 1991; and F erlay et al. 1993), which
may have a large effect on energy absorption, especially considering the high energy
value of FA. Merchen et al. (1997) proposed that fat digestion experiments should utilize
duodenally cannulated animals for observation of duodenal FA proﬁle and calculation of
ruminal and post-ruminal digestibility as common in starch and ﬁber digestion studies.

Unsaturated FA treatment linearly increased digestibility of C18 FA ﬂowing to
the duodenum 7.6 percentage units compared to SAT, and we have previously reported
4.2 percentage unit increase in total tract digestibility with non-cannulated cows on the
same diets (Chapter 3). In the current experiment, post-ruminal C18 FA digestibility of
UNS was not different from control but was decreased by SAT. Decreased FA
digestibility of SAT cannot be directly attributed to lower digestibility of C18:0 as CON
and SAT did not differ in duodenal C1810 composition. Fatty acid digestion across RPF
saturation may be a result of the amount of duodenal FA or DM ﬂow. Schauff and Clark
(1992) reported decreased FA digestibility as the FA content of the diet was increased
with dietary calcium salts of palm oil, but Jenkins (1999) reported linearly increased total

tract FA digestibility when feeding Oleamide from 0 to 5% of the diet. Palmquist (1991)

140

 

 

discussed decreased FA digestibility with increasing duodenal ﬂow and reported drastic
decreases in true and marginal true digestibility with increasing FA intake, noting a 4.4
percentage unit decrease in marginal true FA digestibility (digestibility of each increment
of fatty acid consumed) per 100 g of FA intake. Weisberg et al. (1992a and 1992b)

observed decreased FA digestibility when increasing FA intake from 500 to 1000 g/d of

 

palmitic and stearic acid and when increasing FA intake from tallow. Analysis of the
relationship between FA digestibility and FA intake is confounded by FA form and

saturation used to increase FA intake, making conclusions difﬁcult.

 

The associative effects of amount of feed intake, digesta passage rate and diet
composition on FA digestibility has received little attention. Grum et al. (1996) reported
a large decrease (>23.9 percentage units) in C18 FA digestibility with increased
concentrate feeding with and without tallow. Elliott et al. (1995) observed increased total
tract FA digestibility in diets that replaced ground corn with soyhulls. Weisbjerg et al.
(1992b) did not observe any difference in tallow FA intake at low (8.6 kg) and high (12.6
kg) DMI, although both intake levels were considerably lower than observed in the
current study. In the current study, increasing DMI tended to increase total FA
digestibility of duodenal ﬂow (R2 = 0.10, P < 0.10). Associative effects of dietary

carbohydrates on FA digestibility are not known but may include level of DM intake, and

 

duodenal digesta ﬂow rate and composition.

CONCLUSION
Saturated FA treatment decreased ruminal NDF digestibility possibly because

increased rumination resulted in a faster passage of more rapidly fermentable NDF.

141

Saturated FA treatment also decreased FA digestibility, although it is not possible to
discern if it is because of FA composition or increased ﬂow of FA to the duodenum.
Addition of rumen-protected FA may not increase energy intake because of decreased

DM intake and negative associative effects on ruminal digestion.

142

 

 

Table l. Ingredient and nutrient composition of experimental diets].

 

CON SAT INT UNS
Ingredients ------ % of DM -------
Corn silage2 24.6 24.7 24.7 24.6
Alfalfa silage3 12.6 12.6 12.6 12.6
Ground Corn 28.7 28.8 28.8 28.7
Whole Cottonseed 13.5 13.5 13.5 13.5
Protein mix4 10.5 10.6 10.5 10.5
Mineral vitamin rnix5 4.3 4.3 4.3 4.3
CON Mix6 5.7 0.5 0.2 -
SAT Mix6 — 5.0 2.5 -
UNS Mix6 - - 2.9 5.7
Nutrient

DM 55.6 55.7 55.7 55.7
OM 92.6“ 92.9b 93.1“ 93.1“
Total FA 5.5“ 8.3“ 8.1b 7.8“
Unsaturated FA 3.6“ 3.9“ 4.4b 4.9c
Starch 30.8“ 30.3“b 30.5bc 30.7c
NDF 29.1“ 27.3d 27.5c 27.7b
Indigestible NDF 11.2“ 9.7b 10.0b 9.9c
Forage NDF 16.96 17.0“ 16.9bc 17.0b
CF 16.2“ 16.1b 16.1b 16.1b
Rumen-undegraded CP7 5.1al 4.8d 5.1b 4.9c
% NDF from forage 57.4d 61.4a 60.9b 60.5c
GE MCal/Kg 4.55b 4.72“ 4.72“ 4.71“

 

' Treatments were CON- control with no supplemental rumen-protected fatty acids (FA), SAT- saturated
FA from prilled FA, INT- intermediate saturation as a mix of prilled FA and calcium soaps of LCFA, and
UNS- unsaturated FA fed as Ca soaps of LCFA.

2 Corn silage contained 34.7% DM (as fed), and 43.4% NDF, 8.4% CP, 10.7% indigestible NDF, 24.1%
starch, and 4.8% ash on a DM basis.

3 Alfalfa silage contained 36.3% DM (as fed) and 48.1% NDF, 16.2% CP, 25.7% indigestible NDF, 2.6%
starch, and 9.7% ash on a DM basis.

4 Protein mix contained 74.1% soybean meal, 20.1% corn gluten meal, and 5.8% blood meal.

5 Mineral vitamin mix contained 12.7% sodium bicarbonate, 11.5% limestone, 5.5% salt, 2.2% trace
mineral premix, 2.0% urea, 2.0% dicalcium phosphate, 0.6% vitamin D, 0.48% vitamin A, 0.12% vitamin
E, and 62.9% dry ground corn as a carrier

6 Mix Composition listed in Table 1

7 Rumen-degraded protein estimated using values from NRC (2001).

143

Table 2. Effects of rumen protected fatty acids varying in saturation on digestion of DM

 

 

 

and OM.
Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q

DM
Intake, kg/d 27.1 25.4 24.5 23.0 0.93 <0.001 <0.001 <0.001 0.50
Apparently ruminally digested

kg/d 8.0 6.1 7.1 7.5 0.68 0.05 0.06 0.03 0.65

% 29.7 23.7 29.0 33.2 2.5 0.01 0.64 0.002 0.82
Apparent total tract digested

kg/d 17.7 16.5 16.2 15.3 0.7 0.008 0.003 0.04 0.61

% 65.6 65.1 66.0 66.7 1.2 0.73 0.77 0.28 0.93
OM
Intake, kg/d 5.0 23.6 22.8 21.3 0.87 <0.001 <0.001 <0.001 0.45
Apparently ruminally digested

kg/d 8.5 6.8 7.5 7.8 0.64 0.05 0.03 0.07 0.70

% 34.1 28.6 33.1 37.1 2.4 0.02 0.58 0.002 0.92
Passage to

duodenum, kg/d 16.4 16.7 15.3 13.5 0.76 <0.001 0.06 <0.001 0.75
Apparent post-ruminal digested

kg/d 8.0 8.6 7.6 6.5 0.65 0.07 0.52 0.01 0.97

% of intake 32.3 37.0 33.4 30.3 2.6 0.18 0.63 0.03 0.92

% of duodenal

passage 48.5 51.3 49.6 47.9 2.4 0.69 0.67 0.27 0.99

Apparent total tract digested

kg/d 16.5 15.4 15.2 14.3 0.6 0.01 0.004 0.06 0.52

% 66.3 65.6 66.8 67.4 1.2 0.68 0.85 0.24 0.82

 

l Treatments were CON- control with no rumen-protected fatty acids (FA), SAT-

saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCFA.
2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

144

 

 

Table 3. Effects of rumen protected fatty acids varying in saturation on digestion of total

NDF and potentiallj digestible NDF (pdNDF).

NDF
Intake, kg/d
Ruminally digested
kg/d
%
Passage to duodenum,
kg/d
Post-ruminally digested
kg/d
% of intake
% of duodenal ﬂow
Total tract digested
kg/d

% of intake

pdNDF3

Intake, kg/d

Ruminally digested
kg/d

%
Passage to
duodenum, kg/d

Post-ruminally digested
kg/d

% of intake
% of duodenal
passage

Total tract digested
kg/d

% of intake

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q
7.5 6.8 6.7 6.1 0.6 <0.001 <0.001 0.005 0.37
2.2 1.7 1.9 2.0 0.17 0.13 0.08 0.10 0.87
28.8 24.6 27.8 32.2 2.0 0.04 0.78 0.006 0.80
5.3 5.1 4.9 4.2 0.18 <0.001 0.004 <0.001 0.29
0.81 0.99 1.00 0.55 0.22 0.36 0.89 0.13 0.37
10.8 15.0 14.5 8.9 3.2 0.42 0.59 0.15 0.51
15.0 19.5 20.1 12.8 4.1 0.50 0.61 0.23 0.43
3.0 2.70 2.85 2.56 0.19 0.36 0.18 0.57 0.29
39.9 39.6 42.5 41.1 2.5 0.78 0.67 0.63 0.43
4.5 4.4 4.2 4.0 0.18 0.02 0.03 0.02 0.69
2.2 1.7 1.9 2.0 0.17 0.13 0.08 0.10 0.87
47.8 38.5 43.9 50.5 3.0 0.03 0.30 0.006 0.86
2.3 2.6 2.4 1.9 0.12 0.004 0.92 <0.001 0.56
0.66 0.77 0.95 0.50 0.19 0.36 0.73 0.28 0.17
15.5 18.6 22.8 13.1 4.3 0.40 0.59 0.34 0.19
26.4 28.8 40.5 26.7 0.72 0.47 0.50 0.82 0.16
2.8 2.5 2.8 2.5 0.19 0.38 0.25 0.90 0.19
62.8 57.1 65.7 63.6 3.6 0.31 0.86 0.17 0.19

 

' Treatments were CON- control with no rumen-protected fatty acids (FA), SAT- saturated FA

from prilled FA, INT- intermediate saturation as a mix of prilled FA and calcium soaps of LCFA,
and UNS- unsaturated FA fed as Ca soaps of LCFA.
2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic effect.3
Potentially digestible NDF.

145

.-

 

Table 4. Effects of rumen protected fatty acids varying in saturation on digestion of

 

 

 

starch.
Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q

Intake, kg/d 8.5 7.6 7.4 7.0 0.30 <0.001 <0.001 0.001 0.45
Apparently ruminally digested

kg/d 5.0 4.3 4.6 4.2 0.36 0.02 0.007 0.67 0.11

% 58.2 55.7 61.6 59.7 3.5 0.39 0.78 0.24 0.21
Passage to duodenum,

kg/d 3.5 3.4 2.8 2.8 0.26 0.04 0.04 0.06 0.23
Apparent post-ruminal digested

kg/d 3.0 2.9 2.4 2.4 0.26 0.07 0.09 0.07 0.17

% of intake 35.8 38.8 32.5 34.9 3.6 0.37 0.89 0.26 0.18

% of duodenal ﬂow 84.8 87.1 83.6 86.0 1.8 0.38 0.63 0.59 0.12
Apparent total tract digested

kg/d 8.0 7.2 7.0 6.6 0.3 <0.001 <0.001 0.003 0.59

% 94.0 94.5 94.2 94.5 0.43 0.67 0.35 0.95 0.44

 

1 Treatments were CON- control with no rumen-protected fatty acids (FA), SAT-
saturated FA from prilled FA, IN T- intermediate saturation as a mix of prilled FA and

calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCF A.
2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

146

Table 5. Effects of rumen protected fatty acids varying in saturation on ruminal
fermentation.

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q

Total VFA, mM 140 133 135 138 2.0 <0.05 <0.001 <0.10 0.92
Lactate, mM 0.10 0.50 0.19 0.67 0.17 0.07 0.005 0.45 <0.05
VFA, mol/100 mol

Acetate 52.5 49.3 49.8 50.2 0.50 <0.001 <0.001 0.09 0.87

Propionate 32.1 33.9 34.0 33.4 0.53 0.007 <0.001 0.30 0.34

Butyrate 11.1 1 1.5 11.4 11.8 0.25 0.17 <0.001 0.32 0.26

Branched-chain

VFA 4.7 5.3 4.8 4.7 0.17 0.04 <0.001 0.01 0.42
Acetate: Propionate 1.64 1.46 1.47 1.51 0.04 <0.001 <0.001 0.19 0.61

 

l Treatments were CON- control with no rumen-protected fatty acids (FA), SAT-
saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCF A.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.

147

 

 

 

Table 6. Effects of rumen protected fatty acids varying in saturation on ruminal digestion

kinetics.

 

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q

Ruminal Turnover, rate, %/h

DM 7.8 7.4 7.3 7.6 0.27 0.57 0.32 0.43 0.58

OM 7.8 7.4 7.4 7.7 0.27 0.53 0.27 0.46 0.56

NDF 4.0 3.6 3.8 3.9 0.16 0.30 0.19 0.17 0.92

pdNDF 5.1 4.9 4.8 4.8 0.33 0.92 0.53 0.87 0.86
INDF 3.1 2.5 2.8 3.0 0.14 0.03 0.04 0.02 0.96

Starch 34.5 32.7 31.8 29.5 2.4 0.49 0.25 0.34 0.79

Total FA 6.8 8.1 7.9 7.8 0.32 0.03 0.005 0.60 0.93
Ruminal Passage, rate, %/h

Starch 14.8 14.5 12.5 11.7 1.6 0.29 0.22 0.14 0.71

pdNDF 2.6 3.1 2.7 2.4 0.22 0.16 0.66 0.03 0.81

INDF 3.1 2.5 2.8 3.0 0.15 0.04 0.06 0.02 0.74

Total FA 6.4 7.3 7.4 6.5 0.36 0.14 0.16 0.11 0.26
Ruminal digestion, rate %/h

Starch 19.8 18.0 20.4 17.8 1.8 0.59 0.58 0.91 0.22

pdNDF 2.4 1.8 2.2 2.4 0.2 0.12 0.21 0.04 0.81

Total FA 0.31 0.60 0.74 1.43 0.37 0.17 0.15 0.11 0.54

 

1 Treatments were CON- control with no supplemental rumen-protected fatty acids (FA),
SAT- saturated FA from prilled FA, INT— intermediate saturation as a mix of prilled FA
and calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.
3 Potentially digestible NDF.

148

Table 7. Effects of rumen protected fatty acids varying in saturation on digestion of

 

 

 

energy.
Treatment LS Meansl P 2

CON SAT INT UNS SE TRT RPF L Q
Energy
Intake, Mcal GE/d 122.8 119.8 115.4 107.8 4.4 0.001 0.001 0.001 0.47
Apparent total tract digested
Mcal/d 78.0 75.3 74.0 70.1 3.1 0.07 0.04 0.06 0.59
% 63.7 62.9 64.1 65.1 1.2 0.52 0.80 0.15 0.94

 

l Treatments were CON- control with no rumen-protected fatty acids (FA), SAT-
saturated FA from prilled FA, TNT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated FA fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

149

 

Table 8. Effects of rumen protected fatty acids varying in saturation on digestion of FA.

 

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q

Total FA
Intake, kg/d 1.5 2.1 2.0 1.8 0.07 <0.001 <0.001 <0.001 0.68
Apparently ruminally digested

kg/d 0.06 0.17 0.16 0.32 0.09 0.23 0.15 0.20 0.43

% 4.0 7.3 7.9 18.4 4.9 0.15 0.19 0.09 0.38
Passage to duodenum,

kg/d 1.4 2.0 1.9 1.5 0.12 <0.001 0.003 <0.001 0.25

Apparent post-ruminal digested

kg/d 1.0 1.3 1.3 1.0 0.11 0.11 0.12 0.08 0.31

% of intake 69.1 59.6 63.0 56.7 5.2 0.33 0.12 0.66 0.42

% of duodenal

passage 71.5 63.8 67.9 69.3 2.5 0.11 0.09 0.08 0.62

Apparent total tract digested

kg/d 1.1 1.4 1.4 1.4 0.06 <0.001 <0.001 0.16 0.79

% 73.1 66.9 70.6 75.1 1.6 <0.001 0.15 0.001 0.80

 

1 Treatments were CON- control with no rumen-protected fatty acids (FA), SAT-

saturated FA from prilled FA, IN T- intermediate saturation as a mix of prilled FA and
calcium soaps of LCF A, and UNS- unsaturated FA fed as Ca soaps of LCFA.
2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

150

 

A-

 

 

Table 9. Effects of rumen protected fatty acids varying in saturation on digestion of C16
and C18 fatty acids.

 

 

 

 

Treatment LS Means1 P2
CON SAT INT UNS SE TRT RPF L Q

Total C16 Fatty Acid
Intake, kg/d 0.28 0.50 0.47 0.44 0.02 <0.001 <0.001 0.001 0.96
Apparently ruminally digested

kg/d -0.01 0.05 0.04 0.09 0.03 0.06 0.02 0.23 0.32

% -3.8 9.1 8.0 20.0 6.2 0.05 0.02 0.16 0.34
Passage to

Duodenum, kg/d 0.30 0.45 0.43 0.35 0.03 <0.001 <0.001 0.006 0.28
Apparent post-ruminal digested

kg/d 0.23 0.31 0.30 0.24 0.03 0.08 0.07 0.08 0.38 4

% of intake 81.6 62.8 64.8 56.0 6.5 <0.05 <0.01 0.41 0.47 1

% of duodenal

passage 77.9 68.4 70.1 69.5 2.6 0.06 0.008 0.74 0.71

Apparent total tract digested

kg/d 0.22 0.36 0.34 0.33 0.02 <0.001 <0.001 0.12 0.85

% 77.9 71.9 73.2 76.0 1.6 0.03 0.02 0.04 0.66
Total C18 Fatty Acid
Intake, kg/d 1.0 1.4 1.3 1.2 0.05 <0.001 <0.001 <0.001 0.55
Apparently ruminally digested

kg/d 0.09 0.13 0.14 0.13 0.05 0.30 0.27 0.16 0.53

% 9.6 8.8 10.7 20.3 4.5 0.19 0.46 0.06 0.46
Passage to

Duodenum, kg/d 0.9 1.2 1.2 0.9 0.07 <0.001 0.005 <0.001 0.25
Apparent post-ruminal digested

kg/d 0.64 0.78 0.79 0.66 0.07 0.16 0.15 0.14 0.30

% of intake 64.0 57.3 60.7 55.9 4.7 0.57 0.26 0.82 0.46

% of duodenal

passage 70.4 62.4 67.5 70.0 2.5 0.05 0.15 0.02 0.64

Apparent total tract digested

kg/d 0.75 0.91 0.91 0.89 0.04 <0.001 <0.001 0.73 0.60

% 73.5 66.1 70.9 76.2 1.6 <0.001 0.11 <0.001 0.89

 

' Treatments were CON- control with no rumen-protected fatty acids (FA), SAT-
saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCF A, and UNS- unsaturated FA fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.

 

151

 

CHAPTER 6

Effect of rumen-protected fatty acid saturation on feed intake, and feeding and

chewing behavior of lactating dairy cows.

ABSTRACT
Saturated and unsaturated rumen-protected fat sources were evaluated for effects on feed
intake, meal patterns and chewing behavior. Eight ruminally and duodenally cannulated
cows were used in a replicated 4x4 Latin square design with 21 (1 periods. Treatments
were control and a linear titration of 2.5% added rumen-protected fatty acids (RPF)
varying in unsaturation; saturated (SAT; prilled hydrogenated free FA), 50:50 ratio of
SAT and unsaturated (UNS; calcium soaps of long-chain FA), and UNS. Dry matter
intake for SAT was not different from control while UNS linearly decreased DMI. Wet
weight of ruminal digesta decreased with RPF, and decreased linearly with increasing
UNS. Treatment did not change meal number, meal length or time between meals, but
increasing UNS decreased meal size within RPF. Time spent ruminating was greater for
SAT compared to CON and was linearly increased by SAT. Increasing unsaturated FA
ﬂow to the duodenum decreases feed intake by decreasing meal size, and increasing
saturated FA ﬂow to the duodenum increases rumination time per day by increasing

rumination bout length.

152

 

 

 

INTRODUCTION

High producing dairy cows have high energy requirements that may exceed their
ability to consume dietary energy on some diets, resulting in less than maximum milk
yield. Addition of fat to the diet increases energy density without increasing rumen acid
production, or maintains energy density while increasing ﬁber for stabilization of rumen
fermentation (Allen, 1997). Prilled saturated free fatty acids (FA) and calcium salts of
FA are two manufactured products marketed to minimize effects of fat on ruminal
fermentation. However, calcium salts of FA are not entirely protected in the rumen and
dissociation of the calcium ion allows rumen biohydrogenation of unsaturated FA (Wu et
a1. 1991).

Intake is highly regulated by animal nutrient requirement and metabolic state, and
by the type and temporal pattern of fuels absorbed. A meta-analysis of treatment means
from the literature indicated different hypophagic effects of fat supplements differing in
FA source, form, and type (Allen, 2000). Within commonly fed rumen-protected fat
(RPF) sources, calcium salts of pahn oil linearly decreased DMI with increasing dietary
concentration, while hydrogenated FA did not affect DMI (Allen, 2000). Fatty acids
reaching the duodenum have been proposed to have different hypophagic effects
(Drackley, 1992), and abomasal FA infusion has consistently demonstrated unsaturated
FA hypophagia (Benson and Reynolds, 2001). Although many experiments observe
daily DMI, few have observed feeding and chewing behavior. Daily intake is a function
of meal size and intermeal interval. Prilled and hydrogenated free FA and calcium soaps

of long-chain FA were selected to provide the largest difference in unsaturated FA,

153

 

 

especially poly-unsaturated FA (PUFA) ﬂow to the duodenum possible with commonly
available feed ingredients. Calcium salts of long-chain FA increased duodenal ﬂow of
monounsaturated FA, but failed to increase PUFA ﬂow in this study (Chapter 3). The
objective of this experiment was to determine effects of rumen-protected FA saturation
on feed intake and feeding and chewing behavior of lactating dairy cows. We

hypothesized that increasing UNS would decrease intake.

MATERIALS AND METHODS
This paper is the fourth of four papers in a series from one experiment that
evaluated effects of RPF differing in FA saturation. This paper discusses treatment
effects on DM1 and feeding and chewing behavior, and the companion papers focus on
milk yield, milk FA proﬁle and energy balance (Chapter 3), rumen kinetics of FA
biohydrogenation (Chapter 4), and ruminal and post-ruminal nutrient digestion (Chapter
5). Experimental procedures were approved by the All University Committee on Animal

Use and Care at Michigan State University.

Cows and Treatments

Eight ruminally and duodenally cannulated multiparous Holstein cows (77 :t 8.7
DIM; mean x SD) from the Michigan State University Dairy Cattle Teaching and
Research Center were used in a replicated 4 x 4 Latin square design experiment. Cows
were randomly assigned to treatment sequence. Treatments were a control diet (CON)
containing no added RP- FA or 2.5% added RPF from saturated (SAT - prilled

hydrogenated FA, Energy Booster 100®, Milk Specialties Company Inc., Dundee, IL),

154

 

intermediate mixture of saturated and unsaturated (INT), or unsaturated (UNS) FA (Ca
Soaps of LCFA, Megalac-R®, Church and Dwight Company, Inc., Princeton, NJ).
Treatment periods were 21 d with the ﬁnal 11 d used for sample and data collection.
Surgery was performed at the Department of Large Animal Clinical Science, College of
Veterinary Medicine, Michigan State University. Immediately prior to initiation of the
experiment, empty body weight (ruminal digesta removed) of cows was 516 1 33 kg
(mean 1 SD).

Treatment mix composition and diet composition are reported in Chapter 3.
Experimental diets contained 40% forage (66:33, corn silage: alfalfa silage), 13.5%
whole cottonseed, dry ground corn, premixed protein supplement (soybean meal, corn
gluten meal, and blood meal), a mineral and vitamin mix, and 2.5% added rice hulls
(CON), saturated FA (SAT), 50:50 mix of saturated and unsaturated fat (INT) or

unsaturated FA (UNS). All diets were fed as a total mixed ration.

Data and Sample Collection

Throughout the experiment, cows were housed in tie-stalls and fed once daily
(1100 h) at 115% of expected intake. Amounts of feed offered and orts were weighed for
each cow daily. Samples of all diet ingredients (0.5 kg) and orts from each cow (12.5%)
were collected daily on d 11 to 14 and (1 16-19 and combined into one sample to
represent four days for digestibility analysis ((1 11-14) and four days for feeding behavior
observation (d 16-19).

Feeding behavior was monitored from d 16 to d 19 (96 h) of each period using a

computerized data acquisition system (Dado and Allen, 1993). Data of chewing

155

 

activities, feed disappearance, water consumption and rumen pH were recorded for each
cow every 5 sec. When chewing equipment malﬁrnctioned for an individual cow during
a 24-h period (1100h to 1100h), chewing behavior was deleted for that day. The system
successfully collected 77% of the total chewing behavior data (average 3.1d per cow per
period). The feeding behavior analysis procedure allowed determination of meal size and
length for days missing short periods of chewing data. This intervention allowed analysis
of meal parameters for 88% of the observation days. Feeding behavior was determined
according to Dado and Allen (1993) with the following modiﬁcations. Potential meals
were identiﬁed at the 75th percentile of the running standard deviation of the manger
weight to account for differences in baseline variation between data ﬁles. Minimum meal
size was 1 kg (as fed) and minimum continuous meal length was 30 5. Meals parameters
determined to be outliers (outside the 10th and 90th percentile) were manually veriﬁed and
corrected if determined to be in error. Daily means were calculated for number of meal
bouts per day, interval between meals, meal size, eating time, ruminating time, and total
chewing time. These response variables were calculated as daily means and averaged for
each period. Indwelling rumen pH probes were calibrated daily. Data was discarded for
the probe if calibration drifted 1 0.10 pH units at either pH 4.0 or pH 7.0. Seventy-eight
percent of the cow days met the criteria. Ruminal contents were evacuated manually
through the ruminal cannula at 1350 h (4.5 h after feeding) on d 20 and at 0700 h (2 h

before feeding) on d 21 of each period as described in Chapter 3.

156

Sample and Statistical Analysis

Diet ingredients, orts, rumen contents and duodenal digesta were processed as
previously described (Chapter 3). Overall DM and nutrient intake was calculated as the
means of digestibility (d 11-14) and feeding behavior (d16-19) observations. The ratio of
chewing activity to DM and nutrient intake utilized intake from feeding behavior
observation only. Ruminal pool sizes (kg) of OM, NDF, iNDF, and starch were
determined by multiplying the concentration of each component in DM by the ruminal
digesta DM weight (kg).

Hunger and satiety ratios were calculated according to Forbes (1995) as follows:

Hunger ratio = meal kg DM / premeal interval ; and

Satiety ratio = meal kg DM / postmeal interval.
Ratios were calculated for individual meals and averaged for the 4 d of feeding behavior
data collection.

Energy values were calculated as follows:

DE intake = GEI x GE digestibility [GE digestibility as reported in Chapter 4]

Selection was calculated as concentration of the component in the diet consumed

divided by the concentration of the component in the diet fed.

All data were analyzed using the ﬁt model procedure of JMP® (Version 5, SAS
Institute, Cary, NC) according to the following model:

Yijk=11+Ci+Pj+Tk+eijk
where

11 = overall mean,

157

 

C; = random effect of cow (i = l to 16),
Pj = ﬁxed effect of period (j = 1 to 4),
Tk = ﬁxed effect of treatment (k = 1 to 4),

eiJ-k = residual error.

Period by treatment interaction was evaluated, but was removed from the
statistical model when not signiﬁcant (P > 0.10). Period by treatment interaction was not
signiﬁcant for any variable of primary interest; variables with signiﬁcant interactions are
noted in the tables. Data points with Studentized Residuals greater than three were
considered outliers and excluded from analysis. Few points were excluded in analysis
and rarely more than one per response variable. Preplanned contrasts included the effect
of addition of RPF (CON vs. SAT, INT and UNS), linear effect of increasing
concentration of unsaturated FA [L (SAT vs. UNS)], and quadratic effect of increasing
concentration of unsaturated FA [Q (INT vs. SAT and UNS)]. The preplanned contrasts
did not allow individual comparison of each fat treatment to the control. Protected LSD
and was used for mean separation when the model treatment effect was signiﬁcant.
Pearson correlation coefﬁcients were determined between cow-period observations for
some parameters. Treatment effects, linear and quadratic responses, and correlations
were declared signiﬁcant at P < 0.05, and tendencies were declared at P < 0.10.

Data from two cow-periods were excluded from statistical analysis. One cow
developed clinical mastitis on d 19 of period 3, rumen samples, body weight and body

condition score was not collected for this period. Data previously collected in this period

158

 

was included in our analysis. The cow did not fully recover and data from period 4 was

not used.

RESULTS AND DISCUSSION
Intake

Addition of RPF decreased DMI compared to control (P < 0.001), and increasing
UNS linearly decreased DMI (P=0.02; Table 2). Mean comparison showed that SAT and
INT had no effect on intake but UNS decreased intake compared to CON. Intake of OM,
starch, and FA were similarly decreased. Neutral detergent ﬁber intake was less affected
because of differences in NDF concentration among diets from the inclusion of rice hulls
as dietary ﬁll in balancing diets. Digestible energy intake was decreased by RPF (P =
0.04), and tended to linearly decrease with increasing UNS (P = 0.06).

Allen (2000) reported that 11 out of 24 summarized studies feeding increasing
amounts of calcium salts of palm oil showed a decrease in intake, while 22 of the 24
studies resulted in numerical decreases in intake. Calcium salts of palm oil has a lower
concentration of unsaturated FA and a higher C16:Cl8 FA ratio than the calcium salts of
blended oil used in this experiment. Also reported, hydrogenated FA or triglycerides
resulted in decreased feed intake in only one study and increased feed intake in two out of
21 studies reported (Allen, 2000).

Abomasal infusion of unsaturated fat consistently decreases intake relative to no
fat and saturated fat infusions (Benson and Reynolds, 2001). Drackley et al. (1992) and

Christensen et al. (1994) showed abomasal infusions of increasing unsaturated FA with a

159

 

lower C16:C18 FA ratio decreased DM and energy intake. The RPF treatments in the
current study differed in unsaturated fat concentration while maintaining similar C16:C18
ratios. Bremmer et al. (1998) demonstrated decreased intake with increasing FA
unsaturation with abomasal infusions of FA with the same C16:C18 FA ratio. Lastly,
protected Oleamide FA compared to free oil that is readily biohydrogenated in the rumen
consistently linearly decreased intake (Jenkins, 1998, Jenkins, 2000, and DeLuca and
Jenkins, 2000).

Dietary PUF A were extensively biohydrogenated in the current study, but
decreased biohydrogenation of trans-C18:1 increased duodenal ﬂow of mono-unsaturated
FA (Table 1). Duodenal FA ﬂow of C 1 8:2 did not differ between treatments, and C18:3
ﬂow was linearly decreased with increasing UNS. Intake depression cannot be attributed
to increased intestinal absorption of PUFA. Duodenal ﬂow of both cis-C18:1 and trans-
C18:1 increased with increasing UNS corresponding to the linear decrease in DMI.

Unsaturated FA treatment decreased DM1 and tended to decrease energy intake,
however a larger intake depression would be expected with the large reduction in energy
required from milk fat production. Increased BW gain and the failure to regulate energy
homeostasis may be because of metabolite balance. Intake is regulated by the type and
temporal pattern of fuels available (Allen, 2000) and the interaction of available fuels and
metabolic state. Mammary lipogenic enzyme concentration is decreased during milk fat
depression (Peterson et al., 2003) leading to decreased de novo synthesis of milk fat and
decreased metabolic use of acetate. Mammary nutrient use of other metabolites,

especially gluconeogenic metabolites, is relatively unaffected.

160

 

Neither acetate nor glucose spared by utilization of the acetate as an energy
source are directly oxidized in the ruminant liver to generate a satiety signal (Allen,
2000). Decreasing intake would only decrease availability of propionate and protein
needed for milk production. Partitioning of excess acetate to adipose tissue without
negative feedback allows maintenance of protein and gluconeogenic nutrient intake.
Cows in this experiment were lower in body condition score (mean 1.9) and
replenishment of body energy reserves may have created an acetate demand that limited
accumulation of circulating acetate. We speculate that the extent to which milk fat
depression results in decreased feed intake depends upon gluconeogenic nutrient
absorption from the diet, gluconeogenic metabolite demand by tissues, and the extent to
which acetate spares gluconeogenic metabolites. It is reasonable to speculate that UNS
failed to regulate energy homeostasis because the positive energy balance is excess
acetate, and not excess of a balance of metabolites.

We reported no effect of treatment on intake of eight non-cannulated cows fed the
same diet (Chapter 3), although these cows differed greatly in body condition (~1 BCS
heavier) and did not experience milk fat depression. Differences in biohydrogenation or
metabolic or physiologic response to trans-FA and CLA related to greater body condition
may explain variation in intake response to dietary FA reported in the literature and
between cow groups.

Direct effects of FA saturation on intake are complicated to determine because of
the multiple effects of FA on physiology and metabolism. Fatty acids are recognized as
powerful physiologic modiﬁers affecting endocrine signaling and gene regulation

(Drackley, 2000). These modiﬁcations result in production and nutrient partitioning

161

 

changes, as observed in CLA mediated milk fat depression (Bauman and Griinari, 2003).
Intake is regulated by the effect of type and temporal variation of absorbed fuels (Allen,
2000) and animal metabolic state. The role of FA in regulation of intake is complicated

by the dual roles of FA as fuels and metabolic modiﬁers.

Rumen pools

Increasing unsaturated FA concentration linearly decreased wet weight of rumen
digesta and rumen digesta volume (Table 3). Addition of RPF decreased wet weight and
volume of rumen contents because of a linear decrease by UNS. Treatments did not affect
rumen DM percent but RPF decreased pool size of DM, OM and NDF; DM and OM pool
sizes tended to decrease, and NDF pool size decreased with increasing UNS. Rumen
pools decreased in proportion to intake resulting in no difference in rumen nutrient
turnover of DM, OM or NDF (Chapter 4). The observation that UNS decreased DM1 and
rumen digesta OM pool without affecting turnover of OM provides strong evidence that
the intake depression is not an intestinal brake that slows passage rate leading to
increased digesta pool size and increased distension in the reticulorumen may have
affected intake if physiological response to FA saturation altered the response threshold

in the central satiety center.

Feeding and chewing behavior
Intake is determined by the number and size of meals consumed over a day.
Treatment did not affect total time spent eating or the number of meals, but UNS linearly

decreased meal size by 0.22 kg (P < 0.03; Table 4). There were no treatment effects

162

 

detected for meal length, meal frequency or intermeal interval. Unsaturated FA linearly
increased eating rate. Hunger ratio (meal size over premeal interval) was quadratically
affected by UNS but satiety ratio (meal size over postmeal interval) was not changed.
Addition of RPF and FA saturation had no effect on chewing time per meal, or per kg of
DMI, NDF or forage NDF. The reduction in feed intake by UNS treatment was because
of decreased meal size, which implicates greater stimulation of satiety with UNS.
Heirichs et al. (1982) observed smaller initial meal but increased number of spontaneous
meals with increased FA concentration.

SAT increased time spent ruminating compared to CON and UNS. SAT linearly
increased time spent ruminating up to 50 min per (1 (P < 0.01). This increase in

rumination time was because SAT linearly increased rumination bout length because

there were no treatment effects on number of rumination bouts or interval between bouts.

SAT linearly increased rumination chewing time per bout, and quadratically affected
rumination time per kg of DMI, NDF, and forage NDF. Allen (1997) reported that total
chewing time per day was not related to DMI across treatment means reported in the
literature. In this study, the increased rumination for SAT was not because of increased
DMI because rumination per kg of DM and ﬁber intake was quadratically affected by
SAT. Benson and Reynolds (2001) were unable to detect signiﬁcant differences in
rumination behavior with rapeseed infusion despite large decreases in time spent
ruminating for some individual cows. We have previously observed that SAT increased
time spent ruminating but did not change time spent eating compare to calcium salts of
palm oil when observed by manual observation every ﬁve minutes (Chapter 2). In the

current experiment, the linear increase in rumination with increasing SAT appears to be

163

 

 

because of increased duodenal ﬂow of saturated FA because SAT increased rumination
compared to CON but UNS did not.

Total time spent chewing linearly decreased with SAT, but was not affected by
RPF. Similar to rumination, total time spent chewing per kg of DM1 and NDF were
quadratically affected by SAT. There were no effects of treatment on rumination or
chewing rate, showing that changes in chewing time relate directly to number of chews.
Number of eating and ruminating chews per day followed similar patterns to the time
spent in these activities previously reported. There was no treatment effect on total
eating chews per day but increasing SAT linearly increased total rumination chews per
day.

The changes in rumination behavior may be mediated through feed intake
differences, associative effects on digestion, or stimulation of gut peptides. As
previously mentioned, intake differences cannot explain the changes in chewing behavior
because time spent ruminating per kg of NDF was affected by treatment. In addition,
treatment diets differed only in FA concentration and proﬁle and contained the same base
ration. Diets were not expected to differ in effectiveness of stimulating rumination;
chewing activity is more highly related to forage NDF concentration than total NDF
concentration (Allen, 1997), and forage NDF concentration was the same across
treatments. It is unlikely that increased rumination for SAT was because of increased
distention in the reticulorumen because digesta pool size and volume was numerically
lower for SAT compared to CON.

We have previously proposed that time spent ruminating may be related to

reticular-rumen motility (Chapter 2). Deswysen et al. (1987) reported a strong positive

164

 

 

relationship between the number of rumen contractions and rumination time. We
speculate that less rumination for the UNS treatment compared to SAT is associated with
reduced ruminal motility related to duodenal FA ﬂow. Nicholson and Omer (1983)
showed intestinal infusion of unsaturated FA decreased rumen motility of sheep, and
Grovum (1984) reported almost total cessation of rumen motility after 13 h of intragastric
infusion of unsaturated fat compared to intravenous infusion. UNS increased plasma
concentrations of the gut peptides Cholecystokinin (CCK; Choi and Palmquist, 1996),
and glucagon-lik-peptide-l (GLP—l; Benson and Reynolds, 2001), and direct intravenous
infusions of CCK depressed reticular-rumen motility and intake in sheep (Grovum,
1981). These gut peptide are normally secreted in response to FA ingestion and effect
gut motility (Reidelberger, 1994; and Hellstom and Niislund, 2001). Most experiments
testing stimulation of gut peptide secretion employed PUFA treatments and no fat
controls. The effect of saturated FA on gut peptide secretion and gut motility is not
understood and the mechanism for the increase in rumination time for SAT compared to

CON is unknown.

Nutrient Selection

CON treatment resulted in selection against NDF and total FA and for starch
compared to RPF treatments. The CON diet contained lower total FA concentration and
slightly higher NDF concentration due to inclusion of rice bulls in the space of RPF.
Selection against both NDF and FA for the CON treatment might be explained by
selection against cottonseeds, which are a source of both nutrients. UNS linearly

increased selection against GE and FA. This might be because of palatability differences

165

 

 

in the fat sources, although it is not consistent with the linear increase in eating rate with
increasing UNS. An alternative explanation is that selection against GE concentration
was because of decreased energy requirements experienced with UNS because of milk fat
depression. Differences in selection were very small but highly signiﬁcant. Small
differences may be expected because feeding to 15% orts limits selection in the amount
of each nutrient available, and large difference in ort composition is needed to change the

composition of diet consumed.

Ruminal pH

Daily mean, minimum and maximum pH were not changed by treatment, but pH
range and variance were linearly increased by SAT (Table 5). UNS range and variance
was not different from CON, but SAT increased range and variance. Increased range and
variance for SAT compared to UNS may reﬂect the larger meal size observed with SAT;
increasing meal size increases fermentable organic matter per meal, increasing VFA
production and acid load after the meal. However, meal size was not different for SAT
and CON and we are unable to explain the increased range and variance in ruminal pH

for SAT compared to CON.

CONCLUSION
Increasing dietary energy density with rumen-protected fat may not increase
digestible energy intake. Direct and indirect hypophagic effects of unsaturated FA are
not separable as FA and fuel type modify short and long-term physiology. The reduction

in feed intake from rumen-protected FA that are unsaturated is through decreased meal

166

 

 

 

size without an increase in meal frequency. Addition of saturated FA increases
rumination bout length and rumination time per day, possibly through modiﬁcation of gut

peptide secretion.

167

 

Table 1. Effects of dietary rumen-protected fatty acids on duodenal fatty acid ﬂow.

 

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q

Duodenal Flow, g/d

Cl6:0 290 440 430 350 30 <0.001 <0.001 0.007 0.29

C1820 580 880 680 470 53 <0.0001 0.07 <0.0001 0.89

C18:1 trans 160 170 280 260 24 <0.001 0.003 0.001 0.02

C18:1 cis 75 84 100 100 6.3 <0.001 <0.001 0.003 0.15

C18:2 94 100 99 97 6.6 0.57 0.28 0.37 0.99

C18:3 7.4 7.8 7.6 6.6 0.4 0.16 0.87 0.04 0.41
Duodenal Composition, % Total FA

C16:0 20.4 22.3 22.8 23.4 0.33 <0.0001 <0.0001 0.03 0.88

C1820 41.1 44.6 36.7 30.9 1.4 <0.0001 0.03 <0.0001 0.53

C18:1 trans 11.1 8.5 15.0 18.2 1.4 0.0001 0.07 <0.0001 0.31

C18:1 cis 5.2 4.2 5.4 6.9 0.16 <0.0001 0.11 <0.0001 0.47

C 18:2 6.7 5.1 5.3 6.4 0.28 <0.001 0.003 0.002 0.15

C18:3 0.53 0.39 0.41 0.47 0.02 0.001 <0.001 0.02 0.39

 

1 Treatments were CON- control with no supplemental rumen-protected fat, SAT-

saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.
2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

168

__-.

 

Table 2. Effects of dietary rumen-protected fatty acids on intake of nutrients.

 

 

Intake, kg/d
DM
OM
NDF
Starch
FA
DE’, Meal/d

 

 

Treatment LS Means ' P 2
CON SAT INT UNS SE TRT RPF L Q
27.3 25.7 25.1 24.1 1.0 0.001 <0.001 0.02 0.60
24.4 23.0 22.6 21.6 0.9 0.001 <0.001 0.02 0.61
7.3 6.7 6.6 6.3 0.25 <0.001 <0.001 0.09 0.52
8.2 7.4 7.3 7.0 0.3 <0.001 <0.001 0.04 0.64
1.5 2.1 2.0 1.8 0.07 <0.0001 <0.0001 <0.0001 0.68
78.0 75.3 74.0 70.1 3.1 0.07 0.04 0.06 0.59

 

' Treatments were CON- control with no supplemental rumen-protected fat, SAT-
saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCF A, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF : fat supplement effect, L: linear effect, and Q: quadratic

effect.

Table 3. Effects dietary rumen-protected fatty acids on ruminal nutrient pool.

 

Ruminal wet
Contents, kg

Ruminal contents
Volume, L

Ruminal contents,
%DM

Ruminal pool, kg
DM
OM
NDF

 

 

Treatment LS Means ' P 2
CON SAT INT UNS SE TRT RPF L Q
86.6 85.7 80.7 74.4 3.7 0.041 <0.10 0.02 0.87
101 97 93 89 4 0.06 0.04 0.05 0.94
16.9 17.1 17.4 16.9 0.4 0.72 0.62 0.73 0.35
14.7 13.7 14.0 12.5 0.6 0.02 0.02 0.09 0.12
13.5 12.7 12.9 11.6 0.5 0.02 0.03 0.09 0.10
7.9 7.5 7.4 6.5 0.3 <0.01 0.02 0.01 0.16

 

1 Treatments were CON- control with no supplemental rumen-protected fat, SAT-
saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

169

 

 

Table 4. Effects of dietary rumen-protected fatty acids on meal patterns and water
consumption.

 

Treatment LS Meansl [9 2

 

 

CON SAT INT UNS SE TRT RPF L Q

 

Meals
Bouts / d 10.1 10.2 10.0 10.6 0.6 0.585 0.76 0.37 0.32
Length, min/meal 30.5 29.5 30.1 26.9 2.0 0.30 0.41 0.19 0.27
Interval, min 104 l 11 109 106 7 0.75 0.51 0.80 0.91
Eating rate, kg
DM/min 0.089 0.085 0.086 0.095 0.006 0.16 0.98 0.04 0.33
Meal size, kg
DM 2.51 2.50 2.54 2.28 0.15 0.04 0.48 0.03 0.07
Total FA

Hunger ratio}, kg/min 0.057 0.051 0.058 0.046 0.007 0.15 0.42 0.34 0.05
Satiety ratio3, kg/min 0.056 0.053 0.059 0.051 0.008 0.66 0.81 0.73 0.25

Rumination
Bouts /d 13.3 13.0 13.6 13.5 0.5 0.55 0.88 0.25 0.41
Bout length, min 44.2 48.1 43.0 42.5 1.6 0.01 0.84 <0.01 0.10
Bout interval, min 63.9 59.8 59.2 62.2 3.6 0.55 0.30 0.47 0.52
Ruminating chew
rate, chews/min 63.9 64.9 64.9 63.9 1.5 0.47 0.46 0.23 0.49
Water drunk, L/d 105.9 99.6 99.4 100.4 3.7 0.20 0.04 0.81 0.82
Drinking bouts /d 12.6 9.9 11.3 12.8 1.2 0.02 0.12 0.007 0.53

 

' Treatments were CON- control with no supplemental rumen-protected fat, SAT-
saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS— unsaturated fat fed as Ca soaps of LCFA.
2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.
3 Hunger ratio = meal kg DM / premeal interval.

Satiety ratio = meal kg DM / postmeal interval.

170

 

Table 5. Effects of dietary rumen-protected fatty acids on chewing behavior.

 

 

 

Treatment LS Meansl P 2
CON SAT INT UNS SE TRT RPF L Q

Eating chewing Time, min

/d 257 257 253 246 10.6 0.65 0.61 0.26 0.86

/bout 25.3 25.6 25.6 23.2 1.6 0.33 0.74 0.12 0.35

/kg of DM1 10.1 10.7 10.4 10.6 0.6 0.41 0.18 0.82 0.30

/kg of NDF intake 37 39 38 39 2.4 0.53 0.29 0.65 0.36

/kg of forage NDF

intake 62 65 63 65 4.0 0.37 0.16 0.79 0.28
Ruminating chewing time, min

/d 574 616 568 560 15 0.02 0.65 0.004 0.16

/bout 43.5 47.6 42.1 41.6 1.6 <0.01 0.84 <0.01 0.08

/kg of DM1 23 25 23 24 0.9 0.01 0.04 0.08 0.02

/kg of NDF intake 83 93 85 87 3.2 <0.01 0.04 0.03 0.02

/kg of forage NDF

intake 138 154 141 147 5.5 0.01 0.03 0.08 0.02
Total chewing time, min

/d 833 872 822 805 22 0.08 0.98 0.01 0.41

/kg of DM1 18.2 20.0 18.6 19.2 0.8 0.02 0.04 0.17 0.03

/kg of NDF intake 120 132 122 125 5.4 0.02 0.06 0.07 0.04
Chew Rate

Eating 83.4 81.8 85.9 83.0 3.0 0.44 0.97 0.66 O. 12

Ruminating 64.0 64.9 64.9 63 .9 1.5 0.47 0.46 0.23 0.49
Chews, # / (1

Eating 21500 21200 22000 20600 1500 0.74 0.85 0.66 0.33

Ruminating 37100 40200 37500 36200 1500 0.04 0.53 <0.01 0.51

Total 58800 61200 59400 56700 2400 0.31 0.13 0.07 0.83

 

‘ Treatments were CON- control with no supplemental rumen-protected fat, SAT-
saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic

effect.

171

Table 6. Effects of dietary rumen-protected fatty acids on nutrient selection.

 

Treatment LS Meansl P 2
CON SAT INT UNS SE TRT RPF L Q

 

 

Cannulated
NDF Selection3 95.6 98.8 99.3 99.2 1.4 0.08 0.01 0.80 0.81
INDF Selection3 99.2 100.9 101.2 99.0 1.5 0.37 0.37 0.23 0.34
Starch Selction3 102.1 99.0 99.0 99.8 1.2 0.08 0.01 0.54 0.74
CP Selection3 100.4 100.2 100.6 100.3 0.29 0.77 0.90 0.89 0.31
GE Selection3 99.6 99.9 99.8 99.6 0.08 0.009 0.12 0.003 0.33
TFA Selection3 99.4 101.3 100.9 101.7 0.68 0.10 0.02 0.63 0.43

 

I Treatments were CON- control with no supplemental rumen-protected fat, SAT-
saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.

3Selection: (nutrient concentration consumed / nutrient concentration fed) x 100

 

Table 7. Effects of dietary rumen-protected fatty acids on ruminal pH.

 

 

 

 

Treatment LS MeansI P 2

Daily ruminal pH CON SAT INT UNS SE TRT RPF L Q

Mean 5.97 5.99 6.05 6.01 0.06 0.76 0.50 0.74 0.46
Minimum 5.42 5.34 5.42 5.42 0.05 0.61 0.58 0.29 0.50
Maximum 6.66 6.66 6.67 6.56 0.06 0.33 0.29 0.23 0.35
Range 1.13 1.31 1.25 1.15 0.05 0.02 0.03 <0.01 0.62
Variance 0.1 1 0.14 0.13 0.1 0.01 0.01 <O.1 0.005 0.46
Standard deviation 0.32 0.37 0.35 0.31 0.02 0.01 0.08 0.005 0.50
Time < 6.0, h 12.3 11.5 9.9 10.9 1.3 0.53 0.30 0.72 0.35
Area below 6.0, h 497 470 376 380 73 0.51 0.30 0.38 0.56

 

1 Treatments were CON- control with no supplemental rumen-protected fat, SAT-
saturated fat from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS— unsaturated fat fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.

 

172

CHAPTER 7

CONCLUSIONS AND IMPLICATIONS

Fatty acids (FA) are bioactive compounds that have signiﬁcant effects on animal
physiology and metabolism. Commonly fed FA sources can be used to alter absorbed
FA, feed intake, milk fat synthesis, and energy partitioning. This research showed that
FA proﬁle reaching the duodenum is important in determining intake response to fat
supplementation.

The ﬁrst experiment demonstrated that the decrease in feed intake from rumen-
protected palm oil was not related to milk yield across cows. Calcium salts of palm oil
did not induce milk fat depression and is expected to have increased duodenal ﬂow of
unsaturated FA. Saturation of FA is an important determinant of insulin secretion and
plasma NEF A concentration. Saturated FA increased milk protein and the magnitude of
the response appears to be related to production level, or insulin signaling.

Dietary unsaturated FA may have a large impact on milk fat yield and body
weight gain, depending on production of trans-FA isomers in the rumen. Absorbed fatty
acid proﬁle may a have larger effect on energy balance than does the amount of FA
absorbed because fatty acids can drastically modify production. Milk fat depression
induced by biohydrogenation intermediates decreases milk fat production through
decreased de novo synthesis, reducing the concentration of short and medium chain fatty

acids. Increased body weight gain during milk fat depression may be due to increased

173

 

 

 

availability of acetate while intake is partially maintained to sustain protein and
gluconeogenic metabolite homeostasis.

Prediction of the proﬁle of duodenal FA ﬂow requires a greater understanding of
rumen FA metabolism. Calculation of individual FA biohydrogenation with a simpliﬁed
model allows a more mechanistic description of rumen FA biohydrogenation. Future
ability to predict duodenal FA proﬁle depends on observation and analysis of individual
steps of FA metabolism. Modeling biological systems requires simpliﬁcation and
assumption of unknown or undeterminable events. Assumptions for this model must be
tested in the future. More complex models may be developed to model duodenal ﬂow of
individual trans isomers, although ﬂux through such pathways may require isotope
labeling. Calcium soaps provide limited protection of polyunsaturated FA in the rumen.
Polyunsaturated FA are highly biohydrogenated, but trans-monounsaturated FA
biohydrogenation is limited especially with increased levels of dietary polyunsaturated
FA.

Increased rumination with saturated FA might alter rumen nutrient passage rate,
increasing passage of more highly digestible particles. Increasing passage rate of highly
digestible particle decreases ruminal OM and NDF digestibility due to selective retention
of less digestible feed particle. The impact of rumination on digestion and passage
kinetics has not been explored. Changing the amount of rumination and gut motility may
be very applicable to lower ﬁber diets containing less effective ﬁber. Increasing FA
intake and saturation together decrease FA digestibility, although the effects are
confounded. Fatty acid digestibility may be modiﬁed by associative effects of other FA

and duodenal OM composition and the amount of duodenal ﬂow. Addition of rumen-

174

 

 

 

protected FA may not increase energy intake because of associative effects on ruminal
digestion and decreased DM intake. More controlled experiments of FA digestibility are
needed to titrate the effect of duodenal FA ﬂow and associative effects of digesta on FA
digestibility.

Increasing dietary energy density with rumen-protected fat may not increase
digestible energy intake. Direct and indirect hypophagic effects of unsaturated FA are
not separable because FA are an energy source and modify physiology. The reduction in
feed intake from unsaturated rumen-protected FA is through decreased meal size without
an increase in meal frequency. Addition of saturated FA increases rumination bout
length and rumination time per day, possibly through modiﬁcation of gut peptide
secretion. Stimulation of gut peptide secretion may be tested with duodenal challenges of
different FA to provide a clearer understanding of FA proﬁle on gut peptides without

confoundation of FA intake, biohydrogenation and rumen FA activity.

 

Fatty acids play a much larger role in animal nutrition than simply an energy
source. As Storlein (2000) observed “Oils ain’t oils”. Fatty acids provide an opportunity
to modify ruminal digestion, rumination, milk component yield, body weight gain and
intake through manipulation of a small portion of the diet. It is expected that many of
these effects are mediated by very small concentrations of a FA or FA isomers. Future
research should allow identiﬁcation of effects of individual FA and an understanding of
their mechanism of action. The experiments presented provide strong preliminary
evidence for further research in a number of areas. Saturated FA have large affects on
rumination that has not been previously observed. The effect of saturated FA on

rumination and gut motility should be examined independently of intake with abomasal

175

 

infusion of different FA at the same intake. The effect of fat saturation on gut peptide
secretion should also be examined using abomasal infusion challenges of different FA.
The role of saturated FA in plasma insulin concentration also deserves further research to
determine if plasma concentrations rise because of increased insulin secretion or
decreased clearance. The biohydrogenation model proposed may also provide a
framework to understand associative dietary and rumen environment effects on
biohydrogenation. Lastly, the effect of C18:1 FA isomers on intake and feeding behavior

should be examined.

176

 

APPENDIX

Table 1. Effects of dietary rumen-protected fatty acids on fatty acid intake of non-
cannulated cows

 

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q
Intake, g/d
Cl6:0 270 500 470 450 17 <0.001 <0.001 0.01 0.62
C1820 40 390 220 60 ll <0.001 <0.001 <0.001 0.63
C18:1 240 290 340 400 12 <0.001 <0.001 <0.001 0.66
C18:2 680 690 710 770 26 0.004 0.02 0.002 0.47

C18:3 4O 41 43 47 1.5 0.001 0.02 <0.001 0.56 a

l Treatments were CON- control with no supplemental rumen-inert fat, SAT- saturated
fat from prilled FA, INT- intermediate saturation as a mix of prilled FA and calcium
soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.

 

 

177

Table 2. Effects of dietary rumen protected fatty acids on ruminal pool variance.

 

 

 

Treatment LS Meansl P2
CON SAT INT UNS SE TRT RPF L Q
Before v After Feeding Rumen Proﬁle, Before / After
Total FA 0.82 0.83 0.70 0.72 0.05 0.11 0.22 0.09 0.14
C16:0 0.83 0.83 0.69 0.71 0.05 0.06 0.13 0.06 0.14
C18:0 0.95 0.90 0.85 0.92 0.05 0.32 0.17 0.76 0.23
C18:1 trans 0.84 1.05 0.95 0.88 0.09 0.37 0.24 0.19 0.90
C18:1 cis 0.67 0.74 0.53 0.54 0.08 0.17 0.45 0.08 0.23
C18:2 0.66 0.73 0.51 0.54 0.10 0.30 0.52 0.16 0.24
C18:3 0.48 0.65 0.44 0.47 0.10 0.30 0.66 0.14 0.23

 

' Treatments were CON- control with no supplemental rumen-protected FA, SAT-
saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCF A.

2 TRT: treatment effect, RPF: fat supplement effect, L: linear effect, and Q: quadratic
effect.

Table 3. Effects of dietary rumen-protected fatty acids on ﬂux of ruminal fatty acid
disappearance.

 

 

 

Treatment LS MeansI P2
CON SAT INT UNS SE TRT RPF L Q
Disappearance Flux (g/d)
C18:1 680 630 600 570 29 0.02 0.009 0.07 0.99
C18:1 trans 740 660 590 540 38 0.002 0.001 0.01 0.87
C18:1 cis 160 180 230 240 8 <0.001 <0.001 <0.001 0.07
C18:2 560 540 620 620 25 0.03 0.005 0.02 0.01
C18:3 33 31 38 40 1.4 <0.001 0.007 <0.001 <0.001

 

' Treatments were CON- control with no supplemental rumen-protected FA, SAT-
saturated FA from prilled FA, INT- intermediate saturation as a mix of prilled FA and
calcium soaps of LCFA, and UNS- unsaturated fat fed as Ca soaps of LCFA.

2 TRT: treatment effect, Fat: fat supplement effect, L: linear effect, and Q: quadratic
effect.

178

 

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