EFFECTS OF METABOLIC FUELS ON FEEDING BEHAVIOR AND DRY MATTER
INTAKE OF DAIRY COWS IN THE POSTPARTUM PERIOD
By
Laura Bibiana Gualdrón-Duarte

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Animal Science – Doctor of Philosophy
2017

ABSTRACT
EFFECTS OF METABOLIC FUELS ON FEEDING BEHAVIOR AND DRY MATTER
INTAKE OF DAIRY COWS IN THE POSTPARTUM PERIOD
By
Laura Bibiana Gualdrón-Duarte
Feeding behavior (FB) and dry matter intake (DMI) of ruminants has been linked to
hepatic oxidation of fuels. The type and temporal pattern of fuels absorbed from the
gastrointestinal tract of ruminants and available for oxidation are affected by diet starch
concentration and fermentability. However, research considering the fuels produced from starch
digestion is lacking for cows in the postpartum (PP) period. Our overall hypothesis is that fuels
produced during digestion affect hepatic metabolism and FB differently. To investigate the effect
of anaplerosis of the tricarboxylic acid (TCA) cycle on FB, experiments were conducted using
cows in PP period. Propionic acid (PA, 99.5%) and glycerol (GL, 99.7%) were infused into the
abomasum (AB) in isoenergetic (193 kcal/h) and isomolar (~0.5 mol/h) solutions using a double
crossover design. Infusion of PA decreased DMI compared with GL 17 and 23%, by decreasing
meal size 13 and 15% with isoenergetic and isomolar infusions respectively, and PA decreased
total metabolizable energy intake (MEI, diet plus treatment infusions) compared with GL.
Compared with PA, GL increased plasma glucose and insulin concentrations for isomolar
infusions. Pulse-dose (2 moles) of PA into the AB in a crossover design decreased hepatic acetyl
CoA (AcCoA) content whereas GL increased 32%, resulting in lower content for PA compared
with GL at 30 min (18.0 vs. 36.9 nmol/g), which persisted at 60 min after dosing (21.9 vs. 32.8
nmol/g). These results are consistent with anaplerosis from PA stimulating oxidation of AcCoA
in the liver and satiety compared with GL.
To investigate the effect of fuels from starch digestion on FB, continuous isoenergetic

AB infusions (150 kcal/h) of PA (99.5%, 0.41 mol/h), lactic acid (LA, 88.0%, 0.46 mol/h) or
glucose (GLU, 99.9%, 0.22 mol/h) were studied with cows in PP period in a duplicated 4 x 4
Latin square design. A control (CON, no infusion) treatment was also included. Treatment
solutions were infused at 500 mL/h for 22 h/d and provided ~3.3 Mcal/d. Compared with CON,
PA decreased DMI by 24% (14.3 vs. 18.9 kg/d) and MEI by 13% (34.8 vs. 40.2 Mcal/d) by a
tendency to decrease meal frequency; and LA decreased DMI by 14% (16.3 vs. 18.9 kg/d) by
decreasing meal size 20%, but did not affect MEI. Glucose infusion did not affect DMI or MEI.
While ruminal infusions of sodium acetate have little effect on DMI, infusion of acetic
acid (AA) into the AB of cows in the PP period decreased DMI 64% compared with a sham
control (Gualdrón-Duarte and Allen, unpublished). To understand the mechanism, we
investigated the effects of continuous isomolar infusions (~0.75 mol/h; 0.5 L/h; 8 h) of AA,
sodium acetate (NA) or CON (NaCl) into the rumen (RU) or into the AB on FB, DMI, and
metabolic responses of cows in the PP period in a 6 x 6 Latin square design. Treatments were
infused for the first 8 h following feeding with a rest day between infusion days. During
infusions treatments decreased DMI 30% (4.3 kg vs. 6.1 kg) compared with CON by decreasing
meal frequency but their effects differed; NA-RU did not reduce DMI (7.0 kg), whereas AA-RU
(2.6 kg), AA-AB (3.7 kg) and NA-AB (4.0 kg) decreased DMI compared with CON. Contrary to
the other treatments, NA-RU did not decrease DMI compared with CON, but the reason cannot
be determined from the results available from this study.
Collectively, these experiments provide strong evidence that anaplerotic metabolites for
the TCA cycle decreased DMI and MEI on cows in the PP period consistent with their expected
effects on hepatic oxidation. Depression of feed intake by diets including highly fermentable
starch is likely because of the ability of propionate to stimulate hepatic oxidation.

A mis padres Elsa y Luis, y mis hermanos
José Luis y Jorge Eduardo por ser todo en mi vida.
Y a mi abuelita y Lina, las dos nuevas estrellas
guiándome desde el cielo.

iv

ACKNOWLEDGEMENTS

I would like to say “thank you”:
To my advisor Dr. Michael Allen for his guidance, patience and all the support that has
helped me to improve during the past five years to become a better researcher and scientist. I will
always be grateful for the opportunity he has given me to work in his team which allowed me to
work with wonderful and knowledgeable people especially the technicians, graduate and
undergraduate students.
To my graduate committee members, Dr. Thomas Herdt, Dr. Richard Ehrhardt, Dr. David
Beede and Dr. Michael VandeHaar for their guidance and for helping me during my time at
Michigan State University.
To everyone at the Dairy Cattle Teaching and Research Center, particularly Bob Kreft,
Rob West and especially to Randy, Jake, Fred, Bob, Bruce. Thank you for being my farm-mates
daily for almost one year and then a couple times again.
To Dave Main and Dewey Longuski, from Allen lab, for always having my back. To Jim
Liesman for his time, patience and always lending a keen ear to hear me talk about my home
country. To my lab and office members Paola P., Sarah S., Bill B., Sarah B., Jackie B., Sarah S.,
Katie K., Gabriela M., Rodrigo A., Roni Y and Danilo S who were always ready to help.
To K.C. Main and Dave Main (Main team) without whose help, I wouldn’t have been
able to complete my longest and hardest experiments.
To Karen Beede and Dr. Beede for all your support, love and warmth, especially when I
was new here. It was really meaningful to me.
To all my latino friends (including their significant others). My time here with all of you

v

is something that I will cherish forever. You all brought the Latino flavor that made life in
Michigan truly wonderful.
To my roommates, Juan, Eduardo, Andrea, and especially Norma. And to my little sister
Yan Sun. They became my new family.
To all those who don’t speak English: Ustedes, mi familia y amigos de Colombia fueron
lo más importante y lo que me mantuvo en pie y no me dejo desistir ante las muchas veces tristes
y duras circunstancias que me rodearon. Todos ustedes son muy importantes en mi vida. Los
quiero con todo mi corazón, “primates”, todo el “Clan de las Duarte” y su descendencia, familia
Gualdrón, familia Acero-Camelo y Bernal-Acero, familia Rueda-Uribe, Ximena, Nidia, Laila,
Laurens y a mi Danielito y Samuel.

vi

TABLE OF CONTENTS

LIST OF TABLES ...................................................................................................................... ix
LIST OF FIGURES .................................................................................................................... xi
KEY TO ABBREVIATIONS .................................................................................................... xii
INTRODUCTION ....................................................................................................................... 1
CHAPTER 1. LITERATURE REVIEW ........................................................................................ 4
FEED INTAKE AND FEEDING BEHAVIOR .......................................................................... 4
CONTROL OF FEED INTAKE .................................................................................................. 5
Physical regulation .................................................................................................................. 5
Osmotic regulation .................................................................................................................. 6
Metabolic regulation ................................................................................................................ 7
Endocrine regulation ............................................................................................................ 8
Hepatic control of feed intake ............................................................................................ 10
HEPATIC METABOLISM OF FUELS .................................................................................... 12
HEPATIC CONTROL OF INTAKE IN DAIRY COWS ......................................................... 13
POSTPARTUM COWS............................................................................................................. 16
Fatty acids oxidation .............................................................................................................. 16
Gluconeogenic precursors and effect on DMI ....................................................................... 18
Amino acids ....................................................................................................................... 18
Glycerol.............................................................................................................................. 19
Lactate ................................................................................................................................ 21
Propionic acid .................................................................................................................... 22
STARCH FERMENTABILITY AND EFFECT ON FEED INTAKE IN THE POSTPARTUM
PERIOD ..................................................................................................................................... 24
BIBLIOGRAPHY ...................................................................................................................... 28
CHAPTER 2. INCREASED ANAPLEROSIS OF THE TRICARBOXYLIC ACID CYCLE
DECREASED MEAL SIZE AND ENERGY INTAKE OF COWS IN THE POSTPARTUM
PERIOD ........................................................................................................................................ 37
CHAPTER 3. FUELS DERIVED FROM STARCH DIGESTION HAVE DIFFERENT
EFFECTS ON ENERGY INTAKE AND METABOLIC RESPONSES OF COWS IN THE
POSTPARTUM PERIOD ............................................................................................................. 38
ABSTRACT ............................................................................................................................... 38
INTRODUCTION ..................................................................................................................... 39
MATERIALS AND METHODS ............................................................................................... 40
Animals, Housing, and Care .................................................................................................. 40
Experimental Design, Diets and Treatments ......................................................................... 40
Data and Sample Collection .................................................................................................. 42
vii

Analysis of Samples .............................................................................................................. 43
RNA Extraction and Real-Time Quantitative PCR ............................................................... 45
Statistical Analysis ................................................................................................................ 45
RESULTS .................................................................................................................................. 46
Feed Intake and Feeding Behavior ........................................................................................ 46
Milk Production and Components ......................................................................................... 48
Hepatic Acetyl CoA content and Plasma Hormones and Metabolites .................................. 48
Hepatic gene expression ........................................................................................................ 52
DISCUSSION ............................................................................................................................ 52
CONCLUSION .......................................................................................................................... 57
ACNOWLEDGEMENTS .......................................................................................................... 57
APPENDICES ........................................................................................................................... 58
APPENDIX A: GENE EXPRESSION ...................................................................................... 59
APPENDIX B: BLOOD METABOLITES ............................................................................... 68
BIBLIOGRAPHY ...................................................................................................................... 69
CHAPTER 4. EFFECTS OF ACETIC ACID OR SODIUM ACETATE INFUSED INTO THE
RUMEN OR ABOMASUM ON FEEDING BEHAVIOR AND METABOLIC RESPONSE OF
COWS IN THE POSTPARTUM PERIOD .................................................................................. 74
ABSTRACT ............................................................................................................................... 74
INTRODUCTION ..................................................................................................................... 75
MATERIALS AND METHODS ............................................................................................... 76
Animals, and Care ................................................................................................................. 76
Experimental Design and Treatment Diets ............................................................................ 77
Sample and Data Collection .................................................................................................. 78
Analysis of Samples .............................................................................................................. 79
Statistical Analysis ................................................................................................................ 80
RESULTS .................................................................................................................................. 82
Feed Intake and Feeding Behavior ........................................................................................ 82
Milk Production and Components ......................................................................................... 86
Blood Metabolites and Insulin Concentration ....................................................................... 87
Blood Chemistry .................................................................................................................... 87
DISCUSSION ............................................................................................................................ 89
CONCLUSION .......................................................................................................................... 95
ACKNOWLEDGEMENTS ....................................................................................................... 95
APPENDIX ................................................................................................................................ 96
CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS .............................................. 112
BIBLIOGRAPHY .................................................................................................................... 115

viii

LIST OF TABLES

Table 3.1. Ingredients and nutrient composition of diets fed during blocks 1 and 2 ……………42
Table 3.2. Effects of isoenergetic1 intra-abomasal infusion of glucose (GLU), lactic acid (LA)
and propionic acid (PA) relative to a no infusion control (CON) on feeding behavior and
energy intake for cows in the immediate postpartum period ............................................ 47
Table 3.3. Effects of intra-abomasal infusion of propionic acid (PA), lactic acid (LA) and
glucose (GLU) relative to a control (CON) with no infusion on milk yield and milk
components over the 22 h/d infusion for cows in the immediate postpartum period for
isoenergetic infusions1 ...................................................................................................... 48
Table 3.4. Effect of intra-abomasal infusion of propionic acid (PA), lactic acid (LA) and glucose
(GLU) relative to a control (CON) with no infusion on initial, final and change in plasma
concentration of insulin, glucagon, metabolites and liver acetyl CoA (AcCoA) content
over the 22 h/d infusion for cows in the immediate postpartum period for isoenergetic
infusions1........................................................................................................................... 50
Table 3.5. Primer sequences and accession numbers for genes analyzed with real-time
quantitative PCR ............................................................................................................... 62
Table 3.6. Effect of isoenergetic1 intra-abomasal infusion of glucose (GLU), lactic acid (LA) and
propionic acid (PA) and a no infusion control (CON) on least square means of the CT
differences after infusion relative to before infusion2 on hepatic gene expression for cows
in the immediate postpartum period ................................................................................. 64
Table 3.7. Fold changes (FC)1 on hepatic genes affected by isoenergetic2 intra-abomasal infusion
of glucose (GLU), lactic acid (LA) and propionic acid (PA) relative to a no infusion
control (CON) for cows in the immediate postpartum period .......................................... 65
Table 3.8. Confidence intervals1 of hepatic gene expression fold changes (FC) in genes affected
by isoenergetic2 intra-abomasal infusion of glucose (GLU), lactic acid (LA) and
propionic acid (PA) relative to a no infusion control (CON) for cows in the immediate
postpartum period ............................................................................................................. 66
Table 4.1. Ingredients and nutrient composition of the experimental diet ................................... 78
Table 4.2. Effects of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8 h/d) of
sodium chloride (CON), acetic acid (AA) and sodium acetate (NA) on feeding behavior
and water intake for cows in the immediate postpartum period ....................................... 84
Table 4.3. Effects of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8h/d) of
sodium chloride (CON), acetic acid (AA) and sodium acetate (NA) on milk yield and
milk components for cows in the immediate postpartum period ...................................... 85
ix

Table 4.4. Effects of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8h/d) of
sodium chloride (CON), acetic acid (AA) and sodium acetate (NA) on plasma
metabolites and insulin for cows in the immediate postpartum period ............................ 88
Table 4.5. Effect of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8h/d) of
sodium chloride (CON), acetic acid (AA) and sodium acetate (NA) on blood chemistry
for cows in the immediate postpartum period (samples taken 4 h after starting of
infusions)........................................................................................................................... 91
Table 4.6. Ingredients and nutrient composition of the experimental diet ................................. 101
Table 4.7. Effects of isoenergetic1 intra-abomasal infusions (22h/d) of glucose (GLU), acetic
acid (AA), lactic acid (LA), propionic acid (PL), and isoamount2 intra-abomasal infusion
of propionic acid (PH), relative to a control (CON) with no infusion on feeding behavior
and water intake for cows in the immediate postpartum period ..................................... 105
Table 4.8. Effects of isoenergetic1 intra-abomasal infusions (22h/d) of glucose (GL), acetic acid
(AA), lactic acid (LA), propionic acid (PL), and isoamount2 intra-abomasal infusion of
propionic acid (PH), relative to a control (CON) with no infusion on milk yield and milk
components for cows in the immediate postpartum period ............................................ 107
Table 4.9. Effects of isoenergetic1 intra-abomasal infusions (22h/d) of glucose (GL), acetic acid
(AA), lactic acid (LA), propionic acid (PL), and isoamount2 intra-abomasal infusion of
propionic acid (PH), relative to a control (CON) with no infusion on change in
concentrations of insulin and metabolites in plasma, and liver acetyl CoA (AcCoA)
content over the infusion day for cows in the immediate postpartum period ................. 108

x

LIST OF FIGURES

Figure 3.1. Effects of isoenergetic intra-abomasal infusion of glucose (GLU), lactic acid (LA)
and propionic acid (PA) relative to a no infusion control (CON) on change in plasma
concentration of insulin, metabolites and liver acetyl CoA (AcCoA) content over the 22
h/d infusion for cows in the immediate postpartum period……………………………...68
Figure 4.1. Effects of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8h/d) of
sodium chloride (CON), acetic acid (AA) and sodium acetate (NA) on plasma
metabolites for cows in the immediate postpartum period. Treatments were: continuous
isomolar infusions (0.75 mol/h) of acetic acid (AA, black solid circle), sodium acetate
(NA, solid triangle,) or sodium chloride (CON, solid square) as a control; infused into the
abomasum (AB, solid line) or rumen (RU, broken line) at a rate of 0.5 L/h ……………90

xi

KEY TO ABBREVIATIONS

AA

Acetic acid treatment

AB

Abomasum

AcCoA

Acetyl CoA

BHB

ß-hydroxybutyrate

CON

Control treatment

CP

Crude protein

DE

Digestible energy

DEI

Digestible energy intake

DM

Dry matter

DMI

Dry matter intake

EB

Energy balance

FA

Fatty acids

FC

Fold change

FDR

False discovery rate

GE

Gross energy

GL

Glycerol treatment

GLU

Glucose treatment

GLP-1

Glucagon-like peptide

HOT

Hepatic oxidation theory

LA

Lactic acid treatment

ME

Metabolizable energy

xii

MEI

Metabolizable energy intake

NA

Sodium acetate treatment

NDF

Neutral-detergent fiber

NEFA

Nonesterified fatty acids

NEB

Negative energy balance

OM

Organic matter

OXPHOS

Oxidative phosphorylation

PA

Propionic acid treatment

PP

Postpartum

RR

Reticulo-rumen

RU

Rumen

TCA

Tricarboxylic acid

TMR

Total-mixed ration

VFA

Volatile fatty acids

xiii

INTRODUCTION

The transition period from gestation to lactation in dairy cows causes homeostatic and
homeorhetic changes highly related with the decrease in dry matter intake (DMI) plus increased
energy output with the resulting negative energy balance. The postpartum (PP) period is
characterized by a high incidence of metabolic disorders associated with the severe energy
deficit and excessive lipid mobilization (Bell, 1995; Overton and Waldron, 2004). The decrease
in plasma insulin concentration and insulin sensitivity in adipose tissue close to parturition
promotes lipolysis leading to a lipolytic state (Drackley, 1999). Increased hepatic uptake and ßoxidation of nonesterified fatty acids (NEFA) likely suppress feed intake by providing a
continuous supply of acetyl CoA (AcCoA) for oxidation in the tricarboxylic acid (TCA) cycle,
increasing ATP production and energy status and thus, signaling for satiety. Energy status is
associated to phosphorylation potential and is measured by calculation of adenylate energy
charge ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP]) (Allen et al., 2009).
Starch is the principal energy source used in diets for dairy cows to increase energy
intake and meet the high demand for glucose precursors. However, it has been shown that
increasing ruminal starch fermentability can depress feed intake (Nelson et al., 2011; Albornoz
and Allen, 2016). Diets with high moisture corn decreased DMI 7.6% for mid-lactation cows
(Oba and Allen, 2003a) and 11.4% for cows in the PP period (Albornoz and Allen, 2016),
compared with diets containing the less fermentable dry ground corn. Ruminal starch
fermentability varies greatly depending upon particle size, gelatinization, solubility of endosperm
proteins, type of processing method, and storage method (Kotarski et al., 1992; Larson and
Hoffman, 2008). Ruminal starch fermentation ranged from less than 30% to more than 85%
(Allen, 2000) thus, altering the type and temporal pattern of fuels absorbed. The ability of these
1

fuels for stimulating hepatic oxidation varies and therefore, their effect on DMI varies.
According to the hepatic oxidation theory (HOT), oxidation of fuels in peripheral tissues is
implicated in the control of feed intake as a result of the transmission of signals to brain feeding
centers by hepatic vagal afferents. Fuels absorbed by the liver that anaplerotic of the TCA cycle
may promote oxidation of AcCoA, increase hepatic energy charge signaling for satiety, thus
decreasing intake (Allen et al., 2009).
Substituting cereal grains for forage increases dietary starch content and the production of
propionic acid (Bauman et al., 1971; Sutton et al., 2003). Propionate decreased DMI by
decreasing meal size compared with acetate when infused intraruminally in lactating cows (Choi
and Allen, 1999, Oba and Allen, 2003a), and the hypophagic effect is greater in cows in the early
PP period than in mid lactation (Oba and Allen, 2003b), likely because cows in the PP period are
in a lipolytic state with both elevated plasma NEFA concentration and hepatic AcCoA content
(Allen et al., 2009). Ruminal infusion of propionic acid was more hypophagic for cows in the PP
period with greater hepatic AcCoA concentration (Stocks and Allen, 2012, 2013). Whereas
propionate is hypophagic compared to acetate, comparisons of propionate to other fuels absorbed
and its effect on energy intake of cows during the immediate PP is lacking. Objectives of this
research were to evaluate the effects of fuels resulting from ruminal fermentation and digestion
of starch on DMI and feeding behavior of cows in the PP period. Our central hypothesis is that
these fuels differ in their effects on DMI and metabolizable energy intake consistent with their
capacity to be metabolized by the liver and stimulate hepatic oxidation.
Therefore, this dissertation includes results of five experiments (Exp.) on cows in the PP
period that were conducted to: 1) investigate the effect of anaplerosis of the TCA cycle on
feeding behavior (Exp. 1 to 3); 2) investigate the effects of different fuels derived from starch
2

digestion on feeding behavior (Exp. 4); and, 3) investigate the effects of infusions of acetic acid
or sodium acetate into the rumen or abomasum on feeding behavior (Exp. 5). In the appendices
are presented results of gene expression analyzed as part of Exp. 4; and a preliminary experiment
to Exp. 5 conducted to investigate the effects of fuel derived from starch digestion on feeding
behavior that included the infusion of acetic acid into the abomasum.
Chapter 2 from this dissertation was already published as “Increased anaplerosis of the
tricarboxylic acid cycle decreased meal size and energy intake of cows in the postpartum period”
in Laura B. Gualdrón-Duarte and Michael S. Allen. 2017 J. Dairy Sci. 100:4425-4434.

3

CHAPTER 1. LITERATURE REVIEW

FEED INTAKE AND FEEDING BEHAVIOR
Feed intake is a function of the quantity of food ingested during a meal and the number of
meals per period of time. Feed intake is the most important parameter driving the acquirement of
nutrients and energy required for living and productive processes.
Mechanisms regulating feed intake have a differential temporal effect depending on their
magnitude of duration over time, and involve a combination of short-term and long-term factors.
Short-term regulation is more closely related to body homeostatic regulation and is mostly
influenced by dietary factors, whereas long-term control relies more on homeorhetic adaptations
according to changes in animal factors (Allen, 2014).
Feeding behavior includes the number of meals, the length of meals, and the rate of
eating; strategies that animals can adjust in order to change dry matter intake (DMI). It is known
that some aspects, for instance level of milk production, can modify feeding behavior. Highproducing cows with free access to total mixed rations ate 9 to 14 meals/d compared with lowerproducing cows who ate 7 to 9 meals/d (Grant and Albright, 1995). This behavior can be
modified if other behavioral or management situations are involved in the studies or production
systems.
Dado and Allen (1994) suggested that in dairy cows the control of individual meals and
total DMI within a day are related to different mechanisms depending not only on level of
production but on parity, ruminal capacity and body weight variability among animals as well. A
meal initiates when the animal is hungry and stops when it is satiated. Hence, daily intake is the

4

sum of the mass ingested in several meals regulated, on an individual basis, by hunger and
satiety mechanisms.
CONTROL OF FEED INTAKE
Animal and dietary factors affect mechanisms controlling feed intake. The importance of
the different factors varies over time both within and across days and can be highly influenced by
metabolic state (Allen, 2014).
Physical regulation
Regulation of DMI by physical factors occurs when feed intake is constrained by
distention signals from the gastrointestinal tract and when the time required for chewing is
increased. Physical regulation becomes a major factor regulating DMI with the increase in
energy needs and filling effect of the diets (Allen, 2000).
The filling effect is one of the main dietary factors that regulates feed intake, and is
determined by the quantity and initial bulk density, the forage source, and the digestibility of
forage NDF present in the diet. The pressure tension and mechanoreceptors cause distention.
Distention in the reticulo-rumen (RR) is determined by volume and weight of digesta (Schettini
et al., 1999), the initial water content of the diet, as well as the water retention capacity of digesta
over time (Allen, 2014). For ruminants, the RR has been considered as the major regulatory site
for DMI induced distention (Allen, 1996; Forbes, 2007). Stretch receptors located in the muscle
layer in the wall of the RR and cranial sac of the rumen are stimulated, and signals are sent to the
brain satiety centers via vagal afferent nerves triggering the meal cessation. Despite the
importance of distention in controlling feed intake, the differences in response among cows and

5

physiological state provide evidence that feed intake is controlled through the integration of
various signals converging in satiety centers of the brain (Mbanya et al., 1993).
Osmotic regulation
Osmolality of the RR may range from 250 to 300 mOsm/kg during pre-feeding to as high
as 500 mOsm/kg following the hours after a large meal (Forbes, 2007). It is affected by DM and
water intake and is highly variable depending on the content of mineral salts and fermentability
of OM in the diet (Allen, 2000; Forbes, 2007). Osmolality is decreased water entering the RR
and by absorption from the RR (Scott, 1975). Additionally, Warner and Stacy (1972) reported
that increasing ruminal osmolality by infusion of solutes into the rumen decreased salivary flow
contrary to the increase in salivary secretion when hypotonic solutions are infused, so saliva flow
is involved in the maintenance of ruminal osmolality.
In experiments conducted by Ternouth and Beattie (1971) where ruminal osmolality was
increased by infusing inorganic salts or salts of volatile fatty acids (VFA), DMI was reduced.
They concluded that the observed decrease in feed intake was related to increased osmolality, but
not to the energy content of infusions. This effect can be associated with excitation of epithelial
receptors in the RR (Leek and Harding, 1975). However the same authors indicated that even
though epithelial receptors in the RR can be stimulated by acids, alkali, and hypo- and hyperosmotic solutions, the threshold to cause excitation of those receptors is very rare under normal
physiological conditions. Only undissociated VFA were found to excite those receptors with
thresholds in the range of 40 to 100 mM (Harding and Leek, 1972).
The ruminal infusion of iso-osmotic solutions of NaCl or VFA as Na-salts or acids as
spontaneous meals (Choi and Allen, 1999) showed a reduction in meal size, implying that

6

termination of meal was regulated by osmolality. However, sodium chloride infusions did not
decrease total DMI because the time between meals also was reduced compared with Na salts of
VFA and non-infused cows. Additionally there was a greater extent in the reduction of meal
length, meal size, and DMI with propionate infusion indicating that the type of VFA in addition
to osmolality affected the termination of a meal.
Despite lack of identification of osmoreceptors that are sensitive to the physiological
range of osmolality in the RR, evidence indicates that hyperosmotic solutions administered into
the RR reduced the amount consumed of single meals (Ternouth and Beattie, 1971). Ruminal
osmolality artificially increased greater than 400 mOsm can terminate a meal and markedly
decrease overall intake; but, the local administration of an anesthetic to the RR stops this effect
(Bergen, 1972). However, the big increase in osmolality in the RR could activate a physiological
mechanism different from the direct stimulation of osmoreceptors. When RR osmolality is high
there is an influx of plasma fluids into the RR causing signs of systemic dehydration and
stimulating vasopressin release from the posterior pituitary gland. It is possible that this
hormone plays a role in the depression of intake when salt infusions are administered intraruminally (Forbes et al., 1992).
Metabolic regulation
Metabolic control of feed intake occurs as a result of concentrations of nutrients,
metabolites and (or) hormones that signal the nervous system to cause the animal to start or stop
feeding (McDonald et al., 2002). Metabolic regulation of intake assumes that some factors
related to the metabolism of the feed control feed intake through feedback signals sent to the
brain (Fisher, 2002). Factors, such as the concentrations and flows of nutrients and energy,
including VFA produced via ruminal fermentation (Illius and Jessop, 1996) and mobilization of
7

tissues and subsequent utilization as fuels, may contribute to the signaling to brain feeding
centers (Fisher, 2002). Physical and metabolic controls of intake are likely not mutually
exclusive, but additive (Choi and Allen, 1999).
Endocrine regulation
Ghrelin is a hormone secreted by abomasal tissue that acts on the hypothalamus (Forbes,
2007). Currently, ghrelin is the only known gut peptide that stimulates feed intake, likely by
promoting emptying and increasing passage rate from the rumen (Allen and Piantoni, 2013).
However, the influence of ghrelin on feed intake has been inconsistent (Roche et al., 2008).
Cholecystokinin and the incretin glucagon-like peptide 1 (GLP-1), which also may stimulate
insulin secretion, are both gut peptides involved in short-term regulation of feed intake in
ruminants (Bradford et al., 2008; Relling and Reynolds, 2008). These peptides are secreted by
the enterocytes as a response to the presence of certain nutrients reaching the intestine, and are
involved in decreasing gastrointestinal tract motility (Holst, 1997; Relling and Reynolds, 2007),
thus, decreasing gastric emptying.
Insulin and glucagon, the principal pancreatic hormones controlling fat and carbohydrate
metabolism in nonruminants, have inconsistent effects on regulation of feed intake in ruminants
(Forbes, 2007). Insulin plays a role in long-term dietary intake and body weight regulation in
ruminants. High plasma concentration of insulin indicates energy sufficiency and affects
mobilization of body reserves and oxidation of fuels, and likely provides a negative feedback on
gluconeogenesis (Vandermeerschen-Doize et al., 1983; McCann et al., 1992). However, within a
day changes in insulin and glucagon can play important roles in the partitioning of fuels, and
likely also have an indirect effect on feed intake. Short-term effects of insulin at meals seems to
affect differently intake. Increased plasma insulin concentration after meals might remove fuels
8

from the blood more quickly (Oba and Allen 2000) increasing DMI of cows in the PP period and
in late lactation. Pulsatile insulin secretion within a day probably reduces lipolysis causing a
reduction in plasma NEFA concentration (Oba and Allen, 2003a) reducing hepatic AcCoA
content available for oxidation in the liver decreasing hepatic energy charge and increasing
intake (Piantoni et al., 2015), consistent with the hepatic oxidation theory (HOT). The increase
in plasma insulin concentration within a day also may contribute to increased hunger, although
promoting oxidation of fuels in the liver likely causes satiety (Allen et al., 2005).
Little research has been done on the effect of glucagon and GLP-1 on feed intake. Studies
conducted with sheep (Deetz and Wangsness, 1981; Faulkner and Martin, 1998) showed that
glucagon administered intravenously at physiological rates reduced intake, and that GLP-1
concentration is greater during lactation compared with the dry period; however, cause and effect
is not clear for GLP-1. In brief, the effects of these hormones on feed intake are likely dependent
on their effects on metabolism of fuels available in accordance with the stages of lactation (Allen
et al., 2009).
Leptin is a hormone secreted mainly by adipose tissue and its secretion is proportional to
the increase in adipocyte size (Ehrhardt et al., 2000) and according to Bradford et al. (2006), the
secretion of leptin has a positive relationship (r = 0.61) with body condition score in lactating
cows. Leptin stimulates satiety and may be related to the homeorhetic control of body weight
(Houseknecht et al., 1998).
Cortisol and catecholamines (epinephrine and norepinephrine, principally) are hormones
that have effects in multiple processes. They are produced and released mainly during stressing
conditions such as weaning, loading, transport and calving. These hormones are lipolytic agents;
9

therefore, the stress response results in lipolysis and elevated plasma nonesterified fatty acids
(NEFA) concentrations. Even if adrenergic mechanisms can decrease DMI (Brandt et al., 2007),
the availability of NEFA provides an oxidative substrate for the liver, increasing energy charge
and decreasing DMI through metabolic control (Allen et al., 2009).
Hepatic control of feed intake
The concept that animals eat to balance energy intake with energy expenditure fails to
explain the suppression of appetite and extended negative energy balance (NEB) during the
periparturient period. Instead, the possibility of a common integrated mechanism among
peripheral tissues sensing the energy status based on the metabolism of fuels has gained
acceptance during the last few decades. The liver has been considered the primary sensor organ
integrating all the long- and short-term mechanisms affecting the initiation and termination of
meals (Allen, 2014).
Metabolites derived from the diet or mobilized from body reserves with the ability to be
oxidized in the liver may affect feed intake of animals including ruminants (Allen et al., 2009;
Allen and Bradford, 2012). Research conducted on laboratory species suggests that control of
feeding behavior is related to energy charge in the liver and is integrated synergistically with
other metabolic inhibitory mechanisms by a common signal related to the hepatic energy status
derived from the oxidation of different fuels. These signals are sent to the brain centers that
control feed intake through afferent fibers of the vagus nerve (Anil and Forbes, 1988). Although
enterocytes were proposed as the primary sensors for fuel oxidation and also are innervated by
the vagus nerve (Langhans, 2008), research performed across species, including ruminants,
provides evidence that the liver instead of the enterocytes could be the key organ sensing the
oxidation of fuels (Allen and Bradford, 2012). Hepatic oxidation is the only proposed
10

mechanism that accounts for both differences in fuels absorbed and sites of absorption among
species, and is consistent with feed intake responses to diet. Even if multiple mechanisms are
involved in the control of feed intake, the HOT implicates a peripheral feedback coordination
integrated with the central nervous system via hepatic vagal afferents (Allen and Bradford,
2012).
Friedman and coworkers (Tordoff et al., 1991; Rawson et al., 1994; Horn et al., 2004)
showed that feeding behavior was related to hepatic energy status rather than fuel oxidation and
ATP production per se. Prevention of ATP production in the liver by sequestering of inorganic
phosphate by infusing the fructose analogue 2,5-anhydromannitol, or by inhibiting fatty acid
(FA) oxidation by using methyl palmoxirate and etomoxir stimulated feeding behavior.
Additionally, these experiments confirmed that the response tending to reduce intake likely
originates in the liver for the following reasons: 1) the effect was faster when the infusion was
into the portal vein compared with the jugular vein; 2) radiolabelled 2,5-anhydromannitol
increased in the liver but not in brain tissue; and, 3) vagotomy blocked the response.
Energy status is measured by adenylate energy charge and considers liver ATP content,
ATP-to -ADP ratio, and phosphorylation potential ([ATP]/[ADP][Pi]) (Allen et al., 2009). The
equation to measure energy charge, ([ATP] + 0.5[ADP])/([ATP]+[ADP]+[AMP]) takes into
account the balance between the rate of production of high-energy phosphate bonds from
oxidation of fuels and their rate of utilization by energy consuming reactions. When hepatic
energy charge is reduced, firing rate of hepatic vagal afferents increases, resulting in hunger and
meal initiation. As energy charge increases after a meal, the firing rate decreases, thus signaling
satiety. Albeit hepatic energy charge is likely responsible for changes in the firing rate of the

11

hepatic vagal afferents that control intake, the exact mechanism by which the signal is
transmitted is not fully understood (Allen and Piantoni, 2013).
HEPATIC METABOLISM OF FUELS
Fuels extracted from the blood and oxidized in the ruminant liver include glycerol, amino
acids, lactate and NEFA, most of them mobilized from body reserves through catabolic
processes or from dietary sources; as well as propionate and butyrate produced by fermentation
in the rumen. Plasma NEFA are extracted by the liver from the blood in proportion to their
concentration (Bell, 1979) and generally are oxidized or stored as triacylglycerides for later
oxidation. Glycerol results mostly from lipolysis in adipose tissue, and lactate can be produced
from the Cori cycle or via the absorption of glucose in the small intestine. All the fuels extracted
by the liver have the potential to affect feed intake by inducing satiety (Langhans, 1996;
Friedman, 1998; Allen et al., 2009).
One of the most important differences between ruminants and non-ruminants is that
ruminant liver does not extract glucose from the blood. Hepatic uptake of glucose is negligible in
adult ruminants (Stangassinger and Giesecke, 1986) likely because of the low activity of
glucokinase (Emmanuel, 1981). Consistent with HOT, glucose also does not appear to have a
hypophagic effect in ruminants (Baile and Forbes, 1974; Frobish and Davis, 1977) contrary to
the effect in non-ruminant species. Additionally, hepatic uptake and utilization of plasma acetate
is negligible in ruminants (Seal and Reynolds, 1993) because AcCoA synthetase activity is very
low. Acetate, which is the most abundant fuel resulting from ruminal fermentation, is used
principally as an energy source for peripheral tissues and can spare the utilization of glucose. The
effect of acetate on feed intake in ruminants is likely related to osmotic effect in the rumen
(Allen et al., 2009).
12

For ruminant species, NEFA instead of glucose are the principal fuels oxidized in the
hepatocytes and the primary energy source for the liver. Medium-chain FA and unsaturated C18
FA are delivered and oxidized faster and have a greater hypophagic effect than long-chain
saturated FA (Langhans, 1996). In general, NEFA supply varies both within the day (negatively
related with plasma insulin concentration; Nielsen et al., 2003) as well as throughout lactation, as
the physiological state of cows change (Ospina et al., 2010).
Propionate, glycerol, lactate and some amino acids can either be oxidized in the liver or
used as gluconeogenic precursors. Mobilization, uptake, oxidation in the tricarboxylic acid
(TCA) cycle or gluconeogenesis of these fuels are regulated according to the concentration of
insulin in plasma over the long-term, determined primarily by stage of lactation or energy
balance (EB, Allen and Piantoni, 2013).
HEPATIC CONTROL OF INTAKE IN DAIRY COWS
Concentration of hormones and metabolites in blood, metabolic processes,
responsiveness of tissues to hormones, and mobilization of fuels vary greatly according to
physiological state. These differences may modify the importance of the several factors
controlling feed intake. Control of feed intake in dairy cows by hepatic oxidation likely becomes
more relevant as nutrient requirements and the filling effect of diets decrease, reducing the signal
from gut distension (Allen, 2000). Hepatic oxidation likely controls feed intake to a greater
extent for animals with low nutrient requirements, animals consuming a high-starch diet, or for
animals in a lipolytic state caused by stress or physiological conditions surrounding the
periparturient period. Intake in ruminants fed a high-forage diet or with high nutrient and energy
demands, such as cows at peak lactation, are more likely to be regulated through other
mechanisms related to gut distention signaling than to oxidation of fuels (Allen et al., 2009).
13

According to the HOT,control of feed intake at the dry period may be regulated initially
by the filling effect of the diet, but, as calving approaches changes in hormonal augmenting
lipolysis and consequently shifting the control of feed intake toward hepatic oxidation
mechanisms (Allen et al., 2009). Hormonal changes are mainly directed to reduce insulin
concentration in plasma and insulin sensitivity of adipose tissue and muscle several weeks before
parturition (De Koster and Opsomer, 2013), and to increase growth hormone concentration in
plasma close to calving (Bauman, 1992), Growth hormone is involved in the reduction of
lipogenesis, the decrease in glucose uptake and oxidation, and decreases insulin sensitivity of
adipose tissue and increases nutrient utilization by the mammary gland. Growth hormone alters
the synthesis of lipids in adipose tissue but has no effect on rates of lipid mobilization (Simmons
et al., 1994). During the last weeks before parturition, the aim for nutritionists is to formulate
diets to increase DMI, milk yield, and reduce the incidences of metabolic disorders after calving.
Feeding high-grain diets at the end of lactation and at the beginning of the dry period has been
proposed as a strategy to stimulate insulin production and limit lipolysis after calving. However,
those studies have shown inconsistent results (Grummer, 2008). In fact, feeding low-fiber diets
when plasma insulin concentration and insulin responsiveness in tissues is still high decreases
the potential benefit of the filling effect that may constrain excessive accumulation and
mobilization of fat prepartum and postpartum, respectively. Reducing lipolysis postpartum likely
decreases the pool of hepatic AcCoA available to be oxidized and so increases DM and energy
intake post calving (Allen and Bradford, 2012). Moreover, the filling effect helps to improve
DMI after parturition and prevent the harmful effect of shifting to a more fermentable diet during
the early PP period (Murondoti et al., 2004; Douglas et al., 2006).
At the onset of lactation, energy demands are not met by energy intake and fat reserves
14

are mobilized to provide energy. There is an increased requirement for glucose utilization for
milk production, and propionate is the principal gluconeogenic precursor in ruminants. However,
feeding diets rich in rapidly fermentable starch may exacerbate the problem instead of solve the
imbalance. Diets with high starch concentration and readily fermentable starch sources increase
propionic acid production (Allen, 2000), and increase propionate absorption (Benson et al.,
2002) and liver uptake (Kristensen, 2005). Although propionate follows the gluconeogenic
pathway, it is first metabolized to succinyl CoA, which allows it to enter the TCA cycle. Its
entrance into the TCA cycle likely promotes the oxidation of AcCoA, and thus, increases energy
charge and decreases meal size that may ultimately decrease total DMI and energy intake (Allen
and Piantoni, 2013).
As lactation progresses, milk yield increases and part of the NEFA mobilized from
adipose tissue are redirected to the mammary gland for milk fat synthesis and as such reduce the
NEFA supply to the liver. Metabolic signals controlling intake start to decrease and diets with
high fiber content limit DMI and the supply of gluconeogenic precursors from dietary sources.
During this period, animals respond well to diets high in fermentable starch sources and with
lower forage NDF content (Allen et al., 2009). As lactation continues and milk yield decreases,
metabolic control through the HOT likely begins to dominate intake again as a result of negative
feedback on gluconeogenesis due to increased plasma glucose and insulin concentrations.
Increased insulin sensitivity allows the animal to replenish the body reserves lost during early
and peak lactation (Allen and Piantoni, 2013). Recombinant bovine somatotropin (rbST) favors
partitioning of energy to milk by increasing nutrient uptake for milk synthesis, decreasing the
loss of secretory cells in the mammary gland, increasing gluconeogenesis in the liver, decreasing
uptake of glucose in muscle, and decreasing lipogenesis in positive-EB and increasing lipolysis
15

in negative EB in adipose tissue.
POSTPARTUM COWS
During the last 3 weeks of gestation there are a tremendous increase in nutrient demands
(Bell, 1995), and significant changes in hormone concentrations and general metabolism
adjusting for the new lactation cycle. The insulin concentration of plasma decreases and the
insulin sensitivity of adipose tissue and muscle is reduced (Holtenius et al., 2003). The decrease
in insulin sensitivity is likely caused by several factors interacting synergistically; for instance,
the increase in growth hormone, the release of proinflammatory cytokines from different tissues
(Bell and Bauman, 1997) causing an increase in lipolysis with the consequent elevation of
plasma NEFA concentration . Nonesterified FA are used as fuels for the liver and extrahepatic
tissues in order to spare glucose for fetal development, thus maintaining euglycemia, and as
precursor for FA synthesis in the mammary gland close to parturition (Allen and Piantoni, 2014).
However, excessive lipolysis during the periparturient period can result in increased risk for
metabolic disorders, such as clinical ketosis, fatty liver and a chronic inflammatory reaction, with
the concomitant decrease in DMI aggravating the NEB (Grummer, 2008).
Fatty acids oxidation
Fatty acids are the primary fuels oxidized in the liver. Uptake of NEFA by the liver
increases greatly after calving (Reynolds et al., 2003) as a result of their proportional increase in
concentration in circulation. Fatty acids are oxidized through β-oxidation in the mitochondria or
in the cytosol by peroxisomes or microsomes. Fatty acids also can be re-esterified for later
oxidation or exported as very low-density lipoproteins. However, ruminant liver has less capacity
to export triacylglycerides compare with other species, and excessive lipid mobilization can
cause lipid accumulation that compromises liver function; increasing the risk of metabolic
16

disorders (Kleppe et al., 1988; Zhu et al., 2000).
To undergo β-oxidation, long-chain FA are transported into mitochondria bound to
carnitine acyl transferase. The activity of this enzyme is controlled by various factors such as the
availability of the enzyme and the presence of inhibitory metabolites like malonyl CoA and
methylmalonyl CoA, one of the intermediates in FA synthesis and an intermediate in propionate
metabolism, respectively. The end products of β-oxidation are mostly AcCoA and reduced
cofactors (NADH and FADH2) that are re-oxidized through oxidative phosphorylation
(OXPHOS) for the generation of ATP.
Acetyl CoA is a compound shared in the metabolic pathway for most of the fuels with the
ability to be oxidized, entering the TCA cycle in the citrate synthase reaction to complete
oxidation to CO2 and water. ATP and reducing equivalents also are produced with the latter
being converted to ATP through OXPHOS. The production of CO2 stimulates OXPHOS by
bicarbonate production (Acin-Perez et al., 2009). During the postpartum period, when cows are
in a lipolytic state, liver increases the uptake of NEFA increasing hepatic energy charge as a
result of the complete FA metabolism through β-oxidation, TCA cycle and OXPHOS. According
to the HOT, when feeding behavior is mostly regulated by metabolic control, temporal pattern of
absorption of fuels, mobilization of body reserves and oxidation in the liver can alter meal size
and meal frequency affecting feeding behavior and feed intake. All metabolic processes
stimulating oxidative pathways in the liver promote the increase in hepatic energy charge and
decrease intake (Allen et al., 2009). Excess of AcCoA that cannot be oxidized in the TCA cycle
can be metabolized to ketone bodies and exported from the liver and be used as an alternative
energy source for some extrahepatic tissues.

17

The capacity to oxidize AcCoA in the TCA cycle is regulated by the availability of
intermediates of the cycle. Because the liver is a key organ in the control of metabolism, the
TCA cycle not only contributes to oxidation of metabolites, but also in other biosynthetic
pathways in which intermediates leaving the cycle are mostly converted to glucose or nonessential amino acids. This process is called cataplerosis, and all reactions tending to replace the
TCA cycle intermediates, allowing for its continued function, are called anaplerotic reactions
(Owen et al., 2002). Hence, the regulation of the entrance and exit of intermediates to the TCA
cycle depends upon the metabolic and physiologic state, and the specific tissue or organ
involved. Therefore, the capacity for hepatocytes to oxidize AcCoA is affected by the
concentrations and flux of TCA cycle intermediates as wells as by the concentrations and activity
of the enzymes required for oxidation.
Gluconeogenic precursors and effect on DMI
Availability and partitioning of gluconeogenic precursors and their effects on DMI
depend on the physiological state and glucose requirements of the animal. Quantitatively
important substrates for hepatic gluconeogenesis in the order of hepatic uptake are propionate
(60 to 74%), L-lactate (16 to 26%), valerate and isobutyrate (5 to 6%), alanine (3 to 5%),
glycerol (0.5 to 3%), and other amino acids (8 to 11%) (Aschenbach et al., 2010).
Amino acids
Although skeletal muscle is a reservoir of amino acids, amino acids released after fasting
or during severe periods of NEB, do not reflect the amino acid composition of the skeletal
muscle proteins. Local metabolism in muscle appears to interconvert amino acids specifically to
alanine and glutamine, which therefore represent a disproportional increase in the fraction of
amino acids in blood circulation (Owen et al., 2002). There is an important contribution of
18

alanine (3 to 5%) to the total hepatic uptake of gluconeogenic precursors by the liver in the
postpartum period (Reynolds et al., 2003; Larsen and Kristensen, 2009).
Amino acids are used mainly for protein synthesis, however, when protein synthesis is
limited by deficiency of individual amino acids or amino acids are supplied in excess, the amino
acid surplus is deaminated and the carbon chains can enter the TCA cycle via anaplerotic
reactions and promote oxidation or be oxidized as fuels and likely affect intake. Ammonia and
urea were hypophagic (Mugerwa and Conrad, 1971) and intraruminal infusion of ammonium
salts of VFA compared with potassium salts decreased DMI by decreasing the number of meals
without affecting meal size (Oba and Allen, 2003d); furthermore, the effects were increased
when propionate salts were infused compared with acetate salts (Oba and Allen, 2003b). A
possible explanation of this effect consistent with the HOT is that gluconeogenesis from
propionate is decreased by ammonia, thus increasing the availability of propionate to be
oxidized. In addition, ammonia detoxification through urea synthesis generates additional
anaplerotic carbon sources to be oxidized (Oba and Allen, 2003b). In sum, both explanations
increase hepatic energy charge and decrease intake (Allen et al., 2009).
Glycerol
Glycerol as a feed ingredient was used mostly to replace starch sources or as a possible
alternative treatment for metabolic disorders in periods of NEB. The inclusion of glycerol in
diets ranges from less than 1 up to 15% of DM with responses related to the level of inclusion
and stage of lactation. Inclusion at 3.6% of the DM in mid-lactation cows had no effect on milk
production or composition but it altered concentration of ruminal fermentation end products,
increasing propionate and butyrate at the expense of acetate (Khalili et al., 1997). In a more
recent study on mid-lactation cows, Donkin et al. ( 2009), fed diets containing 0, 5, 10, or 15% of
19

glycerol for 56 days with no difference in production responses except for a slight reduction in
feed intake during the first week of the study for the 15% glycerol inclusion treatment (treatment
x time effect). In a transition period study, DeFrain et al. (2004) reported a 17% decrease in DMI
in the prepartum period and no effect on milk production or DMI after calving when glycerol
was fed at 5.4% of the ration DM from 2 weeks before calving to 3 weeks after calving.
However, Carvalho et al. (2011), replaced high moisture corn with glycerol at ~11% of the
dietary DM from 4 weeks before calving to 8 weeks after calving with no effect on intake. On
the other hand, Bodarski et al. (2005), reported increased milk yield and milk protein content
when feeding glycerol at 3.1% of DM from 3 weeks before calving to 70 days after calving.
It is important to clarify that glycerol is mostly fermented in the rumen when added as a
feed ingredient and has little or no opportunity to reach the liver through portal circulation. When
glycerol is administered as a drench or infused for treatment of ketosis, it ideally should be
absorbed mostly intact from the rumen or escape ruminal fermentation to be absorbed from the
small intestine. Glycerol is metabolized in the liver and enters the cytosolic gluconeogenic
pathway as triose-phosphate without the need of entering the TCA cycle, bypassing the
metabolic steps with limiting enzymes (Johnson, 1954). When glycerol and propylene glycol
were infused into the abomasum, glycerol was more effective at increasing glucose and insulin
concentrations; however, when they were infused into the rumen, propylene glycol was more
effective at increasing insulin and glucose concentrations in plasma. Abomasal infusions of both
precursors tended to decrease ß-hydroxybutyrate concentration in plasma without an effect on
DMI (Piantoni and Allen, 2015). In a second experiment where propylene glycol and glycerol
were dosed into the cranial rumen, ~30 % more glycerol than propylene glycol (5.2 mol vs. 3.8

20

mol) was required to observe similar effects on plasma concentrations of glucose and insulin
with both glucose precursors (Piantoni and Allen, 2015).
Glycerol, an endogenous gluconeogenic source, is mobilized from adipose tissue along
with NEFA during ketosis, starvation or other periods of body fat mobilization. Glycerol
turnover rate is highly increased during ketosis and starvation compared with the fed state
(Aschenbach et al., 2010).
The effect of glycerol on feed intake is consistent with the control of feed intake by the
HOT. At high dietary levels of inclusion, glycerol is fermented to propionate causing
hypophagia, and when it is administered as a drench or infused, some of it likely escapes ruminal
fermentation and is used directly as a glucose precursor in the liver, thus with less hypophagic
effect.
Lactate
Some lactate is consumed via diets containing silages and fermented products, or may be
produced by ruminal fermentation of starch or anaerobic metabolism of glucose by splanchnic
tissues and erythrocytes (Aschenbach et al., 2010). In starvation periods or during the
periparturient period, when cows are in a low glucose status, the release of gluconeogenic carbon
from peripheral tissues is increased and lactate becomes the main endogenous gluconeogenic
substrate (De Koster and Opsomer, 2013; Aschenbach et al., 2010). At the end of gestation the
uterus and placenta are important sources of lactate. In skeletal muscle the expression of lactate
dehydrogenase, the enzyme which catalyze the conversion of pyruvate into lactate is
upregulated, whereas the expression of the main enzymes of the TCA cycle are downregulated
(De Koster and Opsomer, 2013).

21

Lactate is a fuel extracted by the liver at a rate related to the ratio of NAD to NADH in
the hepatic tissue, with less absorption when the ratio is decreased (Allen et al., 2009). During
the postpartum period and starvation periods, when insulin concentrations in circulation are low,
liver uptake of lactate is increased (Reynolds et al., 2003; Brockman and Laarveld, 1986), with
the highest contribution around the second week after calving (Reynolds et al., 2003). Rate of
uptake of lactate decreases as lactation progresses and as intake increases, and gluconeogenesis
becomes more dependent on the flux of propionate to the liver rather than endogenous sources
(Reynolds et al., 2003; Aschenbach et al., 2010).
Lactic acid can supply between 10 to 26% of gluconeogenic substrates (Reynolds et al.,
2003; Larsen and Kristensen, 2009) and lactate enters the TCA cycle through conversion to
pyruvate and then to oxaloacetic acid in an anaplerotic reaction promoting oxidation of AcCoA,
increasing energy charge in the liver and decreasing feed intake (Allen et al., 2009). Oxaloacetic
acid also can leave the TCA cycle through conversion to phosphoenol pyruvate and follow the
gluconeogenic pathway. Lactate is expected to have a smaller effect on decreasing feed intake
than propionate (Allen et al., 2009), considering that extraction of lactate by the liver is less
efficient than propionate (Reynolds et al., 2003).
Propionic acid
Of the VFA produced by ruminal fermentation, only propionate, valerate, and isobutyrate
can serve as glucogenic precursors for net synthesis of glucose. Propionate is the most abundant
of the three-glucogenic acids (15 to 40% of total ruminally released organic acids) and by far the
predominant substrate for gluconeogenesis in ruminants. The production and absorption of
propionate are greatest after feeding; gluconeogenesis in ruminants also is greatest after periods
of high energy intake (Aschenbach et al., 2010).
22

Of the fuels produced during fermentation of the diet, propionate is most likely to
stimulate satiety. Propionate can be produced and absorbed rapidly (Benson et al., 2002) within a
time frame of a meal. Activation with CoA is necessary for metabolism of FA. Propionate is
readily extracted from the blood by the liver as a result of the high activity of propionyl CoA
synthetase in ruminants, and the low activity of hepatic acetyl CoA synthetase could explain, in
part, the differences in hypophagic effects of infusions of propionate and acetate in ruminants
(Ricks and Cook, 1981). Even though butyrate can be oxidized in the liver, its net metabolism is
usually less than propionate or acetate because its ruminal production is lower and it is mostly
oxidized by ruminal epithelia (Weigand et al., 1975).
Propionate is an anaplerotic compound and promotes AcCoA oxidation in the TCA cycle
in the liver unlike acetate (Allen and Piantoni, 2013). Propionate enters the TCA cycle as
succinyl CoA, promoting oxidation of AcCoA and increasing hepatic energy charge, and likely
decreasing the firing rate of the vagus nerve, thus signaling satiety. Total liver denervation in
sheep and splachnic blockade using anesthetic, bilateral splanchnotomy, and hepatic vagotomy
eliminates the hypophagic effect of portal infusion of propionate (Anil and Forbes, 1988).
Isosmotic mixtures of propionate infused continuously into the rumen in lactating cows
linearly decreased metabolizable energy intake by decreasing meal size compared with acetate
(Oba and Allen, 2003e). Similarly propionate infusions at spontaneous meals decreased meal
size compared with acetate (Choi and Allen, 1999), and the degree of hypophagia from propionic
acid increased with increased hepatic AcCoA content (Stocks and Allen, 2012; 2013).
When ruminal propionate production exceeds hepatic gluconeogenic capacity it is likely
that some propionate could be oxidized. When glucose demands are increased (peak of
23

lactation), propionate is mainly used for gluconeogenesis, reducing the possibility for propionate
oxidation within meals, and extending the length and size of meals. In the contrary, when
glucose demand is low (e.g., past peak of lactation), propionate could be oxidized within or
during actual meals, resulting in satiety and smaller meal size (Oba and Allen, 2003c). However,
propionate oxidation during the PP period is less likely because of the high availability of hepatic
AcCoA for oxidation (Allen, 2014) and the consequent down regulation of pyruvate
dehydrogenase complex activity.
STARCH FERMENTABILITY AND EFFECT ON FEED INTAKE IN THE
POSTPARTUM PERIOD
Because starch constitutes up to 35% of the dietary DM for lactating cows, its ruminal
fermentability can have a great effect on the type and pattern of absorbed fuels. The ruminal
fermentation and small intestine digestion of starch is affected by several factors, such as particle
size, physical processing, and amount and solubility of endosperm proteins (Kotarski et al.,
1992; Larson and Hoffman, 2008)). Different grain types and different hybrids from the same
type of grain differ in proportion and solubility of endosperm proteins affecting the rate of
fermentation and site of starch digestion (Allen, 2000). All physical processes (e.g., rolling and
grinding) that increase surface area or chemical treatment (e.g., steaming that gelatinizes starch)
that increase accessibility for ruminal microorganisms increase the rate of starch fermentation
(Owens et al., 1986; Huntington, 1997). The ensiling process also increases fermentability by
solubilizing endosperm proteins, and greater moisture content at the time of ensiling and longer
periods of storage before use of ensiled material can further augment starch fermentability (Oba
and Allen, 2003a).
In accordance with the rate of digestion and passage, dietary starch can be digested in
24

different sections of the gastrointestinal tract in ruminants. The site of digestion determines the
principal end products of starch digestion. It can be fermented partially by microorganisms in the
rumen and large intestine, and digested in the small intestine by pancreatic amylases. Ruminal
fermentability of starch can range from ~25% to 90% (Allen, 2000), increasing the total
production of VFA as fermentability increases. However, when modifying ruminal starch
fermentation, propionic acid is the VFA that varies more when compared with acetic and butyric
acid. Propionic acid production could increase from 6 to 52 moles/d (Allen, 1997) because its
concentration in the rumen can range from 15 to 45% of total fermentation acids based on
organic matter fermented in the rumen (Davis, 1967). Starch that passes through the rumen can
be digested in the small intestine by the action of pancreatic and intestinal enzymes. Digestion at
the level of the small intestine increases the absorption of glucose directly, although most is
metabolized to lactate by intestinal tissues (Reynolds et al., 2003). Depending on the site of
digestion and digestibility of starch, the type of fuel produced and the patterns of absorption are
affected greatly.
Diets with greater ruminal starch fermentability can depress feed and energy intakes.
Increasing the amount of a more fermentable starch source in the diet could reduce feed intake of
cows nearly 3 kg/d, likely as a result of the increased flow of propionate to the liver (Allen,
2000). More fermentable starch sources increase ruminal propionate production compared with
less fermentable starch sources.
Starch fermentability of the diet interacts with the physiological state of the cow and can affect
energy intake differently. High-moisture corn, a more fermentable starch source, decreased feed
intake by 7.6% (20.8 vs. 22.5 kg/d) in early lactation cows compared with dry ground corn, a less
fermentable source, by reducing meal size in diets containing 32% of starch (Oba and Allen,
25

2003a). The decrease in meal size is likely because of a more rapid flux of propionate to the liver
and the stimulation of oxidation in the hepatocyte signaling for satiety (Allen et al., 2009).
However, propionate is more hypophagic for early postpartum cows than cows in mid-lactation.
After parturition, cows are in a lipolytic state with high plasma NEFA concentration, increased
glucose demand and low concentration of insulin in plasma causing an increase in glucogenic
flux. The increase of propionate supply to the liver is likely used for gluconeogenic purposes
more than its oxidation. However, the metabolic pathway of propionate makes it an obligatory
anaplerotic compound and it likely promotes the oxidation of the increased pool of hepatic
AcCoA derived from ß-oxidation, which in turn increases the energy charge in the liver and
induces satiety; thus decreasing meal size and likely total DMI. In a recent study, Albornoz and
Allen (2016) evaluated the effect of starch content (22 and 28%) and fermentability on cows
during the first 3 weeks of lactation. Intake increased by 2.2 kg/d when diets formulated with dry
ground corn (less fermentable starch source) compared with high-moisture corn (more
fermentable starch source) diets (19.7 kg/d vs. 17.1 kg/d, P <0.01). They reported an interaction
between starch content and starch fermentability, where high starch diets tended to increase DMI
3.4 kg/d (20.0 vs. 16.6 kg/d) compared with the low starch diets which increased DMI 1 kg/d
(18.6 vs. 17.6 kg/d).
Therefore, this review presents support that the hepatic oxidation of fuels is involved in
the control of feed intake in the postpartum period and shows that ruminal fermentability of
starch should be an important consideration for diets formulated for cows in the immediate
postpartum period.
Our long-term goal is to understand the metabolism of fuels in the liver and their
integrating effects on feeding behavior to develop feeding strategies to achieve a transition from
26

gestation to lactation with a better EB and with less metabolic problems. Our main objective is to
determine the effects of different fuels derived from ruminal fermentation and digestive
processes during the postpartum period on feeding behavior and energy intake. The results of
this new research will aid in the formulation of diets to increase energy intake during the
postpartum period, reduce risk of metabolic disorders after calving, and improve the efficiency in
nutrient utilization.

27

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28

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36

CHAPTER 2. INCREASED ANAPLEROSIS OF THE TRICARBOXYLIC ACID CYCLE
DECREASED MEAL SIZE AND ENERGY INTAKE OF COWS IN THE
POSTPARTUM PERIOD

We evaluated effects of anaplerosis of the tricarboxylic acid cycle on feeding behavior
and energy intake of cows in the postpartum period using abomasal infusions of glycerol and
propionic acid. Both are three-carbon glucose precursors with similar energy contents but
propionic acid is obligatorily anaplerotic while glycerol is not. Propionic acid decreased meal
size and total energy intake compared with glycerol. For a full text of this work see Laura B.
Gualdrón-Duarte and Michael S. Allen. 2017. J. Dairy Sci. 100:4425-4434.

37

CHAPTER 3. FUELS DERIVED FROM STARCH DIGESTION HAVE DIFFERENT
EFFECTS ON ENERGY INTAKE AND METABOLIC RESPONSES OF COWS IN THE
POSTPARTUM PERIOD
ABSTRACT
Absorbed fuels from the digestion of starch include propionic acid (PA) produced by
ruminal fermentation and glucose (GLU) from intestinal digestion, which is partially
metabolized to lactic acid (LA) by intestinal tissues. Our objective was to evaluate the effects of
these fuels on dry matter intake (DMI) and feeding behavior of cows in the postpartum period.
We hypothesized that these fuels affect feed intake differently and that their effects are related to
their capacity to stimulate oxidation in the liver. Glucose was expected to have little effect on
feed intake because little or no GLU is extracted from the blood by the liver. Whereas, both LA
and PA are anaplerotic and can stimulate oxidation of acetyl CoA in hepatocytes, hepatic
extraction of PA is greater than LA, which depends on cytosolic redox state. Continuous
isoenergetic infusions (150 kcal/h) of PA, LA or GLU or no infusion were administered
abomasally to eight ruminally cannulated multiparous Holstein cows (12.4 ± 6.2 d postpartum)
in a duplicate 4 x 4 Latin square design experiment balanced for carry-over effects. Treatment
sequences were assigned to cows randomly, and treatments included control (CON, no infusion),
PA (99.5%, 0.41 moles/h), LA (88.0%, 0.46 moles/h) and GLU (99.9%, 0.22 moles/h) Solutions
containing treatments were infused at 500 mL/h for 22 h/d and provided ~3.3 Mcal/d. Feeding
behavior was recorded by a computerized data acquisition system. Gross energy digestibility of
the diet was determined for each cow and used to calculate metabolizable energy intake (MEI)
from the diet. Total MEI was calculated as the sum of MEI from the diet plus energy from
infusions. Data were analyzed statistically with a mixed model including the fixed effect of

38

treatment and random effects of block and cow within block. Each treatment was compared with
CON by preplanned contrasts. Compared with CON, PA decreased DMI by 24% (14.3 vs. 18.9
kg/d) and total MEI by 13% (34.8 vs. 40.2 Mcal/d) with a tendency to decrease meal frequency.
Lactic acid decreased DMI by 14% (16.3 vs. 18.9 kg/d) compared with CON by decreasing meal
size 20% but did not affect MEI. Glucose infusion did not affect DMI or MEI. Treatment effects
on DMI and MEI were consistent with their expected effects on hepatic oxidation. Depression of
feed intake in diets containing highly fermentable starch is likely because of the ability of
propionic acid to stimulate hepatic oxidation.
INTRODUCTION
Cereal grains high in starch are included in rations to meet dietary energy requirements of
high-producing dairy cows. However, highly fermentable starch sources can depress feed intake
compared with less fermentable starch sources (Allen, 2000). High moisture corn decreased DMI
8% for mid-lactation cows (Oba and Allen, 2003a) and 11% for cows in the postpartum (PP)
period (Albornoz and Allen, 2016), compared with the less fermentable dry ground corn.
Ruminal fermentability of starch varies greatly from ~30% to ~90% (Allen, 2000) depending
upon genetics, maturity, processing, and storage method of the starch source (Kotarski et al.,
1992; Larson and Hoffman, 2008). This wide variation affects the type and supply of fuels
available for absorption including propionic acid from ruminal fermentation, glucose from
enzymatic hydrolysis of starch that passed from the rumen to the small intestine, and lactic acid
from the partial metabolism of glucose by enterocytes. Once absorbed, metabolism of these fuels
varies with different efficiencies of extraction from the blood by the liver and potential of
stimulating hepatic oxidation. According to the hepatic oxidation theory (HOT) of the control of

39

feed intake, anaplerotic fuels extracted by the liver may promote oxidation of acetyl CoA
(AcCoA) increasing hepatic energy charge contributing to satiety (Allen et al., 2009). Of the
primary fuels absorbed from starch digestion in ruminants, propionate and lactate are anaplerotic
whereas little glucose is extracted from the blood by the liver. However, hepatic extraction of
propionate from the blood is greater than lactate (Reynolds et al., 2003), which is dependent
upon cytosolic redox state in the hepatocyte (Allen et al., 2009) The objective of this experiment
was to evaluate the effects of fuels derived from fermentation and digestion of starch on DMI
and feeding behavior of cows in the PP period. We hypothesized that these fuels differ in their
effects on DMI and ME intake (MEI) and that their effects are consistent with their ability to
stimulate hepatic oxidation, with propionic acid causing the greatest hypophagic effect, followed
by lactic acid, and little effect for glucose.
MATERIALS AND METHODS
Animals, Housing, and Care
All experimental procedures were approved by the Institutional Animal Care and Use Committee
at Michigan State University, East Lansing. Cows were ruminally cannulated at least 45 d before
expected calving date. Each cow was housed in the same individual tie-stall for the duration of
the experiment. Cows were fed once daily (1200 h) at 125% of expected intake and milked in
their stalls twice daily at 0530 and 1730 h.
Experimental Design, Diets and Treatments
Eight multiparous Holstein cows (n = 8, 12.4 ± 4.2 d PP) at the Michigan State
University Dairy Teaching and Research Center were used in a repeated 4 x 4 Latin square

40

design with 2 blocks of cows containing 4 cows each. Cows were blocked by date of parturition;
the first block was conducted during March, 2015 and the second during October, 2015. Cows
within each block were randomly assigned to treatment sequence. Each block was conducted
over an 8-d period with 4 infusion days separated by a washout day. All cows received a
common experimental diet from parturition through the end of the experiment. The experimental
diet (Table 3.1) was formulated to meet or exceed cows estimated requirements according to
NRC (2001) and fed as a TMR.
Treatments were propionic acid (PA; food grade, 99.5%, Kemin Industries, Inc., Des
Moines, IA), L-lactic acid (LA; food grade 88.0%, Sena International Inc., Polo, IL) and glucose
(GLU; ≥ 99.5%, Sigma Aldrich, Chemical Co., St. Louis, MO) infused continuously into the
abomasum, and control (CON; no infusion). Isoenergetic amounts of PA (0.41 mol/h), LA (0.46
mol/h) and GLU (0.22 mol/h) dissolved in distilled water were infused at a rate of 508 mL/h for
22 h/d, providing an average of 3.26 Mcal ME/d (148 kcal/h), approximately 10% of daily MEI
by cows in the postpartum (PP) period. Rate of infusion was selected based on previous studies
in which PA decreased DMI of cows in the PP period compared with acetic acid (Stocks and
Allen, 2012; Oba and Allen, 2003b). Metabolizable energy content of treatments were assumed
to equal their gross energy of 365.0 kcal/mol for PA (Labedeva, 1964), 321.2 kcal/mol for LA
(Saville and Gundry, 1959) and 673.4 kcal/mol (Emery and Benedict, 1911) for GLU. Solutions
were infused into the abomasum using Baxter Flo-Gard 6201 infusion pumps (Baxter Medical
Products, Deerfield, IL) through vinyl tubing (0.95 cm OD, 0.71 cm ID) connected to a Nalgene
bottle (3.8 cm diameter, 8.5 cm long) held in place with a rubber disk as described in GualdrónDuarte and Allen (2017).

41

Ingredients and nutrient composition of diets fed during blocks 1 and 2
Item
Block 1
Block 2
Ingredient, % of DM
Corn Silage
39.6
33.8
Soybean meal
16.1
15.9
Alfalfa silage
14.1
5.54
Ground corn
10.4
17.8
Alfalfa Hay
9.9
9.7
Soybean hulls
5.7
5.5
Grass silage
7.6
1
Vitamin and mineral mix
4.2
4.1
Nutrient composition
DM, %
51.0
55.0
Starch, % of DM
19.8
22.0
NDF, % of DM
35.1
34.2
Forage NDF, % of DM
29.5
27.5
CP, % of DM
16.6
16.0
1

Vitamin and mineral mix contained 25.6% NaCl, 10.0% Ca, 2.0 Mg, 2.0% P, 30 ppm Co, 506 ppm Cu, 20 ppm I,
2220 ppm Fe, 2,080 ppm Mn, 15 ppm Se, 2,030 ppm Zn, 300 KUI/kg vitamin A, 50 KIU/kg vitamin D, 1500
KIU/kg vitamin E.

Data and Sample Collection
Cows were withheld from feed from 1000 to 1200 h daily, and the amount of feed offered
and orts were weighed for each cow daily. Samples (0.5 kg) of all dietary ingredients and the
TMR were collected daily throughout the experiment, and orts for each cow were collected at the
end of each infusion day and stored in plastic bags at -20°C until processed. Feeding behavior
data (feed disappearance and water intake) were recorded continuously for 22 h daily for each
cow during infusions via computer every 5 s, and size, length, and frequency of meals, intermeal
interval, total eating time, and water intake were calculated (Dado and Allen, 1995).
Milk yield was recorded daily at each milking, and milk samples were collected from
each milking and stored with preservative (Bronopol tablet; D&F Control Systems, San Ramon,

42

CA) at 4ºC. Milk samples were analyzed for fat, true protein, lactose, somatic cell count, and
milk urea nitrogen by Michigan DHIA (AOAC, 1997).
Fecal samples were collected every 6 h for 24 h (n = 4) during each day following
infusion days and frozen at -20°C for later analysis to determine diet digestibility. Blood was
sampled every day at the beginning and end of each infusion period from coccygeal vessels and
collected into 4 evacuated tubes, 3 containing K2EDTA and 1 containing potassium oxalate with
NaF as a glycolytic inhibitor, and placed on ice until centrifugation. Tubes were centrifuged at
3,000 x g for 15 min at 5°C immediately after sample collection. A 1-mL aliquot of plasma from
each potassium EDTA tube was stored with 0.05 M benzamidine (final concentration) to prevent
enzymatic degradation of glucagon. Plasma was harvested and frozen at -20°C until analysis.
Liver samples were collected by needle biopsy (Bradford and Allen, 2005) between 1100 h and
1200 h (before feeding) before each infusion and at the end of infusions for each period for
analysis of hepatic AcCoA content. Samples were flash frozen in liquid nitrogen and stored on
dry ice for transportation. Samples were stored at -80°C until further analysis.
Analysis of Samples
Feed ingredients, orts and fecal samples were dried in a 55°C forced-air oven for 72 h
and analyzed for DM concentration. All samples were ground with a Wiley mill (1-mm screen;
Arthur H. Thomas, Philadelphia, PA). Samples were analyzed for ash, NDF, indigestible NDF,
crude protein and starch. Feces were composited by cow by day on an equal DM basis before
analysis. All nutrients are expressed as percentages of DM, determined by drying at 105ºC in a
forced-air oven for more than 8 h. Ash content was determined after 6 h of oxidation at 500°C in
a muffle furnace. . Crude protein was determined according to Hach et al. (1987). Starch was
43

analyzed using an enzymatic method to hydrolyze it to glucose (Karkalas, 1985) after samples
were gelatinized with sodium hydroxide; glucose concentration was measured by a glucose
oxidase method (Sigma Chemical Co., St. Louis, MO). Content of NDF was analyzed according
to Mertens (2002) with the inclusion of amylase and Na sulfite. Indigestible NDF was
determined as NDF residue after 240 h in vitro fermentation (Goering and Van Soest, 1970).
Ruminal fluid was collected from a non-pregnant fistulated dry cow and flasks were reinoculated at 120 h to ensure microbes were viable. Indigestible NDF was used to estimate fecal
output and apparent total-tract digestibility (Cochran et al., 1986). All samples were analyzed for
gross energy (GE) by bomb calorimeter according to the manufacturer’s instructions (Parr
Instrument Inc., Moline, IL). Digestible energy (DE) intake, MEI from the diet and total MEI
was calculated following the procedure described in Gualdrón-Duarte and Allen (2017).
Plasma samples were analyzed using commercial kits for concentration of nonesterified
fatty acids (NEFA; NEFA HR kit, Wako Chemicals USA, Richmond, VA), b-hydroxybutyrate
(BHB; kit no. 2240, Stanbio Laboratory, Boerne, TX). Triglycerides were analyzed with a
commercial kit (L-type Triglyceride M, Wako Chemicals USA, Richmond, VA) modifying the
quantity of sample used (40 µL). Insulin was determined using an ELISA kit (kit no. 10-120101, Mercodia, Uppsala, Sweden). Plasma concentration of metabolites were analyzed
colorimetrically with a microplate reader (SpectraMax Plus 384, Molecular Devices Corp.)
Plasma glucagon concentration was determined using a RIA kit (kit no. GL-32K, Linco Research
Inc., St. Charles, MO). Plasma propionate and lactate concentrations were analyzed by HPLC
(Waters Corp., Milford, MA). Plasma (600 μL) was mixed with 600 μL Ca(OH)2 and 300 mL of
CuSO4 containing crotonic acid as an internal marker in 1.7 ml microcentrifuge tubes. Samples

44

were frozen, thawed and centrifuged at 12,000 × g for 10 min, and supernatant (1000 μL) was
mixed with 42 μL of H2SO4 in 1.5-mL microcentrifuge tubes. Samples were frozen and thawed
twice, and centrifuged at 12,000 × g for 10 min to precipitate and remove protein. Supernatant
was transferred to HPLC vials. The column used was Aminex HPX-87H (catalog number 1250140; 300 x 7.8 mm; Bio-Rad Laboratories, Richmond, CA). Column temperature was 50°C,
and the solvent was 0.015N H2S04. Detection was by refractive index (Waters 410; Millipore
Corp., Milford, MA). Liver acetyl CoA (AcCoA) was determined by HPLC following the King
and Reiss (1985) procedure with modifications by Stocks and Allen (2012). Liver AcCoA
content was expressed per g of wet liver.
RNA Extraction and Real-Time Quantitative PCR
Treatment effects on expression of genes associated with lipid and carbohydrate
metabolism were analyzed. Methods, primers, and results are reported in the Appendix.
Statistical Analysis
Feeding behavior, intake, metabolite and hormone responses, yield of milk and milk
components, and concentration of milk components were analyzed using the Fit Model
procedure of JMP (version 12.0, 2015; SAS Institute Inc., Cary, NC) with the following model:
Yijkl    Si  S (Cj )i  S ( Pk )i  Tl  eijk

where Yijkl = the dependent response variable of interest, μ = overall mean, Si = random
effect of square (i = 1 to 2), S(Cj)i = random effect of cow within square (j = 4), S(Pk)i = fixed
effect of period within square (k = 1 to 4), Tl = fixed effect of treatment (l = 1 to 4), and eijkl =
residual, normally distributed. Treatment effects were declared significant at P ≤ 0.05 and

45

tendencies for treatment effects at P ≤ 0.10. Interactions were declared significant at P ≤ 0.10 and
tendencies for interactions at P ≤ 0.15. All data are expressed as least squares means (LSM) and
standard error of the means (SEM), unless otherwise specified. Contrasts were conducted
between each treatment and the control as pre-planned comparisons.
Eight cows started the experiment; however, one cow from the second block was
removed from the study due to displaced abomasum during the first period and all data from this
animal were excluded before statistical analysis. That cow was replaced with a spare, which
followed the subsequent treatments within its sequence beginning with the second period and
skipping the GLU infusion.
RESULTS
Feed Intake and Feeding Behavior
Results for feed intake and feeding behavior are reported in Table 3.2. Propionic acid and
LA decreased DMI compared with CON whereas GLU had no effect. Lactic acid decreased DMI
by 14% (16.3 vs. 18.9 kg/d, P = 0.02) by decreasing meal size by 20% (1.38 vs. 1.72 kg, P =
0.04) whereas PA decreased DMI by 24.3% (14.3 vs. 18.9 kg/d, P <0.001) by tending to
decrease the number of meals per day (10.6 vs. 12.3 meals; P = 0.09). Similarly, LA and PA
decreased DE intake by 15% and 22%, respectively compared with CON and decreased ME
intake from the diet by 15% and 21%, respectively compared with CON. However, only PA
infusion decreased total ME intake including ME from the infusions and the ME from the diet
compared with CON; PA decreased ME intake 13.2% compared with CON (34.8 vs. 40.1
Mcal/d; P = 0.04).

46

Effects of isoenergetic1 intra-abomasal infusion of glucose (GLU), lactic acid (LA)
and propionic acid (PA) relative to a no infusion control (CON) on feeding behavior and
energy intake for cows in the immediate postpartum period
Significance, P

DMI, kg/d
Feeding behavior (22h/d)
Meal frequency, meals/d
Meal length, min/meal
Meal Size, kg/meal
Eating interval, min
Hunger ratio2
Satiety ratio3
Water intake, L/d
Drink frequency, bouts/day
DM digestibility, %
DE intake (Mcal/d)4
ME intake (Mcal/d)5
Diet
Infusion6
Total7

GL vs. LA vs. PA vs.
CON
CON CON

CON

GLU

LA

PA

SEM

18.9

17.3

16.3

14.3

0.72

0.14

0.02

12.3
23.7
1.72
95.6
0.029
0.036
82.6
15.5
60.6
48.2

12.4
23.6
1.46
89.3
0.025
0.036
72.3
13.2
59.4
43.6

11.9
21.7
1.38
91.4
0.026
0.029
69.0
14.3
60.6
41.1

10.6
24.7
1.54
109.6
0.025
0.042
52.9
10.9
62.7
37.7

0.61
1.81
1.53
6.0
0.004
0.012
3.3
1.94
2.22
2.85

0.91
0.96
0.12
0.51
0.284
0.928
0.07
0.10
0.42
0.12

0.68
0.09
0.32
0.59
0.04
0.26
0.63
0.13
0.30
0.16
0.59
0.69
0.02 < 0.0001
0.33 < 0.01
0.99
0.17
0.02 <0.01

40.1
0
40.1

36.2
3.36
39.6

34.2
3.33
37.5

31.6
3.25
34.8

2.6
0.06
2.65

0.12

0.02

<0.01

0.80

0.28

0.04

< 0.001

1

Isoenergetic infusion, 151 kcal/h.
2
Hunger ratio = weight of meal/pre-meal interval
3
Satiety ratio = weight of meal/post-meal interval
4
Digestible energy (DE) intake (Mcal/d) = gross energy (GE) intake – GE feces.
5
ME intake from the diet was calculate according to NRC (2001). ME (Mcal/kg) = 1.01 x (DE – 0.45). ME intake
(Mcal/d) ME x DMI.
6
ME from the infusion was based on energy density of 0.673 Mcal/mol ME (Emery and Benedict, 1911) for GLU;
0.321 Mcal/mol (Saville and Gundry, 1959) for LA; and 0.365 Mcal/mol (Lavedeva, 1964) for PA.
7
Total ME intake = ME intake diet + ME infusion.

Infusion treatments did not affect any other feeding behavior variable compared with
CON (Table 3.2). Glucose infusion tended (P = 0.07) to decrease water intake and LA and PA
decreased (P = 0.02 and P = < 0.0001, respectively) water intake compared with CON. Propionic
acid decreased (P = < 0.01) the number of drinking bouts per day compared with CON.

47

Milk Production and Components
Milk yield averaged 37.5 kg/d and no differences were observed between infusion and
CON treatments (P > 0.50; Table 3.3). There was a tendency for PA to increase the true protein
content in milk (P = 0.07, 3.11% vs. 2.87%), true protein yield (P = 0.10, 1.19 kg/d vs. 1.08
kg/d), and milk urea nitrogen concentration in milk (9.68 mg/dL vs. 8.38 mg/dL, P = 0.10)
compared with CON. Lactic acid tended to decrease lactose yield (P = 0.08, 1.56 kg/d vs. 1.70
kg/d) compared with CON.
Effects of intra-abomasal infusion of propionic acid (PA), lactic acid (LA) and
glucose (GLU) relative to a control (CON) with no infusion on milk yield and milk
components over the 22 h/d infusion for cows in the immediate postpartum period for
isoenergetic infusions1
Infusion
Significance, P
GL vs. LA vs. PA vs.
CON GLU
LA
PA
SEM
CON
CON
CON
Milk yield kg/d
37.9
38.1
36.3
37.8
2.16
0.92
0.50
0.96
Fat, %
4.58
4.43
4.09
4.63 0.321
0.76
0.30
0.91
Fat yield, kg/d
1.68
1.59
1.47
1.77 0.109
0.61
0.21
0.61
True protein, %
2.87
2.70
2.70
3.11 0.145
0.42
0.56
0.07
True protein yield,
1.08
1.13
0.99
1.19 0.043
0.55
0.16
0.10
kg/d
Lactose,
%
4.50
4.50
4.38
4.72 0.043
0.67
0.56
0.28
Lactose yield, kg/d
1.70
1.71
1.56
1.78 0.044
0.77
0.08
0.32
Solids, %
5.45
5.38
5.21
5.50 0.258
0.81
0.37
0.58
Total solids, kg/d
1.98
1.97
1.97
2.00 0.010
0.90
0.92
0.86
2
MUN , mg/dL
8.38
9.40
8.87
9.68
2.02
0.22
0.52
0.10
1
2

Isoenergetic infusion, 151 kcal/h.
MUN = milk urea nitrogen

Hepatic Acetyl CoA content and Plasma Hormones and Metabolites
Results for hepatic AcCoA content and plasma concentration of hormones and
metabolites before and after infusion, and the differences between them are shown in Table 3.4.
After 22 h of infusion, GLU tended to decrease (P = 0.10, 8.5 nmol/g) and LA decreased (P =

48

0.04, 11.1 nmol/g) liver AcCoA content compared with CON, but PA had no effect compared
with CON.
All three infusion treatments increased plasma insulin concentration compared with
control. Infusion of GLU had the greatest effect, more than doubling plasma insulin
concentration by 0.22 µg/ L (P < 0.0001) whereas, LA and PA increased plasma insulin
concentration by 0.05 µg/L (P = 0.03) and 0.06 µg/L (P = 0.02), respectively. Plasma glucagon
concentration decreased by 9.7 pg/mL after GLU infusion which was greater than CON (P =
0.04) but the decreases for LA and PA infusions were not different from CON.
Glucose treatment decreased plasma NEFA concentration by 242 µEq/L which tended to
differ from CON (P = 0.06) in which plasma NEFA concentration decreased 56 µEq/L over the
same period, whereas, LA and PA did not differ from CON. Propionic acid treatment decreased
plasma BHB by 5.8 mg/dL which was greater (P = 0.02) than the change for CON of 1.3 mg/dL
and both GLU and LA decreased plasma BHB by ~4.4 mg/dL, which tended to be greater than
the change for CON (P ≤ 0.09).
Treatment infusions did not affect plasma concentrations of acetate or lactate compared
with CON. However, propionate concentration increased after PA infusion (P = 0.01), and
tended to increase after GLU infusion (P = 0.09) compared with CON. Glucose treatment
increased (P < 0.01) and LA infusion tended to increase (P = 0.08) plasma glucose concentration
compared with CON.

49

Effect of intra-abomasal infusion of propionic acid (PA), lactic acid (LA) and glucose (GLU) relative to a control
(CON) with no infusion on initial, final and change in plasma concentration of insulin, glucagon, metabolites and liver
acetyl CoA (AcCoA) content over the 22 h/d infusion for cows in the immediate postpartum period for isoenergetic
infusions1
Significance, P
GL vs.
LA vs.
PA vs.
CON
GLU
LA
PA
SEM
CON
CON
CON
Hepatic AcCoA before, nmol/g
26.3
21.4
21.5
23.1
3.03
0.10
0.10
0.27
Hepatic AcCoA after, nmol/g
19.1
15.5
13.5
18.9
2.46
0.38
0.18
0.97
Change in hepatic AcCoA, nmol/g
5.81
-2.66
-5.3
2.5
3.18
0.10
0.04
0.50
Insulin before, µg/L
0.188
Insulin after, µg/L
0.181
Change in insulin, µg/L
-0.007
Glucagon before, pg/mL
148.5
Glucagon after, pg/mL
144.5
Change in glucagon, pg/mL
2.63
Insulin to glucagon ratio before
1.29
Insulin to glucagon ratio after
1.29
Change in insulin to glucagon ratio 0.989
NEFA before, µEq/L
715
NEFA after, µEq/L
659
Change in NEFA, µEq/L
-55.9
BHB before, mg/dL
11
BHB after, mg/dL
9.72
Change in BHB, mg/dL
-1.31
Triacylglycerides before, mg/dL
4.60
Triacylglycerides after, mg/dL
4.94
Change in triacylglycerides, mg/dL 0.338
Glucose pre infusion, mM
2.81
Glucose post infusion, mM
2.87
Change in glucose, mM
0.059

0.208
0.425
0.222
149.8
134.2
-9.7
1.40
3.23
2.46
613
398
-241.7
10.1
5.90
-4.3
5.27
5.06
-0.525
2.88
3.21
0.352

0.187
0.238
0.051
149.6
138.5
-5.45
1.26
1.73
1.67
624
602
-22.3
10.5
6.12
-4.42
5.1
5.02
-0.083
2.75
2.97
0.22
50

0.194
0.254
0.06
149.8
140.3
-5.84
1.37
1.85
1.59
665
532
-117.8
11.1
5.60
-5.55
5.05
4.3
-0.754
2.86
2.95
0.085

0.04
0.034
0.019
8.13
10.2
4.53
0.309
0.397
0.181
103.5
92.3
5.1
1.07
0.582
1.18
1.18
0.923
0.405
0.09
0.06
0.06

0.50
<0.0001
<0.0001
0.84
0.09
0.04
0.57
<0.0001
<0.0001
0.13
<0.001
0.06
0.52
<0.01
0.09
0.05
0.79
0.16
0.25
<0.001
0.01

0.95
0.12
0.03
0.86
0.29
0.14
0.88
0.18
0.007
0.16
0.35
0.71
0.73
<0.01
0.06
0.11
0.85
0.48
0.36
0.19
0.08

0.83
0.05
0.02
0.83
0.46
0.13
0.67
0.10
0.014
0.31
0.05
0.50
0.93
<0.001
0.02
0.15
0.15
0.08
0.41
0.30
0.77

Table 3.4. (cont’d)
Lactate before, mM
Lactate after, mM
Change in lactate, mM
Propionate before, mM
Propionate after, mM
Change in propionate, mM

0.479
0.518
0.039
0.230
0.218
-0.012

0.712
0.515
-0.150
0.244
0.204
-0.047

0.427
0.852
0.425
0.227
0.238
0.012

1

Isoenergetic infusion, 151 kcal/h.

51

0.336
0.574
0.238
0.222
0.259
0.037

0.11
0.136
0.201
0.022
0.02
0.029

0.13
0.99
0.49
0.38
0.51
0.09

0.71
0.12
0.15
0.79
0.32
0.20

0.32
0.79
0.45
0.56
0.05
0.01

Hepatic gene expression
Infusion treatments increased hepatic expression of GLUD1 (glutamate dehydrogenase
1), GPAM (glycerol-3-phosphate acyltransferase) and PCK1 (phosphoenolpyruvate
carboxykinase 1) compared with CON [false discovery rate (FDR) value < 0.05]. Relative
mRNA abundance of GLUD1 was increased 4.0 fold by GLU (P = 0.01), 3.3 fold by PA (P =
0.02) and 2.7 fold by LA (P = 0.04) compared with CON. Relative mRNA abundance of GPAM
was increased 3.1 fold by LA (P = 0.01), 2.5 fold by GLU (P = 0.01) and 2.0 fold by PA (P =
0.05) compared with CON. Relative mRNA abundance of PCK1 was increased 6.0 fold by GLU
(P = 0.03) and 5.4 fold by PA (P = 0.03) but was not affected by LA compared with CON.
Changes in hepatic expression of FBP1 (fructose-1,6-bisphosphatase), GOT2 (aspartate
aminotransferase), PC (pyruvate carboxylase), PCCA (propionyl-CoA carboxylase alpha), and
PPARA (peroxisome proliferator activated receptor alpha) were considered as potential (FDR <
0.10) hepatic genes affected by infusion of fuels and results are reported in the Appendix.
DISCUSSION
Our results indicate that fuels derived from ruminal fermentation and intestinal digestion
of starch result in different effects on DMI and MEI of cows in the PP period compared with a
sham control consistent with their expected effects on hepatic oxidation. According to the HOT,
anaplerotic fuels extracted by the liver may promote oxidation of AcCoA increasing hepatic
energy charge and contributing to satiety (Allen et al., 2009). Of the fuels produced from the
fermentation and digestion of starch, propionate is expected to stimulate hepatic oxidation to the
greatest extent because it is efficiently extracted from the blood by the liver and is obligatorily
anaplerotic, stimulating TCA cycle activity and oxidation of AcCoA (Allen, 2014). Therefore,
the greater reduction in DMI and MEI by PA compared with CON among treatments was
52

expected. Although the change in hepatic AcCoA content over the 22 h infusion period was
similar for PA compared with CON, entry and oxidation in the TCA cycle was likely greater for
PA compared with CON because PA decreased BHB concentration in plasma but not NEFA
concentration compared with CON. Additionally, pulse doses of propionic acid decreased
hepatic AcCoA content at 30 and 60 minutes post-dosing and changes in AcCoA content over a
day do not reflect changes relative to meals as discussed by Gualdron and Allen (2017)
Intraruminal infusion of PA decreased DMI and MEI of cows in the PP period by
decreasing meal size with no effect on meal frequency compared with acetic acid (Stocks and
Allen, 2013). Whereas, we expected PA to reduce meal size compared with CON, the effect of
PA on meal size was not significant and the reduction in DMI and MEI was from combination of
a numerical reduction of 11% in meal size and a tendency for a 14% decrease in meal frequency
compared with CON.
The hypophagic effects of propionate compared with acetate have been well established
by previous experiments reported in the literature (Allen, 2000). While isomolar amounts of
propionate and acetate were generally infused, propionate has greater ME per mole than acetate,
and the effects on total MEI accounting for the ME infused were not measured in most studies.
However, propionate also decreased total MEI from the diet and treatment infusions in lactating
cows compared with acetate when infused intra-ruminally in one study (Oba and Allen, 2003b).
Although both propionic acid and acetic acid can be produced by fermentation of starch, we did
not include acetic acid as a treatment in the present experiment because increased grain feeding
increases ruminal production of propionic acid (Bauman et al., 1971; Sutton et al., 2003) with
little effect on acetic acid production (Davis, 1967; Sutton et al., 2003), and because its effects on
DMI compared with propionic acid have been established previously (Allen, 2000). The effects
53

of propionate and acetate on feed intake are consistent with the HOT because little acetate is
extracted from the blood by the liver (Knapp et al., 1992) and acetate is not anaplerotic and
cannot stimulate hepatic oxidation.
The importance of the anaplerotic effects of fuels was demonstrated by a study in which
propionic acid decreased DMI and MEI of cows in the PP period compared with isoenergetic
infusion of glycerol (Guadrón-Duarte and Allen, 2017). Whereas glycerol is potentially
anaplerotic, its anaplerotic effect is likely greatly diminished when glucose demand is high in the
PP period, entering the gluconeogenic pathway in the cytosol instead of stimulating hepatic
oxidation and satiety (Guadrón-Duarte and Allen, 2017).
Lactate also is anaplerotic, entering the TCA cycle as oxaloacetate through pyruvate, and
can also stimulate oxidation of AcCoA. In support of this, we found that LA infusion decreased
hepatic AcCoA content and tended to decrease plasma BHB concentration. However, extraction
of lactate from the blood by the liver is much less than propionate (Reynolds et al., 2003) likely
because metabolism of lactate to pyruvate is thermodynamically unfavorable when cytolosic
NADH to NAD ratio is elevated (Allen et al., 2009). In addition, pyruvate from lactate might be
used for alanine conversion through transamination. Therefore, the reduction in DMI by LA
compared with control was expected. Whereas LA reduced DMI compared with CON, the effect
was not adequate to significantly reduce MEI when the ME of the diet and infusate were
combined. The reduction in DMI was from a reduction in meal size indicated that its effect was
during the timeframe of meals. The LA treatment tended to increase glucose, consistent with its
use as a glucose precursor and increased plasma insulin concentration. Plasma propionate
concentration was increased 17% by PA over the 22 h infusion, but plasma lactate concentration

54

was doubled (numerical only) consistent with their expected efficiency of hepatic extraction
from the blood.
Infusion of GLU did not affect DMI or MEI compared with CON, consistent with
previous experiments with midlactation cows in which glucose was infused post-ruminally
(Frobish and Davis, 1977; Clark et al., 1977) or intravenously (Dowden and Jacobsen, 1960; AlTrad et al., 2009) in dairy cows. The ruminant liver extracts little glucose from the blood
(Stangassinger and Giesecke, 1986) because activities of glucokinase and hexokinase are low
(Emmanuel, 1981). Therefore, glucose is unable to stimulate hepatic oxidation directly and its
effect on DMI compared with control was as expected. Although a significant effect of glucose
on DMI compared with CON was not detected, the numerical reduction in DMI is likely the
reason for the lack of effect of GLU on MEI compared with CON because the numerical
reduction in MEI of the diet that was similar to the amount of ME infused per day.
Whereas, we did not detect a reduction of DMI by GLU treatment, infusion of graded
amounts of glucose (0 to 1,500 g/d) into the duodenum of lactating cows (~60 d postpartum)
decreased DMI linearly, but did not affect NEL intake when the infused glucose was considered
(Hurtaud et al., 1998) and infusion of 1,500 g of starch hydrolysate into the rumen or abomasum
reduced DMI but increased NE intake of cows in early lactation (24 to 66 d postpartum) when
the infused glucose was considered (Knowlton et al., 1998). Variation in effects of glucose
infusions on DMI and MEI might be from differences in the dose of glucose, physiological state
of cows, differences in diet composition, and length of infusion. Although, the above
experiments were not conducted during the PP period, continuous infusion of glucose (1,500 g/d)
into the abomasum of cows from 4 to 29 days in milk, decreased DMI ~6 kg/d (Larsen and
Kristensen, 2009). We also infused during the PP period but infused a lower dose (~60%) for
55

only one day. Differences for the effects of glucose infusion between these experiments might be
because treatment had a greater effect on increasing plasma insulin concentration for a longer
time which might have down-regulated gluconeogenesis, decreasing cataplerosis of the TCA
cycle, increasing TCA cycle activity and oxidation of AcCoA, and stimulating satiety sooner, as
discussed by Brown and Allen (2013). Effects of glucose infusion on meal size and DMI were
dependent upon the diet in an experiment in which glucose was infused at a rate of 1,000 g/d for
the first 12 d PP (Brown and Allen, 2013). Therefore, depression of DMI by glucose might be
related to its indirect effects on hepatic oxidation, dependent on dose, diet and physiological state
of cows.
Increased expression of hepatic GPAM, the gene coding for the enzyme responsible for
the first and committed step in glycerolipid synthesis, by all infusion treatments was consistent
with effects on increasing plasma insulin concentration because SREBF1 (Mughal et al., 2010), a
nuclear factor which up-regulates GPAM expression is induced by insulin. Infusion of GLU and
PA increased expression of hepatic PCK1 despite increasing plasma insulin concentration.
Insulin has been reported to decrease mRNA abundance of PCK1 in rat liver cells (Granner et
al., 1983) and glucose infusion in cows numerically increased blood insulin concentration 54%
and decreased hepatic mRNA abundance of PCK1 51% compared to water infusion although the
treatments were not directly compared statistically (Zhang et al., 2015). However, in that study
hepatic PCK1 expression was maintained when serum insulin concentration was nearly doubled
in cows infused with propionate (Zhang et al., 2015). Differences in effects of propionate and
glucose infusion on PCK1 expression between the current experiment and the experiment of
Zhang et al. (2015) might be because we infused propionate and glucose at twice the rate (0.41
and 0.22 mol/h, respectively) compared with (0.21 and 0.11 mol/h, respectively), for longer (22 h
56

vs. 8 h) to cows in the immediate PP period compared with mid-lactation. Glutamate
dehydrogenase 1 (GLUD1) converts L-glutamate into alpha-ketoglutarate and plays a key role in
anaplerosis of the TCA cycle. However, the mechanism by which all treatments increased its
expression is not known.
CONCLUSION
Fuels supplied by the ruminal fermentation and intestinal digestion of starch have
different effects on DMI that are consistent with their ability to stimulate hepatic oxidation of
AcCoA. Greater ruminal starch fermentation increases the production of propionic acid, which
decreased intake of dry matter and total metabolizable energy compared with control. However,
the primary fuels from digestion of starch in the small intestine did not decrease metabolizable
energy intake of cows compared with control. Depression of feed intake of cows in the PP period
by diets containing highly fermentable starch is likely because of the ability of propionic acid to
stimulate hepatic oxidation.
ACNOWLEDGEMENTS
We acknowledge the financial support for this project from USDA National Research
Initiative Competitive Grant no. 2014-67015-21708 to MSA, and from Fulbright Colombia and
Colciencias to LGD. We also thank D. G. Main, R. A. Longuski, J. Liesman, R. West, G.
Maldini, K. Kennedy, R. Albornoz, R. Yair and, D. de Sousa (all from Michigan State
University) for technical assistance, G.A. Contreras, C. Strider, Norma Manrique-Carpintero and
J. Landgraf for assistance on gene expression analysis, R. J. Tempelman and J. P. Steibel for
statistical advice, and Kemin, Inc. for supplying the propionic acid treatment.

57

APPENDICES

58

APPENDIX A: GENE EXPRESSION
RNA Extraction and Real-Time Quantitative PCR.
Total RNA was extracted from ~15 mg of hepatic tissue using the RSC simply RNA
Tissue Kit (Promega Corp., Madison, WI) and then purified using the RQ1 RNase-Free DNase
Kit (Promega Corp., Madison, WI). Purity, concentration, and integrity of mRNA were evaluated
using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and an Agilent
Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) system. All samples had a RNA
integrity number > 6. Conversion to cDNA was performed using the Applied Biosystems High
Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA).
Quantitative PCR Analysis
Gene expression of predetermined genes was determined using real-time quantitative
PCR (qPCR) and primers were designed using Primer Express 3.0 (Life Technologies Corp.) and
Lasergene 9 core suite software (DNASTAR) and synthesized commercially (Integrated DNA
Technologies, Coralville, IO). Primer sequences and accession numbers are summarized in
Supplemental Table 3.5. Complementary DNA was synthesized from 100 ng of total RNA as a
template using the High-Capacity cDNA Reverse-Transcription Kit (Life Technologies Corp.),
and amplification efficiency was validated for endogenous control and target genes with polled
cDNA from samples using a 5-fold dilution series. A non-reverse-transcriptase control sample
validated absence of genomic DNA.
For relative gene expression, samples were assayed in triplicate on the Wafergen
Smartchip System (WaferGen Biosystems, Fremont, CA) using cDNA at a primer-efficient
concentration of 500 pg/µL and 10 uL of Power SYBR Green (Life Technologies Corp.), 10 µL
of a forward and reverse primer mix (1 µM each), and 5 µL of sample (0.5 ng of cDNA/µL).
59

The relative gene expression was determined using HPRT1, PGK1 and RPS9 as
endogenous control genes.
Gene expression was analyzed from raw cycles to threshold (CT) values obtained using
Data Assist software (Applied Biosystems) and then corrected for efficiency using the formula
CTcorrected = -log2 (E –CT). The computation of ΔCT for genes of interest pre and post infusion was
calculated using the corrected CT values for genes of interest and the mean of the corrected CT
for the control genes which included HPRT1, PGK1 and RPS9I, according to the formula
ΔCT = CT Target gene pre or post-infusion – CT Mean of control genes, for computing the difference in expression
after infusion was used the following formula, ΔΔCT = ΔCT Target gene post-infusion – ΔCT Target gene preinfusion.

ΔCT and ΔΔCT values were analyzed with the following model using the Fit Model
procedure of JMP (version 12.0, 2015; SAS Institute Inc., Cary, NC):
Yijkl    Si  S (Cj )i  S ( Pk )i  Tl  eijk

where Yijkl = the ΔCT of the gene of interest in each observational unit, μ = overall mean,
Si = fixed effect of square (i = 1 to 2), S(Cj)i = random effect of cow within square (j = 4), S(Pk)i
= random effect of period within square (k = 1 to 4), Tl = fixed effect of treatment (l = 1 to 4),
and eijkl = residual, normally distributed. Treatment effects were declared significant at P ≤ 0.05
and tendencies for treatment effects at P ≤ 0.10. Interactions were declared significant at P ≤ 0.10
and tendencies for interactions at P ≤ 0.15. All data are expressed as LSM ± SEM, unless
otherwise specified. This model is equivalent to the model described by Steibel et al. (2009).

60

Results are presented as fold changes relative to the CON samples. These were calculated
from difference in least squares mean differences according to the formula 2−diff, where diff =
LSmeans(treatment ) - LSmeans(CON), i.e. it is the difference in the least squares means obtained from
the mixed model. After analysis P values for treatment effect were subject to Multiple Test
correction using a local false discovery rate (FDR) value < 0.05. Results on LSM of the CT
differences after infusion relative to before infusion are reported in Supplemental Table 3.6. Fold
changes (FC) on hepatic genes affected by infusion treatments and the confidence intervals for
the fold changes are reported in Supplemental Table 3.7 and 3.8 respectively.

61

Primer sequences and accession numbers for genes analyzed with real-time
quantitative PCR
Gene1
Accession number
Forward Sequence (5’-3’)
Reverse Sequence (5’-3’)
ADH1C
NM_001206387.1
CAGGACTACGAGAAACCCATC
TCCTACAATGACGCTTACACC
CPT1A
XM_019954456.1
TGACGGCTCTGGCACAAG
TTTTAGCTTCAAATACCACTGCCAATC
CS
NM_001044721
ACTAATGCATGTAGTGTGGGTTAGGT
AAGAGCCAGATTCCCACTCTGA
FBP1
XM_019966535.1
GGCACGGGCGAGATGA
CTTTGACCGCGGTGCAA
G6PC
NM_183364
CCATATCCGAAACCAATCAAGAG
GAACAGGCAAGGAGGAGGAGTT
GAPDH
XM_019960295.1
TGCCGCCTGGAGAAACC
CGCCTGCTTCACCACCTT
GCGR
XM_019982395.1
GAGCCCACCAGATGCGTTAT
GATGAGGGTCAGTGTGGACTTG
GLUD1
NM_182652.2
CGCTCTGCCAGGCAAATC
GGTCCAGCCCCAGGTTATACTT
GOT2
XM_019979412.1
GGCTGTGCGCCACTTCAT
TTGGCATAGGACTGGCAGAGA
GPAM
NM_001012282
GAAGTCATGAGGGTGACGAGAAA
TGCCACAACCTGAGCTTACACT
HMGCS1
XM_019982983.1
AAGCTCCGAGAGGATACTCATCA
TCGAAGAGGGAATCTATGGAACTC
HMGCS2
AA112667
CCTGCTGCAATCACTGTCATG
TCTGTCCCGCCACCTCTTC
HNF4A
NM_001015557
GGCGTCCCGCCAGACT
GCACAGGACCCGATGACATC
2
HPRT1
NM_001034035
TGGCGTCCCAGTGAAATCA
CAGCTGGCCACAGAACAAGA
MCEE
NM_001045995
GGGCTGGCCCTGTATGG
GCACTGCGACTGCAACATG
MUT
NM_173939
CTCGCTGCTGGGCACAA
AAGGGCGTTGAGTTCCTTGA
PC
NM_177946
AGGCAAGACGCTGCACATC
GCCCGCCCGGTTGA
PCCA
NM_001083509
GGTCAGGAAATCTGTGTGATTGAA
GTCTTCCCGGCCGTCATA
PCCB
BC109784
CGAATCTGCTGTGACCTGGAT
TCCTCCAAGGGCGCTGTAC
PCK1
NM_174737
CAGCCAAGCTGCCCAAGA
CCGGCCTTGTCCTTTCG
PCK2
XM_019968310.1
CCCCCACCCCTACTCCAA
62

Table 3.5 (cont’d)
PDK4

NM_001101883

PGK12

NM_001034299.1

PPARA

NM_001034036

RPS92

XM_019978604.1

SLC374A

NM_001193045

TGCTCATAGTTTCTGCAAGCCTAGT
GAGGTGGTGTTCCCCTGAGA
TTGGTGCAGTGGAGTACGTGTAA
CAAGGATGTTTTGTTCTTGAAGGA
GGGTCAGCACAAGCCTTCTC
CAGCGCCGAGGAGTCATC
TGTCCCCGCAGATCCTACAC
TCCCGCCTCGACCAAGA
TTTGTTCCGGAGCCCATACT
TTGTCATGCCGTCGTTGGT
GGTGATGAGCCCCAAGTCA

1

ADH1C = alcohol dehydrogenase 1C; CPT1A = carnitine palmitoyltransferase 1A; CS = citrate synthase; FBP1 =
fructose-1,6-bisphosphatase 1; G6PC = glucose-6-phosphatase; GADPH = glyceraldehyde-3-phosphate
dehydrogenase; GCGR = glucagon receptor; GLUD1 = glutamate dehydrogenase 1; GOT2 = aspartate
aminotransferase 2; GPAM = glycerol-3-phosphate acyltransferase; HMGCS1 = HMG-CoA synthase 1; HMGCS2
= HMG-CoA synthase 2; HNF4A = hepatocyte nuclear factor 4 alpha; HPRT1 = hypoxanthine
phosphoribosyltransferase 1; MCEE = methylmalonyl CoA epimerase; MUT = methylmalonyl CoA mutase; PC =
pyruvate carboxylase; PCCA = propionyl-CoA carboxylase alpha; PCCB = propionyl-CoA carboxylase beta;
PCK1 = phosphoenolpyruvate carboxykinase 1; PCK2 = phosphoenolpyruvate carboxykinase 2; PDK4 = pyruvate
dehydrogenase kinase; PGK1 = phosphoglycerate kinase 1; PPARA = peroxisome proliferator activated receptor
alpha; RPS9 = ribosomal protein S9; SLC37A4 = solute carrier family 37 A4.
2
Endogenous control gene.

63

Effect of isoenergetic1 intra-abomasal infusion of glucose (GLU), lactic acid (LA)
and propionic acid (PA) and a no infusion control (CON) on least square means of the CT
differences after infusion relative to before infusion2 on hepatic gene expression for cows in
the immediate postpartum period
Gene3
CON
GLU
LA
PA
SEM
P - value
ADH1C
3.52
0.07
-0.28
0.02
2.285
0.43
CPT1A
0.20
-0.27
0.76
0.53
0.552
0.62
CS
0.08
0.03
-0.39
-0.29
0.480
0.83
FBP1
1.85
-0.35
1.51
0.26
0.670
0.11
G6PC
2.63
0.94
0.79
0.42
1.042
0.40
GAPDH
0.25
0.22
-0.09
0.09
0.307
0.88
GCGR
3.25
0.52
1.30
0.07
1.508
0.29
GLUD1
1.56
-0.44
0.11
-0.14
0.415
0.03
GOT2
1.15
0.22
-0.23
0.10
0.334
0.10
GPAM
1.47
0.12
-0.14
0.45
0.374
0.02
HMGCS1
1.15
-0.45
0.54
0.82
0.667
0.28
HMGCS2
0.36
-0.51
-0.29
-0.08
0.410
0.61
HNF4A
0.98
0.18
-0.20
0.20
0.435
0.51
MCEE
1.39
0.03
0.66
0.47
0.510
0.33
MUT
1.34
0.43
0.39
0.21
0.447
0.36
PC
1.33
0.37
0.55
-0.01
0.369
0.16
PCCA
1.45
0.15
-0.14
0.12
0.473
0.08
PCCB
0.96
-0.12
-0.08
0.24
0.403
0.14
PCK1
2.34
-0.25
1.53
-0.11
0.854
0.06
PCK2
1.12
-0.05
0.41
1.14
0.535
0.43
PDK4
0.69
0.06
0.46
0.26
0.410
0.79
PPARA
1.12
-0.05
0.24
-0.13
0.324
0.10
SLC374A
1.27
0.25
0.04
-0.02
0.551
0.37
1

Isoenergetic infusion, 151 kcal/h.
ΔCT = CT Target gene pre or post-infusion – CT Mean of control genes, for computing the difference in expression after infusion was
used the formula ΔΔCT = ΔCT Target gene post-infusion – ΔCT Target gene pre-infusion.
3
ADH1C = alcohol dehydrogenase 1C; CPT1A = carnitine palmitoyltransferase 1A; CS = citrate synthase; FBP1 =
fructose-1,6-bisphosphatase 1; G6PC = glucose-6-phosphatase; GADPH = glyceraldehyde-3-phosphate
dehydrogenase; GCGR = glucagon receptor; GLUD1 = glutamate dehydrogenase 1; GOT2 = aspartate
aminotransferase 2; GPAM = glycerol-3-phosphate acyltransferase; HMGCS1 = HMG-CoA synthase 1; HMGCS2
= HMG-CoA synthase 2; HNF4A = hepatocyte nuclear factor 4 alpha; MCEE = methylmalonyl CoA epimerase;
MUT = methylmalonyl CoA mutase; PC = pyruvate carboxylase; PCCA = propionyl-CoA carboxylase alpha;
PCCB = propionyl-CoA carboxylase beta; PCK1 = phosphoenolpyruvate carboxykinase 1; PCK2 =
phosphoenolpyruvate carboxykinase 2; PDK4 = pyruvate dehydrogenase kinase; PPARA = peroxisome proliferator
activated receptor alpha; SLC37A4 = solute carrier family 37 A4.
2

64

Fold changes (FC)1 on hepatic genes affected by isoenergetic2 intra-abomasal
infusion of glucose (GLU), lactic acid (LA) and propionic acid (PA) relative to a no infusion
control (CON) for cows in the immediate postpartum period
Confidence interval4
3
Gene
Infusion
FC
Lower
Upper P value
FBP1
GLU
4.60
0.11
2.36
0.04
LA
0.74
1.26
0.40
0.64
PA
0.12
3.01
0.17
1.55
GLUD1
GLU
0.01
3.99
0.16
2.62
LA
0.04
2.73
0.24
1.75
PA
0.02
3.25
0.20
2.11
GOT2
GLU
0.10
1.89
0.37
1.32
LA
0.02
2.60
0.27
1.80
PA
0.06
2.07
0.34
1.43
GPAM
GLU
0.01
2.54
0.28
1.83
LA
0.00
3.05
0.23
2.18
PA
0.05
2.03
0.35
1.46
PC
GLU
0.11
1.95
0.35
1.32
LA
0.19
1.72
0.39
1.16
PA
0.03
2.53
0.27
1.71
PCCA
GLU
0.05
2.04
0.27
1.62
LA
0.02
3.01
0.22
1.96
PA
0.04
2.51
0.26
1.64
PCK1
GLU
0.03
6.00
0.08
2.93
LA
0.45
1.75
0.28
0.85
PA
0.03
5.44
0.09
2.66
PPARA
GLU
0.03
2.24
0.31
1.58
LA
0.11
1.84
0.38
1.29
PA
0.03
2.37
0.30
1.67
FC = 2−diff, where diff= LSM infusion treatment – LSM CON, i.e. it is the difference in the least squares means obtained
from the mixed model.Gene expression fold changes were calculated from LSM differences of the ΔC T values
(ΔΔCT) normalized to the mean of HPRT1, PGK1 and RPS9 housekeeping genes. The CON treatment was the
calibrator for the calculation of ΔΔCT.
2
Isoenergetic infusion, 151 kcal/h.
3
Genes listed in the table were subject to Multiple Test correction using a local FDR value <0.10, FBP1 = fructose1,6-bisphosphatase 1; GLUD1 = glutamate dehydrogenase 1; GOT2 = aspartate aminotransferase 2; GPAM =
glycerol-3-phosphate acyltransferase; PC = pyruvate carboxylase; PCCA = propionyl-CoA carboxylase alpha;
PCK1 = phosphoenolpyruvate carboxykinase 1; PPARA = peroxisome proliferator activated receptor alpha.
4
95% confidence intervals of the ΔΔCT values were transformed to fold changes and calculations are presented in
Supplemental Table 3.8.
1

65

Confidence intervals1 of hepatic gene expression fold changes (FC) in genes affected by isoenergetic2 intra-abomasal
infusion of glucose (GLU), lactic acid (LA) and propionic acid (PA) relative to a no infusion control (CON) for cows in the
immediate postpartum period
log
Lower
FC
FC
Gene4
Infusion
Diff
SEM
P value
Upper CI
FC
FC3
CI
Lower CI Upper CI
FBP1
GLU
-2.20
0.961
0.04
2.20
3.16
-1.24
4.60
0.11
2.36
LA
-0.34
0.981
0.74
0.34
1.32
0.64
1.26
0.40
0.64
PA
-1.59
0.963
0.12
1.59
2.56
-0.63
3.01
0.17
1.55
GLUD1
GLU
-2.00
0.609
0.01
2.00
2.61
-1.39
3.99
0.16
2.62
LA
-1.45
0.639
0.04
1.45
2.09
-0.81
2.73
0.24
1.75
PA
-1.70
0.619
0.02
1.70
2.32
-1.08
3.25
0.20
2.11
GOT2
GLU
-0.92
0.527
0.10
0.92
1.45
-0.40
1.89
0.37
1.32
LA
-1.38
0.534
0.02
1.38
1.91
-0.85
2.60
0.27
1.80
PA
-1.05
0.529
0.06
1.05
1.58
-0.52
2.07
0.34
1.43
GPAM
GLU
-1.35
0.473
0.01
1.35
1.82
-0.87
2.54
0.28
1.83
LA
-1.61
0.483
0.00
1.61
2.09
-1.13
3.05
0.23
2.18
PA
-1.02
0.475
0.05
1.02
1.50
-0.54
2.03
0.35
1.46
PC
GLU
-0.97
0.568
0.11
0.97
1.53
-0.40
1.95
0.35
1.32
LA
-0.79
0.577
0.19
0.79
1.36
-0.21
1.72
0.39
1.16
PA
-1.34
0.569
0.03
1.34
1.91
-0.77
2.53
0.27
1.71
PCCA
GLU
-1.30
0.611
0.05
1.03
1.91
-0.69
2.04
0.27
1.62
LA
-1.59
0.617
0.02
1.59
2.21
-0.97
3.01
0.22
1.96
PA
-1.33
0.615
0.04
1.33
1.94
-0.71
2.51
0.26
1.64
PCK1
GLU
-2.58
1.032
0.03
2.58
3.62
-1.55
6.00
0.08
2.93
LA
-0.81
1.052
0.45
0.81
1.86
0.24
1.75
0.28
0.85
PA
-2.44
1.032
0.03
2.44
3.48
-1.41
5.44
0.09
2.66
PPARA
GLU
-1.17
0.502
0.03
1.17
1.67
-0.66
2.24
0.31
1.58
LA
-0.88
0.513
0.11
0.88
1.40
-0.37
1.84
0.38
1.29
PA
-1.25
0.504
0.03
1.25
1.75
-0.74
2.37
0.30
1.67
95% confidence intervals of the ΔΔCT values were transformed to fold changes
Isoenergetic infusion, 151 kcal/h
3
Fold changes are relative to gene expression in the CON treatment, normalized to the mean of HPRT1, PGK1 and RPS9 housekeeping genes.
4
Genes listed in the table were subject to Multiple Test correction using a local FDR value <0.10, FBP1 = fructose-1,6-bisphosphatase 1; GLUD1 = glutamate
1
2

66

Table 3.8. (cont’d)
dehydrogenase 1; GOT2 = aspartate aminotransferase 2; GPAM = glycerol-3-phosphate acyltransferase; PC = pyruvate carboxylase; PCCA = propionyl-CoA
carboxylase alpha; PCK1 = phosphoenolpyruvate carboxykinase 1; PPARA = peroxisome proliferator activated receptor alpha.

67

APPENDIX B: BLOOD METABOLITES

Effects of isoenergetic intra-abomasal infusion of glucose (GLU), lactic acid (LA)
and propionic acid (PA) relative to a no infusion control (CON) on change in plasma
concentration of insulin, metabolites and liver acetyl CoA (AcCoA) content over the 22
h/d infusion for cows in the immediate postpartum period

68

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69

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CHAPTER 4. EFFECTS OF ACETIC ACID OR SODIUM ACETATE INFUSED INTO
THE RUMEN OR ABOMASUM ON FEEDING BEHAVIOR AND METABOLIC
RESPONSE OF COWS IN THE POSTPARTUM PERIOD

ABSTRACT
Effects of continuous isomolar infusions of acetic acid (AA) or sodium acetate (NA)
infused into the rumen (RU) or into the abomasum (AB) on feeding behavior, dry matter intake
(DMI), and metabolic response of cows in the early postpartum period were evaluated. Six
rumen-cannulated multiparous Holstein cows (11.8 ± 3.9 days in milk) were utilized in a 6 x 6
Latin square design experiment balanced for carryover effects with a 2 x 3 factorial arrangement
of treatments. Treatments were AA and NA, with sodium chloride (CON) as a control, infused at
a rate of ~0.75 mol/h (0.5 L/h) into the RU or AB for the first 8 h following feeding with a rest
day between infusion days. Treatment sequences were assigned randomly to cows. Feeding
behavior was recorded by a computerized data acquisition system and blood was sampled at 0, 4,
and 8 h relative to the start of infusion. We hypothesized that AA is more hypophagic than NA,
and that infusion into the AB is more hypophagic than into the RU. Treatments decreased dry
matter intake (DMI) 30% (4.3 kg/8h vs. 6.1 kg/8h) compared with CON by decreasing meal
frequency but their effects on DMI differed; NA-RU did not reduce DMI (7.0 kg/8 h), whereas
AA-RU (2.6 kg/8 h), AA-AB (3.7 kg/8 h) and NA-AB (4.0 kg/8 h) decreased DMI compared
with CON. There was a residual effect following infusions of AA compared with NA on DMI
for the remainder of the day but treatments did not affect DMI during the rest day. Treatments
increased plasma acetate and ß-hydroxybutyrate concentrations over time (interaction, P < 0.01)
and decreased plasma insulin concentration compared with CON. Plasma glucose concentration
decreased over time after AA-AB infusion compared with other treatments and CON. Plasma
74

nonesterified fatty acid concentration increased over time for AA compared with NA and CON
suggesting the increase in lipolysis to compensate the decrease in DMI. In contrast to the other
treatments, NA-RU did not decrease DMI compared with control but the reason cannot be
determined from the data available from the current study.
INTRODUCTION
Acetic acid is the primary end-product of ruminal fermentation, typically comprising over
50% of the fermentation acids produced in the rumen on molar basis. It is used for fatty acid
synthesis in adipose and mammary tissue and as an energy source by various tissues.
Intraruminal infusions of acetic acid (AA) or sodium acetate (NA) have been used to test their
effects on yields of milk and milk components and feed intake of lactating cows (Rook and
Balch, 1961; Reynolds et al., 1979; Sheperd and Combs, 1998; Urrutia and Harvatine, 2017).
Whereas some studies have infused AA intraruminally (RU) without detrimental effects on DMI
(Rook and Balch, 1961; Choi and Allen, 1999), others have reported a reduction in DMI
(Montgomery et al., 1963; Wilson et al., 1967) or adverse physiological effects on the animals
(e.g. cessation of rumination, decrease in ruminal pH and symptoms of metabolic acidosis
(Montgomery et al., 1963; Sheperd and Combs, 1998).
Our laboratory has used AA alone or partially neutralized with sodium hydroxide
intraruminally as control treatments to study the effects of propionate on feeding behavior of
lactating cows. Infusions up to 28 moles of NA (1.56 mol/h) or ~20 moles of AA (0.75 mol/h)
had no detrimental effect on the animals or reduction on feed intake (Choi and Allen, 1999; Oba
and Allen, 2003a, b, c; Stocks and Allen, 2013, 2014). We considered that propionate is
hypophagic compared with acetate because propionate is anaplerotic and stimulates hepatic
oxidation of acetyl CoA (AcCoA) in the tricarboxylic acid (TCA) cycle whereas acetate does not
75

(Allen et al., 2009). Feeding behavior is affected by osmotic changes in the rumen so we
rationalized that isosmotic infusions of acetate were appropriate controls for the propionate
treatment. All previous experiments to our knowledge have infused AA alone or partially
neutralized with sodium hydroxide into the rumen. However, we recently observed adverse
effects of abomasal (AB) infusion of 20.3 mol/d of AA (4.2 Mcal) at a rate of 0.5 L/h (0.92
mol/h) to cows in the postpartum (PP) period (Gualdrón-Duarte and Allen, unpublished) in
which AA decreased DMI 64% compared with control (no infusion). This was not expected
because intraruminal infusions of AA at this rate did not decrease DMI in previous experiments.
The objective of this experiment was to evaluate the effects of continuous isomolar
infusions of AA or NA infused into the RU or into the AB on feeding behavior, DMI and
metabolic responses of cows in the PP period. We hypothesized that AA would be more
hypophagic than NA, and that infusion into the AB would be more hypophagic than into the RU.
MATERIALS AND METHODS
Animals, and Care
All experimental procedures were approved by the Institutional Animal Care and Use
Committee at Michigan State University, East Lansing. Cows were ruminally cannulated at least
45 d before parturition and housed in individual tie-stalls for the 12-d duration of the experiment.
All cows were fitted with a single jugular catheter 2 d before the beginning of the experiment,
according to Bradford et al. (2006). Catheter patency was checked daily until removed at the end
of the experiment. Cows were withheld from feed at 0800 h and were not allowed access to feed
until all blood samples were collected. Cows were fed once daily (900 h) at 125% of expected ad
libitum intake, and milked twice daily (0530 and 1730 h). The amounts of feed offered and orts

76

were weighed for each cow daily.
Experimental Design and Treatment Diets
Six ruminally cannulated multiparous, lactating Holstein cows (11.8 ± 3.9 DIM; mean ±
SD) at the Michigan State University Dairy Teaching and Research Center were used in a 6 x 6
Latin square design experiment with a 2 x 3 factorial arrangement of treatments. Cows were
assigned randomly to stalls and a treatment sequence balanced for carry-over effects. Infusion
periods were 8-h long with continuous isomolar infusions (0.75 mol/h) of AA (pH 2.3, > 99.7%,
Sigma Aldrich, Chemical Co., St. Louis, MO), NA (pH 5.9, AA + NaOH,) or NaCl (CON, >
99.5%, Sigma Aldrich, Chemical Co., St. Louis, MO) as a control; infused into the AB or RU at
a rate of 0.5 L/h. Concentration of solutions and duration of infusions were 0.75 mol/h and 8 h
respectively to reduce adverse the physiological effects observed in a previous experiment
(Gualdrón-Duarte and Allen, unpublished) when solutions were infused at 0.91 mol/h for 22 h/d
into the AB. Solutions were infused into the RU or AB using Baxter Flo-Gard 6201 infusion
pumps (Baxter Medical Products, Deerfield, IL) through two vinyl tubes (0.95 cm OD, 0.71 cm
ID), the first connected to a Nalgene bottle (3.8 cm diameter, 8.5 cm long) and placed into the
AB and held by a semihard flexible rubber flange as described in Gualdrón-Duarte and Allen
(2017), and the second tube placed into the RU. A recovery period of 40 h was included between
infusions.
All cows received a common experimental diet from parturition through the end of the
experiment. The experimental diet (Table 4.1) was composed of corn silage, ground corn,
soybean meal, alfalfa silage, alfalfa hay, soy hulls, and a vitamin and mineral mix and
formulated to meet requirements for absorbed protein, minerals, and vitamins (NRC, 2001).

77

Sample and Data Collection
Feed offered and refused was measured daily. Cows were blocked from feed from 0800
to 0900 h daily. Samples (0.5 kg) of all dietary ingredients and the TMR were collected daily
throughout the experiment, and orts for each cow were taken at the end of each infusion period
and stored in plastic bags at -20°C until processed. Feeding behavior data (feed disappearance
and water intake) were recorded continuously for 22 h daily for each cow during infusion days
via computer every 5 s, and size, length, and frequency of meals, intermeal interval, total eating
time, and water intake were calculated (Dado and Allen, 1995).
Table 4.1. Ingredients and nutrient composition of the experimental diet
Item
Ingredient, % of DM
Corn silage
Ground corn
Soybean meal
Alfalfa silage
Alfalfa Hay
Soybean hulls
Vitamin and mineral mix1
Sodium bicarbonate
Limestone
Nutrient composition
DM, %
NDF, % of DM
Forage NDF, % of DM
Starch, % of DM
CP, % of DM
Ash, % of DM

Amount
29.9
19.5
16.4
15.1
13.6
1.4
2.0
1.1
1.1
52.4
28.7
24.3
22.0
17.4
8.9

1

Vitamin and mineral mix contained 25.6% NaCl, 10.0% Ca, 2.0 Mg, 2.0% P, 30 ppm Co, 506 ppm Cu, 20 ppm I,
2220 ppm Fe, 2,080 ppm Mn, 15 ppm Se, 2,030 ppm Zn, 300 KUI/kg vitamin A, 50 KIU/kg vitamin D, 1500
KIU/kg vitamin E.

Milk yield was recorded and milk samples were collected daily at each milking and
stored with preservative (Bronopol tablet, D&F Control Systems, San Ramon, CA) at 4ºC for
component analysis (Universal Lab Services, East Lansing, MI). Milk samples were analyzed for
fat, true protein, lactose, and milk urea nitrogen (MUN) by Michigan DHIA (AOAC, 1997).
78

Blood samples were collected immediately before and at 4 and 8 h following the start of
infusions. Blood was sampled from a jugular catheter and collected into 3 evacuated tubes, 2
containing K2EDTA and 1 containing potassium oxalate with NaF as a glycolytic inhibitor, and
placed on ice until centrifugation. Tubes were centrifuged at 3,000 x g for 15 min at 5°C
immediately after sample collection. Plasma was harvested and frozen at -20°C until analysis.
Before and 4 h after the start of infusions, blood samples were taken in a heparinized syringe,
transported to the laboratory and analyzed with a blood gas analyzer.
Analysis of Samples
Diet ingredients and orts were dried in a 55°C forced-air oven for 72 h and were ground
with a Wiley mill (1-mm screen; Arthur H. Thomas, Philadelphia, PA). Samples were analyzed
for CP, starch, NDF, and ash. Crude protein was determined according to Hach et al. (1987).
Starch was analyzed using an enzymatic method (Karkalas, 1985) after samples were gelatinized
with NaOH; glucose concentration was measured by a glucose oxidase method (Sigma Chemical
Co., St. Louis, MO). Content of NDF was analyzed according to Mertens (2002) with the
inclusion of amylase and Na sulfite. Ash content was determined after 6 h of oxidation at 500°C
in a muffle furnace. All nutrients are expressed as percentages of DM that was determined by
drying at 105°C in a forced-air oven for more than 8 h.
Plasma samples were analyzed colorimetrically with a microplate reader (SpectraMax
Plus 384, Molecular Devices Corp.) to determine concentrations of metabolites. Commercial kits
were used to determine concentrations of nonesterified fatty acids (NEFA; NEFA HR kit, Wako
Chemicals USA, Richmond,VA) and β hydroxybutyrate (BHB; kit no. 2240, Stanbio
Laboratory, Boerne, TX), and glucose was determined using a glucose oxidase method (Sigma
Chemical. Co.). Insulin was determined using an ELISA kit (kit no. 10-1201-01, Mercodia,
79

Uppsala, Sweden). Plasma acetate, propionate and lactate concentrations were analyzed by
HPLC (Waters Corp., Milford, MA). Plasma (600 μL) was mixed with 600 μL of 3.6 M Ca(OH)2
and 300 μL of 0.5 M CuSO4 containing crotonic acid as an internal marker in 1.7 mL
microcentrifuge tubes. Samples were frozen, thawed and centrifuged at 12,000 × g for 10 min,
and supernatant (1,000 μL) was mixed with 42 μL of 98% H2SO4 in 1.5-mL microcentrifuge
tubes. Samples were frozen and thawed twice, and centrifuged at 12,000 × g for 10 min to
precipitate and remove protein. Supernatant was transferred to HPLC vials. The column used
was Aminex HPX-87H (catalog number 125-0140; 300 x 7.8 mm; Bio-Rad Laboratories,
Richmond, CA). Column temperature was 50°C, and the solvent was 0.015N H2S04. Detection
was by refractive index (Waters 410; Millipore Corp., Milford, MA). Blood chemistry and gas
analysis was performed in a Stat Profile pHOX Ultra Analyzer (Nova Biomedical Corp.,
Waltham, MA) by Michigan State University Diagnostic Center for Population and Animal
Health (East Lansing) for direct measurement of pH (hydrogen ion selective glass membrane),
partial pressure of CO2 (modified hydrogen ion selective glass membrane), partial pressure of O2
(gas permeable membrane), saturation percentage of O2 (reflectance photometry system), Na+,
K+, Cl-, Ca++, Mg++ (ion selective membrane), glucose, lactate and creatinine (platinum
electrode), and blood urea nitrogen (ammonium ion selective membrane). Values for HCO3, total
CO2, base excess blood, anion gap, osmolality and standard bicarbonate were determined
indirectly by calculation.
Statistical Analysis
Feeding behavior, intake, yields of milk and milk components, concentration of milk
components, and plasma composition were analyzed using the Fit Model procedure of JMP
(version 12.0, 2015; SAS Institute Inc., Cary, NC) using the following model:
80

Yijk    Ti  Pj  Ck  eijk

where Yijk = the dependent response variable of interest, μ = overall mean, Ti = fixed
effect of treatment (i = 1 to 6), Pj = fixed effect of period (j = 1 to 6), Ck = random effect of cow
(k = 6), and eijk = residual, normally distributed.
Plasma concentrations of glucose, NEFA, BHB, acetate, propionate, lactate and insulin
were analyzed using the MIXED procedure of SAS (version 9.4, 2013; SAS Institute Inc., Cary,
NC) with repeated measures, using the following model:
Yijkl = µ +Ti + Pj + H k +Ti H k + Cl + eijkl

where Yijkl = the response variable; µ = overall mean; Ti = fixed effect of treatment (i = 1
to 6); Pj = fixed effect of period (j = 1 to 6); Hk = fixed effect of time (k = 1 to 3); TiHk =
interaction of treatment and time; Cl = random effect of cow (l = 6); and eijkl = residual. Time
was included in the repeated statement. Covariance structure was first-order autoregressive.
Treatment differences at each time point were analyzed using the SLICE option. Cow by
treatment interaction was implicitly used as the error term for treatment. Denominator degrees
of freedom were estimated by using the Between-Within option.
For all statistical models used, residuals were normally distributed as determined by the
Shapiro-Wilk test.
Blood chemistry results at 4 h following the start of infusion were analyzed using the Fit
Model procedure of JMP (version 12.0, 2015; SAS Institute Inc., Cary, NC) with the following
model:

81

Yijk    Ti  Pj  Ck  Cov  eijk

where Yijk = the dependent response variable of interest, μ = overall mean, Ti = fixed
effect of treatment (i = 1 to 6), Pj = fixed effect of period (j = 1 to 6), Ck = random effect of cow
(k = 6), Cov = effect of covariate, and eijk = residual, normally distributed. Data for samples
preceding each infusion were included as covariates to account for differences in baseline
measurements.
Treatment effects were declared at P ≤ 0.05 and tendencies for treatment effects at P ≤
0.10. Interactions were declared significant at P ≤ 0.10 and tendencies for interactions at P ≤
0.15. All data are expressed as least squares means and standard error of the means, unless
otherwise specified. Means were compared with preplanned contrasts.
RESULTS
Feed Intake and Feeding Behavior
Treatments decreased DMI during the infusion period 30% (4.3 kg/8h vs. 6.1 kg/8 h, P <
0.01) compared with CON by decreasing meal frequency (P < 0.05) but their effects on DMI
differed (interaction between type and site of infusion, P < 0.01). The treatment NA-RU did not
reduce DMI during the 8h of infusion (7.0 kg/8h), whereas AA-RU (2.6 kg/8 h), AA-AB (3.7
kg/8 h) and NA-AB (4.0 kg/8 h) decreased DMI compared with CON. . Acetic acid decreased
DMI 32% compared with NA infusions during the 15 h following infusions and the effects of
treatment on DMI for the day of infusion was similar to those observed during the infusion
period. Treatments did not affect DMI (P = 0.22) during the rest day. Treatments decreased
water intake 33% (31.4 L/8 h vs. 47.1 L/8 h, P < 0.01) compared with CON and AA decreased
water intake during the infusion period by 56% (19.1 L/8h vs. 43.7 L/8h, P < 0.01) compared
82

with NA. However, treatments did not affect water intake during the 15h following infusions
(Table 4.2).

83

Table 4.2. Effects of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8 h/d) of sodium chloride (CON), acetic acid
(AA) and sodium acetate (NA) on feeding behavior and water intake for cows in the immediate postpartum period
Infusion
Significance, P
CON- CON- AA- AA- NA- NACON vs. NA vs. AB vs.
SEM
Trt2
Int3
RU
AB
RU
AB
RU
AB
AA+NA
AA
RU
During infusion, /8h
DMI, kg
Meal frequency, meals
Meal size, kg
Water intake, L
Drinking frequency,
bouts
Following
infusion, /15 h
DMI, kg
Water intake, L
Total for infusion day, /23 h
DMI, kg/d
Rest day, /23 h
DMI, kg
Water, L

6.23
4.67
1.37
49.6
7.17

6.05
4.67
1.34
44.6
5.5

2.57
2.83
1.45
17.7
2.93

3.72
2.46
2.04
20.4
3.5

7.02
5.12
1.39
46.2
7.35

4.01
4.08
1.22
41.2
5.6

0.99
0.772
0.315
7.88
1.47

< 0.001
0.15
0.41
< 0.01
< 0.01

< 0.01
0.14
0.46
< 0.01
0.05

< 0.01
0.03
0.13
< 0.01
< 0.01

0.19
0.4
0.48
0.86
0.5

0.01
0.69
0.19
0.56
0.19

6.70
31.5

7.58
30.4

4.08
26.9

5.92
27.0

7.30
32.4

7.43
28.6

1.08
6.04

0.06
0.8

0.22
0.43

0.02
0.32

0.3
0.6

0.37
0.59

13.0

13.6

6.67

9.62

14.2

11.4

1.76

< 0.001

0.01

0.97

0.02

13.6
64.5

14.4
78.4

10.7
66.8

13.1
69.5

14.2
62.9

11.5
72.7

2.67
11.8

0.33
0.92

0.21
0.62

<
0.001
0.54
0.96

0.9
0.46

0.11
0.67

1

Isomolar infusion, 0.75 mol/h
Trt = treatment
3
Int=interaction of acetate type (AA vs. NA) and site of infusion (AB vs. RU).
2

84

Table 4.3. Effects of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8h/d) of sodium chloride (CON), acetic acid
(AA) and sodium acetate (NA) on milk yield and milk components for cows in the immediate postpartum period
Infusion
Significance, P
Significance, P
CON- CON- AAAANANACON
vs. NA vs. AB vs.
SEM
Trt2
Int3
RU
AB
RU
AB
RU
AB
AA+NA
AA
RU
PM milking (at the end of infusions)
Yield, kg
Milk
16.5
18.0
Fat
0.90
0.86
Protein
0.46
0.48
Lactose
0.79
0.87
Composition
Fat, %
5.42
4.75
Protein, %
2.84
2.65
Lactose, %
4.78
4.85
MUN, mg/dL
12.6
11.7
AM milking (12 h after infusions)
Yield, kg
Milk
15.4
15.6
Fat
0.65
0.62
Protein
0.42
0.41
Lactose
0.73
0.75
Composition
Fat, %
4.15
3.98
Protein, %
2.80
2.59
Lactose, %
4.72
4.77
MUN, mg/dL
11.7
12.0

17.0
0.69
0.48
0.81

17.5
0.86
0.46
0.82

16.9
0.88
0.45
0.83

14.9
0.74
0.41
0.73

1.74
0.11
0.04
0.08

0.30
0.16
0.33
0.53

0.37
0.17
0.41
0.42

0.14
0.60
0.10
0.44

0.42
0.89
0.26
0.38

0.19
0.04
0.62
0.23

4.16
2.83
4.76
13.6

4.78
2.65
4.73
13.9

5.24
2.69
4.92
12.3

4.87
2.78
4.92
13.0

0.42
0.08
0.06
0.88

0.15
0.42
< 0.001
0.14

0.24
0.85
0.44
0.06

0.09
0.95
< 0.01
0.11

0.71
0.56
0.67
0.43

0.15
0.08
0.77
0.79

14.0
0.77
0.38
0.64

11.1
0.60
0.31
0.51

14.5
0.58
0.39
0.70

13.2
0.56
0.35
0.63

1.85
0.09
0.05
0.09

0.01
0.02
0.02
< 0.01

< 0.01
0.88
0.01
< 0.01

0.14
0.02
0.33
0.05

0.03
0.04
0.03
0.03

0.38
0.13
0.59
0.50

5.60
2.74
4.60
15.4

5.54
2.72
4.62
14.8

4.09
2.71
4.81
11.6

4.37
2.64
4.75
13.7

0.36
0.08
0.08
0.97

< 0.001
0.90
0.08
0.01

< 0.001
0.92
0.35
0.01

< 0.001
0.49
0.01
0.01

0.68
0.52
0.72
0.41

0.54
0.75
0.52
0.15

1

Isomolar infusion, 0.75 mol/h
Trt = treatment
3
Int=interaction of acetate type (AA vs. NA) and site of infusion (AB vs. RU).
2

85

Milk Production and Components
At the end of the infusion period (PM milking), there was no effect of treatment on milk
yield. Treatments interacted (P = 0.04) to affect fat yield and tended to interact (P = 0.15) to
affect fat concentration with NA increasing, and AA decreasing fat yield when infused in the RU
compared with the AB (Table 4.3). Protein yield tended to be increased by AA compared with
NA (0.47 vs. 0.43 kg, P =0.10) but protein concentration was not affected by treatment. Lactose
concentration was increased by NA compared with AA (4.92% vs. 4.75%, P < 0.01) but lactose
yield was not affected by treatment. Concentration of MUN tended to be increased by AA and
NA treatments compared with control (12.2 vs. 10.0 mg/dl, P = 0.06).
Effects of treatment on milk yield and composition for the AM milking (12 h after
infusion) are reported in Table 4.3. The AA and NA treatments decreased milk yield 15%
compared with CON (13.2 kg vs. 15.5 kg, P < 0.01) and infusion into the AB decreased milk
yield 15% compared with infusion into the RU (12.2 kg vs. 14.3 kg, P = 0.03). Fat yield was
affected by treatment (P = 0.02) with AA-RU tending to increase fat yield compared with AAAB, NA-AB, and NA-RU (interaction, P = 0.13). Treatments increased fat concentration 16%
compared with CON (4.9 vs. 4.1%, P < 0.001) and AA increased fat concentration compared
with NA (5.6 vs 4.2%, P < 0.001). Treatments decreased protein yield 14% compared with CON
(0.36 vs. 0.42 kg, P = 0.01) and infusion into the AB decreased protein yield 14% compared with
RU (0.33 vs. 0.39, P = 0.03). No effect of treatment on protein concentration was observed.
Treatments decreased lactose yield 16% compared with CON (0.62 vs 0.74 kg, P < 0.01), with a
decrease of 13% for AA compared with NA (0.58 vs. 0.67 kg, P = 0.05) and a decrease of 15%
for infusion into the AB compared with RU (0.57 vs. 0.67, P = 0.03). The AA treatment
decreased lactose concentration 3.6% compared with NA (4.61 vs. 4.78%, P = 0.01). Treatments
86

increased MUN concentration 14% compared with CON (13.9 vs. 11.9 mg/dL, P = 0.01), but
their effects tended to differ with AA increasing, and NA decreasing, MUN concentration when
infused into the RU compared with the AB (interaction P = 0.15).
Blood Metabolites and Insulin Concentration
Treatments interacted with time to affect plasma concentrations of glucose (P = 0.01),
NEFA (P = 0.02), BHB (P < 0.01), acetate (P < 0.0001) and propionate (P = 0.03; Table 4.4,
Fig. 4.1). Compared with CON, treatments decreased plasma lactate concentration 8% (P =
0.01), decreased plasma insulin concentration 38% (P = 0.01), increased plasma acetate
concentration 122% (P < 0.001) and tended (P = 0.07) to increase propionate concentration 21%.
However, no differences were detected between infusion treatments (AA vs. NA), site of
infusion (RU and AB), or their interaction for any plasma metabolite or hormone.
Blood Chemistry
Acetic acid decreased pH (7.42 vs. 7.44, P = 0.04), HCO3- (22.0 vs. 25.3 mmol/L, P <
0.01), total CO2 (23.0 vs. 26.4 mm/L, P < 0.01), base excess (-1.10 vs. 1.89 mmol/L, P < 0.01)
and osmolality (273 vs. 278 mOsm/L, P < 0.01) compared with NA but interacted with site of

87

Table 4.4. Effects of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8h/d) of sodium chloride (CON), acetic acid
(AA) and sodium acetate (NA) on plasma metabolites and insulin for cows in the immediate postpartum period
Infusion
Significance, P
CON
NA AB
CON- CON- AA- AA- NA- NASEM Trt2
Time Trt*time
vs.
vs. vs. Int3
AB
RU
AB
RU
AB
RU
AA+NA AA RU
Glucose, mg/dL
NEFA, µEq/L
BHB, mg/dL.
Lactate, mmol
Acetate, mmol
Propionate, mmol
Insulin, µg/L

42.0
601
23.2
0.333
1.46
0.117
0.180

45.3 41.3 39.0 41.2 43.0
527
676
773
604
653
22.0 25.5 30.2 27.8 29.0
0.368 0.337 0.309 0.336 0.309
1.26 2.33 2.98 2.82 3.95
0.108 0.145 0.155 0.125 0.142
0.182 0.105 0.093 0.131 0.119

1

Isomolar infusion, 0.75 mol /h
Trt = treatment
3
Int=interaction of acetate type (AA vs. NA) and site of infusion (AB vs. RU).
2

88

4.2
137
6.7
0.01
0.54
0.03
0.04

0.56
< 0.01
0.01
0.34 < 0.001
0.02
0.59 <0.0001 < 0.01
0.01
0.06
0.71
< 0.01 <0.0001 <0.0001
0.40
0.03
0.03
0.17
0.94
0.43

0.24
0.11
0.10
0.01
< 0.001
0.07
0.01

0.43
0.25
0.89
0.98
0.12
0.39
0.40

0.67
0.73
0.63
0.51
0.18
0.71
0.77

0.41
0.77
0.65
0.97
0.61
0.87
1.00

infusion with decreases for AA and increases for NA when infused into the AB compared with
the RU (interaction P ≤ 0.10). Acetic acid increased anion gap (16.3 vs. 15.1 mmol/L, P = 0.04),
and ionized magnesium (1.33 vs. 1.20 mg/dl, P < 0.01) and decreased sodium (139 vs. 142
mmol/L, P < 0.01) and tended to decreased creatinine (0.946 vs. 0.999, P = 0.08) and ratio of
calcium to magnesium (2.32 vs. 2.43, P = 0.08) compared with NA. Acetic acid decreased blood
urea nitrogen (BUN, 12.3 vs. 13.0 mg/dL) and the ratio of BUN to creatinine (12.7 vs. 13.1)
when infused into the RU, but increased BUN (13.1 vs. 12.5 mg/dL) and BUN/creatinine (14.0
vs. 13.2) when infused into the AB (interaction P = 0.02). Acetic acid and NA treatments
decreased chloride (1.05 vs. 1.08 mmol/L, P < 0.001) and tended to decrease normalized calcium
(4.91 vs. 5.10 mg/dL. P = 0.06) compared with CON. Plasma potassium concentration was not
affected by treatment (P > 0.15).
DISCUSSION
This study was conducted to investigate the mechanism by which abomasal infusion of
AA decreased DMI by cows in the immediate PP period in a previous experiment (GualdrónDuarte and Allen, unpublished). Our objective was to evaluate the effects of continuous isomolar
infusions of AA or NA infused into the RU or into the AB on feeding behavior, DMI and
metabolic response of cows in the PP period.
In agreement with our hypothesis, AA was more hypophagic than NA but NA-AB
decreased DMI whereas NA-RU did not compared with CON. The decrease in DMI by AA was
because AA decreased meal frequency and not meal size compared with NA, indicating that AA
did not affect satiety within the timeframe of meals but delayed hunger. Choi and Allen (1999).

89

*
**

**
*

*

**

*

Figure 4.1. Effects of isomolar intra-ruminal (RU) or intra-abomasal (AB) infusion (8h/d) of sodium chloride (CON), acetic acid
(AA) and sodium acetate (NA) on plasma metabolites for cows in the immediate postpartum period. Treatments were:
continuous isomolar infusions (0.75 mol/h) of acetic acid (AA, black solid circle), sodium acetate (NA, solid triangle,) or
sodium chloride (CON, solid square) as a control; infused into the abomasum (AB, solid line) or rumen (RU, broken line) at a
rate of 0.5 L/h

90

Table 4.5. Effect of isomolar1 intra-ruminal (RU) or intra-abomasal (AB) infusion (8h/d) of sodium chloride (CON), acetic acid (AA)
and sodium acetate (NA) on blood chemistry for cows in the immediate postpartum period (samples taken 4 h after starting of
infusions)
Significance, P
Infusion

pH
pCO2, mm Hg4
pO2, mm Hg5
p50, mm Hg
Total CO2, mm/L
Saturation O2, %
HCO3, mmol/L
sHCO3, mmol/L6
BEb, mmol/L7
BEecf, mmol/L8
Anion gap, mmol/L
Osmolality, mOsm/kg
BUN, mg/dL
Creatinine, mg/dL
BUN/Creatinine
Glucose, mg/dL
Sodium, mmol/L
Potassium, mmol/L
Chloride, mmol/L
Lactate, mmol/L
iCa, mg/dL9
nCa, mg/dL10
iMg, mg/dL
nMg, mg/dL
Ca/Mg
1

CONRU

CONAB

AARU

AAAB

NARU

NAAB

SEM

Trt2

CON vs. NA vs.
AA+NA
AA

AB vs.
RU

Int3

7.43
35.1
43.2
27.1
24.6
77.6
23.5
24.2
0.27
-3.5
14.5
278
13.3
0.982
13.6
44.8
140
3.97
107
0.471
5.21
5.13
1.25
1.29
2.47

7.42
34.1
46.2
27
23.2
81.2
22.2
23.2
-1.08
-1.81
14.5
279
12.7
0.955
13.7
48.1
141
3.86
108
0.458
5.06
5.06
1.37
1.34
2.33

7.43
33.7
48.1
26.7
23.6
83.5
22.6
23.8
-0.31
-2.55
16.3
274
12.3
0.959
12.7
42.2
139
3.86
105
0.447
5.03
4.94
1.28
1.33
2.33

7.41
33.2
46.8
27
22.3
81.2
21.3
22.4
-1.88
-1.04
16.3
271
13.1
0.933
14
43.8
138
3.85
105
0.421
5
4.94
1.38
1.33
2.3

7.43
36.04
44.1
27.1
25.5
78.4
24.4
24.9
1.09
1.86
15.3
277
13.0
1.003
13.1
46.3
141
3.86
105
0.44
5.14
5.05
1.2
1.28
2.46

7.45
37.4
46.4
26.9
27.3
83.2
26.1
26.5
2.69
-0.004
14.9
279
12.5
0.995
13.2
44.5
142
3.51
103
0.471
4.84
4.71
1.19
1.27
2.4

0.009
1.26
2.16
0.23
1.07
2.54
1.03
0.79
0.93
1.1
0.53
1.42
0.333
0.03
0.427
2.26
0.782
0.129
0.778
0.021
0.115
0.122
0.045
0.037
0.064

0.06
0.05
0.48
0.62
0.02
0.30
0.02
0.03
0.03
0.02
0.07
0.01
0.11
0.55
0.02
0.56
0.02
0.18
0.03
0.43
0.18
0.12
0.02
0.66
0.33

0.29
0.76
0.29
0.32
0.27
0.22
0.28
0.22
0.6
0.24
0.02
0.05
0.24
0.87
0.09
0.29
0.14
0.19
< 0.001
0.26
0.12
0.06
0.28
< 0.001
0.6

0.96
0.94
0.79
0.9
0.77
0.56
0.8
0.87
0.99
0.91
0.86
0.83
0.61
0.58
0.01
0.97
0.9
0.16
0.36
0.9
0.11
0.13
0.4
<0.0001
0.5

0.05
0.94
0.33
0.23
0.09
0.1
0.1
0.06
0.08
0.01
0.66
0.1
0.02
0.75
0.02
0.49
0.12
0.18
0.43
0.17
0.2
0.13
0.3
<0.0001
0.86

Isomolar infusion, 0.75 mol /h

91

0.04
0.92
0.23
0.37
< 0.01
0.46
< 0.01
< 0.01
< 0.01
< 0.01
0.04
< 0.01
0.82
0.08
0.51
0.33
< 0.01
0.18
0.27
0.27
0.81
0.58
0.01
0.02
0.08

Table 4.5. (cont’d)
2

Trt = treatment
3Int=interaction of acetate type (AA vs. NA) and site of infusion (AB vs. RU).
4
pCO2 = Carbon dioxide partial pressure
5
pO2 = Oxygen partial pressure
6
HCO3 = standard bicarbonate ion concentration after the blood has been equilibrated to a pCO2 of 40 mmHg
7
Base excess blood
8
Base excess extracellular fluid
9
Ionized
10
Normalized to pH 7.4

92

reported no effect of AA, NA or sodium chloride on meal size of lactating cows when infused
into the rumen at spontaneous meals consistent with the results of the current experiment
However, unlike the present experiment, effects of AA did not affect hunger as indicated by
intermeal interval or DMI compared with NA (Choi and Allen, 1999). Decreased water intake
for AA compared with CON and NA is consistent with results reported by Choi and Allen (1999)
and is likely because of differences in intakes of Na and DM Murphy et al. (1983) reported that
DMI explains most of the variation in water intake which is also correlated with milk production;
and that one gram of additional sodium fed increases 50 ± 23 mL of water.
Studies that have evaluated effects of intraruminal infusions of acetate salts or acetic acid
on DMI have used different treatment conditions, including rate of infusion, solution
concentration, and duration of treatment, and cows varying in physiological state. In general,
infusion of AA or NA do not alter DMI of cattle (lactating or non-lactating) when the dose is 1.2
mol/h or less for up to 6 weeks (Huntington et al., 1983; Maxin et al., 2011; Orskov, 1969;
Reynolds et al., 1979; Rook and Balch, 1961; Storry and Rook, 1965; Wilson et al., 1967;
Urrutia and Harvatine, 2017). However, AA decreases DMI when infused into the rumen at rates
greater than 1.2 moles per h. Montgomery et al. (1963) infused AA at 3.5 mol/h for 4 h into
rumens of lactating and non-lactating cows and found that it greatly decreased DMI; however,
after partial neutralization to pH 5.0, with NaOH, acetate did not alter DMI. Similarly, infusion
of AA at 1.5 mol/h into rumens of mid-lactation cows stopped rumination and induced metabolic
alkalosis within 8 h but had no adverse effects when partially neutralized (Sheperd and Combs,
1998).
Response in feed intake to ruminal infusions of AA might be affected by stage of
lactation or site of infusion. Wilson et al. (1967) reported decreased DMI when AA was infused
93

at rates greater than 0.44 mol/h to cows in the PP period, consistent with results of the present
experiment. Similarly, infusion of AA into the AB of cows in the PP period at 0.91 mol/h
decreased DMI 64% (Gualdrón-Duarte and Allen, unpublished). Feed intake response to
infusions of acetate salts or acetic acid might be affected by site of infusion; AA and NA caused
diarrhea and cessation of eating within 24 h and 48 h, respectively, when infused into the
duodenum of non-pregnant, non-lactating cows (Tyrrell et al., 1979).
Infusion of AA and NA increased plasma acetate over time more than CON, as expected,
but the reason for the wide range response to AA and NA treatments at 8 h post-infusion is not
apparent. Interaction of treatment and time for plasma propionate concentration appears to be
from greater propionate concentration for AA-AB after 8 h of infusion compared to other
treatments but the reasons are not apparent. Effects of treatment on plasma insulin concentration
is consistent with their effects on DMI except that an interaction of AA and NA and site of
infusion was not observed; whereas NA-RU did not decrease DMI, it did decrease plasma insulin
concentration. Effects of treatment on plasma concentrations of NEFA and BHB increased over
time and were generally opposite of their effects on DMI and plasma insulin concentration, as
expected.
The effect of treatments on blood gasses and chemistry showed some differences among
treatments in some of the measurements, but most of them were within the normal range for
dairy cows according to Michigan State University Diagnostic Center for Population and Animal
Health (East Lansing). However all infusions had abnormally high values for anion gap and
abnormally low osmolality and sodium concentration for lactating cows. Differences among the
parameters measured could not explain the effects of treatment on DMI because all values for
NA-RU, which did not reduce DMI, were intermediate among treatments.
94

CONCLUSION
Our results indicate that infusion of Na acetate into the rumen at up to 6 mol/d does not
reduce DMI of cows in the early postpartum period compared with sodium NaCl as an osmotic
control. However, infusion of acetic acid into the rumen or either acetic acid or Na acetate into
the abomasum at similar rates greatly decreased DMI compared with control. Evaluation of
changes in plasma concentrations of metabolites, insulin, blood gasses, and blood chemistry did
not reveal clues for potential mechanisms responsible for different effects of treatments, which
requires further investigation.
ACKNOWLEDGEMENTS
We acknowledge the financial support for this project by USDA National Research
Initiative Competitive Grant no. 2014-67015-21708 to MSA, and from Fulbright Colombia and
Colciencias to L. Gualdrón-Duarte. We also thank D. G. Main, J. Liesman, R. West, G. Maldini,
K. Kennedy, R. Albornoz, R. Yair, D. de Sousa and R. West (all from Michigan State
University) for technical assistance.

95

APPENDIX

96

APPENDIX
EFFECTS OF FUELS DERIVED FROM STARCH DIGESTION ON FEED INTAKE,
FEEDING BEHAVIOR AND METABOLIC RESPONSE OF COWS IN THE
POSTPARTUM PERIOD
ABSTRACT
The objective of this study was to determine the effects of fuels derived of starch
digestion on feeding behavior of cows in the postpartum (PP) period. Continuous isoenergetic
infusions (194 kcal/h) of glucose (GLU, 48.1 g/h), lactic acid (LA, 55.1 g/h), acetic acid (AA,
57.5 g/h), propionic acid (PL, 40.7g/h; and propionic acid (PH, 54 g/h) infused at the mean of the
amount infused for GLU, AA and LA, were administered abomasally to six ruminally cannulated
multiparous Holstein cows (13.7 ± 3.9 days in milk) in a 6 x 6 Latin Square design experiment
balanced for carryover effects. Treatment sequences were assigned to cows randomly, and
treatments included control (CON, no infusion), PL (99.5%, 0.51 mol/h), PH (99.5%, 0.70
mol/h), LA (88.0%, 0.63 mol/h) and GLU (99.9%, 0.29 mol/h). Solutions containing treatments
were infused at 485 mL/h for 22h/d. Feeding behavior was recorded by a computerized data
acquisition system and blood was sampled at 0 and 24 h relative to the start of infusion. Data
were analyzed statistically with a mixed model including the fixed effects of treatment and
period and the random effect of cow; treatments were compared with preplanned contrasts. We
hypothesized that fuel from starch digestion affect feed intake differently according with their
capacity to increase oxidation in the liver and that PH is more hypophagic than PL. Compared
with CON, PH decreased dry matter intake (DMI) by 44% (9.93 vs. 17.6 kg/d) by decreasing
meal size 43% (0.83 vs. 1.46 kg/meal). Glucose, LA and PL infusions did not affect DMI.
Abomasal infusions of AA unexpectedly showed adverse physiological effects with signs for

97

nervous ketosis in some cows and decreased DMI by 64% compared with CON (6.28 vs. 17.6
kg/d). The reduction in DMI was explained by a 32% decrease of meal size (1.00 vs. 1.46
kg/meal) and by a 104% increase of the eating interval (198.8 vs. 97.1 min), but the mechanism
causing this response is not known. Among fuels metabolized in the liver, only PH decreased
intake by decreasing meal size 43% (0.83 vs. 1.46 kg/d), and increased satiety ratio almost twice
(0.034 vs. 0.018) compared with CON. In conclusion increasing starch content and ruminal
fermentability in diets may decrease DMI of cows in the PP period likely by increasing oxidation
in the liver. Further research is required to determine the effect of abomasal infusions of buffered
and unbuffered solutions of AA on feed intake and feeding behavior during the PP period.
INTRODUCTION
The transition period from gestation to lactation in dairy cows causes vast homeostatic
and homeorhetic changes highly related with a decrease in DMI with the resulting negative
energy balance. The decrease in plasma insulin concentration and insulin sensitivity in adipose
tissue close to parturition promotes fatty acid mobilization leading the cows to a lipolytic state
(Drackley, 1999). Hepatic uptake and increased oxidation of nonesterified fatty acids (NEFA)
likely suppress feed intake by increasing the hepatic energy charge signaling for satiety at the
brain feeding centers. According to the hepatic oxidation theory (HOT), the continuous supply
of acetyl CoA (AcCoA) from ß-oxidation of NEFA in the hepatocytes increases the activity of
the tricarboxylic acid (TCA) cycle, increasing ATP production and liver energy charge (Allen et
al., 2009).
Starch is the principal energy source used in diets for dairy cows to increase energy
intake and meet the high demand for glucose precursors. However, it has been shown that
increasing ruminal starch fermentability decreases feed intake (Dann and Nelson, 2011;
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Albornoz and Allen, 2016)
The hypophagic effect of most fermentable starch source is related to the effect of
propionic acid on promoting oxidation of the hepatic pool of AcCoA (Stocks and Allen, 2012;
2013). The fermentability of starch can vary according to many factors as variety, processing,
and growing and harvesting conditions among others (Larson and Hoffman, 2008). Changing
starch fermentability alters the type and patterns of fuels obtained through digestion. The
principal end products of starch digestion are acetic, propionic and butyric acid from ruminal
fermentation, and glucose from pancreatic amylase action in the small intestine. Lactic acid can
be produced both from ruminal fermentation and from partial metabolism of glucose by
enterocytes. After absorption, all this fuels reach the liver through portal circulation. Fuels that
are taken up by the liver and are metabolized increasing energy charge through oxidation
decrease firing rate of hepatic vagus nerve sensing for satiety, thus decreasing intake (Allen et
al., 2009).
Despite the evidence that propionic acid is more hypophagic when cows are in lipolytic
state (Stocks and Allen, 2012; 2013) and during the immediate postpartum (PP) period (Oba and
Allen, 2003b; Stocks and Allen, 2012; 2013), abomasal infusion of end products from starch
digestion on cows in the PP period allow to test for effects of these fuels on feeding behavior and
metabolic response. The objective of this experiment was to evaluate the effect of fuels derived
from starch digestion on feeding behavior and metabolic response of cows in the PP period. We
hypothesized that the fuels from starch digestion metabolized in the liver (propionic acid and
lactic acid) are hypophagic and that propionic acid is more hypophagic than lactic acid.

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MATERIALS AND METHODS
Animal Housing and Care
All experimental procedures were approved by the Institutional Animal Care and Use
Committee at Michigan State University, East Lansing. Cows were ruminally cannulated at least
45 d before calving and housed in individual tie-stalls for the 12-d duration of the experiment.
Cows were fed once daily (1200 h) at 125% of expected intake, and milked twice daily (0530
and 1730 h). The amounts of feed offered and orts were weighed for each cow daily.
Design and Treatment Diets
Six ruminally cannulated multiparous, lactating Holstein cows at the Michigan State
University Dairy Field Laboratory (13.7 ± 3.9 DIM; mean ± SD) were used in a 6 x 6 Latin
square experiment, with 5 infusion treatments and a sham control (CON, no infusion). Cows
were randomly assigned to stalls and a treatment sequence balanced for carry over effects.
Infusion periods will be 22-h long with 24-h recovery periods between infusions. Infusion
treatments were continuous isoenergetic infusions (194.2 kcal ME/h) of acetic acid (AA,
>99.7%, Sigma Aldrich, Chemical Co., St. Louis, MO), propionic acid (PL; food grade, 99.5%,
Kemin Industries, Inc., Des Moines, IA), lactic acid (LA; food grade 88.0%, Sena International
Inc., Polo, IL) and glucose (GLU; ≥ 99.5%, Sigma Aldrich, Chemical Co., St. Louis, MO); and
an isoamount (~ 55 g/h) infusion of propionic acid (PH; food grade, 99.5%, Kemin Industries,
Inc., Des Moines, IA) compared with the other infusion treatments; infused into the abomasum at
a rate of 0.5 L/h. The solutions were infused into the abomasum using Baxter Flo-Gard 6201
volumetric infusion pumps (Baxter Medical Products, Deerfield, IL) through vinyl tubing (0.95
cm OD, 0.71 cm ID, 1.5 m L) connected to a Nalgene bottle (3.8 cm diameter, 8.5 cm long) as
described in Gualdrón-Duarte and Allen ((Gualdrón-Duarte and Allen, 2017).
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The experimental diet (Table 4.6) was composed of corn silage, alfalfa silage, alfalfa hay,
ground corn, soybean meal, soy hulls, and a vitamin and mineral mix and formulated to meet
requirements for absorbed protein, minerals, and vitamins (NRC, 2001).
Table 4.6. Ingredients and nutrient composition of the experimental diet
Item
Ingredient, % of DM
Corn Silage
Soybean meal
Alfalfa silage
Ground corn
Alfalfa Hay
Soybean hulls
Vitamin and mineral mix1
Sodium bicarbonate
Limestone
Nutrient composition
DM, %
Starch, % of DM
NDF, % of DM
Forage NDF, % of DM
CP, % of DM

Amount
39.6
16.1
14.2
10.4
9.9
5.7
2.0
1.1
1.1
53.0
19.8
35.1
29.5
16.6

1

Vitamin and mineral mix contained 25.6% NaCl, 10.0% Ca, 2.0% Mg, 2.0% P, 30 ppm Co, 506 ppm Cu, 20 ppm
I, 2,220 ppm Fe, 2,083 ppm Mn, 15 ppm Se, 2,025 ppm Zn, 300 KUI/kg vitamin A, 50 KIU/kg vitamin D, 1,500
KIU/Kg vitamin E.

Data and Sample Collection
Cows were blocked from feed from 1000 to 1200 h daily, and the amount of feed offered
and orts were weighed for each cow daily. Samples (0.5 kg) of all dietary ingredients and the
TMR were collected daily throughout the experiment, and orts for each cow were taken at the
end of each infusion period and stored in plastic bags at -20°C until processed. Feeding behavior
data (feed disappearance and water intake) were recorded continuously for 22 h daily for each
cow during infusions via computer every 5 s (Dado and Allen, 1995), and size, length, and
frequency of meals, intermeal interval , and water intake were calculated. Hunger ratio was

101

calculated as the weight of meal divided by pre meal interval and satiety ratio was calculated as
the weight of meal divided by post-meal interval (Forbes, 2007).
Milk yield was recorded daily at each milking, and milk samples were collected from
each milking and stored with preservative (Bronopol tablet; D&F Control Systems, San Ramon,
CA) at 4ºC. Milk samples were analyzed for fat, true protein, lactose, and milk urea nitrogen by
Michigan DHIA (AOAC, 1997).
Blood was sampled every day at the beginning and end of each infusion period from
coccygeal vessels and collected into 4 evacuated tubes, 3 containing K2EDTA and 1 containing
potassium oxalate with NaF as a glycolytic inhibitor, and placed on ice until centrifugation.
Tubes were centrifuged at 3,000 x g for 15 min at 5°C immediately after sample collection.
Plasma was harvested and frozen at -20°C until analysis. Liver samples were collected by needle
biopsy (Bradford and Allen, 2005) between 1030 h and 1200 h (before feeding) before each
infusion and at the end of infusions for each period for analysis of hepatic AcCoA content.
Sample were flash frozen in liquid nitrogen and stored on dry ice for transportation. Samples
were stored at -80°C until further analysis.
Analysis of Samples
Feed ingredients and orts samples were dried in a 55°C forced-air oven for 72 h to
determine DM content. All samples were ground with a Wiley mill (1-mm screen; Arthur H.
Thomas, Philadelphia, PA) and analyzed for ash, NDF, crude protein and starch. All nutrients are
expressed as percentages of DM, determined by drying at 105ºC in a forced-air oven for more
than 8 h. Ash content was determined after 6 h of oxidation at 500°C in a muffle furnace. Crude
protein was determined according to Hach et al. (1987). Starch was analyzed using an enzymatic
method (Karkalas, 1985) after samples were gelatinized with sodium hydroxide; glucose
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concentration was measured by a glucose oxidase method (Sigma Chemical Co., St. Louis, MO).
Content of NDF was analyzed according to (Mertens, 2002) with the inclusion of amylase and
Na sulfite.
Plasma samples were analyzed using commercial kits for concentration of NEFA (NEFA
HR kit, Wako Chemicals USA, Richmond, VA), BHB (kit no. 2240, Stanbio Laboratory,
Boerne, TX). Plasma concentration of metabolites were analyzed colorimetrically with a
microplate reader (SpectraMax Plus 384, Molecular Devices Corp.) Liver AcCoA was
determined by HPLC following the King and Reiss (1985) procedure with modifications (Stocks
and Allen, 2012). Liver AcCoA content was expressed per g of wet liver.
Statistical Analysis
Feeding behavior, intake, blood metabolites, yield of milk, milk components,
concentration of milk components, and hepatic AcCoA content, were analyzed using the Fit
Model procedure of JMP (version 12.0, 2015; SAS Institute Inc., Cary, NC) with the following
model:
Yijk    Ti  Pj  Ck  eijk

where Yijk = the dependent response variable of interest, μ = overall mean, Ti = fixed effect of
treatment (i = 1 to 6), Pj = fixed effect of period (j = 1 to 6), Ck = random effect of cow (k = 6),
and eijk = residual, normally distributed. Treatment effects were declared significant at P ≤ 0.05
and tendencies for treatment effects at P ≤ 0.10. Interactions were declared significant at P ≤ 0.10
and tendencies for interactions at P ≤ 0.15. All data are expressed as least squares means and
standard error of the means, unless otherwise specified. Contrasts were conducted between each
infusion treatment and the CON. Six cows started the experiment; however, one cow was
removed from the study due to displaced abomasum after the second period.
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RESULTS
Feed intake and Feeding Behavior
Infusion of unbuffered diluted AA solutions into the abomasum resulted in an unexpected
decrease in intake causing severe ketosis and signs of nervous ketosis and erratic behavior in
some cows. One cow was completely removed from the experiment after AA infusion once the
decrease in intake was still more than 40% of expected normal intake during the day after
infusion. Acetic acid and PH infusions, relative to CON decreased DMI 64% (P < 0.0001) and
44% (P < 0.01) respectively (Table 4.7). The reduction in DMI was because AA decreased meal
frequency 59% (P <0.01), and tended to reduce meal size (1.0 kg/meal vs. 1.46 kg/meal, P =
0.06) compared with CON. The decrease in DMI for PH infusion was explained by the decrease
in meal size 43% (P = 0.01) compared with CON. Only AA increased eating interval in 101.7
min (P = 0.04) compared with CON. Infusions with AA and PH decreased hunger ratio by 75%
(P = 0.01) and 50% (P = 0.05), and satiety ratio by 70% (P = 0.02) and 47% (P = 0.09)
respectively compared with CON. Infusion with PL tended (P = 0.09) to decrease satiety ratio by
47% compared with CON. Reduction in water intake compared with CON follow the same trend
that DMI where AA and PH decreased 61% (P < 0.001) and 48% (P < 0.01) by decreasing drink
frequency 46% (P = 0.01) and 31% (P = 0.08) respectively compared with CON.

104

Table 4.7. Effects of isoenergetic1 intra-abomasal infusions (22h/d) of glucose (GLU), acetic acid (AA), lactic acid (LA), propionic
acid (PL), and isoamount2 intra-abomasal infusion of propionic acid (PH), relative to a control (CON) with no infusion on feeding
behavior and water intake for cows in the immediate postpartum period
Infusion
Significance, P
AA
GL
LA
PL
PH
CO
SE
3
GL AA LA
PL
PH
Trt
vs.
vs.
vs.
vs.
vs.
N
M
CON CON CON CON CON
DMI, kg/d
17.6 16.0 6.28 16.6 15.2 9.93 1.53 < 0.001 < 0.0001 0.45
0.64 0.25 < 0.01
Feeding behavior (22h/d)
Meal frequency,
10.6 11.2 4.34 10.1 10.3 7.94 1.25 0.01 < 0.01 0.74
0.78 0.89 0.12
meals/d
Meal length, min/meal 24.5 21.1 24.6 22.7 21.8 20.4 2.22 0.56
0.97
0.24
0.54 0.35 0.13
Meal size, kg/meal
1.46 1.25 1.00 1.33 1.07 0.83 0.153 0.09
0.06
0.36
0.57 0.10 0.01
Eating interval, min
97.1 97.5 198.8 108.2 108.6 111.4 33.7 0.29
0.04
0.99
0.82 0.81 0.75
Hunger ratio
0.028 0.026 0.007 0.026 0.02 0.014 0.005 0.05
0.01
0.72
0.73 0.28 0.05
Satiety ratio
0.034 0.031 0.01 0.032 0.017 0.018 0.006 0.09
0.02
0.76
0.81 0.09 0.09
Water intake, L/d
77.7 74.6 30.2 70.4 59.4 40.6 8.75 < 0.01 < 0.001 0.75
0.46 0.08 < 0.01
Drink frequency,
10.5 9.95 5.66 11.9 7.83 7.28 1.52 0.05
0.01
0.75
0.43 0.15 0.08
bouts/day
Liters
infused, L/d
0
10.7 10.6 11.1 10.3 10.6 0.19
Moles infused, mol/d
0
6.46 20.3 13.8 11.3 15.3 0.27
Calories infused, kcal/d
0
4300 4233 4430 4124 5601 89.5
1

Isoenergetic infusion, 194.2 kcal/h
Isoamount infusion, ~ 50 g/h
3
Trt = treatment
2

105

Milk Production and Components
Only infusion with AA compared with CON had an effect on milk yield and milk
components (Table 4.8). Milk yield was decreased by 7.2 kg/d (P < 0.01), with an increase in
24% on the percentage of fat (P < 0.01) and a numerical increase of 9.0% on the percentage of
true protein (P = 0.06). Lactose and total solids yield was reduced by 19% (P = 0.02).
Plasma Metabolites and Liver AcCoA Content
Hepatic content of AcCoA and plasma BHB concentration increased 11.6 nmol/g (P =
0.02) and 28.6 mg/dL (P < 0.01) respectively, after infusion with AA compared with CON.
Infusion with GLU compared with CON decreased NEFA 363 µEq/mL (P < 0.01). Plasma
glucose concentration was affected by infusion treatments. Compared with CON, GLU infusion
increased plasma glucose concentration (11.4 mg/dL, P = 0.01) and LA had a numerical increase
of glucose in plasma (6.93 mg/dL, P = 0.08). Infusion with AA decreased plasma glucose
concentration by17.8 mg/dL (P < 0.001) compared with CON.
DISCUSSION
According to the HOT, fuels derived from starch digestion that can be extracted from
blood circulation by the liver (propionate, butyrate and lactate) and be metabolized, have the
potential to increase hepatic energy charge and decrease intake (Allen et al., 2009). The present
study shows the effect of isoenergetic abomasal infusions (except for PH with isoamount ~50 g/h
in relation to other treatments) of fuels derived from starch digestion on feed intake. In the
current research, as expected, abomasal GLU infusion does not affect feed intake or any of the
feeding behavior parameters measured compared with CON. Glucokinase activity in ruminant

106

Table 4.8. Effects of isoenergetic1 intra-abomasal infusions (22h/d) of glucose (GL), acetic acid (AA), lactic acid (LA), propionic
acid (PL), and isoamount2 intra-abomasal infusion of propionic acid (PH), relative to a control (CON) with no infusion on milk
yield and milk components for cows in the immediate postpartum period
Infusion
Significance, P
AA
GL
LA
PL
PH
3
CON
GL
AA
LA
PL
PH SEM Trt
vs.
vs.
vs.
vs.
vs.
CON CON CON CON CON
Milk yield, kg/d
33.3
33.9
26.1
32.0
34.4
32.5 2.48 0.01 < 0.01 0.78 0.58 0.60 0.68
4.73
4.68
5.88
5.01
4.73
4.98
0.3 0.02 < 0.01 0.89 0.46 1.00 0.45
Fat, %
1.63
1.54
1.73
1.57
1.6
1.63 0.17 0.90
0.56
0.59 0.73 0.84 0.98
Fat yield, kg/d
3.01
2.86
3.27
3.05
3.00
2.90 1.93 0.05
0.06
0.26 0.81 0.93 0.37
True protein, %
1.08
0.964 0.968 1.02
1.05
0.94 0.09 0.80
0.33
0.33 0.62 0.78 0.24
True protein yield, kg/d
4.63
4.65
4.46
4.59
4.78
4.69 0.12 0.22
0.17
0.91 0.76 0.28 0.69
Lactose, %
1.62
1.52
1.31
1.51
1.63
1.53 0.13 0.13
0.02
0.43 0.42 0.92 0.46
Lactose yield, kg/d
5.51
5.52
5.31
5.46
5.68
5.57 0.14 0.25
0.17
0.94 0.75 0.30 0.70
Solids, %
1.92
1.81
1.56
1.79
1.93
1.82 0.16 0.14
0.02
0.43 0.42 0.94 0.46
Total solids, kg/d
11.2
10.7
11.3
11.8
11.4
12.2 1.54 0.85
0.95
0.70 0.67 0.90 0.41
MUN, mg/dL
1

Isoenergetic infusion, 194.2 kcal/h
Isoamount infusion, ~50 g/h
3
Trt = treatment
2

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Table 4.9. Effects of isoenergetic1 intra-abomasal infusions (22h/d) of glucose (GL), acetic acid (AA), lactic acid (LA), propionic acid
(PL), and isoamount2 intra-abomasal infusion of propionic acid (PH), relative to a control (CON) with no infusion on change in
concentrations of insulin and metabolites in plasma, and liver acetyl CoA (AcCoA) content over the infusion day for cows in the
immediate postpartum period
Infusion
Significance, P
AA
GL
LA
PL
PH
CON
GL
AA
LA
PL
PH
SEM
Trt3
vs.
vs.
vs.
vs.
vs.
CON
CON CON CON CON
AcCoA start, nmol/g
20.8
21.7 29.68 19.8
19.2
20.6
2.7
0.15
0.04
0.82
0.82 0.70 0.96
AcCoA end, nmol/g
18.2
17.2
38.7
18.5
16.1
21.4
4.64
0.01
< 0.01
0.86
0.95 0.70 0.56
AcCoA change,
-2.55 -4.46 9.06 -1.83 -3.24
0.9
3.79
0.08
0.02
0.67
0.88 0.88 0.45
nmol/g start, mg/dL
Glucose
49.2
45.9
44.3
46.8
45.7
45
3.88
0.73
0.15
0.32
0.48 0.30 0.22
Glucose end, mg/dL
49.5
57.6
28.2
53.5
52.2
52.3
2.04 <0.0001 <0.0001 < 0.01 0.08 0.21 0.20
Glucose change,
-2.03 9.35 -19.8 4.25
4.15
4.90
2.55 <0.0001 < 0.001 0.01
0.11 0.12 0.08
mg/dL start, µEq/mL 436.8 637.3 554.1 660.6 543.3 663.2 88.32
NEFA
0.09
0.17
0.03
0.02 0.21 0.01
NEFA end, µEq/mL
555.4 393.1 696.1 459 553.8 550.4 140.7
0.61
0.42
0.35
0.58 0.99 0.98
NEFA change,
121.8 -240.9 148 -198.4 13.8 -109.6 111.6
0.11
0.87
0.03
0.06 0.50 0.16
uEq/mL
BHB
start, mg/dL
16.3
16.6
17
9.61
15.2
16.9
6.07
0.86
0.91
0.96
0.34 0.88 0.93
BHB end, mg/dL
12.9
4.76
38.4
4.94
3.52
3.37
4.07 <0.0001 < 0.001 0.15
0.16 0.10 0.10
BHB change, mg/dL
-1.21 -9.75 27.4 -2.54 -9.61 -11.4 5.04
< 0.01
< 0.01
0.28
0.86 0.28 0.20
1

Isoenergetic infusion, 194.2 kcal/h
Isoamount infusion, ~50 g/h
3
Trt = treatment
2

108

liver is very low (Emmanuel, 1981) and glucose is spared for other tissues mostly for
biosynthetic purposes. Glucose infusion increased concentration of plasma glucose
and decreased concentration of plasma NEFA likely because lipolysis was reduced. The infusion
of PH (0.7 mol/h) decreases intake 44% compared with CON by reduction in meal size,
consistent with the hypophagic effect of propionic acid reported by previous research (Oba and
Allen, 2003a; Stocks and Allen, 2012; Gualdrón-Duarte and Allen, 2017) and by an increase of
104% in the interval between meals. None of the metabolites measured in plasma or the hepatic
AcCoA content was affected with PH infusion. In the current study PL treatment did not affect
intake, even with the numerical decrease in DMI of 2.4 kg/d, and it was likely because of the
higher variability in intake among cows caused by carry over effect of AA infusion. The lack of
effect on intake could be also related to the 7% less calories infused compared with the expected
amount because of the higher variability in the infusion rate. None of the feeding behavior
parameters, blood metabolites or hepatic AcCoA content were affected, in contrast to GualdrónDuarte and Allen (Gualdrón-Duarte and Allen, 2017) which report decrease in DMI and
differences in feeding behavior and metabolic response infusing the same moles of propionic
acid per day on cows in the postpartum period.
Abomasal infusion of AA unexpectedly resulted on clinical signs of ketosis and
decreased intake. However the reduction in intake is likely through a mechanism that is not
related to hepatic oxidation. Ruminant liver has a very low activity of AcCoA synthetase, the
enzyme necessary to extract acetate from blood for further metabolism in tissues (Allen, 2000).
Some studies have reported harmful effects when buffered and unbuffered solutions of acetic
acid were infused into the rumen (Sheperd and Holcomb, 1998; Wilson et al., 1967;
Montgomery et al., 1963) but others do not report any detrimental consequence (Stocks and

109

Allen, 2012; 2013; Choi and Allen, 1999; Rook and Balch, 1961). The dramatic decrease in
intake (64%) compare to CON was explained by the reduction in number of meals and a
tendency to decrease meal size. There was no effect on the duration of the meals. The decrease
in glucose concentration to hypoglycemic values (28.2 mg/dL) was likely by the decrease in
intake of gluconeogenic precursors and the high glucose demand after calving. Plasmatic NEFA
levels were not affected compared with CON likely because peripheral tissues, including the
mammary gland, extracted the acetic acid infused. However, content of hepatic AcCoA was
highly increased and likely exported as BHB, which could explain the increased plasmatic
concentration of BHB over the physiological normal levels (38.4 mg/dL). The effect in intake
decrease milk yield 7.2 L/d. Total solids yield decrease but protein and fat concentration
increased (9% and 24%, respectively) compared with CON. In previous experiments with longterm intraruminal infusions of sodium acetate has been reported similar increase (20%) in fat
concentration (Sheperd and Holcomb, 1998) or linear increase (6% to 11%) with increased doses
of sodium acetate infused (Urrutia and Harvatine, 2017) but they also report an increase in fat
yield (Sheperd and Holcomb, 1998; Urrutia and Harvatine, 2017).
CONCLUSIONS
It is difficult to make conclusions from the results of this experiment because of the
unexpected dramatic decrease in DMI when acetic acid was infused into the abomasum. This
increased the standard error and likely resulted in a carryover effect of treatment. The
experiment described in Chapter 3 was conducted to compare isoenergetic infusions of glucose,
lactic acid and propionic acid into the abomasum of cows in the postpartum period without the
acetic acid treatment. In addition, the experiment described in Chapter 4 was conducted to better
understand the hypophagic effects of abomasal infusion of acetic acid. This experiment was

110

included in the appendix of this dissertation to document the effects of treatment and because the
numerical differences in DMI among treatments (except acetic acid) were as we hypothesized.

111

CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS
This research shows that fuels absorbed from the gastrointestinal tract can have different
effects on feed intake, likely related to their ability to stimulate hepatic oxidation. Results from
abomasal infusions of isoenergetic and isomolar solutions of propionic acid (PA) and glycerol
(GL); and isoenergetic solutions of fuels derived from starch digestion showed that
gluconeogenic substrates infused into the abomasum have different effects on feeding behavior
and DMI and that the effects are independent of their energy content. Glycerol did not decrease
intake either in isoenergetic or isomolar infusions compared with PA, and PA decreased hepatic
acetyl CoA (AcCoA) content compared with GL when pulse-dosed into the abomasum likely by
increasing oxidation in the tricarboxylic acid (TCA) cycle.
When fuels resulting from starch digestion were infused directly into the abomasum in
isoenergetic solutions they resulted in different effects on DMI and energy intake. Infusion of
glucose (GLU), which is not taken up from the blood by the liver in mature ruminants, did not
reduce intake, whereas lactic acid (LA) and PA, both extracted by the liver and anaplerotic,
decreased DMI while only PA decreased metabolizable energy intake. Hepatic extraction of
propionate is greater than lactate. Absorption of propionate is high because of the high activity of
propionyl-CoA synthase whereas conversion of lactate to pyruvate is dependent of the redox
state of the cell and likely that explains the differences found between both fuels. The
hypophagic effect of PA is likely because it is an obligatory anaplerotic compound and can
stimulate oxidation of hepatic AcCoA content whereas GL and LA are less so. The reduction of
AcCoA content during the timeframe of a meal possibly explained the reduction in meal size
when PA was infused.

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Infusion 20 mol/d of acetic acid into the abomasum as a control treatment for isoenergetic
infusions of fuels dramatically decreased in DMI unexpectedly. Acetate is the principal endproduct from ruminal fermentation but little acetate is extracted from the blood by the liver, so
the observed decrease in intake is from a mechanism unrelated to hepatic oxidation. We
evaluated the effect of isosmotic solutions of acetic acid infused as an acid or as a salt into the
abomasum or into the rumen and we conclude that up to 6 mol/8h of sodium acetate infused into
the rumen did not decrease DMI of cows in the early PP period, and this treatment could be used
as the isosmotic control in studies to evaluate the effect of VFA or other fuels on feed intake.
However the reasons explaining why other treatments decreased DMI are not understood and
require further investigation.
In addition to the measurement of hepatic AcCoA content, still more work is required to
evaluate the effects of PA and other fuels on TCA cycle flux intermediates and flux-controlling
enzymes, and energy charge in the liver. Pulse-dose infusion experiments of fuels, and sampling
at different time points within the timeframe of a meal could give insight into the more active
metabolic pathways or possible metabolic bottlenecks; and changes in ATP to ADP ratio and
phosphorylation potential to explain the changes observed on feeding behavior, DMI and
metabolic responses along the experiments presented. Research to evaluate the effects of feeding
more fermentable starch sources in diets for cows in the PP period is needed. It is important to
conduct studies evaluating the interaction among starch fermentability and starch concentration
in diets fed to cows in the early PP period on feeding behavior, DMI, milk yield and yield
components, and results from those experiments will provide practical information about feeding
strategies to increase DMI after calving.

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The experiments conducted for this dissertation provide strong support for the control of
feed intake through hepatic oxidation. Changing the amount and source of starch for diets alters
the ruminal fermentability and the availability of substrate for intestinal digestion and thus
modifies the type and pattern of fuels from starch digestion. Fuels have different effects on DMI
and feeding behavior consistent with their ability to promote AcCoA oxidation in the liver. More
fermentable starch sources in diets for cows in the immediate PP period increases PA production
and availability for the liver and might reduce energy intake of cows by stimulating hepatic
oxidation.

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