EFFECT OF TYPE AND AMOUNT OF OMEGA-3 FATTY ACIDS IN THE DIETS OF
EXERCISING HORSES
By
Alaina Parsons

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Animal Science
2011

ABSTRACT
EFFECTS OF TYPE AND AMOUNT OF OMEGA-3 FATTY ACIDS
IN THE DIETS OF EXERCISING HORSES
By
Alaina Michelle Parsons
The objective of this study was to determine the effect of the supplementation of differing
sources of dietary long-chain polyunsaturated omega-3 fatty acids on health and performance
parameters of exercising horses. Twenty-four horses were used in a repeated 3 x 3 Latin square
design with 21-d periods. During each period each horse received one of three diets that differed
in omega-3 fatty acid profiles: a soybean oil source (SOY), a flaxseed oil source (FLAX), or an
algae source (ALG). Horses were exercised 5 d/wk for a minimum of 20 min, with body weights
and body condition scores recorded every 7 d, while blood samples, heart rates, and stride
lengths were measured on days 0, 21, 42, and 63. Blood samples were analyzed for various
inflammatory markers and for a complete plasma fatty acid profile. Heart rates were recorded
during a 30-min exercise bout and stride lengths were measured at the walk and trot before and
after exercise. No treatment differences were observed for stride lengths, heart rates, total
plasma omega-3, omega-6, and docosahexaenoic acid concentrations, or several inflammatory
markers measured in plasma and serum. Plasma eicosapentaenoic acid concentrations were
higher in horses on the SOY diet (0.45 ± 0.07 percent of total plasma lipid) than horses on FLAX
and ALG (P < 0.05) and plasma α-linolenic acid was altered in response to treatment (P < 0.05)
with horses on FLAX having the greatest percentage. Serum interleukin-10 concentrations were
lower in SOY than in ALG (P < 0.05). The results of this study indicate that mature exercising
horses free from osteoarthritis do not exhibit physiological anti-inflammatory benefits when
supplemented with a low dose of omega-3 fatty acids over a 21-d period.

ACKNOWLEDGEMENTS
I would like to acknowledge and thank the members of my committee (Dr. Nielsen, Dr.
Orth, Dr. Geor, and Dr. Schott) for their support of this project. The guidance and knowledge
that they have supplied has truly enhanced my learning experience here. It has been such a great
opportunity to work with a group as talented as this. I would also like to thank Roy Hall and
Cooperative Research Farms for their support of this research, as well as several faculty
members at North Carolina State University for their encouragement to attend graduate school
and their support throughout this experience.
This project involved the assistance of so many individuals. Foremost, I would like to
thank Cara Robison for pretty much rocking my world. Her support and contributions in all
stages of this project were unparalleled, and her encouragement in times of frustration was
unmatched (and she makes a pretty mean “Mussolini” pasta!). Credit is certainly due to all of
the undergraduates who made it possible to exercise 120 horses a week, as well as other staff
members who lent support in this project by helping me learn new techniques and invade their
lab space, especially Jim Liesman, Dave Main, Justin Zyskowski, and Courtney Preseault.
I know that everyone always thanks their fellow grad students, but the MSU grads truly
hold a special place in my heart. Their unwavering support, awe-inspiring volleyball skills, and
the ever-encouraging “awkward hallway wave” have truly made my time here at MSU the great
experience that it has been. I would like to especially thank Landon Cross, Sarah Elzinga, and
Becca Rockwell for helping me keep my head on straight and for their constant support, love,
and encouragement. I’m pretty sure that all of our adventures, particularly all of the pool-side
dates, were the primary contributing factor to the retention of my sanity during the past year.

iii

And to my roommates, Keri Jewell and Katie Murtha: thank you for not kicking me out of the
apartment during this process. Thank you for putting up with my crazy hours and scatter-brained
tendencies, but most importantly, thank you for your love and support in all the ups and downs
and crazy antics that we have endured together over the past two years.
And finally I would like to thank my family (mom, pops, Ben, and Ryan) for their
incredible love and support. Even though I know that they hate the fact that I live so far away,
their daily encouragement has always been clearly felt. I have been truly blessed to have had this
opportunity, and I am so grateful for the experiences, friendships, and skills that I have acquired
here.

iv

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................................... viii
LIST OF FIGURES ....................................................................................................................... ix
CHAPTER 1
INTRODUCTION .......................................................................................................................... 1
CHAPTER 2
REVIEW OF THE LITERATURE ................................................................................................ 2
Introduction ........................................................................................................................... 2
Polyunsaturated Fatty Acids.................................................................................................. 3
Classifications .............................................................................................................. 3
Metabolism and Dietary Sources ................................................................................. 3
The Omega-6 to Omega-3 Ratio. .......................................................................................... 6
Omega-3 Fatty Acids in Phospholipids ................................................................................. 7
Phospholipid Incorporation ......................................................................................... 7
Cell Signaling .............................................................................................................. 8
Metabolism. ................................................................................................................. 8
Inflammatory and Immune Responses and PUFA ................................................................ 9
Introduction.................................................................................................................. 9
Immune and Inflammatory Responses ........................................................................ 9
Eicosanoids ................................................................................................................ 10
Biological Effects ................................................................................................................ 14
Metabolic Effects ....................................................................................................... 14
Arthritis ...................................................................................................................... 15
Exercise...................................................................................................................... 15
Insulin ........................................................................................................................ 15
Lipid in Equine Diets .......................................................................................................... 16
Omega-3 Supplementation and Metabolism ............................................................. 16
Equine Lipid Profiles. ................................................................................................ 16
Biological Effects ...................................................................................................... 18
Inflammation and Omega-3 Supplementation in Horses .................................................... 19
Immunity and Inflammatory Responses .................................................................... 19
Arthritis and Exercise ................................................................................................ 21
DHA-Rich Microalgae ........................................................................................................ 22
Conclusions ......................................................................................................................... 23
CHAPTER 3
MATERIALS AND METHODS .................................................................................................. 25
Animal Management ........................................................................................................... 25
Lameness Evaluations ......................................................................................................... 25
Experimental Design and Diets ........................................................................................... 26
v

Treatments ........................................................................................................................... 27
Exercise ............................................................................................................................... 28
Horsemanship Classes ............................................................................................... 28
Training Classes......................................................................................................... 28
Lunge Line Exercise .................................................................................................. 29
Measurements and Sample Collection ................................................................................ 29
Blood Collection and Handling ................................................................................. 29
Stride Length Measurement ....................................................................................... 30
Heart Rate Measurement ........................................................................................... 30
Laboratory Analyses ........................................................................................................... 31
Analysis of Fatty Acids in Feeds and Plasma. ........................................................... 31
Prostaglandin E2 ........................................................................................................ 32
Tumor Necrosis Factor-alpha .................................................................................... 32
Interleukin 10. ............................................................................................................ 33
Interferon-gamma ...................................................................................................... 33
Statistical Analyses ............................................................................................................. 33
CHAPTER 4
RESULTS ..................................................................................................................................... 39
Body Weight ....................................................................................................................... 39
Stride Lengths ..................................................................................................................... 39
Heart Rates .......................................................................................................................... 40
Plasma Fatty Acid Profiles .................................................................................................. 42
Total Protein and Fibrinogen............................................................................................... 43
Prostaglandin E2 .................................................................................................................. 44
Tumor Necrosis Factor-α .................................................................................................... 44
Interleukin 10 ...................................................................................................................... 45
Interferon-γ .......................................................................................................................... 45
CHAPTER 5
DISCUSSION ............................................................................................................................... 50
CHAPTER 6
SUMMARY AND CONCLUSIONS ........................................................................................... 63
APPENDIX A ............................................................................................................................... 67
FAMES Methylation Procedure .......................................................................................... 67
APPENDIX B ............................................................................................................................... 69
Prostaglandin E2 ELISA Procedure .................................................................................... 69
APPENDIX C ............................................................................................................................... 71
Tumor Necrosis Factor-alpha ELISA Procedure ................................................................ 71
APPENDIX D ............................................................................................................................... 73
vi

Interleukin 10 ELISA Procedure ......................................................................................... 73
APPENDIX E ............................................................................................................................... 75
Interferon-gamma ELISA Procedure .................................................................................. 75
APPENDIX F................................................................................................................................ 77
Tumor Necrosis Factor-alpha Assay Reagent Preparation: ................................................ 77
APPENDIX G ............................................................................................................................... 79
Equine Interleukin 10 Assay and Equine Interferon-gamma Assay Reagent Preparation: . 79
LITERATURE CITED ................................................................................................................. 82

vii

LIST OF TABLES

Table 3.1.

Latin square design used for the experiment, where ALG denotes
algae-based supplement, FLAX denotes flaxseed oil-based
supplement, and SOY denotes soybean oil-based supplement. Each
block of three horses was randomly assigned to either Group 1 or
Group 2 and fed in the supplement order provided below. .................................... 35

Table 3.2.

Block, group, classification, age, sex, and osteoarthritis score for each
horse on trial. .......................................................................................................... 36

Table 3.3.

Delivery (g/d) of n-3, n-6, α-linolenic acid (LNA), eicosapentaenoic acid (EPA),
and docosahexaenoic acid (DHA) as a protected fatty acid supplement volume of
908 g/d as fed. ........................................................................................................ 37

Table 3.4.

Selected fatty acids supplied by each feedstuff. ..................................................... 38

Table 4.1.

Body weight (kg) and body condition score (BCS) for all horses within
treatment diets. ......................................................................................................... 46

Table 4.2.

Mean walk stride length (cm) and trot stride length (cm) of horses.
Stride lengths were measured before and after a 20-min specified
exercise on the lunge line to determine a baseline at d 0 and following
21-d supplementation periods for all treatments. ...................................................... 46

Table 4.3.

Mean heart rate (HR) measurements at baseline (d 0) and following
21-d supplementation periods for all treatments. Heart rates are provided
at rest, the maximum HR during a 20-min specified lunge line exercise, and
specific heart rates post-exercise during a 10-min recovery period: immediately
post-exercise (HR 0), 1 min post-exercise (HR 1), 2.5 min post-exercise
(HR 2.5), 5 min post-exercise (HR 5), and 10 min post-exercise (HR 10). .............. 47

Table 4.4.

Effect of feeding horses diets supplemented with three differing sources
and content of n-3 fatty acids on plasma fatty acid profiles after 21-d
supplementation periods and at baseline values (d 0). ............................................... 48

Table 5.1. Comparison of n-3 supplementation sources, amounts of fatty acids, and
length of supplementation (d) across equine studies……………...…..……………..51
viii

LIST OF FIGURES

Figure 2.1. Metabolism of n-6 and n-3 fatty acids (Calder, 2001). ............................................... 5
Figure 2.2. Synthesis of eicosanoids from arachidonic acid and sites of inhibition by
eicosapentaenoic acid. ARA, arachidonic acid; COX, cyclo-oxygenase; EPA,
eicosapentaenoic acid; HETE, hydroxyeicosatetraenoic acid; HPETE,
hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase, LT, leukotriene; PG,
prostaglandin, TX, thromboxanes (Calder, 2002). ..................................................... 12
Figure 4.1. The response of hematological variables to n-3 treatments of soybean oil
(SOY), flaxseed oil (FLAX), or algal DHA (ALG) in equine plasma..……..……...49

ix

LIST OF ABBREVIATIONS

Alpha-linolenic acid………………………………………………………………………. ALA
Arachidonic acid………………………………………………………………………….. ARA
Cyclooxygenase…………………………………………………………………………... COX
DHA-rich microalgae……………………………………………………………………... DRM
Docosahexaenoic acid…………………………………………………………………….. DHA
Docosapentaenoic acid……………………………………………………………………. DPA
Eicosapentaenoic acid…………………………………………………………………….. EPA
Fatty acids………………………………………………………………………………… FA
Interferon-gamma…………………………………………………………………………. INF-γ
Interleukin 1………………………………………………………………………………. IL-1
Interleukin 6………………………………………………………………………………. IL-6
Interleukin 10……………………………………………………………………………... IL-10
Linoleic acid………………………………………………………………………………. LA
Lipopolysaccharide……………………………………………………………………….. LPS
Lipoxygenase……………………………………………………………………………... LOX
Omega-3………………………………………………………………………….............. n-3
Omega-6…………………………………………………………………………………... n-6
Polyunsaturated fatty acids………………………………………………………………...PUFA
Prostaglandin E2…………………………………………………………………………...PGE2
Tumor necrosis factor-alpha……………………………………………………………….TNF-α

x

CHAPTER 1
INTRODUCTION

Recently research in the area of fat supplementation in the diets of horses has been
increasing, particularly concerning long-chain polyunsaturated fatty acids, with omega-3 and
omega-6 fatty acids of particular interest. Many exercising horses suffer from disease and
unsoundness related to joint health, and current research suggests that oral omega-3 fatty acid
supplementation may have beneficial anti-inflammatory effects to mediate these issues.
However, research has been conducted over varying lengths, amounts, and sources of fatty acid
supplementation, with inconsistent physiological responses to treatment. The purpose of the
following study is to determine the effect of supplementation of differing sources and amounts of
dietary long-chain polyunsaturated omega-3 fatty acids across 21-d periods on health and
performance parameters of exercising horses. Greater insight into the biological effects observed
at an optimal level and source of omega-3 supplementation could contribute to the development
of feed recommendations to mediate inflammatory joint factors that can be associated with
lameness among horses and therefore improve their potential for performance. We therefore
hypothesize that when supplied with supplemental sources of omega-3 fatty acids, plasma fatty
acid concentrations will show a change in relation to the fatty acid sources from each diet after a
21-d period. We also hypothesize that horses supplied omega-3 fatty acids from an algal source,
when compared to a soybean oil and flaxseed source, will show the greatest increase in plasma
docosahexaenoic acid concentrations as well as increased stride lengths and decreased heart rates
during exercise as a result of a decrease of pro-inflammatory cytokines and eicosanoids as
measured in the plasma and serum.

1

CHAPTER 2
REVIEW OF THE LITERATURE

Introduction
Fat is commonly supplemented to both human and animal diets to increase the energy
density of the diet. Recently, research concerning individual dietary fatty acids (FA) and their
role as biological regulators has increased. These FA, particularly the polyunsaturated fatty
acids (PUFA), are important constituents of phospholipids in cell membranes and play a major
role in the structure and function of the membrane (Lands, 2005). The FA composition of the
membrane also influences the eicosanoids that are produced and used for intercellular signaling.
Omega-3 (n-3) PUFA confer several health benefits to animals and humans alike, and therefore
have many potential uses in equine nutrition. In other animal species, n-3 supplementation from
fish oil sources increases the serum concentration of eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) in proportion to the amount of fish oil fed (Bjerve et al., 1993;
Ashes et al., 1992). Additionally, eicosanoids produced from n-3 FA precursors are less
inflammatory than those produced from omega-6 (n-6) FA precursors. This alteration in the type
of inflammatory products produced may be useful in treating inflammatory diseases. Omega-3
supplementation can reduce the risk of cardiovascular disease, cancer, chronic airway disease,
chronic arthritis, gastrointestinal disease, and other inflammatory and autoimmune diseases in
humans (Simopoulos, 2008; Goldberg and Katz, 2007; Knapp, 1995).
Compared to other species, relatively few studies have been conducted regarding the
effects of dietary n-3 FA on inflammatory and immune cell functions in vivo concerning the
horse. This review of published literature will examine the metabolism of n-3 and n-6 FA from
2

dietary sources and the role of these FA as potential modulators of inflammatory pathways. The
effects of supplementation in the equine model will also be reviewed, with a focus on blood lipid
profile modifications as well as exercise and inflammatory responses.

Polyunsaturated Fatty Acids

Classifications. Fatty acids are composed of unbranched hydrocarbon chains with an
even number of carbons and are classified as short chain (4 carbon atoms), medium chain (6 to
12 carbon atoms), or long chain (14 or more carbon atoms). Depending on the presence and
number of double bonds in the aliphatic carbon chain of the molecule, FA can also be classified
as saturated (no double bonds), monounsaturated (one double bond), or polyunsaturated (more
than one double bond). Polyunsaturated fatty acids are further classified according to the
position of the first double bond when counting from the methyl end and consist of two major
classes: the n-3 FA, which exhibit a first double bond positioned between the third and fourth
carbons, and the n-6 FA, with the first double bond located between the sixth and seventh
carbons.
Metabolism and Dietary Sources. All members of the n-3 and n-6 classes of PUFA are
derived from their respective precursors. Linoleic acid (LA; 18:2n-6) is desaturated and
elongated to yield arachidonic acid (ARA; 20:4n-6) and other long chain PUFAs, while αlinolenic acid (ALA; 18:3n-3) is desaturated and elongated to yield EPA (20:5n-3) and
docosapentaenoic acid (DPA; 22:5n-3). Docosapentaenoic acid can be retroconverted to EPA
and further metabolized to DHA (20:6n-3). The 20-carbon FA can also be made by
retroconversion of longer chain FAs obtained in the diet (Figure 2.1). The precursor molecules
3

of LA and ALA compete for the same desaturase and elongation enzymes to form the
polyunsaturates, principally ARA and DHA. These end products are incorporated into the 2position of phospholipids via de novo synthesis and acylation-deacylation mechanisms
(Neuringer et al., 1988).
Within each series of FA, elongation and desaturation occur without altering the methyl
end of the molecule. Both LA and ALA are essential FA in mammals, as they lack the
desaturases necessary to convert LA from short chain FA precursors and are unable to
interconvert n-6 and n-3 FA. These enzymes are found in plants, making some plant tissues and
oils rich sources of the 18-carbon FA. Linoleic acid is found in seeds of most plants, except
coconut, cocoa, and palm, and is a primary component of maize, sunflower, safflower, and
soybean oils, contributing over 50% and often up to 80% of the FA content (Calder, 2001a).
Rapeseed, canola, and soybean oils are good sources of ALA, contributing between 5% and 15%
of the FA present. Flaxseed oil is the richest source of ALA, contributing as much as 60% of the
FA content (Calder, 2001a). Alpha-linolenic acid is also found in the chloroplasts of green leafy
vegetables, in animal fat, and in some nuts.
The longer chain members of the n-6 and n-3 families are obtained directly from animal
foods of land or marine origin. Fish and other marine animals are a particularly rich source of
the long chain PUFA, typically containing approximately 18% EPA and 12% DHA (Cleland et
al., 2006). Many marine plants, especially the unicellular algae in phytoplankton, elongate and
desaturate ALA to yield EPA and DHA. These PUFA then get transferred through the food
chain, accumulating in the tissues of some marine mammals and fish, particularly “oily” fish,
such as herring, mackerel, and tuna (Calder, 2001a).

4

Figure 2.1. Metabolism of n-6 and n-3 fatty acids (Calder, 2001).
18:1(n-9)

∆12-desaturase (plants only)

∆15-desaturase (plants only)
18:2(n-6)

18:3(n-3)

∆6-desaturase

∆6-desaturase

18:3(n-6)

18:4(n-3)

20:3(n-6)

20:4(n-3)

∆5-desaturase
20:4(n-6)
Arachidonic
Acid

∆5-desaturase
20:5(n-3)
Eicosapentaenoic
Acid

5

22:6(n-3)
Docosahexaenoic
Acid

While the relationship between ALA intake and EPA incorporation has been shown to be
linear, several studies consistently demonstrate that increased consumption of ALA does not
result in increased proportions of DHA in plasma or cell lipids (Burdge and Calder, 2005).
Although plant sources can theoretically provide EPA and DHA via desaturation and elongation
of ALA, this conversion of ALA to long chain PUFA appears to be limited in human subjects
(Simopolous 2008). Overall these studies demonstrate that although chronically increased
consumption of ALA does result in conversion to EPA, and therefore enhancement of EPA
concentration in plasma and cell membranes, and the estimated proportion of ALA entering this
pathway and being converted to EPA is low, and lies between 8% and 10% in humans. The
extent of conversion of ALA to DHA is less clear, with most studies reporting estimated
fractional conversion at <0.05%, an amount insufficient to increase the concentrations of DHA in
membranes (Williams and Burdge, 2006).

The Omega-6 to Omega-3 Ratio.

As ∆6-desaturation is the rate-limiting step in the bioconversion pathway, high dietary
intakes of n-6 FA have been proposed to be a limiting factor in the conversion of ALA to its
long-chain polyunsaturates. When increasing the amount of dietary n-3 FA relative to n-6 FA in
the diet, n-3 FA are preferentially incorporated into cell membrane phospholipids (Calder,
2001b). Thus modifications of the diet that result in the increase of a particular FA can impact
synthesis of another. For example, a high concentration of ALA inhibits conversion of LA
because both are substrates for the same ∆6-desaturase enzyme. The conversion executed by the
∆5-desaturase is also inhibited by ARA, EPA, and DHA (Bezard et al., 1994). Therefore the

6

ratio of n-6 to n-3 in the diet becomes extremely important, as the relative abundance of these FA
in the diet affects the relative intensity of a particular response by tissue (Lands, 2005).
The suggested dietary ratios range from 4 to 10 for humans (Albertazzi and Coupland,
2002; Sugano, 1996). An imbalanced ratio can have adverse consequences, as mammalian cells
cannot interconvert n-6 and n-3 FA. Excessive amounts of n-6 promotes the pathogenesis of
many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune
diseases, while increased levels of n-3 FA (and hence a lower n-6 to n-3 ratio) exert suppressive
effects. Additionally, an imbalance of n-6 and n-3 in the peripheral blood causes an
overproduction of pro-inflammatory cytokines (Simopoulos, 2008).

Omega-3 Fatty Acids in Phospholipids

Phospholipid Incorporation. While it is known that differing classes of FA will elicit
differing metabolic effects in tissues, they must first be present in the blood to allow for
incorporation. When FA are supplemented in the diet, they are incorporated into the cell
membrane, with the concentration of PUFA in tissue consequently mimicking the dietary FA
when consumed in adequate quantities in a habitual dietary pattern. This is where the chemical
differences between n-3 and n-6 fatty acids are important, as their unique chemical structures
elicit different biological effects. For example, ALA and EPA are found in triglycerides,
cholesteryl esters, and in phospholipids, while DHA is mostly found in phospholipids
(Simopoulos, 2008) and is one of the most abundant components of the brain’s structural lipids.
High concentrations of DHA are also found in the cerebral cortex, retina, testes, and sperm
(Brinsko et al., 2005; Harris et al., 2005; Mueller and Talbert, 1988; Neuringer et al., 1988).
7

Cell Signaling. Once in the tissue, n-6 and n-3 FA form specific lipid-protein membrane
complexes that are needed for cellular structures at specific stages of tissue differentiation and
development, particularly during brain and retina development (Lands, 2005; Neuringer et al.,
1988). Fatty acids released from membrane phospholipids by cellular phospholipases, or those
made available to the cell from the diet or other aspects of the extracellular environment, are
important signaling molecules. They can act either as secondary messengers or substitute for
classic secondary messengers of the inositide phospholipid and the cyclic adenosine
monophosphate signal transduction pathways (Simopoulos, 2008). Omega-3 and n-6 FA can
also act as precursor molecules by forming hormone-like self-healing autacoids, such as
prostaglandins, leukotrienes, and thromboxanes, which signal important adjustments of healthy
tissue in response to stimuli (Lands, 2005).
Metabolism. Polyunsaturated fatty acids also have a role in the regulation of hepatic gene
expression. As the liver plays a central role in whole body carbohydrate and lipid metabolism,
several metabolic pathways may be regulated by PUFA through changes in the activity or
abundance of transcription factors that target carbohydrate, FA, cholesterol, and bile acid
metabolism. The principle action of n-3 FA on hepatic metabolism involves a shift from lipid
synthesis and storage to lipid oxidation. This mechanism may contribute to the regulation of
blood levels of triglycerides and cholesterols, which are important risk factors for chronic
inflammatory diseases (Jump, 2002). Additionally, long chain PUFA supplementation can
affect inflammation and immunity at the level of inflammatory gene transcription, leading to a
highly integrated signaling cascade that can be affected in multiple ways by PUFA status,
leading to changes in the body’s response to tissue damage, pathogen challenge, or disease
(Calder, 2001b).
8

Inflammatory and Immune Responses and PUFA

Introduction. The fatty acid composition of inflammatory and immune cells is sensitive
to change according to the ratio of n-6 and n-3 FA in the body, and therefore provides a link
between dietary PUFA intake and inflammation. Among all of the FA classes, it is the n-3
PUFA that possess the most potent immunomodulatory activities, with EPA and DHA being
more biologically potent than ALA (Calder, 2001b). An increase in EPA results in partial
replacement of ARA in cell membranes, which leads to a decreased production of ARA-derived
mediators (pro-inflammatory prostaglandins, leukotrienes, and related compounds).
Eicosapentaenoic acid is also a substrate that gives rise to mediators that often have different
biological actions or potencies than those formed from ARA (Calder, 2001b).
Immune and Inflammatory Responses. Communication within the immune system is
brought about by direct cell-to-cell contact involving adhesion molecules and by the production
of chemical messengers, chief among these being proteins called cytokines. Cytokines, such as
tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), and interleukin 6 (IL-6), act to
regulate cellular activity and have multiple activities on different cell types. Cytokines act by
binding to specific receptors on the cell surface and induce changes in growth, development, or
activity of the target cell. In addition, these cytokines mediate the systemic effects of
inflammation. Production of appropriate amounts of TNF-α is clearly beneficial in response to
infection, but chronic overproduction can be dangerous, as it is implicated in causing some of the
pathological responses that occur in inflammatory conditions. These pro-inflammatory
cytokines are antagonized by anti-inflammatory cytokines, such as interleukin 10 (IL-10; Calder,
2001b). Other inflammatory chemical messengers, such as prostaglandin E2 (PGE2), have a

9

number of pro-inflammatory effects, including the induction of fever and erythema and
enhancing pain. Prostaglandin E2 is also immunosuppressive, as it inhibits production of TNFα, interleukins, and interferon-gamma (INF-γ).
Several animal studies demonstrate that dietary n-3 supplementation may improve
immune function. These findings indicate that diets rich in EPA plus DHA modulate aspects of
the immune response in vivo (Calder, 2001b), as these are the n-3 PUFA that exhibit the most
potent anti-inflammatory and antioxidant effects (Bloomer et al., 2009; Simopoulos, 2008). As
inflammation forms the foundation for many chronic diseases, n-3 FA supplementation could
offer a form of prevention.
Eicosanoids. Eicosanoids are a secondary group of chemical messengers, synthesized
from PUFA, that act within the immune system and are released in response to a cellular
stimulus. They exert complex control over many bodily systems, primarily concerning the areas
of inflammation and immunity, and provide a link between the PUFA and the inflammatory
pathways. Depending on the type of fatty acid substrate cleaved from cell membranes,
eicosanoids can facilitate or inhibit inflammatory mediators. Eicosanoids include the
prostaglandins, thromboxanes, and leukotrienes. The two essential FA families are converted
into two distinct families of eicosanoids, with those produced from n-6 fatty acids considered to
be pro-inflammatory, while those produced from n-3 fatty acids have reduced less proinflammatory activity. The conversion of tissue ARA controls the signaling of many eicosanoidmediated pathways, with EPA providing the most important competing substrate. The
competition of these fatty acid precursors during metabolism affects the ratios and types of

10

eicosanoids likely to be formed, and will therefore alter the body’s metabolic function (Lands,
2005; Calder, 2001b).
Generally most immune cell membranes contain large amounts of ARA (when compared
to EPA), thus ARA is usually the principal precursor for eicosanoid synthesis. Arachidonic acid
gives rise to such a range of mediators that have opposing effects to one another that the overall
physiological effect will be governed by the concentration of those mediators. Metabolism of
ARA via cyclooxygenase (COX) enzymes gives rise to the 2-series prostaglandins and
thromboxanes, whereas 4-series leukotrienes are produced from ARA by lipoxygenase (LOX)
metabolism. Overproduction of ARA derived eicosanoids has been implicated in many
inflammatory and autoimmune disorders (Albertazzi and Coupeland, 2002). As EPA competes
with ARA for the active sites on these enzymes, increased phospholipid n-3 FA causes the antiinflammatory products of n-3 FA metabolism, (3-series prostaglandins and 5-series leukotrienes)
to increase, with an analogous decrease in the pro-inflammatory products of n-6 FA (Calder,
2001b).
Biosynthesis of prostaglandins and thromboxanes begins when ARA is released from
membranes by phospholipases. This release may occur due to tissue injury, cytokine or growth
factor signals, or other stimuli. Free ARA acts as a substrate for COX and is then metabolized to
an intermediate prostaglandin, which can then give rise to over 16 different 2-series
prostaglandins and thromboxanes depending on available enzymes and cell type (Figure 2.2;
Calder, 2002). Leukotrienes are synthesized primarily by inflammatory cells. Then ARA, once
cleaved from the membrane, is a substrate for LOX, which is found in the nuclear envelope of
certain inflammatory cells. Arachidonic acid is then transformed to an epoxide leukotriene,

11

Figure 2.2. Synthesis of eicosanoids from arachidonic acid and sites of inhibition by
eicosapentaenoic acid. ARA, arachidonic acid; COX, cyclo-oxygenase; EPA, eicosapentaenoic
acid; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX,
lipoxygenase, LT, leukotriene; PG, prostaglandin, TX, thromboxanes (Calder, 2002).

ARA in cell membrane
phospholipids

EPA in cell membrane
phospholipids

Phospholipase A2
Free ARA

COX

5-LOX

PGG2

PGD2

PGJ2

Free EPA

5-HETE

PGI2

TXA2

5-LOX

3-series PG and TX

5-HPETE

PGH2

PGE2

COX

5-series LT

LTA4

LTC4

LTB4

Lipoxin A4
Lipoxin B4

LTD4

LTE4

12

which will then be either hydrolyzed, conjugated with glutathione, or undergo transcellular
metabolism to create bioactive eicosanoids (Calder, 2002).
Animal and human studies show that feeding diets rich in long chain PUFA results in
increased proportions of EPA and DHA in inflammatory cell phospholipids which are
incorporated at the expense of ARA (Calder, 2001a), as EPA inhibits ARA release from
phospholipids and inhibits the oxygenation of ARA by COX (Calder, 2002). Eicosapentaenoic
acid itself can act as a substrate for COX and 5- LOX (Figure 2.2), giving rise to structurally
distinct prostaglandins and leukotrienes which are less potent inflammatory mediators. These 3and 5- series eicosanoids act through the same receptors and target cells as ARA derived
eicosanoids, thus further competing against and inhibiting the pro-inflammatory actions of 2- and
4-series eicosanoids (Calder, 2002). These observations have led to the presumption that n-3 FA
derived from fish oil and other sources are on the whole anti-inflammatory, and could benefit the
health and well being of the horse. Omega-3 FA supplementation decreased these mediators,
namely the production of PGE2 , 4-series leukotrienes, and the pro-inflammatory cytokines
(especially TNF-α).
Many studies have specifically examined the effects of n-3 supplementation on TNF-α
production. Rodents supplemented with ALA via linseed oil have increased production of TNFα by resident macrophages, but show no effect on TNF-α by inflammatory macrophages
(Hubbard et al., 1994; Turek et al., 1991). Animal and human studies show that ex vivo
production of TNF-α following lipopolysaccharide (LPS) stimulation of monocytes is reduced
following introduction of n-3 FA (Browning et al., 2003; Harbige et al., 2003; Calder, 2001b).
In animal studies using primarily EPA for supplementation, lower peak serum TNF-α
13

concentrations are observed (Sadeghi et al., 1999;Hayashi et al., 1998; Tashiro et al., 1998) while
others have reported that dietary fish oil decreases the expression of interferon gamma (IFN-γ)
on murine peritoneal macrophages (Byleveld et al., 1999; Feng et al., 1999). After
supplementation with EPA and DHA, decreased resting levels of inflammatory biomarkers are
seen in exercise-trained men (Bloomer et al., 2009).

Biological Effects
Metabolic Effects. These n-6 and n-3 PUFA competitive interactions affect health either
via influences in membrane structures, and gene expression in addition to eicosanoid actions
(Lands, 2005). Omega-3 FA supplementation can have a number of beneficial metabolic effects
in several species, particularly associated with the previously described anti-inflammatory effects
of EPA and DHA, which may be beneficial in combating many chronic inflammatory diseases
including coronary heart disease, diabetes, arthritis, cancer, osteoporosis, mental health
disorders, dry eye disease, and age-related macular degeneration (Simopoulos, 2008; MacLean et
al., 2006). A large body of evidence exists, including human experimental studies, animal
experimental studies, and cell culture studies, to suggest beneficial effects of long chain n-3
PUFA on the molecular, cellular, and whole-body pathogenic processes of atherosclerosis and
thrombosis, suggesting that the consumption of EPA and DHA may decrease the risk of
cardiovascular disease (Calder, 2006). As n-3 FA increase erythrocyte deformability and are
incorporated into the membranes (De Moffarts, et al. 2007; Portier et al. 2006), they could play a
role in reducing risks for these diseases. Additionally, dietary supplementation of n-3 FA have a
cholesterol-reducing effect in animals (O’Connor et al., 2004; Warner et al., 1989), may
influence both bone formation and bone resorption in rats (Li et al., 1999), and may also have
14

potential as modulators of respiratory diseases involving chronic inflammatory and infectious
processes (Knapp, 1995).
Arthritis. The responses concerning inflammatory mediators following n-3 FA
supplementation have been an area of interest concerning the alleviation of joint pain, as
supplementation with n-3 FA can have a significant effect on synovial membrane production of
eicosanoids. Controlled trials have demonstrated the efficacy of n-3 FA in reducing joint pain
associated rheumatoid arthritis (Goldberg and Katz, 2007). However, it remains to be
determined empirically whether the ability of n-3 FA to reduce pain is due to either suppression
of the inflammation underlying arthritic conditions, or via direct effects on prostaglandins or
possibly cytokines in the spinal cord dorsal horn (Goldberg and Katz, 2007).
Exercise. As the inflammatory response is increased in response to strenuous exercise,
the anti-inflammatory properties of n-3 FA may have beneficial effects during exercise. A
combination of decreased blood lipid concentrations, increased erythrocyte membrane fluidity
and increased insulin sensitivity as a result of n-3 supplementation have the potential to
beneficially alter several physiological responses to exercise, such as decreasing heart rate
(O’Connor et al., 2004; Simopoulos, 1991; Mueller and Talbert, 1988).
Insulin. Omega-3 FA supplementation also increases insulin sensitivity across a variety
of species. Miniature pigs supplemented with n-3 FA have increased insulin sensitivity in
response to an intravenous glucose tolerance test (Behme, 1996). These results are in agreement
with findings from similar studies conducted using rats. Chicco et al. (1996) found that rats
supplemented with fish oil comprising 7% of their diet also exhibit increased insulin sensitivity
in response to a glucose tolerance test. In an additional study, rats supplemented with a similar

15

quantity of n-3 FA for 6 wk exhibit an increase in n-3 FA concentration in the skeletal muscle
and sarcolemma, combined with a 14-fold increase in insulin binding (Liu et al., 1994). These
researchers suggested that the increasing amount of n-3 FA in the cell membrane might alter the
action of insulin and insulin receptors, therefore resulting in an increase of insulin sensitivity.
Increased insulin sensitivity decreases the fasting blood free fatty acids and triglyceride levels in
humans (McCarty, 1998).

Lipid in Equine Diets

Omega-3 Supplementation and Metabolism. Regarding the diets of exercising horses, fat
has traditionally been added as a means of increasing energy density. Different methods can be
used to supplement fat in the equine diet, with common fat sources being oils from soybean,
corn, sunflower, olive, flaxseed, canola, and fish. All of these sources will increase energy
density equally, although their fatty acid profiles vary. However, the most effective n-3
supplementation is done using marine-based oils, as these are the only oils that naturally contain
significant amounts of EPA and DHA, and will therefore elicit the greatest biological effects
(Lands, 2005). As seen in other species, horses also have a limited ability to convert dietary
ALA to significant amounts of EPA or DHA. When horses were supplemented with equal
amounts of n-3 FA from either flaxseed or fish oil, plasma EPA and DHA content only increased
in horses fed encapsulated fish oil (Vineyard et al., 2009; Siciliano et al., 2003), indicating an
inability to obtain sufficient quantities of EPA or DHA from a land-based food source.
Equine Lipid Profiles. While FA are a small fraction of the normal equine diet, their
popularity in equine supplementation is increasing, with several studies investigating the
16

potential for PUFA to modify circulating blood characteristics and FA profiles in the horse.
Luther et al. (1981) were the first to perform work concerning plasma FA concentrations in
horses using gas chromatography, producing more accurate results than any previously reported.
Their results showed that ALA has the highest concentration in equine plasma, followed by
stearic acid (18:0), oleic acid (18:1n-9), and palmitic acid (16:0). Combined, these four FA
make up over 85% of the total plasma FA profile. Concentrations of FA are tissue specific, as
seen when the same analytical technique was applied to harvested equine erythrocytes and the
FA profile changed significantly. In erythrocytes, stearic acid exists in the highest percentage,
followed by palmitic and oleic acid (Luther et al., 1982). Free FA levels not only differ among
parts of the blood, but also fluctuate throughout the day (Orme et al., 1994; Luther et al., 1982;
Luther et al., 1981), with concentrations of oleic and stearic acid varying. These results suggest
that concentrations of FA fluctuate depending on the time of day, prandial state, and dietary
influences in horses.
Dietary supplementation of fish oils increases the concentrations of EPA and DHA in
equine FA profiles proportionally to the quantity fed (McCann et al., 2000; Morris et al., 1991).
In a study conducted by Hall et al. (2004b), horses were supplemented for 14 wk with either fish
oil or corn oil. Horses fed fish oil had increased plasma concentrations of EPA and DHA
compared to horses fed corn oil, accompanied by a corresponding decreased LA and ALA
concentration. O’Connor et al. (2007) supplied exercising horses with a diet of 324 mg oil/kg
BW from either a corn oil source or a menhaden fish oil source for 63 d and observed that horses
receiving the fish oil treatment had increased serum concentrations of both EPA and DHA.
Several studies are in agreement with this, showing a positive correlation of the n-6 to n-3 ratio
in plasma and serum according to diet in horses fed n-3 supplements from marine sources
17

compared to land based n-3 sources (Vineyard et al., 2009; King et al., 2008; De Moffarts et al.,
2007; Khol-Parisini et al., 2007). Plasma EPA and DHA levels increase as soon as three days
following supplementation with n-3 FA in a dose-responsive manner, with a peak level obtained
by seven days when offered at the amount of 40 g/d of EPA/DHA (King et al., 2008).
Biological Effects. Once incorporated into cell membranes, n-3 FA can have many
beneficial metabolic effects in the horse. Mares fed a marine-derived n-3 FA source containing
10.4 g EPA and 8.6 g DHA for a period of 60 d prior to parturition through 21 d post-parturition
had increased milk EPA and DHA at all sampling times. Foals of all PUFA-supplemented mares
had elevated concentrations of EPA and DHA in plasma compared to control mares (Kruglik et
al., 2005). Concerning sperm output and dynamics, beneficial effects have also been observed
with n-3 supplementation in breeding stallions. Supplementation with long chain PUFA
increases sperm output and the percentage of normal sperm (Harris et al., 2005) and also
increases semen DHA concentrations, improves sperm motility (when cooled and stored for 48
hours), and improves total and progressive motility in stallions (Brinsko et al., 2005).
Beneficial changes in response to exercise following n-3 supplementation have also been
shown in both horses as well as humans. Following exercise in humans, a decrease in resting
oxidative stress, inflammatory biomarkers, and an increase in maximal oxygen consumption
occurs when subjects are treated chronically with EPA and DHA (Calder, 2001b; Mori and
Beilin, 2004; Brilla and Landerholm, 1990). Several beneficial effects following n-3
supplementation in exercising horses have also been observed, including an increase in trot stride
length (Woodward et al., 2007), a decrease in serum cortisol following a standardized exercise
test (Howard et al., 2003), and reduced heart rates associated with intense exercise when
supplemented with a fish oil source (O’Connor et al., 2004). Conversely, horses fed soybean oil
18

for 28 d show no difference in heart rate during exercise when compared to a control group of
corn oil fed horses (Howard et al., 2003). The decrease in heart rate may potentially be
attributed to an increase in fluidity in the erythrocyte membrane as a result of EPA and DHA
incorporation (O’Connor et al., 2004). Two additional studies have been done examining the
effects of n-3 FA on erythrocyte deformability in horses (De Moffarts et al., 2007; Portier et al.,
2006), with horses on a fish oil diet having increased concentrations of EPA and DHA in
erythrocyte membranes that delayed the onset of an exercise-induced decrease in erythrocyte
membrane fluidity. Further, exercising rats receiving dietary fish oil also exhibit decreased
resting heart rates, blood pressure, aortic flow, and cardiac output due to a decrease in vascular
resistance (Demaison et al., 2000; Lortet and Verger, 1995).
When O’Connor et al. (2004) examined the effects of fish oil supplementation on
exercising horses, horses in the treatment group had lower plasma glucose during recovery after
a standardized exercise test and tended to have higher glucose to insulin ratios, suggesting an
increase in insulin sensitivity. Following a 12-h fast, the fish oil treated horses also had lower
free FA concentrations and lower serum glycerol concentrations than the control group.

Inflammation and Omega-3 Supplementation in Horses

Immunity and Inflammatory Responses. While the ability of n-3 FA to mediate
inflammatory and immune responses has been well documented in other animals, only a few
studies have examined these factors in horses. When 6 healthy adult horses were fed a diet
containing 8% linseed oil by weight over an 8 wk period, peritoneal macrophages produced less

19

TNF-α in response in vitro when stimulated with endotoxin compared to pre-supplement
measures. It was proposed that TNF-α synthesis was altered by reduced adenylate cyclase
activity as a result of a high ALA diet. This could alter formation of cyclic nucleotides that
control TNF-α synthesis (Morris et al., 1991). In another study by the same research team, an
intravenous n-3 lipid emulsion decreased basal TNF-α production by monocytes, as well as those
cells which were challenged with calcium ionophore. In addition, an n-6 emulsion increased
TNF-α production by monocytes challenged with LPS (McCann et al., 2000). In mature horses
supplemented with fish oil, EPA and DHA serum levels increase, a 78-fold increase in the
production of leukotrienes B5 in neutrophils can be observed, and a decrease of PGE2 is seen in
bronchoalveolar lavage fluid cells (Hall et al., 2004b). This same research team also saw that
equine peripheral blood neutrophils have reduced leukotriene B4 synthesis in response to dietary
fish oil (Hall et al., 2004a). Many other studies have demonstrated the ability of fish oil to act as
a PGE2 antagonist, however, it is important to note that the overall outcome of dietary n-3
supplementation cannot be predicted solely on the basis of abrogation of PGE2 –mediated
effects.
Dietary n-3 supplementation may also improve immune function in both yearling and
mature horses. Yearling horses supplemented with a fish oil/olive oil blend for 42 d had
increased plasma and red blood cell concentrations of EPA and DHA, with a corresponding
decrease in PGE2 production. Yearlings were given a tetanus booster midway through the trial,
and when tested at the end of supplementation, horses on the n-3 diet had higher tetanus titers
than those on the control diet (Vineyard et al., 2009). While studies in humans have linked n-3s
20

with a reduction of bronchial inflammation associated with asthma (Nagakura et al., 2000;
Broughton et al., 1997), horses fed fish oil showed no differences in pulmonary function (KholParisini et al., 2007). However, the horses on this study were evaluated for a period of 20 wk,
and these researchers suggest that the influence of time, environment, and diet interfered with
each other or contributed to the lack of clinical or lung functions due to changes in diet.
Arthritis and Exercise. Arthritis is a condition of acute or chronic inflammation of a joint
and is a condition that can have adverse implications in the performance horse. As with humans,
the effect of supplementation of long chain PUFA on indicators of arthritis in horses has been
examined. Following 75 d of supplementation with fish oil, horses that had been given dietary n3 exhibited an increase in plasma DHA levels with no changes in PGE2 metabolite or TNF-α
(Woodward et al., 2007). However, these horses exhibited longer stride lengths at the trot
following supplementation with fish oil, suggesting a decrease in joint pain. Despite lack of
cytokine and prostaglandin changes following oral supplementation, dietary n-3 PUFA can
reduce the pain associated with arthritis, although the mechanisms by which this occurs are not
known (Goldberg and Katz, 2007).
Munsterman et al. (2005) conducted a study that tested the effects of n-3 FA on synovial
explants from horses following LPS stimulation with an inflammatory agent. Explants treated
with ALA exhibit an increased concentration of ALA in the cell membranes in linear relationship
to the ALA concentration in the media, as well as a decrease in production of PGE2
(Munsterman et al., 2005). Similar results were seen in an in vivo study that examined the PUFA
supplementation of mature horses with arthritis in the leg and foot joints. Horses were grouped
according to severity of arthritis, affected joints, and age, and were supplemented with either a
21

control diet or a diet of 15 g EPA and 19.8 g DHA for 90 d, with synovial fluid collected from an
arthritic joint every 30 d. Following supplementation, treatment group horses exhibited lower
synovial fluid white blood cell counts and lower plasma PGE2 (Manhart et al., 2009).

DHA-Rich Microalgae

While incorporation of EPA and DHA has been clearly seen with fish oil
supplementation in humans and animals, issues with palatability are often reported. Additionally,
fish oil contains varying amounts of ARA, which is a mediator of several pathways and can
interfere with the influences of n-3 FA. Due to these issues, commercial production of DHA-rich
microalgae (DRM) from Schizochytrium sp. has grown in popularity as an n-3 supplement.
Schizochytrium sp. is a heterotrophic eukaryotic organism that makes commercial amounts of
PUFA by fermentation. It makes both DHA and DPA, but does not produce ARA (Lippmeier et
al., 2009), and therefore eliminates the issue of potential interference regarding ARA metabolic
pathways during supplementation. Microalgae are the original source of DHA in the marine
food chain, and previous research has shown that DRM is a safe and stable source of DHA that
can be effectively used to enrich poultry products, pork products (Abril et al., 2003), and milk
produced from dairy cattle (Franklin et al., 1999; Barclay et al., 1998; Barclay et al., 1994).
While issues with supplement palatability and consumption have been observed with fish
oil in horses (Woodward et al., 2007), an alternative source of DHA through a DRM based
supplement has the probability of delivering the same quantities of n-3 FA without
compromising intake values or complications due to ARA. To the author’s knowledge, DRM
supplementation in the horse has yet to be investigated.

22

Conclusions

While supplementation of PUFA has been extensively researched in studies involving
both animal and human subjects, research in the horse is limited, especially with regards to both
the effects of a DRM source of DHA and the effects of oral supplementation on exercise in the
horse. Although it is clear that EPA and DHA supplementation has the ability to alter the
circulating FA profile of the horse as well as elicit biological and metabolic effects, not enough
data currently exists to create feeding guidelines for the horse regarding n-3 supplementation.
Previous trials have used varying quantities over varying periods of time, initially based off of
the suggested dietary ratios for human consumption, and have consequently seen varying
responses in tissue concentrations. King et al. (2008) suggests that 30 to 35 g of EPA and DHA
per day for a 500 kg horse would be ideal, while other studies have used an n-3 supplementation
rate of around 30 g total n-3/d and seen beneficial effects. The proposed study has a focus on
discerning the effects of n-3 supplementation in the exercising horse when supplying a low
quantity of DHA over a short period (when compared to previous research in the horse), which
will contribute to the increased understanding of DHA incorporation into the body.
Many of these effects, particularly the anti-inflammatory properties of EPA and DHA,
could influence performance capability in the horse. The ability to influence parameters such as
stride length through dietary supplementation would obviously have a tremendous impact on the
equine industry as a whole, and certainly warrant further investigation following the Woodward
et al. (2007) study which demonstrated a tendency for stride length changes. Additionally, as a
DRM source of DHA has not yet been examined in the horse, gathering information regarding its
ability to influence metabolic changes in the horse could encourage opportunities for further

23

research and will allow for information regarding the influence of DHA over a comparatively
shorter time period.
As different species tend to respond similarly to n-3 FA supplementation, this allows for
comparison of results between studies and for the application of hypotheses for further
investigations that could uncover additional benefits of DHA supplementation from a DMR
source in the exercising horse, as well as other species. This could have several positive
applications concerning the alleviation of inflammation, benefitting the equine industry.

24

CHAPTER 3
MATERIALS AND METHODS

Animal Management
Twenty-four mature Arabians (15 mares, 9 geldings) with an average body weight of 434
± 94 kg were used in this study. All methods were approved by the Michigan State University
Institutional Animal Care and Use Committee (approval number 11/09-173-00). All animals
were housed in mixed-grass pastures at the Michigan State University Horse Teaching and
Research Center with free access to grass hay, water, and a trace mineralized salt block prior to
the start of the experiment and did not receive any grain supplementation. All horses were being
used in classes at Michigan State University, with the exception of five horses who were not
receiving exercise. All horses were considered to be in moderate fitness condition at the start of
the trial.
Horses were individually housed in 2.7 m x 3 m box stalls bedded with wood shavings
th

th

while on the 63-d study conducted from January 18 through March 28 , 2010, with the
exception of being used for classes or being provided with exercise. Horses were turned out for
approximately one hour in 15 m x 5 m dry lots 2 d/wk and were not given access to pasture or
any nutrients other than those provided in their daily rations.

Lameness Evaluations

Each horse underwent a lameness evaluation prior to the start of the study to determine
pre-existing lameness or gait defects that might affect stride length. Lameness evaluations and
25

flexion tests were performed according to the American Association of Equine Practitioners
Classification (Kester, 1991) by licensed equine practitioners three days prior to the start of the
trial. Horses were visually evaluated at the walk and the trot. Each leg was then palpated,
searching for bone or soft tissue abnormalities from the carpal and tarsal joints downward, with
any preexisting conditions noted and taken into consideration. Flexion tests were performed to
determine a lameness grade for each carpal, tarsal, metacarpophylangeal, metatarsophylangeal,
and femoropatellar joints, with scores recorded. Each joint was given a score ranging from 0 (no
lameness exhibited) to 5 (extreme lameness exhibited). Scores recorded from flexion tests were
added together to give an overall lameness score, which was used to assign horses to treatment
blocks. None of the horses exhibited gait defects secondary to suspected neurological problems
or muscle atrophy secondary to injury or illness.

Experimental Design and Diets

This study used a repeated 3 x 3 Latin square design with 21-d feeding periods. During
each period, each treatment was fed to eight horses, such that by the end of the study each horse
had received all treatments (Table 3.1). Horses were blocked by gender, age, and lameness
score. Each block, consisting of three horses, was randomly assigned to one of two groups.
Within each block, each horse was randomly assigned a classification of either A, B, or C to
determine the order in which treatments would be fed to eliminate any possible treatment carryover effects (Table 3.2). Horses within the same block were provided with equal exercise and
turnout time throughout the project. Prior to the start of the study and on the last day of each

26

period, blood samples, stride lengths, and heart rate changes surrounding a 20-min exercise
protocol were measured.
Horses were weighed using a digital floor scale and were given a body condition score
(BCS) at d -4 prior to the start of the trial, and then weekly for the duration of the project. The
BCS was determined using the Henneke system (Henneke et al., 1983), with observations from
multiple researchers averaged to obtain the most accurate score. Late bloom timothy grass hay
from the same cutting was provided by the Michigan State University Horse Teaching and
Research Center and was offered at 1.5% (as fed) of each horse’s body weight. A textured feed
(Kalmbach Feeds, Inc., Upper Sandusky, OH) was offered at a total of 0.5% (as fed) of each
horse’s body weight when combined with 908 g/d of treatment supplement. Hay and textured
feed amounts were adjusted individually throughout the project to maintain starting body weight
(BW) and BCS as needed based on weekly evaluations.

Treatments

One week prior to the start of the experiment horses were brought into stalls and
acclimated to a diet of approximately 2.2 kg textured feed and approximately 6.5 kg grass hay.
Both grass hay and textured feed were split between two feedings at 0700 and 1600 and horses
had unlimited access to water and a trace mineralized salt block.
During each period, each horse received one of three treatments, all in the form of a
stabilized pelleted supplement providing differing amounts of omega-3 fatty acids from differing
sources: α-linolenic acid from a soybean oil source (SOY; 4 g/d), α-linolenic acid from a
flaxseed oil source (FLAX; 38 g/d), or docosahexaenoic acid from an algae source (ALG; 28
27

g/d). The supplements were balanced to be isocaloric and isonitrogenous and were mixed and
bagged separately by Kalmbach Feeds, Inc. (Upper Sandusky, OH). Supplements were offered
top-dressed at 908 g/d onto the textured feed (Table 3.3). Orts were collected and recorded
before each feeding. Two horses refused the ALG treatment on the first two days of
supplementation. To encourage consumption, the offered amount was reduced to 227 g/d, and
then was incrementally increased to 908 g/d over a 3-d period. No other issues with palatability
were observed.

Exercise

Horses were exercised 5 d/wk during the duration of the project. Exercise consisted of a
specified lunge line exercise protocol or participation in horsemanship or training classes.
Exercise was initiated on d -5 at the start of the acclimation week.
Horsemanship Classes. In horsemanship classes, horses were worked either hunt seat or
western pleasure style. Horses were lunged at the walk, trot/jog, and canter/lope both directions
for approximately 10 min prior to riding. An additional warm-up period under saddle followed,
consisting of a total of 10 min of work at all three gaits both directions of the arena. Horses were
then exercised by performing maneuvers presented to the class as per the instructor. Riding
times increased from 25 to 45 min throughout the project, with the difficulty of maneuvers
increasing according to rider skill.
Training Classes. Training classes consisted of working horses on a lunge line at the
walk, trot, and canter both directions for approximately 15 min at the start of the trial. As horses
progressed with training, they began work in training aids, such as driving lines and side reins,
28

and were later introduced (d 40 and beyond) to a saddle and ridden by students. Exercise times
again increased from 25 to 45 min throughout the project.
Lunge Line Exercise. For horses not used in classes and for days when classes were not
scheduled, horses were exercised for 20 min on a lunge line. Horses were lunged according to
the following protocol: 5 min walk, 5 min trot, 5 min canter, reverse of direction, 5 min trot, and
5 min walk.

Measurements and Sample Collection

Blood Collection and Handling. Blood samples were collected following a 12 h fast
prior to the start of the study and on the last day of each of the three feeding periods via jugular
venipuncture using a 20 G needle and vacuum tubes (BD Vacutainer®, BD, Franklin Lakes, NJ)
to be analyzed for fibrinogen, total protein, interferon-gamma (INF-γ), tumor necrosis factoralpha (TNF-α), interleukin 10 (IL-10), prostaglandin E2 (PGE2), and a complete fatty acid
profile. One 10-mL serum vial containing no additives, one 10-mL serum vial containing
indomethacin solution (for PGE2 analysis) and three 10-mL plasma vials containing K3EDTA as
the anti-coagulant were filled during each collection day. Indomethacin solution was prepared
by making a stock solution containing 0.001 g indomethacin, 300 µL ethanol, and 700 µL PBS.
The stock solution was added to serum vials at a concentration of 10 µg indomethacin/mL serum.
Tubes were inverted several times to assure thorough mixing with contents. Immediately
following collection, one EDTA plasma vial was taken to the Clinical Pathology Laboratory in
the Diagnostic Center for Population and Animal Health (Lansing, MI) and analyzed for

29

fibrinogen and total protein. Total protein was determined using physical analytical methods
(Todd et al., 1974) and fibrinogen was analyzed according to previously established methods
(Williard et al., 1989; Jain, 1986). All other samples were allowed to coagulate for 30 min prior
to centrifugation at 3,000 x g for 15 min (GS-6KR Centrifuge, Beckman, Fullerton, CA). Serum
or plasma was pipetted off the top, placed into duplicate microcentrifuge tubes, and then frozen
at -20° C until further analysis.
Stride Length Measurement. Stride lengths were measured on d 0, 21, 42, and 63. On
these collection days, each horse performed a 20-min exercise on the lunge line according to
previously described protocol. Stride lengths were determined at both the walk and the trot, both
immediately before and immediately following exercise, using previously established guidelines
(Woodward et al., 2007; Hanson et al., 1997). A 2 m x 10 m area of dry dirt on a level arena
surface was raked to erase any previous hoof prints. Horses were led at the walk and the trot
through the raked area on a loose lead to ensure natural head carriage and stride length by
minimizing possible interference by the handler. The same handler led all horses for all
collection days to maximize consistency. Prior to recording measurements, each horse was led
through to allow for desensitization and prevent interference with accurate measurements due to
tenseness or shying. Three full stride lengths were measured from right hind toe impressions.
This measurement was repeated three times for a total of nine measurements averaged for each
gait. All procedures during the measurement of stride lengths were video recorded.
Heart Rate Measurement. Prior to the start of the study and on the last day of
each period, each horse performed a 20-min exercise on the lunge line according to previously
described protocol. During exercise, heart rate was monitored using an equine heart rate monitor
(Polar Electro Oy, Kempele, Finland), with electrodes placed in the hollow of the left withers
30

and behind the elbow on the left side via an adjustable elastic girth (Polar Electro Oy, Kempele,
Finland) and conductive ultrasound gel was used to enhance detection of heart rate by the
electrodes. Heart rate was recorded using the equine CS600X Training Computer and analyzed
using Polar Equine Software (Polar Electro Oy, Kempele, Finland).
Immediately on completion of exercise, each horse stood quietly in the arena for a 10min recovery period. Resting heart rates were recorded prior to exercise, during exercise, and
during the recovery period. Maximum heart rate during exercise, heart rate at the end of
exercise, 1 min post exercise, 2.5 min post exercise, 5 min post exercise, and 10 min post
exercise were recorded. Total area under the curve during the 10-min post exercise recovery
period was also determined.

Laboratory Analyses

Blood samples were analyzed for plasma fatty acid profiles and serum INF-γ, TNF-α, IL10, and PGE2 concentrations. Each sample was analyzed in duplicate, and replicated further if
the coefficient of variation exceeded 10%. Prostaglandin E2 and TNF-α assays in equine serum
have been previously used in clinical studies (Woodward et al., 2007). Other assays are being
validated by this laboratory. A quality control sample of pooled serum was analyzed with each
INF-γ, TNF-α, and IL-10 assay.
Analysis of Fatty Acids in Feeds and Plasma. All hay bales were core-sampled prior to
each period and textured feed and treatments were sampled continuously throughout the
experiment. Feed samples for each period were pooled and analyzed separately (Table 3.4).
31

Feed samples were ground through a 1-mm screen (Cyclotec 1093 Sample Mill, Foss, Eden
Prairie, MN), then 2.5 g of ground sample was weighed into a clean culture tube. Plasma
samples were thawed to room temperature, vortexed, and 1 mL of sample was added to a clean
culture tube. To extract the lipid in samples, 1 mL methanol, 1 mL hexane, and 1 mL deionized
water were added to each tube, the tubes capped with a Teflon septa insert, vortexed for 10 min,
and centrifuged for 10 min at 950 x g. A positive displacement pipette was used to transfer the
top layer of approximately 0.75 mL hexane into a clean, previously weighed, culture tube.
Samples were then evaporated under a nitrogen gas stream and weighed to determine lipid
content. Lipid extracts were then transmethylated to form fatty acid methyl esters (FAME) using
the procedure described by Woodward et al. (2007) (Appendix A).

Prostaglandin E2. Prostaglandin E2 was determined using an enzyme-linked
immunosorbent assay (ELISA; Prostaglandin E2 ELISA Kit, Thermo Scientific Pierce Protein
Research Products, Rockford, IL) following the manufacturer’s instructions. Serum dilution
factor was determined based on the procedures described in Woodward et al. (2007), with slight
modifications. Preliminary testing at a dilution of 1:2 showed several results that were near the
lower limit of the standard curve. Dilution was therefore adjusted to 1:1.25, which provided
readings falling within the standard curve with good correlation (less than 15%; Appendix B).
Tumor Necrosis Factor-alpha. Tumor necrosis factor-alpha was determined using an
ELISA (Equine TNF-α Screening Kit, Thermo Scientific Pierce Protein Research Products,
Rockford, IL) following the manufacturer’s instructions. Serum was diluted at 1:8. Dilutions
were determined by testing a serum sample diluted at 1:2, 1:4, 1:8, and 1:16. These values were
compared to the manufacturer’s suggestion that the samples fall between 15.6 and 1,000 pg/mL.
32

When the samples fell within the range and recovery was the highest, the corresponding dilution
was chosen for all samples (Appendix C).
Interleukin 10. Interleukin 10 was determined using an ELISA (Equine IL-10 DuoSet
ELISA, R&D Systems, Inc., Minneapolis, MN) following the manufacturer’s instructions.
Serum was filtered (Millex®-GP 0.22 µm filter unit, Millipore, Bedford, MA) and diluted on
plate at 1:4 with reagent diluent. Dilutions were determined by testing a neat (no additives)
serum sample and testing dilutions at 1:2, 1:4, 1:8, 1:10, 1:12.5, 1:16, 1:25, 1:32, 1:50, and 1:64.
These values were compared to the manufacturer’s suggestion that the samples fall between 39
and 20,000 pg/mL. When the samples fell within the range and recovery was the highest, the
corresponding dilution was chosen for all samples (Appendix D).
Interferon-gamma. Interferon-gamma was determined using an ELISA (Equine INF-γ
DuoSet ELISA, R&D Systems, Inc., Minneapolis, MN) following the manufacturer’s
instructions. Serum was filtered (Millex®-GP 0.22 µm filter unit, Millipore, Bedford, MA) and
diluted on plate at 1:10 with reagent diluent. Dilutions were determined by testing a serum
sample spiked and diluted neat (no additives), and at dilutions of 1:2, 1:4, 1:8, 1:10, 1:12.5, 1:16,
1:25, 1:32, 1:50, and 1:64. These values were compared to the manufacturer’s suggestion that
the samples fall between 31 and 4,000 pg/mL. When the samples fell within the range and
recovery was the highest, the corresponding dilution was chosen for all samples (Appendix E).

Statistical Analyses

The study was a 3x3 Latin square design with 8 horses per 21-d period, and was balanced
for carryover effects. Analysis was done with SAS 9.2 (SAS Inst. Inc., Cary, NC) using the
33

PROC MIXED procedure. Terms in the model statement included horse, supplement, gender,
age, and the interaction of supplement and age. Horse and period were set as repeated measures.
Means were separated using the PDIFF option within SAS and were adjusted using Tukey’s test.
Additional statistics were analyzed to determine differences from pre-experiment values
to end of period values for all variables. This was to allow for a comparison of each supplement
to pre-supplemented values, using each horse as its own control, and was accomplished by
subtracting pre-experimental values from end of period values. These values were then analyzed
using the PROC MIXED procedure in SAS using the same statistical program as previously
described. For all procedures, significance was declared at P < 0.05 and a trend toward a
significant difference at 0.05 < P < 0.10, with least squares means presented throughout.

34

Table 3.1. Latin square design used for the experiment, where ALG denotes algae-based
supplement, FLAX denotes flaxseed oil-based supplement, and SOY denotes soybean oil-based
supplement. Each block of three horses was randomly assigned to either Group 1 or Group 2 and
fed in the supplement order provided below.
a

Period

Group 1 Supplement
Horse A
Horse B
Horse C

d 0 to d 21
ALG
FLAX
d 21 to d 42
SOY
ALG
d 42 to d 63
FLAX
SOY
a
Blocks of horses in group 1: n=4
b

SOY
FLAX
ALG

Blocks of horses in group 2: n=4

35

b

Group 2 Supplement
Horse A Horse B Horse C
ALG
FLAX
SOY

FLAX
SOY
ALG

SOY
ALG
FLAX

Table 3.2. Block, group, classification, age, sex, and osteoarthritis score for each horse on trial.
Block No.

Horse No.

1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
7
7
7
8
8
8

2
3
4
1
5
6
7
8
9
10
11
13
12
14
15
16
17
18
20
21
23
19
22
24

Group,
Classification
1, A
1, B
1, C
2, B
2, C
2, A
1, B
1, A
1, C
1, A
1, C
1, B
2, B
2, C
2, A
2, C
2, A
2, B
1, B
1, A
1, C
2, C
2, B
2, A

Age (yr)
11
11
9
7
9
10
4
4
4
3
3
3
3
3
3
4
4
4
18
18
17
18
18
12

36

Sex
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
G
G
G
G
G
G
G
G
G

Osteoarthritis
Score
3.5
1
1.5
0
0
1.5
0
0
0
0.5
0
0
0.5
0.5
1.5
0
0
0
3.5
4
3
2
2
0.5

Table 3.3. Delivery (g/d) of n-3, n-6, α-linolenic acid (LNA), eicosapentaenoic acid (EPA), and
docosahexaenoic acid (DHA) as a protected fatty acid supplement volume of 908 g/d as fed.

Fatty Acid
Total n-3 (g)
Total n-6 (g)
n-6:n-3
ARA (g)
ALA (g)
EPA (g)
DHA (g)
ND: Not detectable.

SOY
4.01
27.84
6.93
ND
3.55
ND
ND

Supplement
FLAX
37.76
27.66
0.73
ND
37.68
ND
ND

37

ALG
31.48
19.36
0.61
ND
2.65
0.54
27.86

Table 3.4. Selected fatty acids supplied by each feedstuff.
Treatments
Textured
b
Feed
89.75
10.62
0.15
12.69
4.40
0.18

Timothy
Hay
90.12
3.2
0.54
24.01
1.79
ND

SOY
91.28
6.58
0.25
13.03
3.37
0.16

FLAX
91.82
10.29
0.15
7.99
2.95
0.14

ALG
91.41
9.31
6.71
21.27
1.48
0.30

17.84
49.62
ND
7.83
ND
ND
0.03
ND
6.77

27.77
18.83
ND
17.26
ND
0.38
1.72
ND
0.54

17.26
49.68
0.42
6.75
ND
ND
0.52
ND
6.93

11.45
29.22
0.35
40.33
ND
ND
0.04
ND
0.73

31.46
22.24
0.34
3.13
ND
0.64
0.21
32.95
0.61

50.17

19.27

52.87

29.61

22.90

7.41
35.81
7.63
40.41
Fatty acids are listed as a percentage of the lipid in the sample.

37.24

Measurement
DM
Crude Fat
C14:0
C16:0
C18:0
C24:0
c
Sum SFA
C18:2(n-6)
C18:3(n-6)
C18:3(n-3)
C20:4(n-6)
C20:5(n-3)
C22:5(n-3)
C22:6(n-3)
(n-6) : (n-3)
d
Sum (n-6)

a

b

b

e

Sum (n-3)

b

a

b

Obtained from Kalmbach Feeds, Inc. (Upper Sandusky, OH).

c

Sum of the saturated fatty acids: 14:0 + 15:0 + 16:0 + 18:0 + 20:0 + 22:0 + 24:0.

d

Sum of the (n-6) fatty acids.

e

Sum of the (n-3) fatty acids.

f

ND = not detectable.

38

CHAPTER 4
RESULTS

Body Weight

No difference in BW was seen between d 0 and d 63 for all horses (430 ± 94 kg vs. 440 ±
94 kg, P = 0.45). A difference was observed between BCS at d 0 and d 63 (6 ± 0 vs. 6 ± 0, P <
0.05). Regardless of treatment, no differences were seen for BW or BCS across each 21-d period
(P = 0.58 and P = 0.60, respectively; Table 4.1).

Stride Lengths

Walk and trot stride lengths were measured both before and after a specified 20-min
exercise on the lunge line. Baseline measurements were taken on d 0 and following each 21-d
supplementation period. Stride length data is presented in Table 4.2. Baseline measurements at
the walk pre-exercise and post-exercise did not differ as a result of age (P = 0.38 and P = 0.79,
respectively). When evaluating the change in stride length at the walk from pre- to post-exercise
on d 0, no difference was seen as a result of age (P = 0.81). Baseline trot stride lengths preexercise were not different due to age (P = 0.36). When analyzing stride length change from
pre- to post-exercise on d 0, no difference was seen as a result of age (P = 0.68).
When stride lengths were measured following n-3 FA supplementation periods, no effects
of treatment, age, or treatment × age interaction were seen at the walk pre-exercise (P = 0.91, P
= 0.72, and P = 0.89, respectively) or post-exercise (P = 0.20, P = 0.71, and P = 0.99,

39

respectively). Stride length change from pre- to post-exercise at the walk also did not differ due
to treatment, age, or treatment × age interaction (P = 0.45, P = 0.88, and P = 0.92, respectively).
When trot stride lengths following supplementation with n-3 FA sources were measured,
no treatment, age, or treatment × age interaction effects were seen pre-exercise (P = 0.61, P =
0.28, and P = 0.86, respectively) or post-exercise (P = 0.33, P = 0.40, and P = 0.59,
respectively). Stride length change from pre- to post-exercise at the trot did not differ due to
treatment, age, or treatment × age interaction (P = 0.74, P = 0.10, and P = 0.33, respectively).
When stride lengths from across each supplementation period were analyzed in
comparison to baseline measurements, no differences for treatment, age, or treatment × age
interactions were seen at the walk pre-exercise (P = 0.95, P = 0.75, and P = 0.89, respectively) or
post-exercise (P = 0.21, P = 0.59, and P = 0.99, respectively). Again, no differences were seen
when analyzing stride length change from pre- to post-exercise for treatment, age, or as a result
of a treatment × age interaction (P = 0.50, P = 0.54, and P = 0.95, respectively). Trot stride
lengths continued to show no difference for treatment, age, or treatment × age interactions for
pre-exercise (P = 0.59, P = 0.68, and P = 0.87, respectively), post-exercise (P = 0.36, P = 0.16,
and P = 0.55, respectively), or when analyzing the change from pre- to post-exercise (P = 0.71, P
= 0.10, and P = 0.63, respectively).

Heart Rates

Resting heart rate (HR) was recorded prior to the onset of exercise. Baseline resting HR
did not differ between horses as a result of age (P = 0.91) and was 34 ± 1 beats/min. When
measured following n-3 FA supplementation periods, resting HR did not differ as a result of
40

treatment, age, or treatment × age interaction (P = 0.73, P = 0.33, and P = 0.14, respectively;
Table 4.3). When resting HR from each period of supplementation were analyzed in comparison
to baseline values, no difference as a result of treatment, age, or treatment × age interaction was
observed (P = 0.53, P = 0.78, and P = 0.59, respectively).
Heart rates were monitored during a specified 20-min lunge line exercise while the
maximum HR during exercise was recorded. Baseline maximum HR differed between horses as
a result of age (P < 0.05), ranging from 164 ± 2 beats/min (18-yr old horses) to 185 ± 3
beats/min (10-yr old horses). When measured following supplementation, maximum HR did not
differ as a result of treatment, age, or treatment × age interaction (P = 0.46, P = 0.14, and P =
0.69, respectively). When results from each supplementation period were analyzed in
comparison to baseline values, maximum HR did not differ as a result of treatment, age, or
treatment × age interactions (P = 0.31, P = 0.37, and P = 0.55, respectively).
Following exercise, HR was monitored continuously for a 10-min recovery period.
Specific heart rates were recorded and analyzed: immediately post-exercise (HR 0), 1 min postexercise (HR 1), 2.5 min post-exercise (HR 2.5), 5 min post-exercise (HR 5), and 10 min postexercise (HR 10). The total area under the curve for the entire 10-min recovery period was also
calculated and analyzed (HR AUC). Baseline recovery HR measurements did not differ as a
result of age (Table 4.3), and following n-3 FA supplementation, all recovery HR measurements
showed no differences as a result of treatment, age, or treatment × age interaction (Table 4.3).
When recovery HR measures from each period were compared to baseline values, no differences
were seen in total HR AUC based on treatment, age, or treatment × age interaction (P = 0.15, P =
0.62, and P = 0.19, respectively).

41

Plasma Fatty Acid Profiles

Plasma FA profiles are presented as a percentage of total plasma lipid concentration
(Table 4.4). Plasma n-6 and n-3 FA composition between horses at baseline measurements did
not differ, with the exception of a trend for α-linolenic acid to be highest in 3-yr old horses and
lowest in 4-yr old horses (4.38 ± 1.88 % vs. 2.81 ± 1.85 %; P < 0.10). Before beginning
supplementation, linolenic acid made up the largest percentage of total plasma PUFA in all
horses. After supplementation with all treatments, this observation remained the same.
Baseline values for the total n-3 and total n-6 content of the plasma FA profile did not
differ (P = 0.87 and P = 0.94, respectively). No differences were observed across
supplementation periods for total n-3 or total n-6 (P = 0.64 and P = 0.24, respectively). When
compared to baseline values, total n-3 did not differ as a result of treatment or age (P = 0.60 and
P = 0.74, respectively); however, a trend for a treatment × age interaction existed (P < 0.10). No
differences were seen in total n-6 when compared to baseline measures based on treatment, age,
or as the result of a treatment × age interaction (P = 0.15, P = 0.77, and P = 0.77, respectively).
The calculated ratio of n-6 to n-3 FA in plasma was not different between horses at
baseline measures (P = 0.94) or across supplementation periods as a result of treatment, age, or
treatment × age interaction (P = 0.64, P = 0.56, and P = 0.17, respectively). When analyzing the
change across each treatment in comparison to baseline values, there continued to be no
differences (P = 0.61, P = 0.88, and P = 0.22, respectively).
On d 0, the mean basal concentrations of the primary n-3 FA of interest, EPA and DHA,
did not differ based on age (P = 0.98 and P = 0.88, respectively). A treatment effect was
observed for EPA plasma levels, with horses on SOY having higher EPA levels (0.45 ± 0.07
42

percent of total plasma lipid) than horses on FLAX and ALG (P < 0.05; Table 4.4).
Docosahexaenoic acid levels remained unchanged as a result of treatment or age (P = 0.62 and P
= 0.46, respectively), but a treatment × age interaction was observed (P < 0.05). When
compared to baseline values across treatments, EPA continued to exhibit a treatment effect (P <
0.05), with horses on the SOY treatment showing approximately a forty percent increase in EPA
levels, FLAX having an approximate forty percent decrease from baseline values, and ALG
showing approximately a one percent decrease. When compared to baseline measures, DHA
remained unchanged as a result of treatment or age (P = 0.63 and P = 0.79, respectively), but did
show a trend for a treatment × age interaction (P < 0.10). Plasma α-linolenic acid was also
altered in response to treatment (P < 0.05), with horses on the FLAX treatment having a greater
percentage than horses on either the SOY or ALG treatments (Table 4.4).

Total Protein and Fibrinogen

Plasma total protein and fibrinogen were measured at d 0 to obtain a baseline value and
following each supplementation period. At baseline there was no difference in plasma total
protein as a result of age (P = 0.61). Following supplementation, plasma total protein increased
in horses on the SOY treatment when compared to both FLAX and ALG (6.80 ± 0.07 g/dL; P <
0.05). When analyzed in comparison to baseline values, a treatment effect continued to exist (P
< 0.05) and a trend for an age × treatment interaction was still present (P < 0.10).
Plasma fibrinogen levels did not differ in horses at baseline as a result of age (P = 0.69).
Following supplementation, plasma fibrinogen levels were not different as a result of treatment,
age, or a treatment × age interaction (P = 0.47, P = 0.49, and P = 0.50, respectively; Figure 4.1).
43

When analyzed in comparison to baseline values on d 0, there continued to be no differences (P
= 0.49, P = 0.51, and P = 0.53, respectively).

Prostaglandin E2

When serum PGE2 was analyzed via ELISA on d 0, baseline values did not differ due to
age (P = 0.46). Following n-3 FA supplementation, PGE2 concentrations did not differ between
horses as a result of treatment, age, or an age × treatment interaction (P = 0.34, P = 0.59, and P =
0.43, respectively; Figure 4.1). When changes during each 21-d period were compared to
baseline values, no differences were seen for treatment, age, or as the result of a treatment × age
interaction (P = 0.62, P = 0.23, and P = 0.71, respectively).

Tumor Necrosis Factor-α

Serum TNF-α concentrations did not differ in baseline values as a result of age (P =
0.38). Following n-3 FA supplementation, TNF-α concentrations were not different as a result of
treatment, age, or treatment × age interaction (P = 0.26, P = 0.79, and P = 0.31, respectively;
Figure 4.1). When comparing changes from each 21-d period to baseline values, no differences
were seen for treatment, age, or as the result of a treatment × age interaction (P = 0.16, P = 0.66,
and P = 0.74, respectively).

44

Interleukin 10

At baseline on d 0, no differences were observed as a result of age in serum IL-10
concentrations (P = 0.51). Serum concentrations differed as a result of treatment, with SOY
having lower concentrations of IL-10 than ALG (4,490 ± 5,080 pg/mL vs. 10,610 ± 4,100
pg/mL; P < 0.05). However, there was no effect noted as a result of age or due to a treatment ×
age interaction (P = 0.48 and P = 0.35, respectively; Figure 4.1). When analyzing changes
across each 21-d period in comparison to baseline values, no differences were seen for treatment,
age, or as the result of a treatment × age interaction (P = 0.21, P = 0.45, and P = 0.40,
respectively).

Interferon-γ

Serum INF-γ concentrations were not different among all horses in baseline values as a
result of age (P = 0.42). Following n-3 FA supplementation, INF-γ concentrations did not differ
as a result of treatment, age, or treatment × age interaction (P = 0.46, P = 0.34, and P = 0.62,
respectively; Figure 4.1). When changes across each supplementation period were compared to
baseline values, again no differences were seen for treatment, age, or as the result of an age ×
treatment interaction (P = 0.55, P = 0.51, and P = 0.90, respectively).

45

Table 4.1. Body weight (kg) and body condition score (BCS) for all horses within treatments.

Measurement
Body weight
(kg)

Starting Weight*
†

Change

SOY
436 ± 94
5.0 ± 1.8

Treatment
FLAX
436 ± 94
4.9 ± 1.8

ALG
439 ± 94
0.2 ± 1.8

P-value
0.97
0.12

6±0
6±0
6±0
0.71
0±0
0±0
0±0
0.29
Change
*Representative of weight and BCS on the first day of supplementation of each respective
treatment.
BCS

Starting BCS*
†

†

Representative of the change in body weight or BCS across each 21-d period while on each
respective treatment.

Table 4.2. Mean walk stride length (cm) and trot stride length (cm) of horses. Stride lengths
were measured before and after a 20-min specified exercise on the lunge line to determine a
baseline at d 0 and following 21-d supplementation periods for all treatments.

Measurement
Walk Stride
(cm)

Pre-exercise
Post-exercise
Change*

Baseline
Day 0
158 ± 2
157 ± 2
-1 ± 1

SOY
161 ± 2
161 ± 2
-1 ± 1

Treatment
FLAX
ALG
162 ± 2
161 ± 2
160 ± 2
162 ± 2
-2 ± 1
0±1

P-value
0.91
0.20
0.45

0.61
189 ± 3
192 ± 5
194 ± 5
194 ± 5
0.33
192 ± 3
190 ± 5
194 ± 5
193 ± 5
Post-exercise
0.74
Change*
3±2
-2 ± 2
0±2
-2 ± 2
*Representative of the mean change in stride length for all horses from pre- to post-exercise.
Trot Stride
(cm)

1

Pre-exercise

1

Post-exercise baseline trot measurements showed a trend for a difference for age (P < 0.10).

46

Table 4.3. Mean heart rate (HR) measurements at baseline (d 0) and following 21-d
supplementation periods for all treatments. Heart rates are provided at rest, the maximum HR
during a 20-min specified lunge line exercise, and specific heart rates post-exercise during a 10min recovery period: immediately post-exercise (HR 0), 1 min post-exercise (HR 1), 2.5 min
post-exercise (HR 2.5), 5 min post-exercise (HR 5), and 10 min post-exercise (HR 10).
Baseline
Day 0
Measurement*
SOY
Resting HR
34 ± 1
34 ± 1
1
174 ± 5
160 ± 6
Maximum HR
HR 0
66 ± 3
66 ± 2
ab
HR 1
59 ±3
56 ± 3
HR 2.5
50 ± 2
46 ± 2
HR 5
45 ± 2
44 ± 2
HR 10
41 ± 2
40 ± 2
*Heart rates are expressed as beats/min.
1

Treatment
FLAX
ALG
35 ± 1
35 ± 1
157 ± 6
164 ± 6
67 ± 2
69 ± 2
a
b
55 ± 3
61 ± 3
49 ± 2
50 ± 2
45 ± 2
48 ± 2
42 ± 2
42 ± 2

P-value
0.73
0.46
0.50
0.05
0.32
0.31
0.46

An overall decrease in maximum HR was observed from d 0 to d 63 of the study (P < 0.05).

abc

Values lacking common superscripts differ (P < 0.05).

47

Table 4.4. Effect of feeding horses diets supplemented with three differing sources and content of n-3 fatty acids on plasma fatty acid
profiles after 21-d supplementation periods and at baseline values (d 0).

Fatty Acid*
C18:2(n-6)
C18:3(n-6)

Baseline
Day 0
34.77
0.73
3.85
0.66
0.24
1.83
5.46

SOY
34.44
1.02

FLAX
42.01
0.87

Treatment
ALG
36.17
0.74

0.45
1.71
7.04

0.18
2.31
5.18

0.24
1.84
5.88

Pooled SEM
3.54
0.10
0.44
0.11
0.07
0.50
2.11

36.04

43.26

37.58

3.62

0.24

11.08
12.39
12.35
Sum (n-3)
Ratio (n-6): (n-3)
0.36
0.42
0.37
*Plasma fatty acids are given as a percentage of total plasma lipid content.

10.70
0.33

1.50
0.07

0.64
0.64

1

C18:3(n-3)
C20:4(n-6)
C20:5(n-3)
C22:5(n-3)

2

C22:6(n-3)

3

Sum (n-6)

36.16

ab

a

3.19
0.58

4.06
0.39

a

b

4

b

2.72
0.67

b

abc

Means in the same row not sharing a common superscript differ (P < 0.05)

1

Trend for an age effect on d 0 (P < 0.10).

2
3
4

Age × treatment effect (P < 0.05).
Sum of n-6: C18:2(n-6), C18:3(n-6), C20:2(n-6), C20:3(n-6), C20:4(n-6), C22:2(n-6), and C22:4(n-6).

Sum of n-3: C18:3(n-3), C20:3(n-3), C20:5(n-3), C22:3(n-3), C22:5(n-3), and C22:6(n-3).

48

P-value
0.20
0.12
0.02
0.18
0.03
0.66
0.62

Figure 4.1. The response of hematological variables to n-3 treatments of soybean oil (SOY),
flaxseed oil (FLAX), or algal DHA (ALG) in equine plasma.

0.25

a

Plasma Fibriongen, g/dL

Plasma Total Protein, g/dL

6.9
6.8

b

6.7
b

6.6
6.5

0.1
0.05
0

FLAX

ALG

SOY

ALG

SOY

FLAX

ALG

SOY

Serum IL-10, pg/mL

3000
2500
2000
1500
1000
500
0

FLAX

ALG

15000
10000
5000
0
-5000

SOY

FLAX

ALG

6000
Serum PGE2, pg/mL

2000

5000
4000
3000
2000
1000

1500
1000
500
0
-500

0
SOY
abc

FLAX

20000

3500
Serum INF-γ, pg/mL

0.15

6.4
SOY

Serum TNF-α, pg/mL

0.2

FLAX

ALG

Values lacking common superscripts differ (P < 0.05).
49

CHAPTER 5
DISCUSSION

The effects of n-3 supplementation have been studied extensively in a variety of animals,
and to a lesser extent in the horse. Based on the FA composition of the n-3 supplements used in
this study (Table 3.4), the amount of total n-3 FA intake for a 500-kg horse would be
approximately either 4 g/d if consuming the SOY treatment, 38 g/d if consuming the FLAX
treatment, or 31 g/d if consuming the ALG treatment. It can be seen in Table 3.4 that the ALG
supplement was the only significant source of EPA and DHA in this study.
In comparison to other studies using the horse as a model, the total amount of n-3 FA, as
well as the amount of DHA and EPA provided by our treatments was generally lower and our
21-d supplementation period for each treatment was shorter than those previously reported
(Table 5.1). While previous research has indicated that dietary supplementation of marinederived sources of EPA and DHA can rapidly and significantly increase the circulating
concentrations of these n-3 FA when orally supplemented in significant quantities, the results of
this study did not show a change in response to treatment in plasma concentrations of DHA, total
n-3, or total n-6 (and consequently no change in the n-6 to n-3 ratio).
Stride lengths at the walk and trot both before and immediately following exercise did not
differ in horses at baseline values as a result of age. Across all treatments, no differences were
seen for stride length values at either gait and no difference was seen when stride length changes
from d 0 to d 63 were evaluated. These findings are in contrast to those reported by Woodward
et al. (2007), who saw a tendency for an increase in trot stride length in horses supplemented
with 19.4 g/d of n-3 FA from a fish oil source for 75 d compared to horses supplemented with
50

Table 5.1. Comparison of n-3 supplementation sources, amounts of fatty acids, and length of supplementation (d) across equine
studies.
n-3
Source

Supp.
Length

1
EPA

1
DHA

1
Total n-3

Brinsko et al., 2005

Fish oil

98 d

17.5

3.5

70

De Moffarts et al., 2007

Fish oil

21 d

20.0

10.0

NR

Exercise induced decrease of erythrocyte membrane
fluidity associated with changes in blood oxidative
balance

Study

Results
Increased sperm concentrations, higher average
velocity, increased DHA concentrations in semen fatty
acids

Hall et al., 2004 a, b

Fish oil

98 d

22.9

19.6

49.6

Plasma FA changes, modulation of inflammatory
responses in bronchoalveolar lavage fluid cells and
leukotrienes

Harris et al., 2005

Marine

90 d

NR

NR

29.1

Increased daily sperm output, normal morphology, and
DHA concentrations in sperm plasma membrane

King et al., 2008

Marine

28 d

2
20.0, 40.0

NR

4.8,17.2,
29.2, 53.1

Plasma FA response is dose dependent, EPA and DHA
timelines to appear and clear from plasma

Khol-Parisini et al., 2007

Seal
blubber
oil

70

NR

NR

35.5

Leukocyte FA changes, pulmonary epithelial lining
fluid immune changes, no changes in pulmonary
function or clinical signs

NR

90 d

15.0

19.8

NR

N-3 treated horses had lower synovial WBC
concentrations

Manhart et al., 2009

0, 10.0,

O’Connor et al., 2004,
2007

Fish oil

63 d

20.1

15.2

39.8

Serum FA changes, lower heart rates, serum
cholesterol, and serum glycerol during exercise,
increase in insulin sensitivity

Parsons et al., 2011

Algae

21 d

0.5

27.9

31.5

No plasma FA changes, no physiological or
metabolic changes

Portier et al., 2006

Fish oil

28 d

20 .0

10.0

NR

Decrease in erythrocyte membrane fluidity induced by
exercise

51

Table 5.1 (cont’d)
3
Vineyard et al., 2009

Fish oil,
flaxseed

70 d

NR

NR

23.5

Plasma and RBC FA changes (greater impact with fish
oil), enhanced early inflammatory responses to
phytohemagglutinin injections

Woodward et al., 2007

Fish oil

75 d

1.4

3.2

19.4

Plasma FA changes, tendency for increase of trot
stride length

1
2

Measurements are given in g/d consumed per horse.
Combination of EPA and DHA.

3

Amounts given represent concentrations in fish oil.
NR: Not reported/unable to determine from provided information.

52

corn oil. While these researchers were supplementing a lower amount of DHA than our trial,
they observed changes in plasma FA profiles that reflected their n-3 dietary sources. A favorable
anti-inflammatory response as a result of n-3 supplementation cannot be elicited unless the n-3
FA are present in the blood to be delivered to the body, consequently enhancing the potential for
their incorporation into cell membranes. As Woodward et al. (2007) witnessed an increase in
plasma DHA, as well as a decrease in the n-6 to n-3 ratio, this suggests that the potential
incorporation of DHA into cell membranes may have been involved in their observation of
increased trot stride lengths, whereas our study did not note any changes in plasma levels for
these same variables. Stride lengths reported by Woodward et al. (2007) were overall greater at
both the walk and the trot when compared to our study (approximately 168 ± 4 cm vs. 160 ± 2
cm and approximately 237 ± 7 cm vs. 194 ± 5 cm).
While horses tend to reflect soundness within their stride length (Rogers et al., 2005;
Toutain and Cester, 2004; White et al., 2003), this can potentially be used as a measure of
lameness and osteoarthritic pain (Hanson et al., 1997). However, Woodward et al. (2007) noted
that while an increase in trot stride length was seen, no definite answer for this increase could be
reached from the study due to a lack of difference in overall lameness scores or markers of
inflammation due to treatment.
A recent study by Headley et al. (2011) examined the effects of supplementation of 50
g/d of conjugated linoleic acid over a 6-wk period on markers of stride length in sedentary
horses. No differences in walk or trot stride length were observed in this study, suggesting that
supplementation with higher quantities of n-6 FA has a limited ability to affect stride length in
sound, sedentary horses. Horses on the SOY treatment were receiving the highest n-6 to n-3
ratio in this study. The addition of one treatment (SOY) that had a substantially higher n-6 to n-3
53

ratio than the others in this repeated Latin square design may have negated the potential positive
effects that a low dose of n-3 FA may have had on stride length.
The lack of change in stride length seen in the horses in our study is likely due to both the
absence of circulatory n-3 FA combined with the absence of osteoarthritic disease and ensuing
inflammation and unsoundness in our experimental model. The horses used by Woodward et al.
(2007) exhibited overall higher lameness evaluation scores than the horses used in this study.
While our horses varied in age and lameness scores, and were grouped accordingly, no
differences in stride lengths could be seen as a result of age, group, treatment × age, or treatment
× group interactions, suggesting that our horses had insignificant inflammation and pain to effect
stride length (Hanson et al., 1997).
No differences were observed in this study concerning resting heart rate, maximum heart
rate reached during exercise, or heart rate recovery post-exercise as a result of treatment. These
results do not reflect a previously conducted trial by O’Connor et al. (2004), who saw that horses
receiving fish oil delivering 17 g/d EPA and 13 g/d DHA for 63 d had lower heart rates during
exercise compared to those receiving corn oil. This study also showed an increase in plasma
EPA and DHA (O’Connor et al., 2007) and an increase in erythrocyte deformability in both the
treatment and control diets, which has been cited as a mechanism to lower heart rate (Oostenburg
et al., 1997; Cartwright et al., 1985). When compared to our study, the exercise protocol used by
O’Connor et al. (2004) was conducted over a longer period of time and at a higher intensity. The
standardized exercise test used by O’Connor et al. (2004) was also performed at a higher
intensity than the exercise test used in our study.

54

It is likely that no resting, maximum, or recovery heart rate differences were observed as
a result of treatment in this study due to DHA and EPA invariance in the circulatory profiles
combined with a light exercise training program. Although baseline maximum heart rates
differed as a result of age, with older horses having lower maximum heart rates than younger
horses, this was most likely due to behavioral differences exhibited by the horses on the baseline
testing day. Generally, older horses were much calmer during the lunging exercise on the first
collection day, whereas younger (and less trained) horses generally reacted more strongly, with
inconsistency primarily at the canter, including bucking, shying, and working with excessive
speed, all of which can result in a greater increase in heart rate than a horse performing a relaxed
and consistent gait.
The plasma FA profile will change to reflect dietary FA influences, and in this study a
treatment difference was observed with ALA plasma concentrations, with horses on the FLAX
treatment having higher plasma concentrations of ALA than horses on the ALG treatment. This
response reflected the dietary intake of ALA, with FLAX horses consuming 38 g/d of ALA and
ALG horses consuming 3 g/d of ALA and is in agreement with other studies examining dietary
influence of ALA in the circulatory profiles of the horse (Vineyard et al., 2009; Hall et al.,
2004b; Hansen et al., 2002). However, other studies using the horse as a model have conversely
not observed a change in plasma ALA as a result of dietary n-3 supplementation (King et al.,
2008; Woodward et al., 2007; O’Connor et al., 2004). The discrepancy for these results could lie
in both differing sources of n-3 as well as a potential seasonal influence of FA circulating
concentrations as suggested by King et al. (2008). Their study noted that plasma concentrations
of 22 different FA increased or decreased in all treatment groups, including controls, and
suggested that this was due in part to a change in the FA profiles of seasonal forages. Non55

supplemented horses in their experiment consumed a diet considered to be typical for the average
management practices in the United States (fresh or cured grass forage plus <0.5 kg of whole
oats), which resulted in a plasma n-6 to n-3 FA ratio of approximately 7.5 during the winter and
approximately 6 during the spring. Changes in the ALA concentrations in varying forages,
independent of supplementation, could be responsible for the variation of ALA plasma responses
to treatment in these studies.
Additionally, a treatment effect was seen for plasma EPA in this study, with horses on the
SOY treatment exhibiting a higher concentration of EPA than horses on the FLAX or ALG
treatment, with concentrations that were higher than baseline values. This is a surprising
discovery, since all treatments were low in EPA, with the ALG treatment delivering the highest
quantity of EPA, at a very low amount of 0.5 g/d, and the SOY and FLAX treatments showing
non-detectable quantities of EPA. This shows that a potential elongation of ALA may have
occurred, yielding an increase in plasma EPA. Mantzioris et al. (1994) found that tissue EPA
concentrations increase in a linear fashion after dietary ALA supplementation. This idea is
further supported by the work of Hansen et al. (2002), where they found plasma concentration of
EPA to be markedly increased in horses supplemented with flaxseed oil in comparison to
baseline measures. The horses in the Hansen et al. study had no other dietary sources of EPA, so
it can be assumed that amounts detected in tissue were the result of bioconversion. However, the
difference in EPA concentrations as a result of treatment in our study is most likely not due to
bioconversion. If bioconversion from ALA was the case, then EPA levels should have been
highest in the FLAX supplement, which supplied a significantly higher quantity of ALA than
either the SOY or the ALG supplement. Additionally, the efficiency of the equine model to
elongate ALA to EPA within the body is still questioned (Vineyard et al., 2009; Siciliano et al.,
56

2003), and it would therefore be difficult to conclude that such a small quantity of EPA present
in the circulatory FA profile was absolutely the result of the bioconversion of a small oral
quantity of ALA from the diet. It is more likely that, due to the small values of EPA reported
here, solvent interference or misidentification of peaks by the GC software is responsible for this
difference.
While plasma levels of DHA remained unchanged across treatments, a trend for an age x
treatment interaction was seen. Upon closer examination, this trend only showed differences
between ages in regards to the SOY and FLAX treatment, both of which contained nondetectable amounts of DHA. While there is the limited ability of the body to convert ALA to
EPA, elongation and desaturation of ALA to form DHA is estimated to be fractional, at < 0.05
percent, which is an amount insufficient to increase the concentration of DHA in plasma and
other membranes (Williams and Burdge, 2006; Siciliano et al., 2003), suggesting that the trend
for this interaction is again due to sampling and processing error rather than bioconversion of
ALA.
In contrast to previous studies, we did not see a treatment difference in circulating levels
of DHA, total n-3, total n-6, or the n-6 to n-3 ratio. Several possibilities exist for these
observations. The horses in this study were being supplied a uniform amount of supplement. As
our horses varied widely in body weight, the amount of FA that was being provided on a mg·kg
BW basis also varied. For example, regarding horses on the ALG treatment, the lightest horse
weighed 354 kg, and would therefore be supplied with approximately 79 mg·kg

-1

BW DHA,

while the heaviest horse, weighing 546 kg, would be supplied with approximately 51 mg·kg
BW DHA. The average 434 kg horse would have received 64 mg·kg
57

-1

-1

BW DHA on the ALG

-1

treatment. This variance makes it difficult to evaluate these horses equally. However, no
2

correlation was seen between body weight and DHA levels (r = -0.14, P = 0.27), total n-3 levels
2

2

(r = -0.12, P = 0.34), or when evaluating the n-6 to n-3 ratio (r = -0.14, P = 0.25), indicating
that regardless of weight, horses were receiving a deficient amount of n-3 FA to elicit plasma
changes over a 21-d period. The combination of a shorter supplementation period and lower
dose of n-3 FA when compared to previous literature resulted in very minute changes in the
plasma profile, with a small quantity of various FA being displayed in the plasma. At such small
quantities, these values could easily be altered by sample contamination, incomplete extraction
and methylation of FAMES, or misidentification of peaks by the GC software. This is further
supported by our baseline data. Although we know little about the naturally occurring
concentrations of n-3 in the horse, the primary source of DHA must come from a marine source
in the diet. When our baseline values are examined, there is a mean basal concentration of DHA
present in the plasma. While this value is low, it should be at non-detectable levels, indicating
that there is some degree of error in the processing and analysis.
Others have fed an amount of DHA from a fish oil source in similar quantities to what
was supplied by the ALG treatment in this trial (Vineyard et al., 2009; King et al., 2008;
Woodward et al., 2007) and have seen increases in DHA in the plasma profile. As DHA cannot
be synthesized in the body, even a small amount of the FA can affect plasma concentrations, as
especially seen in the Woodward et al. (2007) study. However, all of these studies fed low
quantities of DHA for periods of time much greater than the 21-d periods of our study (Table
5.1). Although overall plasma n-3 FA concentration will change linearly with the amount
supplemented (King et al., 2008; Spearman et al., 2005; Hall et al., 2004b), these studies have

58

been conducted with much higher overall n-3 FA and DHA/EPA supplementation than the data
represented here.
King et al. (2008) saw an increase in circulating EPA and DHA in mares supplemented
with varying amounts of both FA following 3 d of supplementation, with a dose-related peak
level by 7 d of feeding. Plasma EPA and DHA demonstrated a steep decline from peak values 9
d after cessation of feeding, were elevated for up to 2 wk post supplementation, and were not
different than pre-supplemented levels at 42 d post-supplementation. This rate of plasma
clearance generally agrees with results of fish oil supplementation of yearling horses (Vineyard
et al., 2009), where plasma DHA, but not EPA, remained elevated beyond baseline through 5 wk
post-supplementation. Harris et al. (2005) reported a longer time (30 d) to attain peak n-3 FA
plasma concentrations after supplementation with a similar compound, but in a smaller
population of horses. As EPA and DHA plasma values were not reflected in this study, it can be
assumed that when feeding low quantities of these n-3 FA, they must be fed for longer than 21 d
to see significant changes in the plasma profile.
It is possible that feeding similar quantities of various FA may require a wash-out period
to eliminate the possibility of a carry-over effect from the previous treatment. Although our
model was statistically tested for any existing carry-over effect (none were observed), previous
literature suggests otherwise. In the King et al. (2008) study, it took a 24-d period to increase the
total n-3 concentration (and consequently reduce the n-6 to n-3 ratio) in the plasma. Additionally
EPA and DHA levels have existed at elevated levels from baseline measurements for up to 2-wk
post-supplementation (King et al., 2008) or 5-wk post-supplementation (Vineyard et al., 2009) in
horses. With residual levels reported at even low dosages of DHA and EPA, it is possible that
when using a repeated Latin square design with short repeated periods, a low quantity of DHA
59

was not being offered long enough for consumption to make an impact on plasma concentration
of DHA and was potentially being influenced by residual effects from the other treatments on the
trial.
To the author’s knowledge, this is the first study to examine the effects of DHA from a
DRM source in the horse. Docosahexaenoic acid from algal sources effectively enriches poultry
and pork products as well as milk produced from dairy cows (Abril et al., 2003; Franklin et al.,
1999; Barclay et al., 1998). Few, if any, studies have looked at the effect of DHA supplied from
a microalgae source on plasma lipid profiles in production animals. Algal DHA sources increase
plasma DHA and total n-3 FA in circulatory profiles in human vegetarians (Conquer and Holub,
1996), however little is known as to the effects of a DRM source of DHA in an animal model
similar to the horse. It could be possible that the bioavailability for absorption of algal DHA is
slightly differently than DHA offered in the source of fish oil or seal blubber, as previously
reported, and therefore needs to be supplied in a higher concentration to have the same biological
effectiveness.
A lower n-6 to n-3 ratio allows for n-3 to be preferentially incorporated into tissues, and
as this ratio lowers, more n-3 FA are present in the plasma for incorporation into erythrocytes
(Bojensen and Bojensen, 1998). As no differences in this ratio were observed in this study, the
likelihood of n-3 incorporation into bodily tissues and membranes is unlikely. This ratio change
is normally attributed to a decline in the percentage of n-6 FA rather than a substantial increase
in n-3 FA. King et al. (2008) suggests that feeding a level of 30 to 35 g/d of EPA/DHA would
be sufficient to shift the n-6 to n-3 ratio to a n-3 favorable state. As our treatments supplied a
maximum of 28.4 g/d of EPA/DHA, the lack of detection of a ratio shift would support this
suggestion.
60

Plasma fibrinogen is a positive marker in the acute phase of inflammation (Auer et al.,
1989). In this study, no differences were seen in fibrinogen changes as a result of treatment.
The horses on our study were on a light exercise program that was not designed at an intensity to
induce inflammation. Additionally, n-3 was not present in circulation for incorporation into
these metabolic pathways. Plasma total protein in the horse may increase by greater than 25% in
response to inflammation, infection or trauma. As a result, these proteins are used as quantitative
markers in general health screenings, diagnosis of disease, and as prognostic indicators
(Eckersall, 2008). In this study an overall increase in plasma total protein was seen for horses on
the SOY treatment when compared to the ALG and FLAX treatments. Numerically, these
changes are minute when realizing that the average plasma protein concentration in healthy adult
horses ranges from approximately 6.0 to 8.5 g/dL (Duncan and Prasse, 1986). As fibrinogen,
included in the total plasma protein count and representing the greatest proportion of plasma
protein synthesized during an acute phase inflammatory response, levels were not determined to
be different, the difference in total protein levels is not noteworthy in the context of this research.

Regarding other inflammatory metabolites, we saw no changes in PGE2, TNF-α, or INFγ. The values recorded for all of these makers were consistently low, with several being too low
to read accurately on the standard curve. As these values were so low, our standard errors were
markedly high. As overproduction of these markers were not present in our horses in general,
this may suggest that they did not have a need for a reduction of them, hence one potential
reason why we did not observe a treatment effect on this study. However, IL-10 showed no
differences at baseline measurements, but did show treatment differences, with IL-10
concentrations in serum being lower for the SOY treatment than the ALG treatment. However,
when analyzed as compared to baseline values, no treatment effect existed. Additionally, IL-10
61

is a known inhibitor of both TNF-α and INF-γ, so it would be expected that a difference
observed in IL-10 concentration would also reflect a difference in TNF-α production (Calder,
2001b). As no treatment differences were seen for either of these metabolites, it is likely that an
accuracy error occurred in regards to the ELISA, as there was not an overproduction of IL-10.
This can be supported by the high standard errors seen with this analysis.
For all of the metabolites measured, fasting blood samples were taken following a 12-h
period where the horses were in stalls overnight. As these inflammatory markers are whole-body
indicators of inflammation, they are rapidly cleared from the blood in times where inflammation
is not present. As our horses were on a light exercise schedule which was not strenuous enough
to induce inflammation, it is likely that these metabolites were simply not present in detectable
quantities at the time of collection. The ELISAs used for analysis are designed to evaluate high
levels of these metabolites, and as most of our measures fell near the bottom end of the standard
curve (or were non-detectable), these resulted in high standard errors for all assays performed.
In conclusion, this study found that feeding either 4 g/d, 38 g/d, or 31 g/d of total n-3
with varying FA profiles for 21-d periods does not elicit changes in the plasma FA profile of the
horse. Additionally, feeding these quantities of n-3 and n-6 FA over a total 63-d period without
a wash-out period did not result in plasma FA concentrations, total n-3 or n-6 concentrations, or
alter the n-6 to n-3 ratio. Further, no changes in stride lengths, heart rates, or inflammatory
metabolites in both plasma and serum showed no treatment differences, likely as a result of the
absence of n-3 FA in the plasma, and therefore other cell membranes within the body, and due to
a lack of inflammatory disease as a result of osteoarthritis or exercise in our model.

62

CHAPTER 6
SUMMARY AND CONCLUSIONS

Our research has shown that the mature exercising horse does not show physiological
responses to a supply of 4 g/d, 38 g/d, or 31 g/d of total n-3 with varying FA profiles over 21-d
periods. No changes in the plasma FA profile of the horse, stride length at the walk or trot, heart
rate before, during, or post-exercise, or inflammatory metabolites in both plasma and serum were
elicited as a result of n-3 supplementation.
Previous research has suggested feeding a minimum of 30 to 35 g/d of EPA and DHA to
elicit a rapid change in the plasma profile of the horse (King et al., 2008). Our study supports
this claim, as no changes in plasma profiles after a 21-d period were observed in horses being
supplemented with 28.4 g/d of EPA and DHA. Based on the results from this experiment, it
would be recommended that if feeding EPA/DHA at an amount lower than 30 to 35 g/d, a
supplementation period of longer than 21-d would be required to observe differences in the
plasma FA concentrations as a reflection of diet. For an experimental design similar to the work
presented here, it would be recommended that a higher dose of EPA/DHA be offered.
As overproduction of inflammatory markers was not present in our horses in general, this
suggested that they did not have a need for the reduction of anything, regardless of n-3
supplementation. To better test the effects of n-3 supplementation on joint health in exercising
horses, inflammation as a result of exercise should be stimulated, with a more intense exercise
protocol than that exhibited here. In hindsight, a post-exercise blood sample rather than a resting
blood sample may have provided a better indicator of inflammatory responses or changes during

63

exercise to allow for more accurate detection by the ELISAs and would be recommended for
future studies.
To our knowledge, this is the first study to supplement n-3 FA in the form of DRM to
exercising horses. As nothing is known about potential differences in bioavailability of a DRM
source of DHA in the horse, additional research is needed with an amount greater than 28.4 g/d
for a 21-d period of EPA/DHA to validate the effects of a DRM DHA source on the metabolic
effects of DHA in the horse.
More research into n-3 effects on joint health would have to be performed before
conclusions regarding the effect of n-3 on stride length could be decisively made. While others
have observed stride length differences (Woodward et al., 2007), these were not reflected in our
study, but this was likely due to an inadequate supply of EPA and DHA to elicit changes in
synovial tissues. Further studies should examine the effect EPA and DHA on stride length when
supplied in an amount of 28.4 g/d over a continuous period greater than 21 d to allow for
changes in the chondrocyte. Also, as our horses showed no overproduction of inflammatory
markers, a more accurate measure of the effect of n-3 supplementation on stride length may be
determined by evaluating horses that would be suspected to have elevated levels due to injury or
disease to witness possible improvements or influences of a n-3 dietary source.
Additionally, further research investigating the time for FA to appear and clear from
circulation would be beneficial. Although rapid changes in FA profiles have been seen, there is
substantial variation among studies and varying amounts of supplementation have been used.
Unfortunately, in vivo animal studies are notoriously variable in this area. In addition to possible
species or breed differences, variation in response to fish oil or PUFA supplementation may stem

64

from genetic variation as pointed out by a recently published human study. Genetic differences
were observed in TNF-α production in response to endotoxin (Grimble et al., 2002). This
variability was determined to be due to polymorphisms in the promoter regions of TNF-α gene.
Identifying these potential differences in clearance and appearance rates would allow us to better
identify a minimum value of EPA/DHA supplementation in the mature, healthy horse over a
certain time period.

65

APPENDICES

66

APPENDIX A

FAMES Methylation Procedure

Following the lipid extraction procedure described using 0.5 g of ground feed sample, or
1 mL plasma, samples were methylated to obtain FAMES. One milliliter of hexane including a
C19:0 internal standard (1 mg standard/mL hexane) and 3 mL of 10% methanolic HCl (prepared
by adding acetyl chloride to methanol at a 1:5 ratio) were added to each sample. Samples were
layered with nitrogen gas, capped, vortexed, and placed into a 90° C water bath for 2 h. Once
removed from the water bath, samples were cooled to room temperature and 10 mL of a 6%
potassium carbonate solution (prepared by adding 60 g K2CO3 to 1 L deionized water) were
added. Samples were vortexed and centrifuged for 5 min at 950 x g, the top layer transferred to a
clean borosilicate test tube, and approximately 1 g sodium sulfate was added to each sample. If
the sample contained color, approximately 0.5 g of activated charcoal was added to it. Again,
samples were vortexed and centrifuged for 5 min at 950 x g. The hexane layer was transferred to
a previously weighed test tube, evaporated under a nitrogen gas stream, and reweighed to
determine lipid content. Samples were then reconstituted with hexane so that the concentration
ranged between 0.25 to 0.40 g lipid/mL hexane.
The reconstituted samples were vortexed and transferred to a gas chromatography (GC)
auto sampler vial and sealed with Teflon septa inserts. Samples were stored at -20° C until
transferred to the Michigan State University Diagnostic Center for Population and Animal Health
for analysis by GC (Clarus 500 with an autosampler; PerkinElmer, Waltham, MA) equipped with
a flame ionization detector, and a 100-m SP-2560 fused silica capillary column (0.25 mm i.d. x
67

0.1 µm film thickness; Supelco, Bellefonte, PA). Chromatograms were obtained and analyzed
using Empower Chromatography Data Software (Waters Corp., Milford, MA) and were
examined for the presence of FAME by comparing the peak retention times. The individual
FAME results were calculated as the concentration (mg/mL) in plasma and as a percentage of the
total plasma FA. The fatty acids summed to obtain total n-3 FA are as follows: C18:3n3,
C20:3n3, C20:5n3, C22:3n3, C22:5n3, and C22:6n3c. The fatty acids summed to obtain total n6 FA are as follows: C18:2n6, C18:3n6, C20:2n6, C20:3n6, C20:4n6, C22:2n6, and C22:4n6c.

68

APPENDIX B

Prostaglandin E2 ELISA Procedure

Prostaglandin E2 was determined using an enzyme-linked immunosorbent assay (ELISA;
Prostaglandin E2 ELISA Kit, Thermo Scientific Pierce Protein Research Products, Rockford, IL)
following the manufacturer’s instructions. Serum dilution factor was determined based on the
procedures described in Woodward et al. (2007), with slight modifications. Preliminary testing
at a dilution of 1:2 showed several results that were near the lower limit of the standard curve.
Dilution was therefore adjusted to 1:1.25, which provided readings falling within the standard
curve with good correlation (less than 15%)
The standard curve was prepared by transferring 900 µL reagent diluent to one tube and
500 µL reagent diluent to an additional eight tubes. One hundred microliters of stock PGE2
standard (50,000 pg/mL) was added to the first tube and serially diluted over the remaining eight
tubes in 500 µL aliquots. The concentrations of PGE2 in the diluted standard tubes were 5,000,
2,500, 1,250, 625, 313, 156, 78, 39, and 19 pg/mL, respectively. Once all standards and samples
were ready, the plate was prepared as follows: 150 µL of reagent diluent was added to the nonspecific binding well, 100 µL of reagent diluent was added to the maximum binding well, 100
µL of each standard was added to specified wells, and 100 µL of each sample was added to each
additional well. Samples were then diluted with 100 µL of reagent diluent on plate (1:1.25
dilution factor). Fifty microliters of Alkaline Phosphatase conjugated PGE2 (PGE2-AP) were

69

added to each well, excluding the total activity and blank wells, and 50 µL of PGE2 antibody
were added to each well except the blank, total activity, and non-specific binding wells. The
plate was then covered with a plate sealer and incubated at room temperature (22 to 25° C) on a
plate shake for 2 h at approximately 500 rpm. The contents of all wells were then aspirated,
washed four times with 400 µL of wash buffer, aspirated again, and firmly tapped on a lint free
paper towel to remove any remaining wash buffer. Five microliters of PGE2-AP was added to
the total activity well and 200 µL of substrate solution was added to every well. The plate was
then covered and incubated for 45 min at room temperature before being read at 405 nm optical
density on a Spectra Max 340 plate reader (Molecular Devices Corp., Sunnyvale, CA). Fifty µL
of stop solution was added to every well and the plate was immediately read at 580 nm.

70

APPENDIX C

Tumor Necrosis Factor-alpha ELISA Procedure

Tumor necrosis factor-alpha was determined using an ELISA (Equine TNF-α Screening
Kit, Thermo Scientific Pierce Protein Research Products, Rockford, IL) following the
manufacturer’s instructions. Serum was diluted at 1:8. Dilutions were determined by testing a
serum sample diluted at 1:2, 1:4, 1:8, and 1:16. These values were compared to the
manufacturer’s suggestion that the samples fall between 15.6 and 1,000 pg/mL. When the
samples fell within the range and recovery was the highest, the corresponding dilution was
TM

chosen for all samples (Appendix C). Flat bottom 96-well plates were used (MaxiSorp

,

Nalge Nunc International, Rochester, NY) and were coated with coating antibody (CA) prior to
use. Coating antibody was diluted 1:100 in a 0.2 M carbonate/bicarbonate buffer, pH 9.4 (110
µL CA added to 10.89 mL of buffer; Appendix A) and 100 µL was added to each well. The
plate was covered with a plate sealer and incubated at room temperature for 18 h, the CA
solution aspirated, and 300 µL of blocking buffer (4% BSA, 5% sucrose in PBS) added to each
well. The plate was covered, incubated for 1 h at room temperature on a plate shaker at 500 rpm,
and blocking buffer was then aspirated.
The lyophilized standard was reconstituted with 1.12 mL of reagent diluent to produce a
stock standard with a concentration of 10,000 pg/mL. The standard curve was prepared by
transferring 900 µL reagent diluent to one tube and 500 µL reagent diluent to an additional six
tubes. One hundred microliters of stock TNF-α standard was added to tube 1 and serially diluted
over the remaining six tubes in 500 µL aliquots. The concentrations of TNF-α in the diluted
71

standard tubes were 1,000, 500, 250, 125, 62.5, 31.25, and 15.6 pg/mL, respectively. Once all
standards and samples were ready, the plate was prepared as follows: 100 µL of each standard
was added to specified wells, 100 µL of reagent diluent were added to the blank wells, and 12.5
µL of each sample was added to each additional well. Samples were then diluted with 87.5 µL
of reagent diluent on plate. The plate was covered with a plate sealer, incubated for 2 h on a
plate shaker, the contents of all wells were aspirated, the plate washed five times with 400 µL of
wash buffer, wells aspirated again, and firmly tapped on a lint free paper towel to remove any
remaining wash buffer.
Biotinylated antibody reagent was diluted with reagent diluent to prepare a 1:100 µg/mL
concentration and was added (100 µL) to each well, the plate sealed and incubated for 1 h at
room temperature on a plate shaker at 500 rpm, the contents aspirated, and the plate was washed
five times with wash buffer. One hundred microliters of streptavidin-HRP reagent (diluted 1:400
in reagent diluent) was added to each well, followed by a 30-min covered incubation at room
temperature and another five washes with wash buffer. Next, TMB substrate solution (100 µL)
was added to each well and an enzymatic color reaction was allowed to develop in the dark at
room temperature for 20 min before being read at 650 nm optical density on a Spectra Max 340
plate reader (Molecular Devices Corp., Sunnyvale, CA). One hundred microliters of stop
solution was added to every well and the plate was immediately read at 450 nm and 550 nm
optical density. The 550 nm readings were subtracted from the 450 nm readings to correct
optical imperfections in the microplate and give the correct absorbance levels.

72

APPENDIX D

Interleukin 10 ELISA Procedure

Interleukin 10 was determined using an ELISA (Equine IL-10 DuoSet ELISA, R&D
Systems, Inc., Minneapolis, MN) following the manufacturer’s instructions. Serum was filtered
(Millex®-GP 0.22 µm filter unit, Millipore, Bedford, MA) and diluted on plate at 1:4 with
reagent diluent. Dilutions were determined by testing a neat (no additives) serum sample and
testing dilutions at 1:2, 1:4, 1:8, 1:10, 1:12.5, 1:16, 1:25, 1:32, 1:50, and 1:64. These values were
compared to the manufacturer’s suggestion that the samples fall between 39 and 20,000 pg/mL.
When the samples fell within the range and recovery was the highest, the corresponding dilution
TM

was chosen for all samples. Flat bottom 96-well plates were used (MaxiSorp

,Nalge Nunc

International, Rochester, NY) and were coated with CA (goat anti-equine IL-10) prior to use.
Capture antibody was reconstituted with 1 mL 1X PBS solution to produce a stock solution
(concentration of 144 µg/mL) and then a working concentration of 0.8 µg/mL (61.1 µL of CA
added to 10.939 µL 1X PBS solution; Appendix B). One hundred microliters of CA was added
to each well, the plate was covered with a plate sealer, incubated at room temperature for 18 h,
the CA solution aspirated, and then washed five times with 400 µL per well of wash buffer
(0.05% Tween-20 in PBS). Plates were then blocked by adding 300 µL of reagent diluent (1%
BSA in PBS) to each well, the plate covered and incubated for 75 min on a plate shaker,
aspirated, then washed five times with 400 µL per well of wash buffer.
The standard (20,000 pg/mL recombinant equine IL-10) was reconstituted with 0.5 mL of
reagent diluent to produce a stock standard. The ten point standard curve was prepared by 2-fold
73

serial dilutions in reagent diluent. The concentrations of IL-10 in the diluted standard tubes were
20,000, 10,000, 5,000, 2,500, 1,250, 625, 313, 156, 78, and 39 pg/mL, respectively. Once all
standards and samples were ready, the plate was prepared as follows: 100 µL of each standard
was added to specified wells, 100 µL of reagent diluent were added to the blank wells, and 25 µL
of each sample was added to each additional well. Samples were then diluted with 75 µL of
reagent diluent on plate, the plate covered with a plate sealer, incubated for 2 h on the plate
shaker at 500 rpm, well contents aspirated, washed five times with 400 µL of wash buffer,
aspirated, and firmly tapped on a lint free paper towel to remove any remaining wash buffer.
Biotinylated antibody reagent was diluted with reagent diluent to prepare a 100 ng/mL
working concentration (61.1 µL of antibody into 10.939 mL of reagent diluent) and was added
(100 µL) to each well, the plate sealed and incubated for 2 h on the plate shaker, contents
aspirated, the plate washed 5 times with wash buffer, and wash buffer aspirated. One hundred
microliters of streptavidin-HRP reagent (made by adding 55 µL streptavidin-HRP reagent to
10.945 mL reagent diluent) was added to each well, followed by a 20-min covered incubation on
the plate shaker and another five washes with wash buffer. Next, the working substrate solution
(prepared by mixing equal parts of Color Reagent A and Color Reagent B) was added to each
well (100 µL), and an enzymatic color reaction was allowed to develop in the dark at room
temperature for 20 min before being read at 620 nm optical density on a Spectra Max 340 plate
reader (Molecular Devices Corp., Sunnyvale, CA). Fifty microliters of stop solution was added
to every well and the plate was immediately read at 450 nm and 570 nm optical density. The 570
nm readings were subtracted from the 450 nm readings to correct optical imperfections in the
microplate and give the correct absorbance levels.

74

APPENDIX E

Interferon-gamma ELISA Procedure

Interferon-gamma was determined using an ELISA (Equine INF-γ DuoSet ELISA, R&D
Systems, Inc., Minneapolis, MN) following the manufacturer’s instructions. Serum was filtered
(Millex®-GP 0.22 µm filter unit, Millipore, Bedford, MA) and diluted on plate at 1:10 with
reagent diluent. Dilutions were determined by testing a serum sample spiked and diluted neat
(no additives), and at dilutions of 1:2, 1:4, 1:8, 1:10, 1:12.5, 1:16, 1:25, 1:32, 1:50, and 1:64.
These values were compared to the manufacturer’s suggestion that the samples fall between 31
and 4,000 pg/mL. When the samples fell within the range and recovery was the highest, the
corresponding dilution was chosen for all samples. Flat bottom 96-well plates were used
TM

(MaxiSorp

, Nalge Nunc International, Rochester, NY) and were coated with CA (goat anti-

equine INF-γ) prior to use. Capture antibody was reconstituted with 1 mL 1X PBS solution to
produce a stock solution (concentration of 72 µg/mL) and then a working concentration of 0.4
µg/mL (61.1 µL of CA added to 10.939 µL 1X PBS solution; Appendix B). One hundred
microliters of CA was added to each well, the plate was covered with a plate sealer, incubated at
room temperature for 18 h, the CA solution aspirated, and then washed five times with 400 µL
per well of wash buffer (0.05% Tween-20 in PBS). Plates were then blocked by adding 300 µL
of reagent diluent (1% BSA in PBS) to each well, the plate covered and incubated for 75 min on
a plate shaker, aspirated, then washed five times with wash buffer.
The standard (4,000 pg/mL recombinant equine INF-γ) was reconstituted with 0.5 mL of
reagent diluent to produce a stock standard. The eight-point standard curve was prepared by 275

fold serial dilutions in reagent diluent. The concentrations of INF-γ in the diluted standard tubes
were 4,000, 2,000, 1,000, 500, 250 125, 62, and 31 pg/mL, respectively. Once all standards and
samples were ready, the plate was prepared as follows: 100 µL of each standard was added to
specified wells, 100 µL of reagent diluent were added to the blank wells, and 10 µL of each
sample was added to each additional well. Samples were then diluted with 90 µL of reagent
diluent on the plate, the plate covered with a plate sealer and incubated for 2 h on the plate
shaker at 500 rpm, wells aspirated, washed five times with wash buffer, aspirated again, and
firmly tapped on a lint free paper towel to remove any remaining wash buffer.
Biotinylated antibody reagent was diluted with reagent diluent to prepare a 400-ng/mL
working concentration (61.1 µL of antibody into 10.939 mL of reagent diluent) and was added
(100 µL) to each well, with the plate then sealed and incubated for 2 h on the plate shaker. After
incubation, the contents were again aspirated and the plate was washed five times with wash
buffer. One hundred microliters of streptavidin-HRP reagent (made by adding 55 µL
streptavidin-HRP reagent to 10.945 mL reagent diluent) was added to each well, followed by a
20-min covered incubation on the plate shaker and another five washes with wash buffer. Next,
the working substrate solution (prepared by mixing equal parts of Color Reagent A and Color
Reagent B) was added to each well (100 µL), and an enzymatic color reaction was allowed to
develop in the dark at room temperature for 20 min before being read at 620 nm optical density
on a Spectra Max 340 plate reader (Molecular Devices Corp., Sunnyvale, CA). Fifty microliters
of stop solution was added to every well and the plate was immediately read at 450 nm and 570
nm optical density. The 570 nm readings were subtracted from the 450 nm readings to correct
optical imperfections in the microplate and give the correct absorbance levels.

76

APPENDIX F

Tumor Necrosis Factor-alpha Assay Reagent Preparation:

Equine TNF-α Screening Kit, Thermo Scientific Pierce Protein Research Products, Rockford, IL
Carbonate/bicarbonate Buffer Preparation (0.2 M buffer, pH 9.4):

1. Measure 21.2 g Na2CO3 into 1 L deionized water.
2. Measure 16.8 g NaHCO3 into a separate 1 L flask of deionized water.
3. Mix 19 mL of the Na2CO3 solution and 81 mL of the NaHCO3 solution.
4. The pH of this solution should be close to 9.3. Adjust to 9.4, adding a bit more NaOH as
needed to make the solution more basic.
Storage and Stability:
•

Store at 2 to 10° C. Warm to room temperature prior to using. Stable for one month.

Wash Buffer Preparation (0.05% Tween-20 in D-PBS, pH 7.4):

1. Prepare 10X PBS stock solution.
a. Combine the following: 80 g NaCl, 2 g KCl, 11.5 g Na2HPO4, and 2.04 g
KH2PO4. Bring to a volume of 1 L using deionized water.
2. Prepare wash buffer 10X stock solution.
a. Combine 49.75 mL 10X PBS stock solution with 250 µL Tween-20. Store in 50
mL conical tubes at 2 to 10°C.
77

3. Prepare wash buffer.
a. Thaw wash buffer 10X stock wash buffer to room temperature. In a clean glass or
plastic container dilute the wash buffer concentrate to 1:30 with distilled or
deionized water. Add 16.7 mL of wash buffer to 483.3 mL of water. Mix
thoroughly.
Storage and Stability:
•

Make fresh daily. Approximately 500 mL of wash buffer is needed per plate per
assay.

Reagent Diluent Preparation (4% BSA in D-PBS, pH 7.4):

1. Prepare 1X PBS solution.
a. Combine the following: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2PO4, and 1.5
mM KH2PO4. Bring to a volume of 1 L using deionized water.
2. Prepare 4% BSA solution.
a. Add 2 g BSA to 50 mL 1X PBS solution.
Storage and Stability:
•

Store at 2 to 10°C for up to a month. Approximately 50 mL is needed per plate per
assay.

78

APPENDIX G

Equine Interleukin 10 Assay and Equine Interferon-gamma Assay Reagent Preparation:

Equine IL-10 DuoSet ELISA, R&D Systems, Inc., Minneapolis, MN
Equine INF-γ DuoSet ELISA, R&D Systems, Inc., Minneapolis, MN
1X PBS Solution Preparation

1. Combine the following: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2PO4, and 1.5 mM
KH2PO4.
2. Bring to a volume of 1 L using deionized water.

Wash Buffer Preparation (0.05% Tween-20 in PBS, pH 7.4):

1. Prepare 10X PBS stock solution.
a. Combine the following: 80 g NaCl, 2 g KCl, 11.5 g Na2HPO4, and 2.04 g
KH2PO4. Bring to a volume of 1 L using deionized water.
2. Prepare wash buffer 10X stock solution.
a. Combining 49.75 mL 10X PBS stock solution with 250 µL Tween-20. Store in
50 mL conical tubes at 2 to 10°C.
3. Prepare wash buffer.

79

a. Thaw wash buffer 10X stock wash buffer to room temperature. In a clean glass or
plastic container dilute the wash buffer concentrate to 1:9 with deionized water by
adding 50 mL of 10X wash buffer stock solution to 450 mL of deionized water.
Mix thoroughly.
Storage and Stability:
•

Make fresh daily. Approximately 500 mL of wash buffer is needed per plate per
assay.

Reagent Diluent Preparation (1% BSA in PBS, pH 7.4):

1. Combine 10X PBS stock solution with deionized water at a 1:9 concentration (ex. 50 mL
10X PBS stock solution and 450 mL deionized water).
2. Add 5 g BSA, stirring slowly to avoid excessive foaming.
sStorage and Stability:
•

Store at 2 to 10°C for up to a month. Approximately 50 mL is needed per plate per
assay.

80

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