<%BANNER%>

Dietary Omega-3 Fatty Acid Supplementation and its Effect on Plasma and Cell Membrane Composition and Immune Function in...

Permanent Link: http://ufdc.ufl.edu/UFE0022769/00001

Material Information

Title: Dietary Omega-3 Fatty Acid Supplementation and its Effect on Plasma and Cell Membrane Composition and Immune Function in Yearling Horses
Physical Description: 1 online resource (206 p.)
Language: english
Creator: Vineyard, Kelly
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: horse, immune, omega3, omega6
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A series of four experiments were conducted to determine how omega-3 (n-3) fatty acid (FA) supplementation affects the FA composition of plasma and cell membranes, FA clearance rate after cessation of supplementation, and different aspects of innate and acquired immune function in horses. Sources of n-3 FA used in these studies included fish oil (FISH; rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA)) and/or flaxseed (FLAX; rich in alpha-linolenic acid). The final study utilized high fat diets (12% of daily DE from fat) created by adding either corn oil (rich in omega-6 (n-6) FA) or a blend of olive oil and FISH. Omega-3 FA were fed at a rate of 6?9 g n-3/100 kg BW for 42 ? 70 d. All studies included a non-supplemented control. FISH supplementation consistently resulted in a greater (P < 0.05) proportion of EPA, DHA and total n-3 FA in plasma, red blood cell and white blood cell membranes compared to other treatments. Similar effects were not observed when an equivalent amount of n-3 FA was provided from FLAX. Changes in FA composition occurred more rapidly in plasma compared to cell membranes. Plasma FA remained altered 5 wk after the cessation of n-3 supplementation, but plasma and cell membrane FA were no different from controls 8 wk after the removal of n-3 FA. Omega-3 FA did not affect lymphocyte proliferation or neutrophil function. PGE2 production by stimulated lymphocytes was not affected when n-3 FA were included as part of a low fat diet, but was reduced (P < 0.05) when either n-3 or n-6 FA were part of a high fat diet. Serum antibody titers in response to a novel vaccine were not affected by n-3 FA in a low fat diet, but were elevated (P < 0.05) in response to a tetanus booster when either n-3 or n-6 FA were supplied in a high fat diet. Horses fed FISH or FLAX had an earlier (P < 0.05) increase in skin thickness after intradermal phytohemagglutinin injection compared to non-supplemented controls. Collectively, these results demonstrate that n-3 FA do not negatively affect immune function and can support select immune responses in healthy horses.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kelly Vineyard.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Warren, Lori.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022769:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022769/00001

Material Information

Title: Dietary Omega-3 Fatty Acid Supplementation and its Effect on Plasma and Cell Membrane Composition and Immune Function in Yearling Horses
Physical Description: 1 online resource (206 p.)
Language: english
Creator: Vineyard, Kelly
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: horse, immune, omega3, omega6
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A series of four experiments were conducted to determine how omega-3 (n-3) fatty acid (FA) supplementation affects the FA composition of plasma and cell membranes, FA clearance rate after cessation of supplementation, and different aspects of innate and acquired immune function in horses. Sources of n-3 FA used in these studies included fish oil (FISH; rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA)) and/or flaxseed (FLAX; rich in alpha-linolenic acid). The final study utilized high fat diets (12% of daily DE from fat) created by adding either corn oil (rich in omega-6 (n-6) FA) or a blend of olive oil and FISH. Omega-3 FA were fed at a rate of 6?9 g n-3/100 kg BW for 42 ? 70 d. All studies included a non-supplemented control. FISH supplementation consistently resulted in a greater (P < 0.05) proportion of EPA, DHA and total n-3 FA in plasma, red blood cell and white blood cell membranes compared to other treatments. Similar effects were not observed when an equivalent amount of n-3 FA was provided from FLAX. Changes in FA composition occurred more rapidly in plasma compared to cell membranes. Plasma FA remained altered 5 wk after the cessation of n-3 supplementation, but plasma and cell membrane FA were no different from controls 8 wk after the removal of n-3 FA. Omega-3 FA did not affect lymphocyte proliferation or neutrophil function. PGE2 production by stimulated lymphocytes was not affected when n-3 FA were included as part of a low fat diet, but was reduced (P < 0.05) when either n-3 or n-6 FA were part of a high fat diet. Serum antibody titers in response to a novel vaccine were not affected by n-3 FA in a low fat diet, but were elevated (P < 0.05) in response to a tetanus booster when either n-3 or n-6 FA were supplied in a high fat diet. Horses fed FISH or FLAX had an earlier (P < 0.05) increase in skin thickness after intradermal phytohemagglutinin injection compared to non-supplemented controls. Collectively, these results demonstrate that n-3 FA do not negatively affect immune function and can support select immune responses in healthy horses.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kelly Vineyard.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Warren, Lori.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022769:00001


This item has the following downloads:


Full Text

PAGE 1

1 DIETARY OMEGA 3 FATTY ACID SUPPLEMENTATION AND ITS EFFECT ON PLASMA AND CEL L MEMBRANE COMPOSITION AND IMMUN E FUNCTION IN YEARLING HORSES By KELLY ROBERTSON VINEYARD A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE U NIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Kelly Robertson Vineyard

PAGE 3

3 T o m y best friend and husband, Mort, for h is unwa vering encouragement, support, and love

PAGE 4

4 ACKNOWLEDGMENTS I have been extremely fortunate to have worked under the guidance of Dr. Lori Warren, who has been an exceptional mentor role model, and friend over the past four years. Her dedication to science and research inspires me, and she has helped mold m e into the person I am today. Dr. Warren always has an educated and tactful solution for even the toughest of problems, and h er high standards of perfection will stay with me for the rest of my li fe I am also grateful to my committee members ( Dr. Steeve Giguere, Dr. Pete Hansen, Dr. Lee McDowell, and Dr. Lokenga Badinga ) for their support and time devoted to reviewing my dissertation Dr. Steeve Giguere deserves special recognition not only for hi s assistance with my immunological analyses, but also for his veterinary expertise when treating my horse Togey after he became ill with pneumonia during my qualifying exams. I would also like to acknowledge the contributions made by JBS United, Inc. and Milling Company for the donation of the encapsulated fish oil and milled flaxseed supplements respectively In addition, Omega Protein, Inc. provided the fish oil and partial funding for my final research project, for which I am very grateful. T here have been many people at the University of Florida who have been instrumental in the completion of my research. First and foremost, Jan Kivipelto deserves my sincere appreciation for her assistance with fatty acid and other lab analyses and for her fr iendship and support over the past 7 years Others who had generously offered their time to help me with lab procedures include Dr. Cynda Crawford, Dr. Stephanie Jacks, Elise Lee, Dr. Clare Ryan, and Werner Col l ante I am also grateful to J.G. Vickers, Dre w Cotton, Sarah Dilling, and Beth Stelzleni my fellow graduate students, who never hesitated to assist with data collection when asked I am lucky to have had the opportunity to work with such great people. The summer workers who helped on my research tri als made the day to day work more enjoyable and many

PAGE 5

5 thanks are in order for Katie Watson, Lindsay Clark, Jen Smugeresky, Analese Peters, Charly Cochran, Sarah White, and Sarah Simpson Also, the crew at the Horse Research Center and Horse Teaching Unit w ere always there to lend a helping hand if necessary and I appreciate all of the assistance offered over the years by Joel McQuagge, Justin Callaham, Steve Vargus, w ho served as my research subjects and provided me with much enjoyment and often a little extra excitement. Many thanks go to my Jamie Foster, Sindy Interrante, Summer Houghton, Becky Williams Cristina Caldari Torres and Jackie Wah rmund who have made life in graduate school more bearable. Last ly, but perhaps most importantly, I am especially appreciative for the support of my wonderful family. Words cannot express the deep gratitude I have for constant encouragement in all of my endeavors. My parents, Charlotte and Steve Robertson, have paved the way for my success, and the ir sacrifices and unconditional love are truly appreciated. I am fortunate to have strong women role models in my family, my mother and grandmothers that with faith in God, I can accomplish anything I set out to do I also owe thanks to my brother Jonathan and my extended family, too numerous to name, for at least pretending to understan d why I found it necessary to stay in graduate school for so many years.

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ......................... 10 LIST OF FIGURES ................................ ................................ ................................ ....................... 11 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 13 ABSTRACT ................................ ................................ ................................ ................................ ... 16 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 18 2 REVIEW OF LITERATURE ................................ ................................ ................................ 22 Fat Metabolism in the Horse ................................ ................................ ................................ ... 22 Fatty Acid Structure and Nomenclature ................................ ................................ .......... 22 Digestion and Metabolism of Dietary Fat ................................ ................................ ....... 24 Benefits of Feeding Fat to Horses ................................ ................................ ................... 27 Fatty Acid Synthesis and Degradation ................................ ................................ ............ 28 Polyunsaturated Fatty Acids ................................ ................................ ................................ ... 30 Omega 6 Fatty Acids ................................ ................................ ................................ ....... 30 Omega 3 Fatty Acids ................................ ................................ ................................ ....... 32 Competition Between Omega 6 and Omega 3 Fatty Acids ................................ ............ 35 Eicosanoid Biosynthesis ................................ ................................ ................................ .. 36 Overview of the Equine Immune System ................................ ................................ ............... 40 Innate Immune Function ................................ ................................ ................................ .. 40 Neutrophils ................................ ................................ ................................ ............... 41 Macrophages ................................ ................................ ................................ ............ 42 Natural killer cells ................................ ................................ ................................ .... 44 Mast cells ................................ ................................ ................................ .................. 45 Eosinophils and basophils ................................ ................................ ........................ 46 Dendritic cells ................................ ................................ ................................ .......... 46 Acquired Immune Function ................................ ................................ ............................. 47 Humoral immune responses ................................ ................................ ..................... 47 Cell mediated immune responses ................................ ................................ ............. 48 Immunomodulatory Effects of Omega 3 and Omega 6 Fatty Acid Supplementation ........... 50 Immune Cell Membrane Composition ................................ ................................ ............ 51 Eicosanoid Production ................................ ................................ ................................ ..... 52 Cytokine Production ................................ ................................ ................................ ........ 55 Lymphocyte Proliferation ................................ ................................ ................................ 57 Phagocytosis and Oxidative Burst ................................ ................................ ................... 59 Antibody Production ................................ ................................ ................................ ....... 61

PAGE 7

7 Gene Regulation and Expression ................................ ................................ ..................... 62 Omega 3 Fatty Acid Supplementation in the Horse ................................ ............................... 64 Blood and Other Physiological Responses to Omega 3 Supplementation ...................... 66 Effects on Immune Function ................................ ................................ ........................... 71 3 EFFECT OF DIETARY OMEGA 3 FATTY ACID SOURCE ON PLASMA AND RED BLOOD CELL FATTY ACID COMPOSITION AND IMMUNE FUNCTION IN YEARLING HORSES ................................ ................................ ................................ ............ 77 Abstract ................................ ................................ ................................ ................................ ... 77 Introduction ................................ ................................ ................................ ............................. 78 Materials and Methods ................................ ................................ ................................ ........... 80 Horses ................................ ................................ ................................ .............................. 80 Dietary Treatments ................................ ................................ ................................ .......... 80 Sample Collection ................................ ................................ ................................ ........... 81 Feedstuff, Plasma and Red Blood Cell Fatty Acid Composition ................................ .... 82 Lymphocyte Proliferation ................................ ................................ ................................ 83 PGE 2 Produc tion ................................ ................................ ................................ .............. 84 Intradermal Skin Test ................................ ................................ ................................ ...... 84 Statistical Analysis ................................ ................................ ................................ .......... 85 Results ................................ ................................ ................................ ................................ ..... 85 Plasma and Red Blood Cell Fatty Acid Composition ................................ ..................... 86 Lymphocyte Proliferation ................................ ................................ ................................ 88 PGE 2 production ................................ ................................ ................................ .............. 88 Intradermal Skin Test ................................ ................................ ................................ ...... 88 Discussion ................................ ................................ ................................ ............................... 89 Plasma and Re d Blood Cell Fatty Acids ................................ ................................ ......... 89 Immune Responses ................................ ................................ ................................ .......... 91 Implications ................................ ................................ ................................ ..................... 94 4 CLEARAN CE OF POLYUNSATURATED FATTY ACIDS FROM HORSE PLASMA AND RED BLOOD CELLS AFTER SUPPLEMENTATION ........................... 101 Abstract ................................ ................................ ................................ ................................ 101 Introduction ................................ ................................ ................................ ........................... 102 Materials and Methods ................................ ................................ ................................ ......... 102 Horses ................................ ................................ ................................ ............................ 102 Dietary Treatments ................................ ................................ ................................ ........ 103 Sample Collection ................................ ................................ ................................ ......... 104 Feedstuff, Plasma and Red Blood Cell Composition ................................ .................... 104 Statistical Analy sis ................................ ................................ ................................ ........ 106 Results ................................ ................................ ................................ ................................ ... 106 Supplementation Period ................................ ................................ ................................ 106 Washout Period ................................ ................................ ................................ ............. 107 Discussion ................................ ................................ ................................ ............................. 110

PAGE 8

8 5 EFFECT OF FISH OIL SUPPLEMENTATION ON INNATE AND ACQUIRED IMMUNE FUNCTION IN YEARLING HORSES ................................ ............................. 119 Abstract ................................ ................................ ................................ ................................ 119 Introduction ................................ ................................ ................................ ........................... 120 Materials and Methods ................................ ................................ ................................ ......... 122 Horses ................................ ................................ ................................ ............................ 122 Dietary Treatments ................................ ................................ ................................ ........ 123 Sample Collection and Processing ................................ ................................ ................ 123 Feedstuff, Plasma, Red Blood Cell, and White Blood Cell Fatty Acid Analysis .......... 124 Preparation of Bacterial Targets ................................ ................................ .................... 126 Ne utrophil Function ................................ ................................ ................................ ....... 126 Antibody Response to Vaccination ................................ ................................ ............... 127 Statistical Analysis ................................ ................................ ................................ ........ 128 Results ................................ ................................ ................................ ................................ ... 128 Plasma, Red Blood Cell, and White Blood Cell Fatty Acid Composition .................... 128 Neutrophil Function ................................ ................................ ................................ ....... 130 Antibody Response to Vaccination ................................ ................................ ............... 130 Discussion ................................ ................................ ................................ ............................. 130 Plasma, Red Blood Cell, and White Bl ood Cell Fatty Acids ................................ ........ 131 Neutrophil Function ................................ ................................ ................................ ....... 132 Antibody Response to Vaccination ................................ ................................ ............... 133 Implications ................................ ................................ ................................ ................... 134 6 EFFECT OF HIGH FAT DIETS AND FAT SOURCE ON IMMUNE FUNCTION IN YEARLING HORSES ................................ ................................ ................................ .......... 141 Abstract ................................ ................................ ................................ ................................ 141 Introduction ................................ ................................ ................................ ........................... 142 Materials and Methods ................................ ................................ ................................ ......... 143 Horses ................................ ................................ ................................ ............................ 143 Dietary Treatments ................................ ................................ ................................ ........ 14 3 Sample Collection and Processing ................................ ................................ ................ 144 Feedstuff, Plasma and Red Blood Cell Fa tty Acid Analysis ................................ ......... 145 Preparation of Bacterial Targets ................................ ................................ .................... 146 Neutrophil Function ................................ ................................ ................................ ....... 147 Prostaglandin E 2 Analysis ................................ ................................ ............................. 148 Lymphocyte Proliferation ................................ ................................ .............................. 148 Tetanus Antibody Titers ................................ ................................ ................................ 149 Statistical Analysis ................................ ................................ ................................ ........ 149 Results ................................ ................................ ................................ ................................ ... 150 Plasma and Red Blood Cell Fatty Acid Composition ................................ ................... 150 Neutrophil Function ................................ ................................ ................................ ....... 152 Prostaglandin E 2 Production ................................ ................................ .......................... 152 Lymphocyte Proliferation ................................ ................................ .............................. 152 Tetanus Antibody Titers ................................ ................................ ................................ 153 Discussion ................................ ................................ ................................ ............................. 153

PAGE 9

9 Plasma and Red Blood Cell Fatty Acids ................................ ................................ ....... 154 Lymphocyte Proliferation ................................ ................................ .............................. 155 Neutrophil Function ................................ ................................ ................................ ....... 156 PGE 2 Production ................................ ................................ ................................ ............ 157 Tetanus Antibody Titers ................................ ................................ ................................ 158 Implications ................................ ................................ ................................ .......................... 159 7 SUMMARY AND CONCLUSION S ................................ ................................ ................... 166 APPENDIX A PROCEDURE FOR PERIPHERAL BLOOD MONONUCLEAR CELL ISOLATION (CHAPTER 3) ................................ ................................ ................................ ...................... 169 B PROCEDURE FOR RED BLOOD CELL ISOLATION (C HAPTER 3) ............................ 171 C PROCEDURE FOR PERIPHERAL BLOOD MONONUCLEAR CELL ISOLATION (CHAPTERS 5 AND 6) ................................ ................................ ................................ ........ 173 D PROCEDURE FOR EXTRACTION AND METHYL ATION OF FATTY ACIDS FOLCH METHOD ................................ ................................ ................................ ............... 174 E PROCEDURE FOR FATTY ACID EXTRACTION AND METHYLATION JENKINS METHOD (CLEMSON 2 STEP) ................................ ................................ ........ 178 F PROCEDURE FOR LYMPHOCYTE PROLIFERATION ................................ ................. 180 G PROCEDURE FOR PGE 2 ANALYSIS ................................ ................................ ............... 182 H PROCEDURE FOR PREPARATION OF BACTERIAL TARGETS ................................ 183 I PROCEDURE, OPTIMIZATION, AND VALIDATION DATA FOR NEUTROPHIL FUNCTION ASSAY ................................ ................................ ................................ ............ 184 J PROCEDURE FOR SERUM IgG SUBCLASS TITER DETERMINATION BY ELISA 189 LIST OF REFERENCES ................................ ................................ ................................ ............. 190 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 205

PAGE 10

10 LIST OF TABLES Table page 2 1 Polyunsaturated fatty acids and their abbreviations ................................ ........................... 24 2 2 Fatty acid composition of common feedstuffs utilized in horse diets ............................... 34 3 1 Nutrient and fatty acid composition of the grain mix concentrate and bahiagrass pasture making up the basal diet, and the milled flaxseed and encapsulated fish oil supplements ................................ ................................ ................................ ........................ 96 3 2 Fatty acid composition of plasma before (d 0), during (d 35), and after 70 d of supplementation with milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) ................................ ................................ ................................ ..... 97 3 3 Fatty acid composition of red blood cells before (d 0), during (d 35), and after 70 d of supplementation with milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) ................................ ................................ ................................ ..... 98 4 1 Fatty acid composition of plasma before (baseline) and after 10 wk of supplementation with milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) and during the 8 wk washout period ................................ ........ 114 4 2 Fatty acid composition of red blood cell membranes before (baseline) and after 10 wk of supplementation with milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) and during the 8 wk washout period ............................... 115 5 1 Nutrient and fatty acid composition of the grain mix concentrate and hay making up the basal diet, and the oats and encapsulated fish oil supplement ................................ ... 136 5 2 Plasma, red blood cell, and white blood cell fatty acid composition in yearling horses receiving no n 3 fatty acid supplementation (CON) or encapsulated fish oil (FISH) ..... 137 5 3 Antigen specific IgGa production (mean optical densitySE) by non supplemented (CON) and encapsulated fish oil supplemented (FISH) horses in response to a bovine vaccine ................................ ................................ ................................ ............................. 138 6 1 Nutrient and fatty acid composition of the grain mix concentrate and Bahiagrass pasture making up the basal diet, and the fish/olive oil blend and corn oil supplements ................................ ................................ ................................ ...................... 161 6 2 Plasma and red blood cell fatty acid composition before (d 0) and after (d 42) supplementation with corn oil (CORN), fish/olive oil blend (FISH), or no supplementation (NON) ................................ ................................ ................................ ... 162

PAGE 11

11 LIST OF FIGURES Figure page 2 1 Triglyceride structure (saturated) ................................ ................................ ....................... 23 2 2 Essential fatty acid elongation and desaturation. Adapted from Yaqoob and Calder, 2007. ................................ ................................ ................................ ................................ ... 32 2 3 Local and systematic effects of selected inflammatory cytokines secreted by activated macrophages. Adapted from Janeway et al., 2005. ................................ ............ 44 2 4 Eicosanoid production from ARA and EPA. COX, cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; PG, prostaglandin; TX, thromboxane. Adapted from Calder, 2003 ................................ ................................ ................................ ...................... 53 2 5 Mechanisms by which n 3 PUFA may influence gene expression. Adapted from Miles and Calder, 1998. ................................ ................................ ................................ ..... 63 3 1 Proliferative respons es of PBMC from horses supplemented with a milled flaxseed (FLAX), encapsulated fish oil (FISH), or not supplemented (CON) for 70 d. .................. 99 3 2 Production of PGE 2 by stimulated PBMC from horses recei ving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) for 70 d. ............... 99 3 3 Change in skin thickness in response to intradermal injection of PHA in horses receiving mi lled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) ................................ ................................ ................................ ... 100 3 4 Change in the area of swelling in response to intradermal injection of PHA in horses receiving milled flaxse ed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) ................................ ................................ ................................ ... 100 4 1 Plasma and red blood cell linolenic acid response to 10 wk of supplementation and during an 8 wk washout period in horses receiving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (NON) ................................ .......... 116 4 2 Plasma and red blood cell eicosapentaenoic acid response to 10 wk of supplementation and during an 8 wk washout period in horses receiving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (NON) ............. 117 4 3 Plasma and red blood cell docosahexaenoic acid response to 10 wk of supplementation and during an 8 wk washout period in horses receiving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (NON) ............. 118

PAGE 12

12 5 1 Relationship between plasma and red blood cell (RBC) arachidonic acid (ARA) concentration after 56 d of supplementation with either encapsulated fish oil or no supplementation ................................ ................................ ................................ ............... 139 5 2 Relationship between plasma and red blood cell (RBC) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentration after 56 d of supplementation with either encapsulated fish oil or no s upplementation ................................ .......................... 139 5 3 Representative scatter (A) and dot (B) plots generated by flow cytometric evaluation of neutrophil function ................................ ................................ ................................ ...... 140 6 1 Representative scatter (A) and dot (B) plots generated by flow cytometric evaluation of neutrophil function ................................ ................................ ................................ ...... 163 6 2 Production of PGE 2 by stimulated PBMC from horses receiving no supplement (NON), a fish/olive oil blend (FISH), or corn oil (CORN) for 42 d ................................ 163 6 3 Proliferative responses of PBMC in horses receiving no supplementation (NON), or supplemented with corn oil (CORN) or a f ish/olive oil blend (FISH) for 42 d. .............. 164 6 4 Tetanus specific IgG titers at d 42 in horses supplemented with corn oil (CORN), fish oil (FISH), or no supplementation (NON) ................................ ................................ 164 6 5 Tetanus specific IgG titers at d 42 in response horses supplemented with corn oil (CORN), fish oil (FISH), or no supplementation (NON) ................................ ................ 165

PAGE 13

13 LIST OF ABBREVIATIONS ACC acetyl co e nzyme A carboxylase ADF acid detergent fiber ADG average daily gain ALA linolenic acid ARA arachidonic acid BW bodyweight ConA Concanavalin A COX cyclooxygenase CP crude protein d day(s) DE digestible energy DGLA dihomo linolenic acid DHA docosahexaenoic acid DMEM DPA docosapen taenoic acid EFA essential fatty acid EPA eicosapentaenoic acid FA fatty acid FAS fatty acid synthase FBS fetal bovine serum GLA gamma linolenic acid h hour(s) i.m. intra muscular IFN interferon

PAGE 14

14 IL interleukin LA linoleic acid LCFA long cha in fatty acid LOX lipoxygenase LPS lipopolysaccharide LSM lymphocyte separation medium LT leukotriene min minute(s) mL milliliters mo month(s) L microliters n omega NDF neutral detergent fiber nuclear factor NK natural killer PBS pho sphate buffered saline PDIFF predicted difference PG prostaglandin PHA phytohemagglutinin PPAR p eroxisome proliferator activated receptor PUFA polyunsaturated fatty acid RBC red blood cell T C cytotoxic T cell TG triacylglycerols TGF transforming growth factor

PAGE 15

15 T H helper T cell TNF tumor necrosis factor TX thromboxane WBC white blood cell wk week(s) yr year(s)

PAGE 16

16 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillme nt of the Requirements for the Degree of Doctor of Philosophy DIETARY OMEGA 3 FATTY ACID SUPPLEMENTATION AND ITS EFFECT ON PLASMA AND CELL MEMBRANE COMPOSITION AND IMMUNE FUNCTION IN YEARLING HORSES By Kelly Robertson Vineyard December 2008 Chair: Lori K. Warren Major: Animal Sciences A series of four experiments were conducted to determine how omega 3 (n 3) fatty acid (FA) supplementation affects the FA composition of plasma and cell membranes, FA clearance rate after cessation of supplementation, and different aspects of innate and acquired immune function in horses. Sources of n 3 FA used in these studies included fish oil (FISH; rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA)) and/or flaxseed (FLAX; rich in alpha linolenic acid). The final study utilized high fat diets (12% of daily DE from fat) created by adding either corn oil (rich in omega 6 (n 6) FA) or a blend of olive oil and FISH. Omega 3 FA were fed at a rate of 6 9 g n 3/100 kg BW for 42 70 d. All studies included a non su pplemented control. FISH supplementation consistently resulted in a greater (P<0.05) proportion of EPA, DHA and total n 3 FA in plasma, red blood cell and white blood cell membranes compared to other treatments. Similar effects were not observed when an eq uivalent amount of n 3 FA was provided from FLAX. Changes in FA composition occurred more rapidly in plasma compared to cell membranes. Plasma FA remained altered 5 wk after the cessation of n 3 supplementation,

PAGE 17

17 but plasma and cell membrane FA were no diff erent from controls 8 wk after the removal of n 3 FA. Omega 3 FA did not affect lymphocyte proliferation or neutrophil function. PGE 2 production by stimulated lymphocytes was not affected when n 3 FA were included as part of a low fat diet, but was reduced (P<0.05) when either n 3 or n 6 FA were part of a high fat diet. Serum antibody titers in response to a novel vaccine were not affected by n 3 FA in a low fat diet, but were elevated (P<0.05) in response to a tetanus booster when either n 3 or n 6 FA were supplied in a high fat diet. Horses fed FISH or FLAX had an earlier (P<0.05) increase in skin thickness after intradermal phytohemagglutinin injection compared to non supplemented controls. Collectively, these results demonstrate that n 3 FA do not negati vely affect immune function and can support select immune responses in healthy horses.

PAGE 18

18 CHAPTER 1 INTRODUCTION Throughout their lives and athletic careers, horses are subjected to many types of immunological challenges. Psychological stress in response to separation from the dam at due to a rise in blood cortisol levels (Hoffman et al., 1995; Cassil et al., 2006) Weaning also typically occurs at an age when the foa susceptibility to disease (Tizard, 2004) As young horses are prepared for sale, o r as they enter training and competition, they are often housed indoors in stalls in close proximity to other horses, which increases the likelihood of pathogenic exposure and transmission. In addition, strenuous training, as well as transport over long di stances, has been associated with depressed immune function (Stull and Rodiek, 2000; Folsom et al., 2001; Nesse et al., 2002; Robson et al., 2003) Another form of immunological challenge is gener ated by inflammato ry conditions such as osteoarthritis, respiratory disease, or allergic dermatitis. In the equine industry, lameness due to joint injury and disease is the most prevalent cause of diminished athletic function and wastage in racing horses (Goodrich and Nixon, 2006) Inflammatory airway disease is prevalent in horses in training, especially young racehorses, and after lameness, it is the second most common cause of lost training days (Rossdale et al., 1985; Wood et al., 2005) As horses age they can also develop heaves, which is character ized by chronic airway inflammation and obstruction. Seasonal equine pruritic dermatitis associated with the bites of Culicoides spp. is a common ailment suffered by many horses across the globe, and is one of the most common allergic skin diseases suffere d by horses in Florida (Friberg and Logas, 1999) All of these conditions are characterized by an inappropriate or amplif ied reaction to inflammatory stimuli

PAGE 19

19 and often involve a cascade of molecular signals that create a positive feedback loop resulting in chronic inflammation. Although veterinary medical treatment will most often provide symptomatic relief for these medical conditions and horse management strategies can be adjusted to reduce the impact of various psychological and physiological stressors the use of nutritional supplementation may be a viable strategy to support medical treatment and to attenuate inflammati on and strengthen the immune system. Two nutrients of particular interest for their roles in the modulation of inflammatory and immune responses are the omega 6 (n 6) and omega 3 (n 3) fatty acids. These cannot make them and therefore, they must be provided in the diet. These fatty acids serve as important components of cell membranes and as precursors for bioactive molecules known as eicosanoids, including prostaglandins, thromboxanes and leukotrienes. A large body of evidence in humans and lab animals suggests that supplementation with n 3 fatty acids may exert immunomodulatory effects and be useful in the prevention and/or treatment of heart disease, thrombosis, hypertension, renal disease, arthritis, i nflammatory disorders, autoimmune disease, and cancer (Calder et al., 2002; Gurr et al., 2002) Feeding marine derived fats such as fish oil, which are rich in eicosapentaenoic acid and docosahexaenoic acid, result s in the partial replacement of arachidonic acid in cell membranes with eicosapentaenoic acid and docosahexaenoic acid, leading to decreased production of inflammatory mediators (Calder, 2003) Clinica l studies have demonstrated that supplementation with fish oil, a source rich in n 3 fatty acids, can relieve some of the symptoms of rheumatoid arthritis and asthma in humans (Broughton et al., 1997; Cleland and Ja mes, 2000) Flaxseed is another rich source of n linolenic acid, and has also been shown to

PAGE 20

20 modulate inflammation, albeit to a lesser extent than fish oil (Wallace et al., 2003; Zhao et al., 2007) The practice of providing supplemental fat to the equine diet has become increasingly popular in the horse industry primarily because it is a safe way to increase the energy density of the ration without increasing the total amount of fe ed necessary to meet energy requirements. Fat supplementation has also been shown to have various positive effects on exercise metabolism and other physiological parameters (Harris, 1998) Fats, however, are not a uni form material and those fats added to equine rations differ in their fatty acid composition. Fat sources commonly added to commercial grain products include rice bran, corn oil, and soybean oil that are high in n 6 fatty acids These fat sources are more a ccessible, more stable, and less expensive than sources high in n 3 such as flaxseed (lins eed) and fish oil. Flaxseed has been traditionally supplemented to horses for many years in relatively small quantities as a supplement to improve the appearance of the hair coat, but supplementing fish oil has not been common due to its reduced availability, at least until recently. Investigation of the effects of dietary n 3 fatty acids on inflammatory mediators has received some attention in the horse; however, man y of these studies have included a small numbers of horses and typically did not include a non fat supplemented control treatment group in the design. Some of these reports indicate that the anti inflammatory effects observed in humans may also be detected in the horse (Henry et al., 1990; Hall et al., 2004a; Hall et al., 2004b) On the other hand, the effect of these fatty acids on overall immune function, including aspects of both innate and acquired immunity, h as received little attention Because horses are routinely fed fat composition of supplementary fat exerts on the immune system is important.

PAGE 21

21 T he objectives of th e four stud ies presented in this dissertation were to 1) determine the effect of different sources of n 3 FA on plasma, red blood cell, and white blood cell fatty acid composition; 2) determine the clearance rate of dietary n 3 fatty acids after supplementation has b een discontinued; and 3) identify effects of dietary fat and specifically n 3 and n 6 FA, on aspects of innate and acquired immune function through the measurement of the response to intradermal skin test, PGE 2 production, lymphocyte proliferation, neutro phil function, and antibody production. In all studies, a non fat supplemented control group of horses was utilized in order to make comparisons to responses observed in fat supplemented horses. The hypothesis of these studies was that the source of fat pr fatty acid composition of plasma, red blood cells, and white blood cells, and dietary n 3 fatty acids will affect immune function differently than n 6 fatty acids. In addition, determination of the natu re of the immunomodulatory effects will be elucidated by results obtained in these studies. Knowledge of how n 3 supplementation affects immune function may encourage feed manufactures to include greater proportions of n 3 rich fat sources in commercial fe ed formulations. In addition, supporting the immune system through nutrition is a viable strategy that would benefit the horse industry through decreased veterinary expenses and reduction of lost training/competition t ime and its associated revenue.

PAGE 22

22 CHAPT ER 2 REVIEW OF LITERATURE Fat Metabolism in the Horse Fatty Acid Structure and Nomenclature Fats belong to a class of water insoluble organic compounds known as lipids, and their primary biological functions include energy storage, cell membrane structure, and participation in signaling mechanisms within and between cells. Lipids can be classified as either glycerol or non glycerol based, and the non glycerol based lipids include cholesterol, waxes, terpenes, cerebrosides, and sterols (Gurr et al., 2002) Glycerol based lipids include glycolipids, phospholipids, and the class of m olecules known as triacylglycerols (TG) (also referred to as triglycerides ), which are lipid s tructures composed of three fatty acid (FA) molecules attached through an ester bond to a single glycerol backbone (Figure 2 1). T G are the primary means by which FA are consumed in the diet, although some non esterified FA can be found in dietary fat sour ces as well. FA are defined as aliphatic monocarboxylic acids and can be classified as either saturated (no double bonds), monounsaturated (one double bond), or polyunsaturated (two or more double bonds). The structure of a FA consists of carbon, hydrogen and oxygen atoms arranged in a carbon chain with a carboxyl group at one end and a methyl group at the other. Short chain FA are defined as having less than 6 carbons, medium chain with 6 16 carbons, and long chain having greater than 16 carbons (Gurr et al., 2002) Most FA derived from plant or non ruminant animal sources contain an even number of carbon atoms Bacteria, however, are able to synthesi ze odd numbered F A Consequently, ruminant animal fat contains FA that contain both an odd and even number of carbons due to the activity of bacteria present in the rumen.

PAGE 23

23 Figure 2 1. Triglyceride structure (saturated) There are several different FA nomenclature syst ems This dissertation will employ the widely used short notation system that d escribes the composition of the FA by the number of carbons contained in the chain, the degree of saturation of the molecule, and the location of the first double bond relative to methyl end of the molecule (e.g., C18:2n 6) The last carbon at the methyl end of the chain is designated carbon The omega nomenclature system, although older, is commonly used when describing essential FA. In this system, the position of the first double bond counted from the methyl end now commo is termed example, a FA containing 18 carbon atoms and 2 double bonds with the first double bond counted from the methyl end found between the 6th and 7th carbon would be designated as C18:2n 6. In contrast, the official systematic nomenclature system for FA designates the position

PAGE 24

24 official systematic name for the same C18:2n 6 FA described above octadecadienoic acid. Table 2 1 contains a list of polyunsaturated fatty acid (PU FA ) structures and nomenclature. Table 2 1 Polyunsaturated fatty a cids and their abbreviations Common Name Systematic Name Abbreviation Structure Linoleic a cid octadecadienoic acid LA C18:2n 6 linolenic acid octadecatrienoic acid GLA C18:3n 6 linolenic acid octadecatrienoic acid ALA C18:3n 3 Dihomo linolenic acid eicosatrienoic acid DGLA C20:3n 6 Arachidonic acid eicosatetraenoic acid ARA C20:4n 6 Eicosapentaenoic acid eicosapentaenoic acid EPA C20:5n 3 Docosapentaenoic acid docosapentaenoic acid DPA C22:5n 3 Docosahexaenoic acid docosahexaenoic acid DHA C 22:6n 3 Digestion and Metabolism of Dietary Fat TG or oils at room temperature, depending on their biochemical makeup. TG with a high proportion of short chain FA and/or unsaturated FA are more likely to be found as a liquid at room temperature, while TG containing more saturated FA will exist as solids due to their higher melting point. Fat digestion in the horse begins in the mouth and stomach, but most digestio n occurs in the small intestine. As the horse ingests feed, it chews to reduce particle size and produces saliva to lubricate the food bolus and facilitate swallowing. It is currently believed that horse saliva contain s no enzymes (Ellis and Hill, 2005) and, therefore fat hydrolysis does not begin until it which presumably begins the process of fat hydrolysis (Cunha, 1991) However, ingesta remains

PAGE 25

25 in the stomach for only a relatively short period of time, and the majority of fat digestion occurs once the contents of the stomac h empty into the duodenum. Unlike humans and many other mammals, horses do not possess a gall bladder for the storage and secretion of bile (Cunha, 1991) In the horse, bile is continuously secreted into the lumen of the upper small intestine directly from the liver, and pancreatic lipase is also secreted directly into the small intestine from the pancreas. Bile salts are necessa ry for fat emulsification, a process which increases the surface area of the ingested fat and allows pancreatic lipase to readily hydrolyze the TG molecules. The predominant digestive enzyme found in pancreatic tissue is lipase, followed by amylase (necess ary for starch digestion) and in smaller concentrations, the proteolytic enzymes elastase, trypsin, and chymotrypsin in small concentrations (Lorenzo Figueras et al., 2007) The specific mechanisms responsible for fat absorption across the small intestinal wall and subsequent transport to the liver and other parts of the body h ave not been specifically elucidated in the horse. It is currently assumed that horses digest, absorb, and process lipids utilizing the same mechanisms as other monogastrics. Once e mulsification of dietary fat occurs in the small intestine t he fat is hyd rolyzed by the pancreatic enzymes lipase, cholesterol esterase, and phospholipase A 2 into a mixture of FA, 2 monoglycerides, cholesterol, and phospholipids. These products, combined with bile salts, form mixed micelles and are delivered to the surface of t he intestinal wall. Facilitated by cytosolic FA binding protein (FABPc), the end products of lipid digestion diffuse across the brush border membrane, leaving the bile salts behind. Once inside the enterocyte, short and medium chain FA are transported dir ectly into the hepatic portal vein. Long chain FA (LCFA) and 2 monoglycerides are repackaged into TG cholesterol is esterified, and lyso phospholipids are

PAGE 26

26 converted to phosphatidylcholine while still in the enterocyte T hese products are then packaged i nto chylomicrons, which are large lipoproteins that act as carriers of absorbed lipids in a form that is stabilized for transport in the aqueous environment of the blood. Chylomicron assembly requires the incorporation of apolipoprotein B into the outer phosp holipid membrane, and the final product is characterized by a TG core stabilized with a surface monolayer of phospholipids, unesterified cholesterol, and protein. Chylomicrons are released from the enterocyte into the lymphatic system via tiny lymph vessel s called lacteals. From there, they enter the circulatory system via the thoracic duct and are distributed to the rest of the body (Gurr et al., 2002) I n the horse, att empts to isolate chylomicrons in adult ponies have been unsuccessful, and the question remains whether or not mature horses form true chylomicrons at all (Watson et al., 1991) Under normal con dit i on s, t he majority of TG stored in the body are found in adipose tissue. However, under conditions of fat mobilization induced by starvation, exercise, or stress, the liver TG content increases significantly. The hormones glucagon, epinephrine, and ACTH stimulate hormone sensitive lipase, which catalyzes lipolysis and facilitates the subsequent energy production from FA cleaved from the glycerol backbone of the TG molecule. In a m eta analysis, Kronfeld et al. (2004) reported that the true digestibility of added fats approaches 100% in the horse, but low er amount s of total fat in th e diet will result in decreased fat digestibility possibly due to the slower rate of fat hydrolysis by lipases at low substrate concentrations. The upper limit of fat inclusion in the diet appears to be 15 20% of total DE (Harris et al., 1999; Bush et al., 2001) as the addition of soybean oil at higher levels has been shown to inhibit fiber utilization when substituted for iso energetic amounts of non structural carbohydrates (Jansen et al., 2000; Jansen et al., 2002) This inhibition may be due to a specific negative effect of soybean oil in particular. Zeyner (2002) reported that soybean oil fed a t 0.7 g

PAGE 27

27 oil/kg BW daily had no adverse effects on fat digestibility but decreased fiber digestibility was observed in horses fed 1 g soybean oil/kg BW daily. Therefore, the NRC has recommended an upper limit of 0.7 g/kg BW daily for the inclusion level of soybean oil in equine diets (NRC, 2007) Recently, identification of the specific mechanism by which soybean oil inhibits fiber digestibility was attempted. It w as hypothesized that LA may play a role, as LA has been shown to inhibit growth of microorganisms in culture (Galbraith et al., 1971) However, when dietary palm oil was replaced by soybean oil in the diet of matur e horses, total tract digestibility of NDF, ADF, and cellulose were not affected, and the group mean digestibility of the fiber fractions were in fact higher for the ration containing soybean oil (Jansen et al., 2007) In addition, the infusion of linoleic acid into cecum fistulated ponies did not affect fiber digestibility and the authors concluded that decreased fiber fermentation after feeding soybean oil is unrelated to the increased intake of LA and further studies are necessary to unravel the mechanism (Jansen et al., 2007) Benefits of Feeding Fat to Horses The practice of supplementing fat to the equine diet has become increasingly common in the horse industry. Fat can be in a variety of ways, such as top dressing oil supplementing with high fat feed ingredients like rice bran or flaxseed, or feeding a commercially available fat added grain mix The FA composition of different fat sources varies greatly Fat sources such as corn o il, soybean oi l, and rice bran are rich in the omega 6 (n 6) FA linoleic acid (LA; C18:2n 6), while sources such as flaxseed ( linseed ) oil and fish oil contain higher amounts of the omega 3 (n 3) FA linolenic acid (ALA; C18:3n 3). Both LA and ALA are essential FA (E FA ) that cannot be synthesized in the body and must be supplied in the diet. LA is the primary E FA found in fat added commercial feeds and supplements. Sources high in n 6 FA are typical ly more readily available, more stable, and less expe nsive than sources high in n

PAGE 28

28 3 FA However, fresh pasture serves as a relatively good source of n 3 FA, as it is relatively high in ALA as compared to LA. T he most common reason for supplementing horses with fat is to increase the energy density of the ration as fats contain approximately 2.25 times the available energy by weight as carbohydrates. Increasing energy intake utilizing fat rather than carbohydrates reduces the risk of carbohydrate overload i n the hindgut. Other benefits to feeding a fat added diet include reduced reactivity and excitability in horses compared to horses fed a high starch diet (Holland et al., 1996) potential for decreased heat production during exer cise (Kronfeld, 1996) and metabolic adaptations favoring the utilization of fat as an energy source during exercise (Dunnett et al., 2002) Replacing starch as an energy source with fat is also recommended for horses suffering from metabolic conditions such as insulin resistance and polysaccharide storage myopathy, as this practice results in decre ased glycemic and insulin emic response following a meal (Williams et al., 2001; Ribeiro et al., 2004; Zeyner et al., 2006) Fatty Acid Synthesis and Degradation The FA that can be found and metabolized in the body arise from either the diet or from de novo synthesis. The pathways of FA storage and synthesis predominate during the well fed state and are promoted by insulin, whereas mobilization and oxidation of stored FA predominate during the fasted state physical exercise, and stress and are promoted by glucagon and epinephrine. The primary tissues engaged in FA synthesis are liver, adipose, and the mammary gland. The three substrates required for biosynthesis of FA are acetyl CoA, malonyl CoA, and NADPH. Acetyl C oA is formed in the mitochondrial matrix from pyruvate (the end product of glycolysis) (Gurr et al., 2002) Acetyl CoA is translocated to the cytosol as a part of a citr ate

PAGE 29

29 CoA. A cetyl CoA carboxylase (ACC), a t ype I biotin containing enzyme, catalyzes the formation of malonyl CoA from acetyl CoA M alonyl CoA is the source of t he majority of the carbons in the FA chain. The NADPH necessary for FA synthesis is generated by the citrate shuttle and the pentose phosphate pathway. The group of enzymes known collectively as FA synthases (FAS) are also critical for FA biosynthesis. Typ e III synthases, also known as elongases, catalyze the addition of 2 carbon units to preformed acyl chains at the carboxyl end. The typical end product of mammalian FA synthesis is palmitic acid (C16:0). Further elongation of C16:0 occur s under certain ci rcumstances. Many cell membrane lipids contain longer chain FA, especially those found in the tissues of the cardiac and immune systems. The formation of LCFA is catalyzed by elongase enzymes, which lengthen preformed FA that have been synthesized endogeno usly or have been consumed in the diet. Desaturation of LCFA occurs when double bonds are introduced through the action of a family of microsomal for desaturation of an 18 carbon FA at the n 3 or n 6 positions T herefore LA and ALA are must be consumed in the diet in order to carry out essential biological functions. One important function of the elongation and desaturation process is the conversion o f E FA to other long chain PUFA that play important roles in the maintenance of growth, rep roduction, and good health and mediate potent physiological effects (Gurr et al. 2002) named depending on which oxidat ion is to occur, the FA is oxidation is in the mitochondria, but it can also occur at other subcellular sites such as microbodies (peroxisomes or glycosomes) (Gurr et

PAGE 30

30 al., 2002) Because CoA cannot cross the mitochondrial membrane, its entry into the mitochondria is mediated by a process known as the carnitine shuffle. This process is characterized by the transfer of long chain acyl groups from CoA to carnitine catalyzed by the enzyme carnitine: palmitoyl transferase. The resulting acylcarnitine has the ability to cross the mitochondrial membrane. Once inside the mitochondria, a cyclic process involving four enzymes facili tate s a stepwise removal of acetyl CoA fragments from the carboxyl end of the molecule. oxidation is acetyl CoA, which is used in the TCA cycle to yield energy in the form of ATP. Polyunsaturated Fatty Acids P UFA are characterized by more than one double bond contained in a FA structure consisting of 18 or more carbons. In animals, endogenous desaturase enzymes allow for the desaturation proces s. As noted above, t synthesis of 18 carbon n 3 and n 6 FA are not found in animal tissues, but they are present in plants and algae. LA ( n 6 ) and A LA (n 3) are two of the most prevalent FA found in plants. The biological activity of the elongated versions of these FA are especially notable for the potent physiological effects they have in a variety of cells and tissues in mammals Omega 6 Fatty Acids Three of the most physiologically significant n 6 FA are LA, linolenic acid (GLA; C18:3n 6), and arachidonic acid (A R A; C20:4n 6) (Figure 2 2) LA cannot be synthesized in the body, but it serves as the precursor to other n 6 FA that are capable of being produced in the body. GLA is produced as an intermediate in desaturase, and it is then rapidly elongated to dihomo linolenic acid (DGLA; C20:3n 6) DGLA can be incorporated into cell phospholipids (Kapoor and Huang, 2006) DGLA can also

PAGE 31

31 ARA ; however, the extent of this reaction is dependent on dietary an d environmental factors such as high chole sterol intake, aging, or stress (Huang et al., 1996; Kapoor and Huang, 2006) DGLA and ARA are precursors of the 1 and 2 series eicosanoids prostaglandin (PG) and thromboxane (TX) respectively (Huang et al., 1996) Eicosanoids are a large family of signaling molecules derived from 20 carbon FA molecules and include PG, TX leukotrienes (LT) and related metabolites. The primary roles of eicosanoids are to act as mediators of inflammation in the body, and because most cell membranes contain a greater concentration of ARA than other 20 carbon FA ARA is us ually the principal precursor for eicosanoid synthesis (Calder and Grimble, 2002) Eicosanoid production an d its relation to dietary FA are discussed in more detail later in this chapter. Many p lant seeds and oils are rich sources of n 6 FA C orn, soybean, and sunflower oils contain over 50% LA and are commonly fed to horses (Table 2 2 ). Rice bran and rice bran oil, also commonly fed to horses, contain approximately 40% LA. In humans, consumption of n 6 FA greatly exceeds that of n 3 FA by a 20 25:1 margin (James et al., 2000; Calder and Grimble, 2002) Increased consump tion of n 6 FA as compared to n 3 is also true in horses due to increased consumption of cereal grains, oil seeds, and supplemental plant oils rich in n 6 rather than n 3 FA.

PAGE 32

32 Figure 2 2 Essential fatty acid elongation and desaturation. Adapted from Ya qoob and Calder, 2007. Omega 3 Fatty Acids Similar to LA, ALA is also considered an EFA ALA is classified as an n 3 FA due to the first double bond located three carbons away from the methyl terminus. Some rich sources of ALA in horse diets include fresh grass, flaxseed and flaxseed oil (Table 2 2 ). Other than ALA, the most physiologically relevant n 3 FA are eicosapentaenoic acid (EPA; C20:5n 3), docosapentaenoic acid (DPA; C22:5n 3), and docosahexaenoic acid (DH A; C22:6n 3). ALA can give rise to EPA in the endoplasmic reticulum through the same pathway that LA is converted to ARA (Figure 2 2) Because the same enzymes are

PAGE 33

33 required in this metabolic pathway, there is competition between n 6 and n 3 FA for the u tilization of these enzymes (Calder and Grimble, 2002) ALA is preferentially converted to EPA through this pathway, as animal studies have indicated that there is a 1.5 to 3.0 fold higher (Sprecher et al., 1995; Sprecher, 2000; Hussein et al., 2005) Although n 3 FA have a higher affinity for desaturase enzymes, n 6 FA are generally more prevalent in cell membranes and are still capable of securing a significant portion of the desaturase enzymes. EPA gives rise to DPA and DHA through further enzyme dependant elonga ti on and desaturation steps. EPA and DHA can be incorporated into inflammatory cell phospholipid membranes, partially at the expense of ARA (Calder, 2006) EPA serves as the precursor to eicosanoids of the 3 and 5 series, which are less potent inflammatory mediators than eicosanoids of the 2 and 4 series derived from ARA (Calder, 2006)

PAGE 34

34 Table 2 2 Fatty acid composition of common feed stuffs utilized i n hors e diets Fatty Acids % of total fat ty acids Feed component Crude Fat (%) C16:0 C18:0 C18:1 C18:2n 6 LA C18:3n 3 ALA C20:4n 6 ARA C20:5n 3 EPA C22:6n 3 DHA Feeds Textured grain concentrate 1 4.1 16.1 2.3 27.4 49.7 4.6 0 0 0 Oats 1 5.7 18.5 0 34 .2 45.3 1.8 0 0 0 Fresh B ahiagrass 1 2.3 18.8 2.7 2.4 17.6 57.7 0 0 0 Bermudagrass hay 1 2 30 3.9 3.8 23.0 36.8 0 0 0 Rice bran 2 23.3 19.3 1.7 37.9 33.5 1.1 0 0 0 Flaxseed 1 37.7 5.6 2.7 14.1 16.5 61.2 0 0 0 Encapsulated fish oil 1 22.8 23.0 5.2 8.3 6.8 2.6 1.0 14.4 11.5 Oils Corn oil 1 100 12.9 1.7 26.2 57.7 1.3 0 0 0 Soybean oil 3 100 10.3 3.8 22.8 51.0 6.8 0 0 0 Olive oil 1* 100 14.2 2.5 70.4 10.6 1.2 0 0 0 Canola oil 3 100 4.8 1.6 53.8 22.1 11.1 0 0 1 Fish oil 1 100 22.9 3.3 2.9 4.5 2.5 0.9 15.4 7.2 1 From studies in this dissertation 2 (Spears et al., 2004) 3 (NRC, 2007) Not typically utilized in ho rse diets, but was used in this dissertation

PAGE 35

35 Competition Between Omega 6 and Omega 3 Fatty Acids The first step in the conversion of LA to ARA and ALA to EPA is desaturase This reaction is considered to be the rate limiting step for ARA and EPA synthesis. Because the same enzymes are utilized in the elongation and desaturation pathways of both LA and ALA, competition between the two FA exists for utiliz ation of these enzymes Some reports indicate that there is a preferential affinit 3 FA (Drevon, 1992) but other studies indicate live r microsomes show little substrate specificity for analogous n 3 and n 6 FA during the process of long chain PUFA and phospholipid biosynthesis (Sprecher, 2000) In addition, LA is typically present in higher concentrations in cellu lar pools, and the results in a greater conversion to n 6 PUFA (Burdge and Calder, 2005) Other enzymes essential to the e and various elongases (Figure 2 2). One way in which PUFA elongation and desaturation may be regulated is through reciprocal inhibition of the enzymatic steps involved in elongation and desaturation (Holman, 1986) For example n 3 FA appear to inhibit the elongation o f LA to ARA under normal basal conditions (Contreras and Rapoport, 2002) On the other hand, Emken (1994) reported that a diet high in LA may reduce the conversion of ALA to EPA by up to 4 0% In humans, results obtained from tracer studies using stable isotopes indicate that the conversion of ALA to EPA and EPA to DPA in adult men range from betwe en 0.2 6 % and 0.12 6%, respectively, and the synthesis of DHA from EPA is highly constrained at 0.05% or less (Burdge, 2006) However, the conversi on of ALA to EPA and DHA in women has been shown to be substantially greater (2.5 fold and 4200 fold, respectively) than in men of similar age (Burdge and Wootton, 2002; Burdge et al., 2002) One possible explanati on for the increased synthesis of EPA and DHA from ALA in women is the influen ce of estrogen on the

PAGE 36

36 elongation/desaturation pathway. In women who were using an oral contraceptive containing ethylnyloestradiol, DHA synthesis was almost 3 fold greater than in those who were not on birth control (Burdge and Wootton, 2002) The greater capacity for ALA conversion in women m ay be due in part to increased fetal demands for DHA during the third trimester of pregnancy (Burdge, 2006) as DHA is essential for proper development of the nervous system and plays a role in retinal function (Lauritzen et al., 2001) Eicosanoid Biosynthesis carbon FA derivative. PG were the first biologically active eicosanoids to be identif ied, and the structures of the E and F families (PGE and PGF) were the first to be defined (Gurr et al., 2002) Other eicosanoids that ar e derived from 20 carbon FA include TX prostacyclins, LT and hydroxy derivatives. The ability of the ARA chain to fold allows the arrangement necessary for subsequent ring formation to occur which is a common structural characteristic of all eicosanoids (Gurr et al., 2002) The action of various phospholipase enzymes, namely phospholipase A 2 facilitates the mobilization of ARA and EPA from cell membrane phospholipids so that they may serve as substrates for the synthesis of eicosanoids. The first key enzyme necessary for PG formation is PG endoperoxide synthase, also known as cyclooxygenase (COX). There are two families of COX enzymes that have been well described Th e action of both COX 1 and COX 2 on ARA or EPA will result in the generation of PG. COX 1 enzymes are constitutively expressed in most mammalian tissues and cells and utilized in normal homeostatic mechanisms, whereas COX 2 enzymes are highly inducible and generally present at very low levels unless increased by one of many types of stimuli including cytokines, endotoxins, and growth factors (Chandrasekhar an and Simmons, 2004) One of the

PAGE 37

37 best know non steroidal anti inflammatory (NSAID) drugs and a classic inhibitor of COX enzymes is aspirin (salicylic acid). Aspirin competes with ARA for binding to the COX active site, and this binding of aspirin and COX is an irreversible reaction. Ibuprofen is another NSAID that inhibits PG synthesis, but its binding to the COX enzyme is reversible (Gurr et al., 2002) Aspirin and ibu profen are non specific inhibitors of both COX 1 and COX 2 enzymes. The therapeutic anti inflammatory actions of these NSAIDs are a result of COX 2 inhibition, while the unwanted side effects such as gastric ulceration are due to the inhibition of COX 1 (Radi and Khan, 2006) Selective COX 2 inhibitors suc h as celecoxib and valdecoxib have been recently developed to specifically target COX 2 enzymes without impairing the action of COX 1, reducing the incidence of unwanted side effects. The second step in PG synthesis involves the production of PGH through the activity of peroxidase enzymes. PGH is the key intermediate for the subsequent conversion to the active prostaglandins PGD, PGE, and PGF. PGD is the main COX product of mast cells PGD 2 (derived from ARA) inhibits platelet aggregation, increases platel et cAMP content, and can act as a peripheral vasoconstrictor, pulmonary vasoconstrictor, and bronchoconstrictor. PGE 2 (derived from ARA) has a number of pro inflammatory effects, including fever induction, increased vascular permeability, vasodilation, enh anced pain perception, and increased edema (Calder, 2006) Macrophages and monocytes produce large amounts of PGE 2 and neutrophils produce moderate amounts upon stimulation (Calder and Grimble, 2002) PGE 2 can also be categorized as having immunosuppressive or anti inflammatory effects, due to its ability to inhi bit lymphocyte proliferation, suppress natural killer cell activity, decrease the production of pro inflammatory 4 series LT and inhibit the production of pro inflammatory cytokines tumor

PAGE 38

38 necrosis factor 6, and interferon (Calder and Grimble, 2002; Calder, 2006) PGF is another eicosanoid which can be produced by macrophages and monocytes, but its primary role is in the regulation of reproductive functions through the initiation of luteolysis (Gurr et al., 2002) T hromboxanes were first discovered i n thrombocytes T he most biologically significant member of this family of eicosanoids is TXA 2 which is derived from PGH. TX A 2 produced by platelets plays a major role in platelet aggregation and induces smooth muscle contraction (vasoconstriction) and cell adhesion to the blood vessel wall (Gurr et al., 2002) These mechanisms are important for the repair of blood vessel damage, but in cases of chronic or extensive damage, they can result in inappropriate blood clot formation which could lead to a stroke or heart attack. Vascular endothelial cells were discovered to produce another derivative of PGH, known as prostacyclin (PGI 2 ). This eicosanoid has the opposite effect of TX A 2 as it promotes vasodilation and has an anti aggregatory effect on platelets (Gurr et al., 2002) The actions of TX and prostacyclins are antagonistic, and their ratio affects the regulation of blood pressure and clot formation in the body. L eukotrienes are not derived from PGH, but rather the direct action of 5 lipooxygenase (5 LOX) on ARA or EPA. Neutrophils, monocytes, and macrophages produce LT B, while mast cells, basophils, and neutrophils produce LTC, LTD, and LTE. S ynthesis of LT only occurs in intact cells following a rise in intracellular calcium due to stimulation by extracellular stimuli (Gurr et al., 2002) LTB 4 increases vascular permeability, is a potent chemotactic agent for neutrophils and eosino phils, induces release of lysosomal enzymes, and stimulates the production of inflammatory cytokines such as tumor necrosis factor TNF ) interleukin 1

PAGE 39

39 ( IL 1 ) and interleukin 6 ( IL 6 ) (Calder, 2006) LTC 4 and LT D 4 are more potent than LT E 4 but they all increase bronchoconstriction, increase vascular permeabili ty, and promote hypersensitivity (Gurr et al., 2002; Calder, 2006) The actions of LT play a major role in the physiological responses characteristic of asthma, immediate hypersensitivity reactions, and acute infla mmatory reactions. In horses, LT are thought to play a major role in the pathogenesis of recurrent airway obstruction (also known as heaves) (Robinson et al., 1996) During the process of LT formation, the immediate product of 5 LOX activity on ARA substrates is hyd roperoxy eicostatetraenoic acid (HPETE ). HPETE can undergo three further reactions: reduction to an alcohol forming a hydroxyeicosatetraenoic acid (HETE), lipoxygenation to yield a dihydroxyeicosatetraenoic acid (diHETE), or a dehydration to form a LT molecule (Gurr et al., 2002) The LT molecule itself can also un dergo further reactions to form other diHETEs. These HPETEs, HETEs, and diHETEs are all intermediates in LOX pathways, but they also have their own interacting effects and interdependent actions. For example, the hydroxy FA 15 HETE is considered to have bo th pro and anti inflammatory effects, as it potentiates smooth muscle contraction and stimulates mucus secretion, but it also has been shown to inhibit 5 LOX, which inhibits LT production, and block LTB 4 induced neutrophil chemotaxis (Robinson et al., 1996) When LOX acts on EPA, a different hydroperoxy product, hydroperoxy pentaenoic acid (HPEPE) is formed and serves as the precursor for the less inflammator y 5 series LT Collectively, the se eicosanoids play a significant role in immune function and although they can have both synergistic and antagonistic effects, it is the ir balance which dictates the nature of the final biological response to external stimu li.

PAGE 40

40 Overview of t he Equine Immune System functions through a complex variety of cells and mechanisms that coordinate to identify and eliminate foreign pathogens Innate immunity provides protection in a non specific manner, while acquired (adaptive) immunity has the capability for specific recognition of pathogens and immunological memory. Innate immunity involves physical barriers to infection ( e.g. skin), soluble factors in the blood ( e.g. complement), and the actions of phagocy tic and other effector cells. The acquired immune response involves lymphocytes that bear highly specific cell surface receptors and are only capable of eliminating cells they recognize These two branches utilize different mechanisms for pathogen removal but they are depend e nt upon one another for optimal function. Innate Immune Function Innate immunity serves as a basic and non specific barrier to the establishment of infection. Horses are faced with a variety of pathogenic organisms on a daily basis, a nd the task of the innate immune system is to prevent these organisms from establishing themselves and causing disease. Some cells of the innate immune system have the ability to immediately recognize molecules associated with foreign invaders so that they can destroy them. Other cells release key molecules that serve to support the recognition and elimination of foreign microorganisms. Epithelial surfaces of the body, such as skin and the gastrointestinal, respiratory, and urogenital tracts, make up an add itional first line of defense against infection (Janeway et a l., 2005) The innate immune system lacks any form of memory and as a result the duration and intensity of inflammatory and other associated responses remain the same no matter how often the horse is faced with a specific invader (Tizard, 2004) This insures that the horse is always ready for an immediate response against infection There are several types of cells

PAGE 41

41 primarily associated with innate immune function, and each plays a uniq ue role in host protection. Neutrophils Polymorphonu c lear neutrophilic leukocytes (PMNs), or neutrophils, are relatively short lived cells with a life span of 8 12 h in the circulation, that are abundant in blood and are capable of phagocytosis (Cotter, 2001) Neutrophils make up approximately 50% of the blood leukocyte population in the horse (Tizard, 2004) Neutrophils have the ability to extravasate from the blo od circulation through epithelial tissues in order to reach the site of microbial invasion. Neutrophils are recruited to the site of tissue damage through the action of chemotactic molecules secreted by various cells at the site of injury, including eicosa noids, bacterial peptides, activated complement, and cytokines. Once in the presence of the pathogen, specific receptors or antibody coated bacteria and undergo either complement or an tibody mediated phagocytosis. Ingestion can also occur if the neutrophil has the ability to directly bind bacteria through cell surface receptors such as mannose receptors or integrins. Once the neutrophil has ingested the bacteria, it must then destroy i t through the initiation of two distinct processes : respiratory burst followed by the release of antimicrobial molecules from intracellular granules T he respiratory burst is characterized by the generation of hydrogen peroxide and its subsequent reaction with intracellular halide ions (primarily Cl ) catalyzed by myeloperoxidase to produce hypochlorite (OCl ), the major product of neutrophil oxidative metabolism (Tizard, 2004) OCl kil ls bacteria by oxidizing bacterial proteins, but it can also damage healthy cells. Neutrophils contain large amounts of glutathione that can reduce these oxidants and prevent excessive damage to surrounding healthy cells. After the respiratory burst,

PAGE 42

42 lysos omal enzymes, antimicrobial peptides, and other substances are released from the cytoplasmic granules of the neutrophil and function to digest and limit the growth of any remaining bacteria. In addition to serving as the first line of defense against invad ing microorganisms, neutrophils are also capable of synthesizing cytokines, including TNF interleukin IL ) and interleukin 8 ( IL 8 ) in response to inflammatory stimuli and during certain chronic inflammatory disorders (Kasama et al., 2005) The actions of these and other cytokines will be discussed in more detail later in this chapter. Because the respiratory bu rst is an essential event for the killing of phagocytized pathogens, methods to assess neutrophil function should ideally measure the ability of a single neutrophil to undergo phagocytosis and initiate the subsequent oxidative burst. Many techniques have b een described that measure phagocytosis and oxidative burst independently of each other in isolated neutrophils. Commercially available kits are available which make this a relatively simple process. However, the process of neutrophil isolation requires re latively large amounts of blood and can represent a source of variation in the assay (Salgar et al., 1991) In addition, measuring phagocytosis and oxidative burst independently of each other may not offer a physiological ly relevant measurement. Flow cytometry is emerging as the method of choice for the measurement of many different cell functions and characteristics, and techniques that assess phagocytosis induced oxidative burst in whole blood are now being utilized to gain a better understanding of neutrophil function (Smits et al., 1997; K ampen et al., 2004) Macrophages Another population of phagocytic cells integral to innate immune function are macrophages present in the tissues of the body. Macrophages are mononuclear cells derived from circulating monocytes which have left the bloodst ream and have migrated into the tissues where

PAGE 43

43 they can become functional. Macrophages differ from neutrophils in that they typically have a slower response time, have greater antimicrobial properties, and have the ability to induce acquired immune response s. Similar to neutrophils, macrophages destroy bacteria through both oxidative and non oxidative mechanisms, but one population of macrophages (M1) in the horse also has the ability to synthesize inducible nitric oxide synthase that catalyzes the productio n of nitric oxide (NO) (Tizard, 2004) When NO reacts with a superoxide anion, highly toxic oxidants are produced that allow for efficient killing of a variety of foreign pathogens. Macr ophages have the ability to undertake sustained and repeated phagocytic activity, unlike neutrophils which are relatively short lived. Another important function of macrophages is their ability to produce pro inflammatory mediators in response to stimulati on. These mediators include the cytokines TNF 6, and the eicosanoids PG and LT (Janeway et al., 2005) These inflammatory mediators influence many different cells and tissues throughout the body to initiate inflammation and activate the acquired immune response (Figure 2 3).

PAGE 44

44 Figure 2 3 Local an d systematic effects of selected inflammatory cytokines s ecreted by activated macrophages. Adapted from Janeway et al., 2005. Natural killer cells Natural killer (NK) cells are a unique category of lymphocytes that are activated by interferons and contrib ute to innate host defense against viruses and other intracellular pathogens (Janeway et al., 2005) The mechanism utilized by NK cells to kill an infected cell is the same as the mechanism utilized by cytotoxic T (T C ) cells in an acquired immune response Unlike T C cells, however, r ecognition of target cells by NK cells occurs via non specific invariant receptors

PAGE 45

45 that characterize infected cell surfaces. NK cells serve to provide protection during the early phase of viral infection before antigen specific T C cells can be generated to clear the infection. Mast cells The majority of mast cells are located throughout the body in connective tissue, under mucosal surfaces, in the skin, and around nerves (Tizard, 2004) They play a key role in innate im munity, as they have the ability to release a complex mixture of inflammatory molecules that are stored in intracellular granules upon stimulation. There are several types of stimuli that can initiate mast cell granule release. The most potent of stimuli i s the binding and cross linking of IgE antibodies ( which are firmly bound to the mast cell membrane ) to specific antigen. This triggers a massive release of all of the granule content s (primarily histamine) and inflammatory mediators from the mast cell in to the surrounding tissues. The acute inflammatory response that follows is characteristic of type I hypersensitivity, also commonly referred to as an allergic reaction. Because the binding and subsequent cross linking of IgE antibody on the mast cell surf ace is a primary stimulator for release of its cell contents, the mast cell can be considered a cell type which plays roles in both innate and acquired immunity. The release of histamine from mast cell granules mediates the local increase in vascular perme ability that leads to the recruitment of neutrophils, macrophages, and other effector cells to the site. Other stimuli that trigger a more attenuated degranulation of mast cells include bacteria, bacterial products, and small peptides released by dead and dying cells. Mast cells also have the ability to synthesize and release lipid mediators of inflammation and secrete cytokines that serve to further enhance the local inflammatory response (Janeway et al., 2005)

PAGE 46

46 Eosinophils and basophils E osinophils and basophils are classified as granulocytes along with neutrophi ls Both types of cells contain cytoplasmic granules containing various enzymes, toxic proteins, and other vasoactive molecules. In contrast to neutrophils, basophils are preferentially recruited to sites of allergic inflammation and eosinophils appear to be involved in defense against parasites. Eosinophils are attracted to sites of mast cell degeneration and can be found in large numbers in tissues undergoing type I hypersensitivity reactions (Tizard, 2004) They have the capacity to phagocytize small particles, but their primary function is to destroy large extracellular particles through degranulation in response to IgE coated parasites, antigen bound IgE, chemokines, and other mediato rs. Eosinophils also have the capacity to produce LT and cytokines including IL 6 and TNF extravascular tissues, except when attracted by some T cell derived chemokines (Tizard, 2004) The cytoplasmic granules contained within the basophil are similar to that found in mast cells. Dendritic cells The dendritic cell similar to the macrophage, functions as a sen tinel cell for innate immunity. It does have the capability to eliminate foreign invaders through phagocytosis, but its primary function is to present antigen to T lymphocytes (T cells) ( Tizard, 2004) Thus, this cell type is also important for bot h the innate and acquired immune response The capac i ty of the dendritic cell to serve as an antigen presenting cell is much more efficient than the other two types of antigen presenting cells, macrophages and B cell s. Activated dendritic cells secrete cytokines that influence both innate and acquired immune responses, and they can determine how the immune system responds to the presence of foreign microorganisms (Janeway et al., 2005)

PAGE 47

47 Acquired Immune Function Innate immunity is an essential prerequisite for the initiation of an acquired immune response This is because lymphocytes key cells involved in the acquired immune response innate immune response. The inn ate response is essential for host survival, and animals that cannot mount an effective innate immune response will quickly succumb to infection and die. However, the innate immune system does not have the ability to recognize and destroy an infinite diver sity of antigens or to retain that ability. Only the acquired immune response can provide protection through specific recognition of antigens that may elude non specific defenses of innate immunity. In addition, the host response against a particular antig en strengthens during each subsequent exposure. The result is a complex and sophisticated system that provides the ultimate in host protection. Humoral immune responses Lymphocytes classified as B cells secrete antibodies, which are simply the secreted fo rm of the B cell receptor, and they serve as the primary mediator of humoral immunity. previously encountered pathogens through a complex clonal differentiation and e xpansion process, and antibodies play an important role in this process (Janeway et al., 2005) When nave B cells in lymphoid tissue are initially exposed to a pathogen via an antigen presenting cell, such as the dendritic cell, and co stimulated by an antigen specific helper T (T H ) cell, they differentiate into a ntibody secreting B cells called plasma cells. These plasma cells secrete antibodies that bind pathogens or their toxic products in the extra cellular spaces of the body. This primary response can take several days, and no circulating antibody may be detec table for

PAGE 48

48 up to a week (Tizard, 2004) Eventually, antibodies will appear in the serum and peak by 10 20 days post infection before they decline and disappear over a period of wk Upon subsequent exposure to the same antigen, th is lag period last s for no longer than 2 3 days and the amount of antibody in serum rises rapidly to a high leve l before declining slowly These specific antibodies may be detectable for months or even years aft er second exposure. A third expos ure to the antigen generates an even faster and more prolonged antibody response. The ability of the immune system to strengthen its response to repeated exposure to antigen forms the basis of vaccination. Vaccination is th e most efficient and cost effective way to control the incidence of infectious disease in domestic animals (Tizard, 2004) Horses are commonly vaccinated to protect against diseases whic h are endemic in the general horse population or that have a high several factors, including antigen content of the vaccine, type of adjuvant, and vaccination protoc ol (Holmes et al., 2006) Most vaccines require an initial series in which protective immunity is generated, followed by subsequent booster vaccines administered at inter vals necessary to maintain protective immuni ty at adequate levels. Assessment of serum antibody titers in response to vaccination has long been utilized as a relatively uncomplicated way to Cell mediated immune responses Cell mediated im munity can be defined as immunity that can be media ted only through lymphocytes and antigen presenting cells T cells are primarily responsible for the cell mediated immune response, which is dependent upon the direct interaction between the T cell and an antigen bearing cells recognized by the T cell T cells only recognize foreign antigens as peptide

PAGE 49

49 fragments bound to major histocompatability complex proteins. T C (CD8) cells have the most direct action, as they have the abili ty to kill an infected cell t hrough mechanisms involving enzymes that initiate the cleavage of host and viral DNA (Janeway et al., 2005) There are two types of T H (CD4) cells that play a more subtle role by means of effector cell activation T H 1 cells are involved in cell mediated cytotoxic and inflammatory reactions, and thorough the product ion of IL 2 and IFN macrophages, NK cells, and T C cells to kill bacteria residing in interior vesicles (Mossmann and Sad, 1996) The other type of T H cell, the T H 2 cell is speci alized to promote antibody production by B cells, especially IgE, and enhances eosin ophil proliferation and function. The T H 2 response is associated with strong allergic responses and the primary cytokines produced by T H 2 cells that mediate this response are IL 4 and IL 5 (Mossmann and Sad, 1996) The differentiation of T H towards the T H 1 or T H 2 phenotype is under direct cytokine regulation as t he developm ent of T H 1 cells is promoted by IL 12 and IFN while IL 4 promotes the development of T H 2 cells (Calder et al., 2002) There are several techniques tha t can be used to measure cell mediated immunity in animals. Intradermal injections with an antigen are the most commonly utilized in vivo technique, as measurement of the local inflammatory response is relatively simple. Injection with the lectin phytohema gglutinin (PHA) provokes a local inflammatory reaction characterized by infiltration with T cells (Tizard, 2004) ability to mou nt a cell mediated response without the need for prior sensitization, but the interpretation of this non specific response may be difficult (Tizard, 2004) One commonly employed in vitro test measures the proliferation of T cells in response to stimulation, typically

PAGE 50

50 with a T cell specific mitogen such as PHA or Concanavalin A (ConA). Measuring cytokine release by T cell s is another method of assessing one aspect of the cell mediated resp onse. Although these assays can be used to measure some aspects of cell mediated immunity, they cannot provide a complete picture on their own. However, when used in concert with other techniques, they can serve apacity to mount a cell mediated immune response. This discussion provided only a basic overview of immune function, as its complexity precludes an in depth and thorough discussion. When attempting to determine the effects of a single external factor suc h as nutrition on immune function, it is important to consider than no single assay or measurement is able to provide a clear answer to such a multifaceted question. In order to characterize the effects of nutrition on immune function, several different a spects of the immune response must be investigated. In addition, results obtained in studies must be interpreted carefully and with consideration of the diverse nature of the immune system and the variety of external factors which can influence the immune response Immunomodulatory Effects of Omega 3 and Omega 6 Fatty Acid S upplementation Fatty acids play diverse yet essential roles in all cells. They serve as substrates for energy metabolism, structural components of the cell membrane, precursors for eico sanoid synthesis, signaling molecules within and between cells, and regulators of gene expression (Yaqoob and Calder, 2007) The link between immune function and di etary omega FA lies within the incorporation of these dietary FA into immune cell membranes, and how a modified membrane

PAGE 51

51 Immune Cell Membrane Composition The FA content of peripheral blood mononuc lear cells (PBMC), a subset of immune cells that include all lymphocytes and monocytes, is often measured to confirm successful incorporation of dietary FA into immune cell membranes. In humans, the average content of ARA in PBMC is approximately 20% of to tal FA, EPA is 0.8%, and DHA is 2% (Miles et al., 2004a) It has been well documented in humans that dietary supplementation with either n 6 or n 3 FA results in altered PBMC membrane FA composition. For example, s uppl ementation of healthy human subjects with 2.1 g EPA + 1.1 g DHA/d from fish oil for 12 wk resulted in a four fold increase of PBMC EPA, a significant increase in DHA, and a slight but significant increase in ARA that reached a maximum plateau after 28 d of supplementation (Yaqoob et al., 2000) Similarly, increased ARA intake resulted in a higher proportion of ARA in PMBC (Thies et al., 2001b) Typically, the increase of n 3 FA in PBMC content occurs at the expense of n 6, especially ARA (Calder, 2007) Compared to investigations concerning EPA and DHA supplementation, relatively few studies have been performed that investigate the effect of LA on immune cell membrane composition (Calder 2001) Yaquoob et al. (2000) reported that supplementation with sunflower oil (77.2% LA) for 12 wk resulted in no alterations of PBMC PUFA content. This lack of effect may be explained by the fact that the avera ge human diet already contains large proportions of LA. It is possible that in humans, a significant dietary intervention is necessary to alter the PBMC FA profile using LA. Fewer studies have investigated the effect of dietary FA supplementation on PMBC F A composition in animals but the results agree with responses observed in humans. Thies et al. (1999) investigated the effect of feeding different oils to growing pigs on PBMC FA composition. In that study, supplementation with 5% sunflower oil for 40 d resulted in higher

PAGE 52

52 ARA and lower EPA and DHA in PBMC when compared to pigs supplemented with 5% fish oil. Similarly, fish oil supplementation in rats increased EPA and decreased ARA in PBMC compared with non supplemented controls, and linseed oil also increased EPA and decreased ARA in PBMC although to a lesser extent (Brouard and Pascaud, 1990) Because immune cell membranes contain larger amounts of ARA relative to EPA or DHA, ARA typically serves as the principal precursor for eicosa noid synthesis (Calder et al., 2002) produce eicosanoids, depending on the extent of modification. Direct supplementation with marine derived oils rich in EPA and DHA appear to be the most effective way to accomplish modification of membrane FA content, primarily due to the limited capacity for humans and other mammals to further elongate and desaturate ALA to longer chain PUFA. Eicosanoid production, however, is not the only immune cell function that is affected by dietary FA supplementation. In the following sections, different aspects of immune function that c an be affected by FA supplementation are first discussed as they relate to humans and other species, followed by a separate discussion of studies that have specifically been performed in the horse. Eicosanoid P roduction Much of the work to investigate the effect of n 3 FA supplementation on immune function has focused on eicosanoid production as these potent mediators of inflammation arise from the 20 carbon precursors ARA and EPA located in the cell membrane (Figure 2 4) Among the mix of eicosanoids pro duced by immune cells, PGE 2 and LTB 4 production by monocytes, macrophages, and neutrophils are the most predominant, depending on the specific cell type and the nature of the stimulus (Calder, 2006, 2007) In addit ion to mediating pro inflammatory effects such as increased vascular permeability and vasodil a tion, PGE 2 has been show n to induce

PAGE 53

53 the production of the inflammatory cytokine IL 6 and upregulate its own production through the induction of COX 2 (Bagga et al., 2003) LTB 4 also induces inflammation, as it is a potent chemotactic agent for leukocytes, increases vascular permeability, promotes hypersensitivity, induces bronchoconstriction, and promotes the production of inflammatory cytokines like TNF 1, and IL 6 (Calder, 2006) Recent studies have demonstrated, however, that PGE 2 may also have some anti inflammatory effects, as it inhibits the 5 LOX enzyme necessary f or LTB 4 production (Levy et al., 2001) Figure 2 4 Eicosanoid production from ARA and EPA. COX, cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; L T, leukotriene; PG, prostaglandin; TX, thromboxane Adapted from Calder, 2003. As previously discussed, increased consumption of dietary n 3 FA, particularly EPA and DHA, leads to increased incorporation of these FA into immune cell membranes, partially at the expense of ARA Trebble et al. (2003b) demonstrated that in healthy men supplemented with fish oi l supplying 0.3, 1, and 2 g EPA +DHA/d for four consecutive wk PGE 2 production by stimulated PBMC decreased in proportion to PBMC EPA content and increased in proportion to

PAGE 54

54 PBMC ARA content. In a similar study that investigated the dose response effect of EPA supplementation in both young and old men, increased incorporation of EPA in PBMC membranes was associated with decreased PBMC PGE 2 production (Rees et al., 2006) DHA supplementation at 6 g/d for 90 d resulted in a 60 75% decrease in the production of both PGE 2 and LTB 4 by PBMC (Kelley et al., 1999) Supplementation with 1.5 g ARA /d, on the other hand, increased PGE 2 and LTB 4 production in young men (Kelley et al., 1998) In human subjects with asthma, fish oil supplementation supplying 3.2 g EPA + 2 g DHA/d for three wk decreased LTB 4 and increased LTB 5 production by PBMC compared to asthmatic subjects consuming a placebo supplement (Mickleborough e t al., 2006) These authors concluded that fish oil supplementation could be utilized as a nonpharmacologic intervention for asthmatic subjects with exercise induced bronchoconstriction It has also been suggested that a lthough EPA is considered as a COX substrate for the synthesis of PGE 3 a less potent inflammatory mediator than PGE 2 its production occurs with very low efficiency or does not occur at all (James et al., 2000) An alternate approach to increasing EPA content in PBMC is feeding flaxseed or flaxseed oil, which contain approximately 55 60% ALA. Wallace et al. (2003) reported that supplementing flaxseed oil to human subjects successfully increased EPA content o f PBMC but did not affect PBMC function. Similarly, supplementation with 9.5 g ALA/d or 1.7 g EPA+DHA/d for 6 mo in human subjects increased PBMC EPA from baseline levels, but DHA decreased in the ALA supplemented group while it increased in the EPA+DHA su pplemented group (Kew et al., 2003a) Although ALA does appear to have the capacity to alter EPA PBMC content, the amount of ALA necessary to increase EPA concentration is relatively high and does not appear to alt er PBMC membrane PUFA concentration to the extent where eicosanoid

PAGE 55

55 production is appreciabl y affected H owever ALA has been show n to affect other immune cell function s In cattle supplemented with flaxseed, PGE 2 production by PBMC was not affected (Lessard et al., 2003; Lessard et al., 2004) In contrast, feeding pigs a diet containing 7% fish oil decreased PGE 2 production by alveolar macrophages (Fritsche et al., 1993) In addition, supplementation with fish oil, sunflower, and canola oil to growing pigs for 40 d resulted in decreased PGE 2 p roduction in cultured blood when compared to non supplemented pigs (Thies et al., 1999) I n dogs consuming fish oil, LTB 5 production by stimulated neutrophils was seven times higher than in dogs consuming corn oil (Hall et al., 2005) Cytokine Producti on Synthesis of inflammatory cytokines is regulated in part by PGE 2 and the 4 series LT and can also be affected by n 3 FA supplementation (Calder, 2006) Like eicosanoid production, the assessment of the eff ects of n 3 FA supplementation on cytokine production has been widely studied in humans and other animals Cytokines are proteins that serve as chemical messengers in the regulation of cell activities. Cytokines can influence the cell that produced them an d/or the activities of other cells. Cytokines bind to specific receptors on the target cell to induce changes in its growth, development, or activity. TNF 6 are two important cytokines produced by monocytes and macrophages that relay signals to facilitate a host of inflammatory outcomes The production of these cytokines is beneficial in response to infection, but inappropriate and exc essive production is a characteristic of pathological inflammatory conditions (Calder, 2001) There are mechanisms in place to counteract the actions of pro inflammatory cytokines, including the production of anti inflammatory cytokines such as IL 4, IL 10, TGF

PAGE 56

56 antagonist ic and soluble cytokine inhibitors that work to maintain an appropriate balance in the body. Inclusion of n 3 FA in the diet has been shown to affect the production of a range of diffe rent cytokines. In a study where the amount of supplemental fish oil was incrementally increased over a 12 wk period (up to 2 g EPA+DHA/d for the final 4 wk period) in healthy human males, production of both TNF 6 by stimulated PBMC was significan tly lower after the supplementation period (Trebble et al., 2003a) In addition, both TNF 6 production was negatively correlated with EPA concentration in PBMC p lasma and erythrocytes. Other studies supp lementing >2.4 g EPA+DHA/d have shown similar decreases in TNF in humans (Endres et al., 1989; Meydani et al., 1991; Caughey et al., 1996) Another study found that IL 6 but not TNF production by PBMC decreas ed after humans consumed 0.94 and 1.9 g EPA+DHA/d for 12 wk (Wallace et al., 2003) Thies (2001a) reported that a more modest amount of fish oil supplementation ( 1 g EPA + D HA /d) did not a lter either TNF 6 production by PBMC. Other studies have also shown that supplementation with 0.55 3.4 g EPA+DHA/d did not affect production of TNF 1, or IL 6 (Cooper et al., 1993; Calder, 2001) Th oil supplementation suppress TNF a less er or no effect. In addition, it can be difficult to compare studies due to different experimental protocols used, particularly those involving cell culture and cytokine assays (Calder, 2001) Not only can n 3 FA supplementation affect inflammatory cytokine production, but it has also bee n shown to affect other cytokines which play a role in T cell development and function. The suppression of IFN 2 production in both rodents and humans supplemented with

PAGE 57

57 fish oil has been demonstrated (Meyda ni et al., 1993; Gallai et al., 1995; Wallace et al., 2001) But in direct contrast to these observations, others have reported no effect of fish oil supplementation o n the production of these T cell cytokines (End res et al., 1993; Yaqoob et al., 2000) IFN H 1 cells and plays a role in macr ophage activation and suppression of T H 2 development, while IL 2 is a another cytokine produced by T H 1 cells that is required for the proliferation of T C cells and is central to the development of th e acquired immune response (Janeway et al., 2005) In mice, fish oil supplementation significantly increased the T H 2/T H 1 ratio through the direct suppression of T H 1 development and not through decreased in IFN production (Zhang et al., 2005) It appears that more than one mechanism may be responsible for the alter ations in T cell functions observed in response to dietary n 3 FA supplementation. In animals, similar results have been noted in regards to both the production of inflammatory cytokines and the T H 1 cytokines IFN 2. Fish oil feeding to rodents decreased production of TNF 6 by macrophages (Yaqoob and Calder, 1995; Wallace et al., 2000) In dogs supplemented with 1.75 g of EPA + 2.2 g DHA/kg of diet the serum of act ivity of IL 1 and IL 6 was significantly decreased 6 h after lipopolysaccharide ( LPS ) challenge when compared to dogs supplemented with sunflower oil, but TNF affected (LeBlanc et al., 2008) In contrast, Kearns et al. (1999) reported that supplementing fish oil to create a 5:1 n 6:n 3 ratio did not affect IL 1, IL 6 or TNF production in dogs In general, for both animal a nd human studies, it appears that a dose level of > 2g EPA+DHA/d is required to significantly alter cytokine production by T cells (Sijben and Calder, 2007) Lymphocyte Proliferation The effects of dietary n 3 FA supplementation on lymphocyte prolif eration (LP) have been reported in many studies T he measurement of the proliferative capacity of lymphocytes serves a

PAGE 58

58 good measure of their overall function and health. Agents used to stimulate proliferation can include either mitogens or specific antigen s, depending on whether or not the host has been previously sensitized to the antigen. Mitogen stimulation is non specific and targets the entire population of culture d lymphocytes. The mitogens Con A and PHA stimulate T cells, pokeweed mitogen stimulates a mixture of T and B cells, a nd bacterial lipopolysaccharide stimulates B cells (and monocytes). In most cases, cells to be utilized in proliferation assays are isolated from the subject as a purified preparation of whole blood ( i.e., PBMC) and in the hors e, the mixture contains both lymphocytes and monocytes in an approximate ratio of 10:1 (Lumsden et al., 1980; Fraser et al., 1991) Data from human studies inv estigating the effect on n 3 FA supplementation on LP are conflicting. In healthy human volunteers, Meydani et al. (1991) reported that providing 2.4 EPA+DHA/d decreased LP in older women, but not in younger women Similarly, in h uman subjects aged 55 7 5 yr 1 g EPA+DHA/d decreased LP compared to subjects consuming capsules containing either 2 g ALA/d or 0.6 g ARA /d. When either 3.5 g ALA/d or up to 1.9 g EPA+DHA/d was supplemented to healthy young adults, no effect on LP was observed (Wallace et al., 2003) While there is evidence in the literature to indicate that dietary n 3 FA can decrease LP more evidence exists to suggest that there is no significant effect when studies were performed in healthy human subje cts (Sijben and Calder, 2007) Many confounding factors exist for these studies, such as age, gender, culture conditions, and antioxidant supplementation status, which complicate the interpretation of results. Similar to what has been observed in h umans, conflicting lymphoproliferative responses have also been observed in animals supplemented with n 3 FA In cattle, Lessard et al. (2003)

PAGE 59

59 reported that flaxseed supplementation resulted in decreased LP in comparison to supplementation with either soybeans or Megalac (calciu m salts of palm oil). In contrast, the addition of 1.5% fish oil to a corn based diet increased LP in cattle when compared to cattle consuming the same base diet with no supplemental oil (Wistuba et al., 2005) In poultry, when chicks wer e supplemented with either 5% fish oil or flaxseed oil, LP decreased in both groups when compared to chicks supplemented with 5% sunflower oil (Wang et al., 2000) No effect was observed on LP when 7% fish oil was supplemented to the diet of pigs for 21 d (Liu et al., 2003) When interpreting and comparing results from different studies, it is important to consider the conditions of the study, including amount of FA supplemented, composition of the control treatments, and proliferation culture conditions. Phagocytosis and Oxidative B urst Neutrophil phagocytosis and subsequent oxidative burst is essential for an optimal innate immune response. In vitro studies have demonstrated that dietary induced alterations in neutrophil PUFA content can be associated with altered phagocytic capacity (Calder, 2007) Increased cell membrane PUFA content results in increased uptake of ta rget material by the neutrophil due to the altered physical nature of the membrane (Calder et al., 1990) However, the killing capacity of the neutrophil is also an important determinant of function, and measuring both phagocytosis and oxidative burst is i mportant in order eliminate foreign invaders. Similar to what has been observed for LP, results from dietary studies pertaining to neutrophil function are contradictory. This may partially be due to the methodological approach utilized in the study. Studies that measure the percentage of neutrophils that undergo phagocytosis might not detect a n effect of PUFA supplementation, because it is unlikely that

PAGE 60

60 supplementation will induce previousl y inactive cells or completely block active cells from engaging in the process (Calder, 2007) A measurement of the extent of phagocytic activity, i.e. the amount of target material that the neutrophil has engulfed, might represent a more sensitive measure of the effect of PUFA supplementation on neutrophil function. In a study of 150 non su pplemented individuals, Kew et al. (2003b) reported that neutrophil phagocytosis and oxidative burst was positively correlated with membrane n 3 FA content and negatively correlated with n 6:n 3 in the membrane This suggests that increasing membrane EPA and DHA content would enhance neutrophil function. However, this effect has rarely been demonstrated in human dietary intervention studies. On e study reported a 62% increase in phagocytic activity and a concomitant increase in the rate of reactive oxygen species production after 2 mo of supplementation with 3 g EPA+DHA/d (Gorjao et al., 2006) In contrast, m ost investigations have shown n 3 supplementation at levels ranging from 4.5 9.5 g ALA/d or 1 4.9 g EPA+DHA/d hav e no effect on either phagocytosis or oxidative burst (Thies et al., 2001a; Kew et al., 2003a; Kew et al., 2004; Miles et al., 2004a) One study demonstrated that supplementing either 2.7 g EPA/d or 4.05 g EPA/d to older men (>55 yr ) decreased neutrophil respiratory burst (Calder et al., 2002) In animals, m ost of the work that has investigated the ability of PUFA supplementation to affect phagocytosis has been performed with macrophages rather than neutrophils. In 7 d old rabbi ts, feeding 5 g/kg BW fish oil decreased superoxide and hydrogen peroxide production by macrophages, but not their capacity to under go phagocytosis (D'Ambola et al., 1991) In rodents, feeding 8% cod liver oil decreased macrophage generation of superoxide anions, hydrogen peroxide, and nitrite radicals compared to a diet supplemented with 5% co conut oil (Joe and

PAGE 61

61 Lokesh, 1994) Thies et al. (1999) examined the capacity of neutrophils from pigs fed 5% fish oil to phagocytize E. coli and found that feeding fish oil significantly decreased the percentage of neutrophils engaged phagocytosis. In contrast, another study in mice found that feeding up to 4.4 g DHA /d had no effect on neutrophil function, but feeding the same amount of EPA increased the ability of neutrophils to undergo phagocytosis (Kew et al., 2003c) The lack of consistent observations in both humans and animals prevents a clear conclusion from being drawn regarding the effect of dietary PUFA on neutrophi l function. Antibody P roduction B cells are responsible for producing specific antibodies in response to antigen stimulation. There are several regulatory mechanisms that can influence the capacity of the B cell to produce and secrete antibodies, including the nature of T cell stimulation, cytokine influence and efficiency of antigen presentation. Few studies have been performed in humans that specifically investigate the effect of PUFA suppl ementation on immunoglobulin production. Miles et al. (2004b) reported that supplementation with 2 g EPA/d to healthy young males resulted in lower circulating IgE and higher IgG 2 in comparison to unsupplemented males A decline in IgE occurred in all treatment groups during the study and may have been due to seasonal changes in the exposure to a stimulus that promotes IgE production. The increase in IgG 2 only occurred in the EPA supplemented group, and may be related to the promotion of T H 2 cytokine production and is suggestive of improved immun i ty. More research has been performed in animals to characterize the effect of PUFA supplementation on humoral immune response, but results have been conflicting. Sijben et al. (2001) performed a s tudy in poultry examining the effect of PUFA supplementation on antibody production in response to different antigens. The conclusion of that study was that effects on

PAGE 62

62 antibody production were different between antigens of a different nature and that the i nteraction of n 6 and n 3 FA is more important than the individual effects of those FA. Another study found no differences in antibody response to vaccination for infectio u s bronchitis virus in chickens supplemented with either corn or fish oil (Korver and Klasing, 1997) Similarly no effect on the primary and secondary antibody response to ovalbumin injection has been observed in cattle fed flaxseed (Lessard et al., 2003; Lessard et al., 2004) I n contrast an increased antibody response t o sheep red blood cell vaccination was observed in 1 d old chicks supplemented with 7% fish oil (Fritsche et al., 1991) In addition, chickens supplemented with 70 g sunflower oil (69% LA) per kg diet mounted an increased IgG antibody response to both pr imary and secondary immunization with bovine serum albumin (Parmentier et al., 2002) These observations suggest that effects on humoral immunity may be dependent on several factors, including the type of antigen u tilized for vaccination and the type and amount of PUFA supplemented to the diet. Gene Regulation and Expression It has been well documented that FA can affect the expression and regulation of genes involved in lipid metabolism (Sampath and Ntambi, 2005) More recently, studies have begun to focus on what effects PUFA may have on inflammatory gene expression and how this might affect immune function. In general, changes in immune cell membrane PUFA content can ulti mately lead to the modification of gene expression and s everal mechanisms have been proposed that may be responsible for this modification. On e mechanism of a structural nature is via alterations in lipid rafts, which are specialized microdomain s in the c ell membrane enriched in cholesterol, sphingolipids, and glycolipids where different membrane proteins preferentially associate. These rafts facilitate the production of signaling molecules and the interaction between

PAGE 63

63 T and B cells, and membrane PUFA modi fications can cause protein displacement that results in altered function (Yaqoob and Calder, 2007) Other proposed mechanisms involve the modification of intracell ular transduction mechanisms that can alter activation of transcription factors and subsequent gene expression (Calder, 2007) and subsequent up or down regulation of genes related to immune function have been demonstrated, and these r elationships are outlined in Figure 2 5 Figure 2 5 Mechanisms by which n 3 PUFA may influence gene expression. Adapted from Miles and Calder 1998.

PAGE 64

64 It is apparent that the regulation of immune function by PUFA is mediated through a variety of proces ses. These complex relationships sometimes make it difficult to predict specifically how dietary PUFA intake will influence an individual aspect of immune function. In addition, differences due to amount and type of fat supplemented, assay conditions, spec ies, and many other exogenous variables will affect study outcome. This underlines the importance for carefully designed studies to identify the effects of PUFA supplementation on immune function in the species of interest. Omega 3 Fatty Acid Supplementati on in the Horse Until recently, s tudies that have investigate d n 3 FA supplementation in horses have been lacking. Compared to studies performed in humans and other species there has been relatively little horse specific research pertaining to PUFA supple mentation and immune function Most of the research conducted in horses thus far has placed the greatest emphasis on determining the ability of dietary n 3 FA to attenuate the production of inflammat ory mediators E ffect s on other aspects of innate and acq uired immune function have not been investigated. Because the practice of feeding fat added diets has become commonplace in the industry, determining how this fat tial associated risks and/or benefits brought about by fat supplementation. The primary sources for supplementary n the whole seed, ground or as oil) and fish oil (fed as oil or as a dry encapsulated fish oil product). Flaxseed has historically been fed by horsemen for decades, due to its reputation for creating a shiny hair coat It is generally well accepted by horses, and unlike fish oil, is not associated with odors that horse owners may find unplea sant. There has been great debate among horseman as to the ideal way to feed flaxseed. Feeding whole flaxseed is a safe and acceptable

PAGE 65

65 practice, however some insist that the flaxseed should be ground and/or stabilized in order to reap the full benefits of flaxseed supplementation The practice of grinding and stabilizing flax may indeed be beneficial, as grinding disrupts the small, hard outer seed coat, making the seed contents potentially more available for digestion in the small intestine Feeding ground flax at a 15% inclusion rate in the diet of layer hens significantly increased the total n 3 FA content of egg yolks in comparison to feeding the same amount of whole flax (Aymond and Van Elswyk, 1995) When 5% whole or ground flaxseed was fed, however, t here was no difference in egg yolk n 3 content. Stabilization of ground flaxseed is intended to reduce the susceptibility of PUFA oxidation. Manufacturers achieve this through different methods, including selecting mature and evenly colored seeds for use in the milling process, the addition o f antioxidants, or by employing further processing methods to protect the fat in the seed. ill et al. (2002) reported that evenly colored seeds are a marker for a highly stable milled flaxseed product, as a flaxseed mixture containing 25% dark colored seeds had peroxide levels (indicative of fat oxidation) over 100 times greater than a mixture containing 2% dark colored seeds. O thers claim that flaxseed must first be cooked before it can be fed to horses. Flaxseed contains cyanogenic glycosides which are nitrogenous secondary plant metabolites de rived from amino acids that can interact with enzymes contained within the seed to release cyanide (Oomah et al., 1992) For this rea son, it is common practice in the field to boil flaxseed before feeding it to the horse in order to release the highly volatile cyanide. However, it is highly unlikely that harmful levels of cyanide are released upon ingestion of flaxseed by the horse, due to the ability of stomach acid to inactivate the enzymes contained within the seeds (O'Neill et al., 2002)

PAGE 66

66 Feeding fish oil products to horses ha s sometimes been met with resistance by owners due and the increased expense and limited availability of fish oil as compared to other oil sources. Some m anufacturers that market fish oil specifically for horse consumption have been able to successfully reduce or even eliminate the and perhaps more importantly the owners, may find more palatable. The popularity of feeding fish oil preparations to horses has grown in recent years, and availability of these products has improved. Many companies currently offer fish oil specifically ma de for horses, including OmegaEquis (Kentucky Equine Research, Versailles, KY). Currently, there are only a few dry encapsulated fish oil supplements commercially manufactured BioNutrition, Sheridan, IN) and DHA EQ (Med Vet Pharmaceuticals, Eden Prairie, MN ). Blood and Ot her Physiological Responses to Omega 3 Supplementation In order to determine the effects of flaxseed supplementation o n plasma concentration, Hansen et al. (2002) fed horses a 10% flaxseed oil enriched complete pellet and grass hay in a ratio of 80:20 pellet:hay for 16 wk The flaxseed oil portion of the diet would have supplied a n estimated 106 g n 3 /100 kg BW At the end of the feeding period, horses consuming the flaxseed oil had increased plasma LA, ALA, and EPA but no difference in ARA or DHA, compared to horses consuming no supplemental fat. On the other hand, Siciliano et a l. (2003) reported that feeding a ground flaxseed sup plement that provided 5.56 g n 3 /100 k g BW did not affect plasma ALA content after 28 d of supplementation. In a study that supplemented pregnant mares with either rapes eed (31% LA, 12% ALA ; supplying 35.4 g n 6/100 kg BW and 7.4 g n 3/100 kg BW ) or linseed (25% LA, 46% ALA ;

PAGE 67

67 supplying 32.8 g n 6/100 kg BW and 16.6 g n 3/100 kg BW ), it was reported that milk from mares supplemented with linseed were higher in ALA (Duvaux Ponter et al., 2004) In addition, f oals from linseed supplemented mares had higher plasma ALA 48 h a fter birth compared to foals from rapeseed supplemented mares. Similarly, Spearman et al. (2005) reported the ALA content of milk was greater in mares supplemented with a 1:1 linseed/corn oil blend providing 66.1 g n 6 and 28.9 g n 3 / 100 kg BW compared to mares fed corn oil, but it was not different than milk from mares not supplemented with oil. Foals suckling mares fed the linseed/oil blend also had higher plasma ALA than foals from mares fed corn oil. Stelzleni et al. (2006) reported higher milk ALA in ma res supplemented with flaxseed (providing 6 g n 3/ 100 kg BW) compared to non supplemented mares, and foals suckling those mares had significantly higher plasma ALA than foals nursing mares that were fed encapsulated fish oil or no additional fat. In addit ion to plasma and milk FA composition, the effect of dietary fl axseed s upplementation on the FA composition of monocyte membrane s has also been reported. After 8 wk of feeding a complete pelleted ration supplemented with 8% linseed oil (estimated to provid e 55.2 g n 3 /100 kg BW) monocyte membrane LA and ARA w ere lower in horses supplemented with linseed compared to unsupplemented horses (Henry et al., 1990) The ALA and EPA membrane concentrations in monocyte membr anes were numerically greater in linseed supplemented horses but not significantly different between the two groups. In a different study, intravenous infusion with a 20% lipid emulsion rich in n 3 or n 6 FA for 7 d was shown to elicit changes in monocyte lipid composition that reflected the incorporation of parenteral FA 8 h after infusion and persisted for 7 d (McCann et al., 2000)

PAGE 68

68 Supplementing horses with fish oil has also been shown to alter blood lipid compo sition. (2007) fed horses fish oil supplying 6 g n 3/100 kg BW or an equal amount by weight of corn oil and reported that fish oil resulted in increased serum EPA and DHA decreased serum TG and de creased serum cholesterol concentration compared to horses supplement ed with corn oil Results of a nother study were interpreted to claim cholesterol lowering effects of n 3 FA supplementation in horses, but the design of the study brings the interpretatio n of the results into question. In this study, soybean oil (7% ALA, 51% LA) was used as the source of n 3 FA F eeding a diet supplemented with 10% soybean oil estimated to provide 51 g n 6 and 7 g n 3/100 kg BW as described in a companion study (Wilson et al., 2003) decreased serum cholesterol concentrations in comparison to feeding a diet supplemented with 10% corn oil (Howard et al., 2003) The number of horses utilized in th e study was very small (n=9) and the FA composition of the oils and erum were not reported making it difficult to attribute the ir results to the n 3 FA content of the soybean oil as opposed the effect of the increased n 6 content of corn oil or the soybean oil itself In a study providing 9.9 g n 3/100 kg BW by and ARA concentration in plasma was increased after 12 wk compared to horses supplemented with 3% corn oil (Hall et al., 2004b) When supplementing horses with a fish oil supplement that supplied approximately 5.4 g EPA+DHA/ 100 kg BW ( plus 5 g D tocopherol acetate and 5 g copper orotate daily) de Moffarts et al. (2007) reported similar increases in plasma EPA and DHA and a decrease in plasma n 3:n 6 when compared to placebo supplemented horses aft er 3 wk of supplementation.

PAGE 69

69 In addition to fish oil, seal blubber oil has also been fed to horses as a source of n 3 PUFA (Khol Parisini et al., 2007) Compared to fish oil, seal blubber oil contains a higher prop ortion of DPA which is the elongation product of EPA just prior to desaturation to DHA (Fig 2 2) Studies in rats have found that after absorption, n 3 FA derived from seal blubber oil are preferentially bound to the sn 1 and sn 3 positions of the glycero l backbone, potentially increasing their availability to lipoprotein lipase in contrast to fish oils that contain n 3 FA attached mainly at the sn 2 position (Christensen and Hoy, 1996) In horses supplemented for 10 wk with seal blubber oil that supplied approximately 7.3 g EPA+DHA/100 kg BW, plasma LA decreased and plasma ARA, EPA, DPA, and DHA increased compared to horses supplemented with sunflower oil (Khol Parisini et al., 2007) In addition, horses supplemented with seal blubber oil had lower LA and higher ARA and EPA in leukocyte membranes A trend for seal blubber oil to increase leukocyte DHA content was noted but the oil had n o effect on leukocyte DPA. The authors s tated this latter finding suggests that horses have t he ability to convert the high level of DPA in seal blubber oil to DHA, as occurs in humans (Khol Parisini et al., 2007) Encapsulated fish oil is another source of EPA and DHA that has successfully been fed to horses. It differs from traditional liquid fish oil s in that it comes in the form of a dry pellet and contains approximately 23% fat. The encapsulation process is utilized to stabilize the PUFA of the fish oil and to improve palatability King et al. (2008) fed three different levels of a protected FA supplement to horses supplying 10, 20, and 40 g EPA+DHA/d (estimated to provide 2 g, 4 g, or 8 g EPA+DHA/100 kg BW, resp ectively) i n order to determine how the circulating FA profile was influenced by supplementation Plasma EPA and DHA increased in a dose responsive manner, and plateaued

PAGE 70

70 after 7 d of supplementation. After the cessation of supplementation, EPA and DHA bega n to decline after 9 d and plasma FA had almost reached pre supplementation concentrations by 42 d. In a companion study, it was reported that red blood cell EPA and DHA content did not increas e in supplemented horses until a fter 23 d of supplementation a nd remained elevated 59 d following the cessation of supplementation (King et al., 2005) Stelzleni et al. (2006) reported that feeding encapsulated fish oil to lactating mares at a rate of 6 g n 3/100 kg BW increased plasma EPA, DHA, and total n 3, whic h was also reflected in their milk and in the plasma of their foals. These results agree with a similar study that found supplementing mares with 19 g EPA+DHA/d (estimated to provide 3.5 g EPA+DHA/100 kg BW) increased plasma EPA and DHA, decreased plasma A RA and ALA, and increased foal plasma EPA and DHA when compared to mares consuming corn oil (Kruglik et al., 2005) The e ffect of fish oil supplementation on reproductive function ha s also been evaluated in horses In a study performed in pregnant mares, encapsulated fish oil supplementation increased not only plasma EPA and DHA, but also increased the time to first postpartum ovulation and increased follicle retention during the first postpartum estrus period (Poland et al., 2006) I n stallions, DHA is important to normal spermatozoa function and t he deficie ncy of long chain n 3 FA especially DHA in the spermatozoa plasma membrane is one marker of impaired fertility in men (Conquer et al., 1999) Harris et al. (2005) fed stallions an encapsulate d fish oil product that provided 29.1 g n 3 /d (estimated to supply 5.8 g n 3/100 kg BW) and reported a n increased daily sperm output a higher percentage of mo rphologically normal sperm and increased s perm plasma membrane DHA concentration after 90 d of s upplementation. Based on dramatic improvements observed in individual horses that initially had very poor semen characteristics,

PAGE 71

71 the study authors concluded that stallions with poor quality ejaculates may benefit from n 3 FA supplementation. S imilar result s were observed when stallions were supplemented with 18.8 g n 3/d (estimated to supply 3.8 g n 3/100 kg BW) of a DHA rich encapsulated fish oil product (25% DHA, 5% EPA) (Brinsko et al., 2005) In that study, the concentration of sperm ejaculate and semen DHA content w ere greater after 14 wk of supplementatio n compared to the semen characteristics of non supplemented stallions. In addition, both the total and progressive motility of sperm after 48 h of cooling and storage was improved by fish oil supplementation. The authors of this study concluded that supple ment ing highly fertile stallions may not be warranted, but stallions of marginal fertility or ones with semen that does not tolerate cooling and storage well would likely benefit the most from supplementation. Effects on Immune Function One of the first s tudies to investig at e n 3 FA supplementation and immune cell function in the horse reported that monocyte procoagulant activity and TXB 2 production decreased by 51% and 71%, respectively, but LTB 4 did not change after 8 wk of supplementation with 8% linsee d oil (p roviding an estimated 55.2 g n 3 /100 kg BW) (Henry et al., 1990) In a companion study, Morris et al. (1991) reported a decrease in endotoxin induced TNF macrophages after 8 wk of linseed oil su ppl ementation (estimated to supply 55.2 g n 3 /100 kg BW) compared to that seen pr ior to the initiation of supplementation A major flaw of this study, however, was that there was no control group included in the design, so results must be interpreted with cau tion. More recently, Hall et al. (2004a; 2004b) sup plemented horses with 3% fish oil (estimated to supply 9.9 g n 3/100 kg BW) or 3 % corn oil and measured several indices of inflammation Supplementation of horses with fish oil resulted in greater production of LTB 4 and LTB 5 by

PAGE 72

72 neutrophils and reduced PG E 2 production by bronchoalveolar lavage ( BALF ) cells compared to observations in horses fed corn oil. No differences were found between treatments for the delayed t ype hypersensitivity skin test response to keyhole limpet hemocyanin (KLH), antibody production to KLH in response to vaccination, and TNF The authors concluded that fish oil could have value in the treatment of equin e recurrent airway obstruction (RAO) due to the decreased PGE 2 production by BALF that was observed in supplemented horses. In a study specifically designed to examine the effects of n 3 supplementation in horses with RAO, no improvements in several measur es of pulmonary function and no improvement in clinical signs were observed after supplementation with seal blubber oil for 10 wk (providing approximately 7.3 g EPA+DHA/100 kg BW) in comparison to horses fed sunflower oil (rich in LA) (Khol Parisini et al., 2007) Skjolass Wilson et al. (2005) observed lower PGE 2 production by leukocytes when mares were fed encapsulated fish oil (estimated to supply 3.5 g n 3/100 kg BW) compared to mares fed corn oil but did not observe differences in neutrophil phagocytosis or oxidative burst fu nctions between treatment groups T he acute phase protein fibrinogen has been utilized to assess non specific inflammation after strenuous exercise and at rest in horses supplemented with oils rich in n 6 or n 3 FA. Wilson et al. (2003) fed horses either a non fat supplemented control diet, 10% corn oil, or 10% soybean oil (estimated to supply 10.4 g n 3/100 kg BW) for 4 wk and found that corn oil supplementation resulted in higher fibrinogen l evels at rest and after exercise F ibrinogen levels did not differ between h orses fed soybean oil o r no supplemental fat. Although in vitro methods of measuring immune function are important for gaining basic knowledge of how dietary n 3 FA can affect i nflammatory processes, evaluation of in vivo

PAGE 73

73 response is essential in order to determine clinical significance. O nly a handful of equine studies have attempted to quantify the anti inflammatory effects of n 3 FA supplementation using an in vivo model. Henr y et al. (1991) observed a longer mean whole blood recalcification time and activated partial thromboplastin time in response to endotoxin infusion when horses received a diet containing 8% linseed oil (estimated t o provide 55.2 g n 3/100 kg BW) but the re was no difference in the production of inflammatory eicosanoid s or the occurrence of clinical signs of discomfort, including transient anorexia, ileus, sweating, and fine muscular fasciculations. In addition, ther e were no differences between treatment groups for rectal temperature, heart rate, or respiratory rate measured up to 24 h post infusion (Henry et al., 1991) In a small pilot study six horses known to have Culico ides hypersensitivity were injected with Culicoides antigen and it was shown that lesion size was reduced after flaxseed supplementation that supplied 55 g n 3/100 kg BW (O'Neill et al., 2002) In contrast to these findings, Friberg and Logas (1999) reported in a study of 17 horses with Culicoides hypersensitivity that 6 wk of supplementation with 200 mL linseed oil (providing 22.3 g n 3/100 kg BW) did not alter the l evel of pruritis and l esional surface as compared to horses supplemented with an equivalent amount of corn oil However, the study did not include a non fat supplemental control treatment, so these results must be interpreted accordingly. Some researchers have attempted to determine if feeding PUFA influences the immunoglobulin content of colostrum and subsequent ly the foal plasma. In one study, mares were supplemented with either a high fat and fiber or high sugar and starch concentrate throughout gestatio n and lactation, and colostrum IgG levels were higher in the mares fed fat and fiber than in mares fed sugar and starch (Hoffman et al., 1998) Kruglik et al. (2005) specifically

PAGE 74

74 investigated the effect of n 3 FA supplementation on colostrum immunoglobulin content. In that study, it was reported that mares fed encapsulated fish oil providing approximately 3.5 g n 3/100 kg BW had higher colostrum IgG than mares fed corn oil, but no differences were noted in foal plasma IgG content between treatment groups. Duvaux Ponter et al. (2004) reported no effect on foal plasma IgG when mares were supplemented with linseed that provided approximately 16.6 g n 3/100 kg BW as opposed to rapeseed (which is approxima tely 9% ALA) Stelzleni et al. (2006) supplemented mares with flaxseed or encapsulated fish oil in amounts to provide 6 g n 3/100 kg BW and found no effect on foal plasma IgG, although mares fed fish oil did exhibit lower colostrum IgG. Collectively, t hese results suggest that fa t supplementation has the potential to affect the immunoglobulin content of colostrum, but more studies should be performed to confirm these findings One highly promoted effect of n 3 FA supplementation in the equine feed industry has been its ability to benefit horses with arthritis. These claims are primarily based on findings from research performed in humans with rheumatoid arthritis that suggest fish oil supplementation may improve symptoms (Cleland et al., 198 8; Lau et al., 1993; Berbert et al., 2005) Studies in horses, however, are extremely limited and the results must be interpreted with caution, as the etiology of rheumatoid arthritis and degenerative arthritis are very different. In a study where horses were supplemented with either encapsulated fish oil providing 15 g EPA+DHA/d (resulting in an estimated 3 g n 3/100 kg BW) or with 49 g corn oil for 75 d, horses supplemented with encapsulated fish oil tended to have a longer trot stride than horses supple mented with corn oil (Woodward et al., 2005) Due to the lack of significant results and the fact that no other indices of inflammation were repo rted no strong conclusions c an be made

PAGE 75

75 from the observations in this study. One in vitro experiment performed on equine synovial explants reported that treatment w ) increased ALA content of the explant cell membranes and inhibited PGE 2 production after an LPS challenge (Munsterman et al., 2005) However, it is unclear how the ALA dose used in this experiment relates to physiological levels o f dietary ALA that could potentially reach the synovial fluid. These results, although useful as preliminary data, do not offer strong evidence for the attenuation of arthritic inflammation in horses supplemented with n 3 FA. One study has attempted to mea sure in vivo effects of n 3 FA supplementation on synovial fluid. Manhart et al. (2007) supplemented 16 arthritic horses with either encapsulated fish oil providing 34.8 g EPA+DHA/d (estimated to supply 7 g n 3/100 kg BW) or a control treatment with no supplemental fat for 90 d. Horses fed the fish oil had a reduction in total white blood cells ( WBC ) in synovial fluid of arthritic joints decreased plasma PGE 2 concentration and exhibited a tendency toward reduced fibrinogen concentrations. The authors concluded that n 3 FA supplementation would benefit horses with arthritis ; however, the inflammatory markers measured in this study were non specific in nature and no improveme nt in clinical signs was reported. In order to definitively claim beneficial effects of n 3 FA supplementation for arthritic horses, more carefully designed research needs to be carried out. The lack of consistency between studies in the literature highli ghts the need for additional investigation into the effect of FA supplementation on overall immune function in the horse Differences between studies may be attributable to several factors, including the type of n 3 FA supplemented, the amount supplemented length of supplementation, and the immunological response variables measured. Unfortunately, the use of oils with a high LA content as the control

PAGE 76

76 treatment in many of these studies does not provide clear support for the attenuation of inflammation in re sponse to dietary n 3 FA supplementation. Corn and sunflower oils are high in n 6 FA, therefore it is difficult to determine whether the n 6 FA (provided by corn or sunflower oil) or the n 3 FA (provided by fish oil, flaxseed, or linseed oil) contributed t o the physiological response when two such dietary treatments were compared. Nonetheless, data in humans and other species provide convincing evidence that the FA composition of fats play an important role in immune function. Therefore, t he objectives for the studies included in this dissertation set out to determine : 1) if fish oil and flaxseed supplemented in amounts to provide the same amount of total n 3 FA affects plasma and red blood cell FA composition differently ; 2) the effect of fish oil supplemen tation on plasma and red and white blood cell membrane FA composition ; 3 ) the clearance rate of dietary n 3 FA in plasma and red blood cells after the cessation of supplementation and 4) how aspects of both innate and acquired immune function are affected by n 3 FA supplementation as part of either a low or high fat diet.

PAGE 77

77 CHAPTER 3 EFFECT OF DIETARY OM EGA 3 FATTY ACID SOURCE ON PLASMA AND RED BLOOD CELL FATTY ACI D COMPOSITION AND IM MUNE FUNCTION IN YEA RLING HORSES Abstract In order to determine the effec t of different sources of dietary omega 3 (n 3) fatty acids (FA) on plasma and red blood cell (RBC) FA composition and immune response 18 Quarter Horse yearlings were randomly and equally assigned to one of three treatments: encapsulated fish oil (FISH, n =6), milled flaxseed (FLAX, n=6), or no supplementation (CON, n=6). FISH contained 15 g eicosapentaenoic acid (C20:5n 3; EPA) and 12.5 g docosahexaenoic acid (C22:6n linolenic ac id (C18:3n 3; ALA) per 100 g FA Horses had free access to bahiagrass pasture during the active growing season and were individually fed a grain mix concentrate at 1.5% BW/d. FISH and FLAX were mixed into the concentrate in amounts to provide 6 g total n 3/100 kg BW. Horses were fed their r espective treatments for 70 d Blood sampl es were obtained on d 0 and d 70 f or determination of plasma and red blood cell ( RBC ) FA composition and for isolation of peripheral blood mononuclear cells (PBMC). PBMC were stimulated with Concanavalin A and phytohemagglu tinin (PHA) for determination of lymphocyte proliferation (LP). PBMC collected on d 70 were also challenged with lipopolysaccharide (LPS) to determine PGE 2 production. On d 70, horses were injected intradermally with PHA, and skin thickness and area of swe lling were evaluated over a 48 h period to assess in vivo inflammatory response. Treatment did not affect BW gain, which averaged 0.6 0.03 kg /d Horses fed FISH had a higher proportion of EPA, DHA, and sum n 3 in plasma and RBC (P<0.05) Plasma arachidon ic acid was higher (P<0.05) and linoleic acid and ALA in FISH was lower (P<0.05) compared to

PAGE 78

78 FLAX and CON. Dietary treatment did not affect LP or PGE 2 production. Across treatments, peak increase in skin thickness was observed between 4 8 h after PHA inj ection. At 4 h post injection, FISH and FLAX had a greater increase in skin thickness than CON (P<0.05) and FISH had a larger area of swelling than CON at 4 h (P<0.05). Skin thickness remained greater (P<0.05) in FLAX than CON 6 h after injection. Although fed to supply a similar level of n 3 FA FISH had a greater impact on plasma and RBC n 3 FA content than FLAX. However, supplementation with both FISH and FLAX resulted in a more pronounced early inflammatory response to PHA injection as compared to unsup plemented horses. Introduction Recent research in humans and animal s suggests that dietary omega 3 (n 3) fatty acid (FA) supplementation may exert immunomodulatory effects, most notably through altered inflammatory mediator production (Calder and Grimble, 2002) Specifically i n the horse, fish oil supplementation increased production of the eicosanoids leukotr iene B 4 and B 5 by peripheral blood neutrophils over that observed in horses fed corn oil (Hall et al., 2004b) Supplementation of horses with l inseed oil decreased endotoxin induced tumor necrosis factor ( TNF ) by peritoneal macrophages compared to production before supplementation (Morris et al., 1991) In addition, linseed oil supplementation decreased thromboxane B 2 production but had no effect on leukotriene B 4 production by monocytes compared to unsupplemented horses (Henry et al., 1990) To date, there have been no studies in the horse that have compared the immun omodulatory effects of fish oil and flaxseed. Additionally, many studies in the horse have used corn oil supplementation as t he control treatment. Because corn oil is high in om ega 6 (n 6) FA it is difficult to determine whet her it was the n 6 or n 3 FA that contributed to the

PAGE 79

79 differences observed in physiological response when the two treatments were compared. Utilizing a non fat added but isocaloric, control diet in experiments that aim to identify effects of fat supplementation is important to determine if the physiological effects that occur in the body are due to the increased overall dietary fat intake or from the specifi c FA supplemented to the diet. The n 3 FA in fish oil are primarily comprised of the long chain PUFA eicosapentaenoic acid (EPA; 20:5n 3) and docosahexaenoic acid (DHA; 22:6n 3) whereas t he primary n 3 FA contained linolenic acid (ALA; 18:3n 3). These two ingredients are the primary means by which n 3 FA can be supplemented in equine diets. When incorporated into cell membranes, EPA and DHA have more potent immunomodulatory effects than ALA due to thei r ability to alter membrane fluidity and cell signaling cascades and EPA a n eicosanoid precursor. The capacity for the horse to bioconvert ALA to EPA and DHA through elongation and desaturation has not been determined, but in humans, the bioconve rsion rate of ALA to EPA is less than 10% and ALA to DHA is less than 0.10% (Williams and Burdge, 2006) Cao et al. (2006) compared the effects of flaxseed and fish oil supplementation on plasma and red blood cell ( RBC ) FA composition in human subje cts and reported that fish oil, but not flaxseed supplementation for 8 wk increased plasma EPA and DHA. Flaxseed supplementation resulted in a 33% increase of RBC EPA above pre supplementation levels and did not affect RBC DHA, while fish oil increased RB C EPA by 300% and DHA by 42% In horses, supplementation with equal amounts of n 3 FA provided from flaxseed or encapsulated fish oil, the proportion of plasma n 3 FA only increased in horses fed encapsulated fish oil (Siciliano et al., 2003; Stelzleni et al., 2006) In addition, studies in the horse have demonstrated that feeding n 3 FA in the form

PAGE 80

80 of fish oil significantly increases the concentration of circulating EPA and DHA in plasma (Hall et al., 2004b; O'Connor et al., 2007; King et al., 2008) Although flaxseed is supple mented to horses with the intention of supplying the horse with beneficial n 3 FA, it is unclear if the ALA present in flaxseed will significantly affect plasma and RBC n 3 FA content. The objective of this experiment was to test the hypothesis that supple mentation with encapsulated fish oil will have a greater impact on plasma and red blood cell (RBC) membrane FA composition than flaxseed supplementation. Furthermore, immune response was measured to determine if n 3 supplementation affects lymphocyte proli feration, PGE 2 production, or response to intradermal phytohemagglutinin (PHA) injection and if response is dependent upon the source of n 3 FA supplemented. Materials and Methods Horses Eighteen Quarter Horse yearling fillies (n=9) and geldings (n=9) with a meanSE age of 14.60.2 m o and initial BW of 391.5 5.2 kg were utilized in this study All experimental protocols were approved by the Institutional Animal Care and Use Committee at the Uni versity of Florida During the trial, fillies and geldings wer e housed separately in two adjacent 8 ha pastures at the Institute of Food and Agriculture Sciences Hors e Research Center in Ocala, FL Dietary Treatments Horses were blocked by sex and age and randomly and equally assigned to one of thre e dietary treatmen ts for 70 d : encapsulated fish oil (FISH, n=6; JBS United, Sheridan, IN), milled Angusville, MB, Canada ), or no supplementation (CON, n=6). The basal diet consisted of a no n fat added grain mix concentrate (Gest O Lac ; OBS Feeds, Ocala, FL) fed individually in feeding pens at 1.5% BW split into two meals at 0700 and

PAGE 81

81 1500 h The daily amount of supplement was equally divided between the morning and evening meals and mixed into the basal grain ration. Based on previous r esearch that demonstrated an observable response to n 3 FA supplementation (O'Connor et al., 2004) FISH and FLAX were provided in amounts to provide 6 g total n 3/100 kg BW Horses were allowed free choice grazing access to bahiagrass ( Paspalum nota tum ) for the duration of the trial (May 2005 August 2005) which was during the active growing season. Horses had been maintained on a diet consisting of the same basal grain mix and pasture for at least 6 mo prior to the start of the study. The nutrient and FA composition of the feeds and supplements are presented in Table 3 1. Sample Collection T o allow adequate time for sample processing h orses were equally divided into three sampling groups with treatments balanced between groups and BW measurement s and blood samples were obta ined over three consecutive d ays Bodyweight measurements and blood samples were obtained prior to ( d 0) and after 35 and 70 d of supplementation On each day of sampling, a pproximately 40 mL of b lood was collected from each ho rse by jugular venipuncture into evacuated tubes containing the anticoagulant sodium heparin (Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ) Tubes were continually mixed by gentle inversion until processing. Blood samples were centrifuged for 10 mi n at 1000 x g at 22C (room temperature). Plasma was removed and stored at 80 C until later analysis. The buffy coat was removed, diluted with PBS, and slowly layered over lymphocyte separation medium (LSM; MP Biomedicals, Solon, OH) in order to isolate p eripheral blood mononuclear cells (PBMC ; see Appendix A ). PBMC were counted and re suspended in freezing media consisting of 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide and stored in liquid nitrogen. The RBC remaining in the blood sample

PAGE 82

82 were i solated by repeated centrifugation and washing with normal saline until al l residual plasma was removed and stored at 80C until later analysis (s ee Appendix B) Feedstuff, Plasma and Red Blood Cell Fatty Acid Composition To analyze FA composition of the diets feed samples were lyophilized (FreeZone 6 Liter Freeze Dryer System, Labconco Corp, Kansas City, Mo) and ground and representative samples consisting of 2 g grass, 0.5 g grain mix, or 0.2 g flaxseed or encapsulated fish oil were utilized for FA ext raction For analysis of blood FA 2 mL of plasma and 4 mL of RBC were frozen at 20 C in 4 mL Wheaton polypropylene Omni vials, lyophilized, sealed with snap caps and stored at 20 C. A mixed reference standard containing 33 FA methyl esters (FAME) (GLC 4 61, Nu Chek Prep, Elysian, MN) was reconstituted in 10 mL hexane. FA in freeze dried feedstuffs, plasma and RBC were extracted and methylated using the procedure of Folch et al. (1957; see Appendix D) A CP 3800 Ga s Chromatograph with a CP 8400 autosampler and i njector, a split injection port, flame ionization detector (Varian, Inc, Walnut Cr eek, CA), and a 100 m CP SIL 88 fused silica capillary column (0.25 mm i.d. x 0.2 mm film thickness; Varian, Inc, Walnut Creek, CA), were used for the analysis of individual FA. An injection of 1.0 L was split 1:20 and the He carrier gas maintained at 1.0 mL/min. Column temperature was held at 120C for 1 min following injection, increased at the rate of 5C/min to 190C and held at that temperature for 30 min, then increased at the rate of 2C/min to 220C and held for 50 min. The injector temperature was set at 250C and the detector at 255C. Identification of 21 FAME ( C8:0, C10:0, C12:0, C14:0, C14:1, C16:0, C16:1 n 7 C17:0, C17:1, C18:0, C18:1n 9, C18:2n 6, C18:3n 3, C20:0, C20:1, C20:2, C20:4n 6, C20:5n 3, C22:0, C22:5n 3, and C22:6n 3) from the chrom atograms were determined by comparing peak retention

PAGE 83

83 times with those from the mixed reference standard. The inclusion of an internal standard (C19:0) was used to verify FA extraction efficiency in plasma and RBC samples The retention times of each indivi dual FA in the mixed FAME standard were verified by use of reference standards ( Nu Chek Prep, Elysian, MN ) containing a single FA population diluted to concentrations expected to be found in blood and feedstuffs The FA present in the extracted sample were quantified (g/L) by multiplying the g/L of an individual FA standard by the area of that individual FA found in the sample, then dividing that by the area of the individual standard. Percent of each FA in the sample was calculated by dividing the g/ L of each FA by the total g/L FA in the sample then multiplying that by 100. The sum of n 6 FA in feedstuffs, plasma or RBC was calculated by adding the percentages of linoleic acid (LA; C18:2n 6) and arachidonic acid (ARA; C20:4n 6) present in each sam ple, and the sum of n 3 FA was calculated by the addition of ALA, EPA, docosapentaenoic acid ( DPA ; C22:5n 3) DHA. Lymphocyte Proliferation Frozen PBMC were thawed gradually and immediately washed and resuspended in ( DMEM ) supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, and penicillin streptomycin (100 IU/mL and 100 g/mL, respectively). Cell viability was determined to be greater than 80% using trypan blue exclusion. Proliferative response to mitogen stimulation w as assessed with a nonradioactive colorimetric assay, which has been shown in many species (including horses) to closely correlate with the conventional radioactive [ 3 H]thymidine incorporation assay (Ahmed et al., 1 994; Witonsky et al., 2003; see Appendix F) Aliquots of 100 L of the cell suspension (1 10 6 cells/mL) were stimulated in triplicate with 100 L of either Concanavalin A (Con A, 2 g/mL) phytohemagglutinin (PHA, 25 g/mL), or culture media (no mitogen control). Cells were incubated at 37C in 6% CO 2 for

PAGE 84

84 a total of 72 h. Alamar Blue (20 L) was added to each well 18 h prior to the end of incubation. Fluorescence was determined with a fluorometer (Synergy HT; BioTek Instruments Inc., Winooksi, VT) at wa velengths of 530 nm excitation, 590 nm emission. The change in fluorescence was calculated by subtracting the mean of the non stimulated control cells from the mean of the stimulated cells. PGE 2 P roduction Fresh PBMC (1 x 10 6 cells/ mL ) were isolated from w hole blood samples collected on d 70 using a commercially System HISTOPAQUE 1077, Sigma Aldrich, St. Louis, MO) PBMC were washed and resuspended in Roswell Park Memorial Institute media 1640 su pplemented with 10% FBS and 1% antib iotic a ntimycotic (Invitrogen Corp., Carlsbad, CA). Cells were challenged with lipopolysaccharide (LPS, 10 ng/ mL ) at a final concentration of 1 10 6 cells/well with either 10 ng LPS/well or culture media as a control (final volume = 1 mL /well) and incubat ed at 37C with 5% CO 2 for 24 h (see Appendix G) PGE 2 production in cultur e supernatant was analyzed using a commercially available kit (Correlate EIA, Assay Designs, Ann Arbor, MI). Intradermal Skin Test On d 70, hair on the left side of the neck was sh aved in a 4 x 4 cm square, and horses were administered an intradermal injection of a phytohemagglutinin solution (PHA; lectin from Phaseolus vulgaris Sigma Aldrich, Inc., St. Louis MO) to test the local inflammatory response To make the suspension, 25 m g PHA was reconstituted in 16.7 mL PBS to create a final concentration of 1.5 mg PHA/mL The volume of the injected PHA suspension consisted of 100 L, which delivered 150 g PHA At an adjacent location on the left side of the neck, 100 L PBS was injecte d to serve as a control. Skin thickness measurements at the injection site s were

PAGE 85

85 obtained by pinching the skin between the thumb and forefinger and measuring the skin fold thickness in mm with an electronic digital micrometer (Marathon Watch Company, Ltd., Ontario Canada) prior to injection ( h 0) and at 2, 4, 6, 8, 12, 24, and 48 h post injection. At the same time, the area of swelling (mm 2 ) was evaluated by measuring the length and width of the inflamed area at the injection site. A baseline measurement f or area of swelling was taken immediately after intradermal PHA injection. The change in skin thickness and area of swelling after PHA injection was calculated by subtracting the measurement taken at baseline (0 h) from the measurement taken at each time p oint. Statistical Analysis Differences in plasma and RBC FA content lymphocyte proliferation, increase in skin thickness, and area of swelling were analyzed using the MIXED procedure of SAS (Version 9.0 SAS Institute Inc., Cary, NC) with repeated measur es PGE 2 production by stimulated PBMC was analyzed using the MIXED procedure of SAS. The effects of treatment, sex, time, and treatment x time interaction were evaluated as fixed effects. Horse within treatment was a random effect. The PDIFF option of the LSMEANS statement of PROC MIXED was used to compare treatment means. Bodyweight measurements were analyzed using the GLM procedure of SAS with repeated measures. Differences were considered significant at P<0.05. Results All horses remained healthy over t he course of the 70 d experiment Weight gain averaged 0.6 0.03 kg/d and was not affected by dietary treatment, but ADG was higher (P<0.05) in geldings (0.70.03 kg/d) than fillies (0.50.03 kg/d). After a 5 d adaptation period during which the amount o f supplement offered was incrementally increased, the majority of horses readily consumed both the milled flaxseed and the encapsulated fish oil supplement s During the first

PAGE 86

86 w ee k one horse from each of the FLAX and FISH treatment groups initially refused to consume their respective supplements However, both horses resumed consumption during wk 2 of the supplementation period. Plasma and Red Blood Cell Fatty Acid C omposition Plasma FA composition was affected by time (P<0.01) and time x treatment interact ion (P<0.05) (Table 3 2) In horses consuming the CON and FLAX treatments, plasma LA, ALA, sum n 6, and sum n 3 increased (P<0.05) and ARA decreased (P<0.05) from d 0 to d 35; however, these FA were not different from d 0 at d 70. EPA and DHA were not dete ctable in plasma before or during the 70 d supplementation period in horses receiving CON or FLAX. On d 35, plasma in horses supplemented with FISH was higher (P<0.05) in LA, ALA, EPA, DHA, sum n 3, and sum n 6 and lower (P<0.05) in ARA compared to plasma at d 0. On d 70, plasma EPA, DHA, and sum n 3 remained elevated above pre supplementation levels, whereas ARA increased (P<0.05) above and LA ALA, and sum n 6 decreased below (P<0.05) d 0 values in horses fed FISH. Although no differences were detected be tween treatments prior to the start of supplementation, several plasma FA were altered in response to treatment (Table 3 2). After 35 d of supplementation, horses fed FISH had lower (P<0.05) plasma LA and sum n 6 than FLAX, whereas the se FA were intermedia te in CON horses. In addition, plasma ALA was lower (P<0.05) and plasma EPA, DHA, and sum n 3 were higher (P<0.05) in horses fed FISH compared to CON and FLAX. There were no differences in plasma ARA between treatments at d 35. After 70 d of supplementatio n, horses fed FISH had higher plasma A R A, EPA, DHA, and sum of n 3 (P<0.05) and lower plasma LA and ALA (P<0.05) than both FLAX and CON. Horses supplemented with FISH also had lower plasma sum n 6 than FLAX on d 70 (P<0.05). Similar

PAGE 87

87 to that observed on d 3 5, no differences in plasma FA, sum n 6, or sum n 3 were observed between horses fed FLAX or CON on d 70. The FA composition of RBC was affected by time (P<0.01) (Table 3 3). In all treatments, the proportion of LA and sum n 6 in RBC was unchanged at d 35, but lower (P<0.05) at d 70. The ALA and sum n 3 concentration of RBC increased at d 35 (P<0.05) and remained above pre supplementation levels at d 70 in all treatments. The proportion of ARA in RBC at d 35 and d 70 did not differ from d 0 in any treatment ; however ARA was higher (P<0.05) at d 35 compared to d 70. A time x treatment interaction was detected for RBC EPA (P<0.01), DHA (P<0.01) and sum n 3 (P<0.05). EPA and DHA were not detectable in RBC at any time in CON horses or those fed FLAX. In horses f ed FISH, EPA and DHA increased from d 0 to d 35 ( P<0.05) EPA remained elevated in RBC of FISH horses at d 70, while a further increase in DHA occurred between d 35 and d 70 (P<0.05). Sum n 3 increased (P<0.05) in RBC of all horses at d 35 and remained ele vated in horses fed FISH at d 70 (P<0.05), whereas sum n 3 declined to levels that were no different from pre supplementation concentrations in CON and FLAX. No differences in RBC FA composition were detected among treatments prior to the start of suppleme ntation. After 35 d of supplementation, differences in RBC n 3 FA composition were detectable as h orses fed FISH had higher EPA, DHA, and sum n 3 in RBC than CON and FLAX (P<0.05) (Table 3 3) After 70 d of supplementation, RBC n 3 FA composition continue d to reflect dietary n 3 FA intake as FISH had higher RBC EPA DH A and sum n 3 than FLAX and CON (P<0.05). Treatment had no effect on the proportion of LA, ALA, ARA, or sum n 6 in RBC.

PAGE 88

88 Lymphocyte P roliferation Across treatments, PBMC positively responded to ConA and PHA stimulation; however, proliferative responses were not different among treatments prior to the start of supplementation, nor after 35 or 75 d of supplementation. Figure 3 1 shows the proliferative response of lymphocytes to ConA and PHA at d 70. PGE 2 production Treatment with LPS increased (P<0.05) PGE 2 production by PBMC from CON, FISH, and FLAX horses compared to the control but treatment had no effect on PGE 2 production after 70 d of supplementation (Figure 3 2). Intradermal Skin T est A noticeable skin swelling response was evident within 2 h f ollowing intradermal PHA injection The peak increase in skin thickness was observed 4 6 h post injection (Figure 3 3 ). At 4 h post injection, both FISH and FLAX had a greater increase in skin thic kness over CON (P<0.05). S kin thickness was still greater 6 h after injection in FLAX than CON (P<0.05). There were no differences among treatments beyond 6 h post injection, and skin swelling continued to decrease over the 48 h period. At 48 h post inject ion, skin thickness was still slightly increased from baseline measurements (P<0.05). The change in skin thickness after PBS injection was minimal and did not differ among treatments. The peak change in skin thickness occurred at 4 h post injection, with a 1.29 mm increase over baseline, and it continued to decline through the remaining 48 h period. The baseline measurement for area of swelling was taken immediately after intradermal PHA injection. After 2 h the area of swelling was greater than the baseli ne measurement in all horses (P<0.05) At 4 h post injection, the change in area of swelling was greater (P<0.05) in

PAGE 89

89 FISH than in CON (Figure 3 4) There were no differences among treatments beyond 8 h post injection, and area of swelling continued to decre ase over the 48 h period. At 48 h post injection, area of swelling was still slightly increased from baseline measurements (P<0.05). The area of swelling measured after PBS injection was minimal and did not differ among treatments. The area of skin swellin g was greatest immediately after the injection (113.3 mm 2 ), and declined to 0 mm 2 at 6 h post injection. Discussion This study demonstrates that feeding an encapsulated fish oil product to horses providing 6 g n 3/100 kg BW daily for 70 d increases the lon g c hain PUFA EPA and DHA in both plasma and RBC. In contrast no changes in the EPA or DHA composition of plasma or RBC were observed when an equal amount of n 3 FA was provided by flaxseed suggesting that horses have a limited ability to bioconvert dieta ry ALA to significant amounts of EPA or DHA. Nonetheless, the early local inflammatory response to a stimulatory challenge was increased in both groups of horses consuming supplementary n 3 FA Plasma and R ed Blood Cell Fatty Acids Previous studies in the horse have demonstrated that plasma FA composition is affected by marine derived dietary n 3 FA supplementation. Hall et al. (2004b) reported increased EPA and DHA and decreased LA, ALA and n 6: n 3 ratio in the plasma of horses fed fis h oil. In a study by King et al. (2008) horses were fed an encapsulated fish oil product similar to the one utili zed in this trial. After 28 d of supplementation, these horses displayed an increase in plasma A R A, EPA, and DHA similar to that observed in the current investigation. Supplementing the same amount of n 3 FA in form of ALA rich flaxseed failed to alter plasma sum n 3 or ALA concentration. These findings agree with Siciliano et al. (2003) who

PAGE 90

90 reported that flaxseed supplementation at a rate of 5.6 n 3/100 kg BW for 28 d did not result in an increase in plasma total n 3 content in mature horses. In contrast, Hansen et al. (2002) reporte d an increase in plasma ALA when horses were fed diets containing 10% flaxseed oil This amount of flaxseed would provide approximately 10 6 g n 3/1 00 kg BW which is more than 17 fold greater than the amount provided in the current study (6 g n 3/100 kg BW ) or by Siciliano et al. (2003) and much higher than that likely to be utilized in the horse industry. The current study took place during the summer when pasture grass was in the active growth stage, resulting in an abundance of grass available for grazing. Assuming horses consumed 1% BW of pasture as forage, with the remainder provided by the grass. Plasma ALA in CON horses also increased and was not different than the proportion of ALA in FLAX horses. P erhaps the amount of ALA provided by preventing any further increase in dietary ALA intake that wou ld be reflected in the plasma. T he FA composition of RBC membranes is a good indicator of long term dietary FA intake due to the longer half life of RBC as compared to plasma phospholipids (Arab, 2003) In addition, RBC are easily accessible because large quantities can be readily isolated from the blood. The FA composition of RBC may also be a good indicator of the FA composition of other cells of the body, including immune cells. In general, plasma and RBC long chain n 3 FA composition were both highly dependent upon the presence of EPA and DHA in the diet. Only horses supplemented with FISH had significant amounts of EPA and DHA in both plasma and RBC and both were detectable after 35 d of supplementation However, RBC DHA concentration continued to rise through 70 d of supplementation. A minute amount of EPA and

PAGE 91

91 DHA were measured in RBC of a few horses not supplemented with FISH, but neither EPA n or DHA were presen t in the plasma of CON or FLAX horses. The other differences noted among treatment groups for plasma LA, ALA, ARA and sum n 6 were no t reflected in the RBC FA This may be due to increased individual variations in RBC FA content or the presence of a more tightly regulated mechanism that regulates cell membrane lipid composition (Thewke et al., 2000) Immune Responses Proliferative responses of lymphocytes are used as an indicator of a functional response to differe nt types of external stimuli. Dietary FA can affect the functional response of a lymphocyte by altering the cell membrane FA composition. Altered composition of membrane lipids can have an effect on lipid raft function, signal transduction, and gene expres sion, all of which can ultimately affect lymphocyte function (Calder, 2007) Much conflicting evidence exists in the literature regarding the effects of n 3 and n 6 dietary supplementation on lymphocyte proliferation. In humans, supplementation with more than 3.3 g EPA+DHA /100 kg BW daily (and in some studies up to 20.7 g EPA+DHA /100 kg BW) has been reported to decrease lymphocyte proliferation (Calder and Grimble, 20 02) However, sup EPA+ DHA/100 kg BW daily did not affect lymphocyte proliferation in healthy human subjects (Kew et al., 2003a) In the current study, horses supplemented with FISH con sumed 5.4 g EPA+ DHA/10 0 kg BW d aily and at this level of supplementation, PBMC response to ex vivo stimulation with the T cell mitogens Con A and PHA was not affected. The interpretation of proliferation assay results can sometimes present a challenge as both an increase and a decrease could be viewed as favorable. In general, a decrease in lymphocyte proliferation would be considered favorable if the subjects suffered from an inflammatory

PAGE 92

92 condition but may be considered unfavorable in healthy subjects, as decreased lymphocyte activity may compromise host defense (Miles et al., 2006) In addition, the relevance of an increase or decrease in proliferation is dependent upon which treatment responses are bei ng compared. For example, Thies et al. (1999) reported that the proliferation of lymphocytes in weaned pigs supple mented with fish oil for 40 d was less than in pigs supplemented with canola oil. In contrast, an increased proliferative response to mitogen stimulation was observed in beef calves fed a corn bas ed supplement with 1.5% fish oil compared to calves fed the corn based supplement alone (Wistuba et al., 2005) The discrepancies among these findings are likely due to several factors, including species differences, amount of fat supplem ented, different treatment groups to which comparisons were being made, and variations in experimental conditions. The lack of a different response among treatment groups in the current study indicates that lymphocyte functions were neither enhanced nor in hibited by the addition of n 3 to the diet. It has been well documented in humans that dietary supplementation with n 3 FA can alter PBMC membrane FA composition and the increase in n 3 FA typically occurs at the expense of n 6 FA, especially ARA (Yaqoob et al., 2000; Calder, 2007) The EPA in cell membrane s competes with ARA for enzymes necessary to produce eicosanoids as EPA is a COX substrate for the synthesis of PGE 3 a less potent inflammatory mediator than PGE 2 although its production occurs with very low efficiency (James et al., 2000) Skjolaas Wilson et al. (2005) observed a reduced level of PGE 2 production by neutrophils in response to LPS in horses supplemented with 3.5 g n 3/100 kg BW encapsulated fish oil Similarly, Hall e t al. (2004a) reported decreased PGE 2 production by LPS stimulated bronchoalveolar lavage fluid cells from horses supplemented with fish oil providing 9.9 g n 3/100 kg BW compared to horses

PAGE 93

93 supplemented with corn o il. In contrast, supplementation with flaxseed or fish oil had no effect on PGE 2 production by PBMC in horses in the current study. Although FA composition of immune cells was not determined, there were no differences among treatments for RBC A R A content a fter 70 d of supplementation. In the current study, the proportions of EPA and DHA, but not ARA, were higher in RBC of horses fed FISH after 35 and 70 d of supplementation. PGE 2 is a potent lipid mediator of inflammation derived from A R A present in the cel l membrane. A direct relationship exists between the A R A content of immune cell membranes and the ability of those cells to produce PGE 2 and it has been established that increased EPA membrane content can be y to produce PGE 2 (Calder, 2007) An increase in PGE 2 syn thesis may only be observed if there is an increased proportion of A R A in the immune cell membrane On the other hand, there may be a threshold level that EPA must reach in the cell membrane before it can begin to inhibit ARA derived eicosanoid production, and the rate and amount o f EPA incorporated into cell membranes would be dependent upon the level and duration of EPA supplementation. In addition, it is possible that variations in the basal diets or specific assay conditions contribute to the conflictin g results observed between this study and others Despite the lack of treatment effect on PGE 2 production, n 3 supplementation appear ed to alter the inflammatory response in vivo All horses displayed an early inflammatory response to PHA injection, with p eak skin thickness observed 4 6 h post injection and peak area of swelling observed slightly later This early response indicates that the inflammatory response was mediated by cells of the innate immune system, primarily neutrophils and macrophages. A sim ilar early response to PHA injection has been observed in cattle, and microscopic evaluation

PAGE 94

94 of skin biopsies confirmed that neutrophil and macrophage infiltrations, but not lymphocytes, were significantly greater in PHA injected sites than in saline injec ted sites (Hernandez et a l., 2005) At 4 h post injection, FISH and FLAX had a greater increase in skin thickness than CON, and FISH had greater area of swelling than CON. The increase in skin thickness remained higher in horses fed FLAX than in non supplemented horses at 6 h po st injection. Feeding horses diets with 3% added fat (from fish oil or corn oil ) for 8 wk increased TNF production and phagocytic activity of BALF cells regardless of fat source (Hall et al., 2004a) TNF otent inflammatory cytokine released by macrophages in the endothelium in response to an activating stimulus. TNF vascular permeability. Although it was not measured in the cur r ent study, it is possible that i nfiltrating macrophages increased their secretion of TNF neutrophils, contributed to the increased early inflammatory response observed in horses fed both FISH and FLAX. Implications Althou gh fed to supply the same level of total n 3 FA fish oil (rich in EPA and DHA) had a greater impact on plasma and RBC n 3 FA composition than milled flaxseed (rich in ALA). When horses are allowed access to fresh pasture during the growing season, supplem enting with flaxseed does not appear to further increase plasma or RBC ALA content above that of horses receiving no n 3 supplementation Fresh grass is rich in ALA, which may have overshadowed that provided by flaxseed. Flaxseed added to high grain diets, particularly when coupled with mature hay, may have a greater impact on plasma and RBC FA composition. Because RBC DHA continued to rise from d 35 to d 70, this indicates that time necessary for complete

PAGE 95

95 incorporation of dietary n 3 FA into cell membranes may extend beyond that necessary for plasma. Despite the alterations in cell membrane composition, feeding horses fish oil at a rate of 6 g n 3/100 kg BW did not result in decreased PGE 2 production as compared to horses fed FLAX or CON. In horses consumi ng both the FISH and FLAX treatments, a more pronounced early inflammatory response to PHA injection was observed. The mechanism responsible for this effect is not clear, as this measure of inflammatory response was relatively non specific. Further study i s warranted to determine the effects of n 3 FA supplementation on other immune response variables, especially the regulation of inflammatory cells and their function along with identifying optimal levels of n 3 FA supplementation that support optimal immun e function in the horse.

PAGE 96

96 Table 3 1. Nutrient and fatty acid composition of the grain mix concentrate and bahiagrass pasture making up the basal diet, and the milled flaxseed and encapsulated fish oil supplements Nutrient 1 Concentrate Pasture 2 Flaxseed E ncapsulated Fish Oil DM, % 89.0 20.9 91.5 91.2 Crude fat, % 4.2 2.7 37.7 21.5 CP, % 16.6 16.3 22.9 11.8 NDF, % 25.3 60.2 40.0 9.9 ADF, % 12.3 35.1 19.0 5.9 Ca, % 0.8 0.5 0.2 0.3 P, % 0.7 0.4 0.6 0.2 Fatty acid 3 C18:2 n 6 48.1 17.1 16.5 6.8 C1 8:3 n 3 3.0 51.0 61.1 2.6 C20:4 n 6 0 0 0 1.0 C20:5 n 3 0 0 0 14.4 C22:5 n 3 0 0 0 2.3 C22:6 n 3 0 0 0 11.5 Other fatty acids 4 48.9 31.9 22.4 61.4 1 With the exception of DM, all values are presented on 100% DM basis. 2 Mean of 3 monthly pasture sample s collected during the trial. 3 Fatty acids expressed as a percentage of total fatty acids.. 4 C12:0, C14:0, C16:0, C16:1n 7, C18:0, C18:1n 9.

PAGE 97

97 Table 3 2. Fatty acid composition of plasma before (d 0), during (d 35), and after 70 d of supplementation with milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) Fatty Acid 1 Treatment d 0 d 35 d 70 SEM P v alues Trt Time Trt*time C18:2 n 6 CON 48.7 b 51.9 a,x,y 48.2 b,x 0.4 0.05 <0.01 0.03 FLAX 48.2 b 52. 6 a, x 49.3 b,x 0.8 FI SH 48.2 b 50. 6 a, y 46.7 c,y 0.5 C18:3 n 3 CON 3.2 b 5.9 a,x 3.6 b,x 0.2 0.01 <0.01 0.10 FLAX 3.6 b 5.3 a,x 3.7 b,x 0.4 FISH 3.1 b 4.2 a,y 2.6 b,y 0.2 C20:4 n 6 CON 2.0 a 1.5 b 2.1 a,y 0.1 0.06 <0.01 0.52 FLAX 2.0 a 1.5 b 2.1 a,y 0.1 FISH 2.1 b 1.7 c 2.4 a ,x 0.1 C20:5 n 3 CON ND ND y ND y 0 <0.01 <0.01 <0.01 FLAX ND ND y ND y 0 FISH ND b 1. 6 a, x 1. 4 a, x 0.1 C22:6 n 3 CON ND ND y ND x 0 <0.01 <0.01 <0.01 FLAX ND ND y ND x 0 FISH ND c 1.2 b,x 1.6 a,x 0.1 Sum n 6 CON 50.7 b 53.5 a,x,y 50.2 b,x,y 0.4 0 .09 <0.01 0.06 FLAX 50.3 b 54.1 a,x 51.4 b,x 0.8 FISH 50.4 b 52.3 a,y 49.1 c,y 0.4 Sum n 3 CON 3.2 b 5.9 a,y 3.6 b,y 0.2 <0.01 <0.01 <0.01 FLAX 3.6 b 5.3 a,y 3.7 b,y 0.4 FISH 3.1 c 7.3 a,x 5.6 b,x 0.2 1 Fatty acids expressed as a percentage of total fatty acids. a,b,c Means in the same row not sharing a common superscript differ (P<0.05). x,y Within a fatty acid, means in the same column not sharing a common superscript differ (P<0.05) ND=not detected.

PAGE 98

98 Table 3 3. Fatty acid composition of red blood cells before (d 0), during (d 35), and after 70 d of supplementation with milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) Fatty Acid 1 Treatment d 0 d 35 d 70 SEM P v alues Trt Time Trt*time C18:2 n 6 CON 42.4 a 45.7 a 34.6 b 1.8 0.94 <0.01 0.99 FLAX 42.2 a 46.1 a 35.5 b 1.6 FISH 43.2 a 45.7 a 35.7 b 1.3 C18:3 n 3 CON 1.4 c 2.6 a 1.8 b 0.2 0.31 <0.01 0.24 FLAX 1.3 c 2.6 a 1.9 b 0.2 FISH 1.2 b 2.0 a 1.5 b 0.1 C20:4 n 6 CON 2.6 a,b 2.8 a 2.3 b 0.1 0.97 <0.01 0.84 FL AX 2.8 a 2.8 a 2.1 b 0.1 FISH 2.6 a,b 2.9 a 2.3 b 0.1 C20:5 n 3 CON ND ND y 0.1 y 0.1 <0.01 <0.01 <0.01 FLAX ND ND y ND y 0 FISH ND b 1.2 a,x 1.4 a,x 0.2 C22:6 n 3 CON ND ND y ND y 0 <0.01 <0.01 <0.01 FLAX ND ND y 0.3 y 0.1 FISH ND c 0.8 b,x 1.3 a,x 0.1 Sum n 6 CON 45.0 a 48.5 a 36.9 b 1.8 0.94 <0.01 0.99 FLAX 45.0 a 48.9 a 37.6 b 1.7 FISH 45.8 a 48.6 a 38.0 b 1.4 Sum n 3 CON 1.4 b 2.6 a,y 2.0 a,b,y 0.2 0.05 <0.01 0.02 FLAX 1.3 b 2.6 a,y 2.2 a,b,y 0.2 FISH 1.2 b 4.0 a,x 4.3 a,x 0.5 1 Fatty aci ds expressed as a percentage of total fatty acids. a,b,c Means in the same row not sharing a common superscript differ (P<0.05). x,y Within a fatty acid, means in the same column not sharing a common superscript differ (P<0.05) ND=not detected.

PAGE 99

99 Figure 3 1 Proliferative responses of PBMC from horses supplemented with a milled flaxseed (FLAX), encapsulated fish oil (FISH), or not supplemented (CON) for 70 d. Figure 3 2 Production of PGE 2 by stimulat ed PBMC from horses receiving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplement ation (CON) for 70 d.

PAGE 100

100 Figure 3 3. Change in skin thickness in response to intradermal injection of PHA in horses receiving mil led flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) Figure 3 4. Change in the area of swelling in response to intradermal injection of PHA in horses receiving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON)

PAGE 101

101 CHAPTER 4 CLEARANCE OF POLYUNS ATURATED FATTY ACIDS FROM HORSE PLASMA AN D RED BLOOD CELLS AFTE R SUPPLEMENTATION Abstract Eighteen Quarter Horse yearlings were monitored following the cessation of supplementation w ith flaxseed or fish oil in order to determine the time period necessary to clear dietary omega 3 (n 3) fatty acids (FA) present in the plasma an d red blood cells (RBC). Horses were randomly and equally assigned to one of three treatments: encapsulated fis h oil (FISH, n=6), milled flaxseed (FLAX, n=6), or no supplementation (CON, n=6) and supplemented for 10 wk. Horses had free access to bahiagrass pasture and were individually fed a grain mix concentrate at 1.5% BW/d. FISH and FLAX were mixed into the conc entrate in amounts to provide 6 g total n 3/100 kg BW. Horses were monitored for an additional 8 wk after supplementation ended to determine FA washout. Blood samples were obtained before (wk 0) and after 10 wk of n 3 supplementation and at 2, 3, 5, and 8 wk post supplementation and analyzed for plasma and RBC FA composition. Prior to the start of n 3 supplementation (wk 0), linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or total n 3 FA among treatments. After 10 wk of supplementation, EPA, DHA and total n 3 in plasma and RBC were elevated (P<0.05) in horses fed FISH compared to FLAX or CON. At 2 wk post supplementation, plasma DHA and RBC EPA, DHA and total n 3 in horses fed FISH we re still above baseline levels (P<0.05). After 5 wk, plasma EPA and DHA were still elevated in horses fed FISH (P<0.05), but RBC EPA and DHA had returned to baseline levels. At 8 wk post supplementation, there were no differences in plasma or RBC n 3 FA am ong treatments; however, plasma and RBC ALA and arachidonic acid were lower and linoleic acid was higher in plasma and lower in RBC compared to pre

PAGE 102

102 supplementation levels. For the purposes of designing research trials involving n 3 FA supplementation, a w ashout period of at least 8 wk is likely needed when using n 3 FA supplements containing EPA and DHA. Introduction Interest in omega 3 (n 3) fatty acid (FA) supplementation in the horse has increased because of potential anti inflammatory and other health benefits (Duvaux Ponter et al., 2004; Hall et al., 2004a; Hall et al., 2004b) Feeding horses flaxseed or fish oil, which are rich in n 3 FA, increases the n 3 con tent of plasma within 14 d and red blood cell (RBC) membrane content within 28 d after supplementation is initiated (Siciliano et al., 2003; Stelzleni et al., 2006) Little is known about how long these FA remain elevated in plasma or RBC membranes after supplementation has ended. Only one study has been performed in horses to characterize the plasma clearance rate of EPA and DHA following encapsulated fish oil supplementation, but it did not measure RBC clearance (King et al., 2008) The knowledge of how much time is necessary for complete clearance of dietary derived plasma an d RBC n 3 FA would be useful to over research trials and to determine how long n 3 FA may exert their effects when supplementation is interrupted or terminated. The objective of this study was to determine the time necessary for the complete clearance of n 3 FA from plasma and RBC membranes once fish oil or flaxseed supplementation is discontinued. Materials and Methods Horses Eighteen Quarter Horse yearling fillies (n=9) and geldings (n=9) with a meanSE ag e of 14.60.2 mo and an initial BW of 391.5 5.2 kg were utilized for this study. During the 10 wk supplementation phase, fillies and geldings were housed separately in two adjacent 8 ha

PAGE 103

103 bahiagrass ( Paspalum notatum ) pastures at the Institute of Food and Agricultural Science s Horse Research Center in Ocala After the 10 wk supplementation period, horses were moved to the Institute of Food and Agricultural Sciences Horse Teaching Unit in Gainesville, FL where fillies and geldings were housed separately in 2 .4 ha bahiagrass pastures and supplemented with C oastal bermudagrass ( Cynodon dactylon ) hay for the remaining 8 wk of the study. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Florida. Dieta ry Treatments As part of a previous study to assess the effects of flaxseed or fish oil supplementation on plasma and red blood cell FA composition (Chapter 3), horses were blocked by sex and age and randomly and equally assigned to one of three dietary tr eatments for 10 wk: encapsulated fish oil Angusville, MB, Canada ), or no supplementation (CON, n=6). The basal diet consisted of a non fat added grain mix concentrate (Ges t O Lac; OBS Feeds, Ocala, FL) fed individually in feeding pens at 1.5% BW split into two meals at 0700 and 1500 h. The daily amount of supplement was equally divided between grain meals and mixed into the basal grain ration. Based on previous research tha t demonstrated an observable response to n et al. 2004), FISH and FLAX were provided in amounts to provide 6 g total n 3/100 kg BW linolenic acid (ALA; C18:3n 3) + 3.0 g eicosapentaenoic acid (EPA;C20:5n 3) + 2.4 g docosahexaenoic acid (DHA; C22:6n 3)/100 kg BW for horses fed FISH and 6 g ALA/100 kg BW for horses fed FLAX). Horse s were allowed free choice grazing access to bahiagrass for the duration of the sup plementation period (May 2005 July 2005)

PAGE 104

104 which was during the active growing season. The nutrient and FA composition of the feeds and supplements were presented in the pre vious chapter (Table 3 1). After the supplementation period ended and horses were moved to the new location, they were fed the same concentrate at the same feeding rate as used during the supplementation period, adjusted as necessary to maintain BW, for t he duration of the washout period (July 2005 September 2005). The daily concentrate was split into two equal sized feedings offered in individual feeding pens at 0700 and 1600 h. Free choice coastal bermudagrass hay was offered to supplement the pasture as the availability of fresh forage began to decline at the end of the growing season. Sample Collection Blood samples were obtained prior to the start of supplementation (baseline), after +10 wk of supplementation, and at 2, 3, 5, and 8 wk post supple mentation. Blood was collected by jugular venipuncture into evacuated tubes containing the anticoagulant sodium heparin (Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ). Blood samples were centrifuged for 10 min at 4 000 x g at 22C (room temperature) Plasma was removed and stored at 80C until later analysis. The RBC remaining in the blood sample were isolated by repeated centrifugation at room temperature and washing with normal saline until all residual plasma was removed and stored at 80C until later analysis. Feedstuff, Plasma and Red Blood Cell Composition To analyze FA composition of the diets feed samples were lyophilized (FreeZone 6 Liter Freeze Dryer System, Labconco Corp, Kansas City, Mo) and ground, and representative samples consisting of 2 g grass, 0.5 g grain mix, or 0.2 g flaxseed or encapsulated fish oil were utilized for FA extraction. For analysis of blood FA, 2 mL of plasma and 4 mL of RBC were frozen at

PAGE 105

105 20 C in 4 mL Wheaton polypropylene Omni vials, lyophilized, sealed with sna p caps and stored at 20 C. A mixed reference standard containing 33 FA methyl esters (FAME) (GLC 461, Nu Chek Prep, Elysian, MN) was reconstituted in 10 mL hexane. FA in freeze dried feedstuffs were extracted and methylated using the procedure of Folch et al. (1957; see Appendix D) FA in freeze dried plasma and RBC were methylated using the two step procedure with a 10 min incubation in sodium methoxide as described by Jenkins et al. (2001; see Appendix E) A CP 3800 Ga s Chromatograph with a CP 8400 autosampler and i njector, a split injection port, flame ionization detector (Varian, Inc, Walnut Creek, CA), and a 100 m CP SIL 88 f used silica capillary column (0.25 mm i.d. x 0.2 mm film thickness; Varian, Inc, Walnut Creek, CA), were used for the analysis of individual FA. An injection of 1.0 L was split 1:20 and the He carrier gas maintained at 1.0 mL/min. Column temperature was h eld at 120C for 1 min following injection, increased at the rate of 5C/min to 190C and held at that temperature for 30 min, then increased at the rate of 2C/min to 220C and held for 50 min. The injector temperature was set at 250C and the detector at 255C. Identification of 21 FAME ( C8:0, C10:0, C12:0, C14:0, C14:1, C16:0, C16:1 n 7 C17:0, C17:1, C18:0, C18:1n 9, C18:2n 6, C18:3n 3, C20:0, C20:1, C20:2, C20:4n 6, C20:5n 3, C22:0, C22:5n 3, and C22:6n 3) from the chromatograms were determined by comp aring peak retention times with those from the mixed reference standard. The inclusion of an internal standard (C19:0) was used to verify FA extraction efficiency in plasma and RBC samples The retention times of each individual FA in the mixed FAME standa rd were verified by use of reference standards ( Nu Chek Prep, Elysian, MN ) containing a single FA population diluted to concentrations expected to be found in blood and feedstuffs The FA present in the extracted

PAGE 106

106 sample were quantified (g/L) by multiplyi ng the g/L of an individual FA standard by the area of that individual FA found in the sample, then dividing that by the area of the individual standard. Percent of each FA in the sample was calculated by dividing the g/L of each FA by the total g/L FA in the sample then multiplying that by 100. The sum of n 6 FA in feedstuffs, plasma or RBC was calculated by adding the percentages of linoleic acid (LA; C18:2n 6) and arachidonic acid (ARA; C20:4n 6) present in each sample, and the sum of n 3 FA was c alculated by the addition of ALA, EPA, docosapentaenoic acid ( DPA ; C22:5n 3) DHA. Statistical Analysis Differences in plasma and RBC FA content were analyzed using PROC MIXED with repeated measures in SAS (Version 9.0, SAS Institute Inc., Cary, NC). The e ffects of treatment, sex, time, and treatment x time interaction were evaluated as fixed effects. Horse within treatment was a random effect. The PDIFF option of the LSMEANS statement of PROC MIXED was used to compare treatment means. Comparisons were made among treatments at each time point, within a treatment compared to baseline, and within a treatment compared to +10 wk of supplementation. Differences were considered significant at P<0.05. Results Supplementation Period Plasma and RBC FA content measure d at each time point are presented in Tables 4 1 and 4 2 and in Figures 4 1, 4 2, and 4 3. For horses consuming the CON and FLAX treatments, there were no changes in plasma LA, ALA, ARA, EPA, DHA, sum n 6, or sum n 3 from baseline to +10 wk of supplementat ion (Table 4 1). In addition, no changes were observed in LA, ARA, EPA, DHA, or sum n 6 in RBC of horses fed the CON or FLAX treatments; but, ALA and sum n 3 in RBC were elevated (P<0.05) over baseline levels after 10 wk of supplementation (Table 4

PAGE 107

107 2). In horses fed FISH, LA and sum n 6 decreased (P<0.05) and EPA, DHA, and sum n 3 increased (P<0.05) in both plasma and RBC in response to supplementation. ARA increased in plasma (P<0.05), but not RBC, in horses supplemented with FISH (P<0.05). No changes in p lasma or RBC ALA were found in response to 10 wk of FISH supplementation. Washout Period Plasma and RBC FA content measured during the 8 wk washout period are presented in Tables 4 1 and 4 2 and in Figures 4 1, 4 2, and 4 3. After the cessation of supplem entation, plasma LA increased in all treatments and remained higher (P<0.05) than baseline and +10 wk values throughout the 8 wk washout period. In contrast, LA increased in RBC at 2 wk in CON and FISH horses, but not FLAX. At 3 wk, LA concentration in R BC began to decline in all treatments (P<0.05) and remained lower than baseline and +10 wk for the remainder of the washout period. Horses that had consumed the CON diet had higher plasma LA at 5 wk post supplementation than FLAX and FISH (P<0.05). In all treatments, plasma ARA declined below baseline and +10 wk levels at 2 wk (P<0.05) and remained lower throughout the 8 wk washout period (P<0.05). Similarly, RBC ARA declined in all treatments below baseline and +10 wk levels at 3 wk (P<0.05) and remaine d lower throughout the 8 wk washout period (P<0.05). At 3 wk, horses fed FISH had higher RBC ARA than CON and FLAX (P<0.05). The sum of n 6 FA in plasma increased and remained higher than baseline and +10 wk throughout the washout period in all treatments (P<0.05). In contrast, RBC sum n 6 increased at 2 wk in FISH horses (P<0.05), but not in CON and FLAX. At 3 wk, sum n 6 concentration in RBC began to decline and remained lower than baseline and +10 wk for the remainder of the

PAGE 108

108 washout period in all trea tments (P<0.05). Horses that had consumed the CON diet had higher plasma sum n 6 at 5 wk post supplementation than FLAX and FISH (P<0.05). In CON horses, plasma ALA declined to below baseline and +10 wk levels at 5 wk (P<0.05) and remained lower at 8 wk (P<0.05). In these same horses, ALA in RBC increased above baseline and +10 wk at 2 wk (P<0.05), but then declined below +10 wk at 3 wk and below baseline levels at 5 wk. In horses fed FLAX, plasma ALA declined below baseline and +10 wk at 3 wk, incre ased slightly at 5 wk, then declined at 8 wk (P<0.05). In RBC of FLAX horses, ALA increased from +10 wk to 2 wk (P<0.05), then decreased to baseline levels at 3 wk (P<0.05) and was not detectable at 5 wk. In horses fed FISH, plasma ALA remained steady until 5 wk when it decreased to below baseline levels (P<0.05) and further declined to below +10 wk levels by 8 wk. Similar to the other treatments, ALA increased in RBC at 2 wk in horses fed FISH, but declined to baseline levels at 3 wk and was undet ectable at 5 wk. RBC ALA was undetectable in all horses at 8 wk. At 2 wk post supplementation, horses fed FISH had lower RBC ALA than FLAX (P<0.05), and at 3 wk FISH had lower RBC ALA than CON and FLAX (P<0.05). Horses fed FLAX had higher plasma ALA tha n CON and FISH at 5 wk post supplementation (P<0.05). EPA was not detectable in plasma and RBC of horses fed CON or FLAX during either the supplementation or washout period, with the exception of a small amount measured in RBC of CON horses at +10 wk of s upplementation. In horses fed FISH, plasma EPA was lower at 2 and 3 wk compared to +10 wk levels (P<0.05) and remained slightly above baseline at 5 wk (P<0.0 5). EPA was not detectable at 8 wk in the plasma of horses that had been fed FISH. In RBC of ho rses fed FISH, EPA decreased at 2 and 3 wk below +10 wk levels (P<0.05), and

PAGE 109

109 returned to baseline and was not detectable at 5 and 8 wk. Horses fed FISH had higher plasma and RBC EPA than CON and FLAX at wk 2 and 3 wk (P<0.05) and higher plasma EPA at wk 5 (P<0.05). DHA was not detectable in plasma and RBC of horses fed CON or FLAX during either the supplementation or washout period, with the exception of a small amount measured in RBC of FLAX horses at +10 wk of supplementation. In horses fed FISH, p lasma DHA decreased below +10 wk levels at 2 and 3 wk (P<0.05 ) From 3 wk to 5 wk plasma DHA increased slightly (P<0.05) but was not detectable at 8 wk in horses that had been fed FISH. In RBC of horses fed FISH, DHA remained elevated at 2 wk, but h ad declined by 3 wk (P<0.05), and was not detectable at 5 or 8 wk (P<0.05). Horses fed FISH had higher DHA in plasma and RBC than CON and FLAX horses at 2 and 3 wk (P<0.05) and higher pla sma DHA at 5 wk (P<0.05). In horses fed FISH, the sum of n 3 FA in plasma decreased below +10 wk levels at 2 wk (P<0.05) and remained there at 3 and 5 wk until it declined furthe r to below baseline levels 8 wk following the cessation of supplementation (P<0.05). In horses fed FLAX and CON, the sum of n 3 FA in plasma did not differ between +10 wk and 2 wk. Plasma sum n 3 decreased in FLAX horses at 3 wk, increased slightly at 5 wk, and decreased to below baseline at 8 wk (P<0.05). For horses fed the CON diet, the sum of n 3 in plasma decreased to below base line and +10 wk levels at 5 and 8 wk (P<0.05). In all treatments, the sum of n 3 in RBC was not different between +10 wk and 2wk. In FLAX and CON horses, the sum of n 3 in RBC decreased to below +10 wk levels at 3 wk (P<0.05), decreased to below baseli ne levels at 5 wk (P<0.05), and remained lower at 8 wk (P<0.05). In horses fed FISH, the sum of n 3 in RBC remained above baseline at 3 wk, but n 3 FA were not detectable at 5 and 8 wk in RBC. The

PAGE 110

110 sum of n 3 FA in plasma was higher in FISH and FLAX th an CON at 5 wk (P<0.05). The sum of n 3 in RBC was higher in horses fed FISH at 2 and 3 wk compared to FLAX and CON (P<0.05). The proportion of saturated FA steadily increased in the RBC of all horses during the washout period, with C16:0 and C18:0 leve ls increasing by 65% and 101%, respectively, of that observed after 10 wk of supplementation. By comparison, the saturated FA content of plasma remained the same over the same time period (data not shown). Discussion This study demonstrates that the time p eriod necessary for complete plasma and RBC PUFA washout after 10 wk of supplementation with flaxseed and encapsulated fish oil is 6 8 wk. In addition, it appears that seasonal changes in fresh forage FA content, as well as forage source, affect the concen tration of PUFA in plasma and RBC, irrespective of dietary n 3 supplementation. As grass hay replaced fresh forage in the diet of horses during the 8 wk washout period, the proportion of LA increased in plasma and decreased in RBC. In addition, both ALA an d ARA decreased in plasma and RBC over the 8 wk washout period. These changes in the proportions of LA, ALA, and ARA in RBC may also be due to the steady increase in the proportion of saturated FA in RBC observed in all horses during the washout period. Th e EPA and DHA supplied in the fish oil appeared to have t he greatest impact on plasma and RBC n 3 FA composition. In FISH fed horses, EPA and DHA returned to baseline levels after 5 wk in RBC, but plasma remained elevated through 5 wk, but not 8 wk, post s upplementation. This is in agreement with King et al (2008) who reported that plasma EPA and DHA in horses supplemented with approximately 8 g EPA+DHA/100 kg BW/d for 28 d remained elevated above baseline levels for 6 wk after supplement withdrawal. Similarly, after

PAGE 111

111 dogs were fed fish oil for 8 wk, serum EPA remained elevated for 3 wk and serum DHA remained elevated for 7 wk after the cessation of supplementation (Hansen et al., 1998) In the current study, EPA and DHA remained elevated for a longer period of time in plasma compared to RBC. In contrast t o these findings, Cao et al. (2006) observed a delayed washout of EPA and DHA from RBC when compared to elimination of these FA from p lasma in human subjects. In that study, 8 wk was necessary for RBC EPA and DHA to return to baseline, but only 2 wk was needed for plasma clearance. The explanation for the difference in plasma and RBC n 3 FA distribution and clearance between horses and h umans is not clear, but may be due to differences in RBC turnover or lipid mobilization in growing horses versus adult humans. Supplementation with flaxseed did not result in any significant changes to plasma or RBC PUFA composition compared to unsuppleme nted horses. The content of ALA in RBC increased in both the FLAX and CON treatments in response to 10 wk of supplementation, and it continued to increase for an additional 2 wk in all horses after supplementation ended. At 3 wk, RBC ALA had returned to b aseline in all treatments and was not detectable 8 wk after supplementation ended. Plasma ALA content, on the other hand, did not change over the course of the supplementation period. In all horses, there was a steady decline in plasma ALA during the washo ut period. However, at 5 wk plasma ALA in FLAX fed horses, although not different than post supplementation levels, was higher than plasma ALA in CON and FISH. The reason for the slightly elevated plasma ALA in horses fed FLAX 5 wk after its withdrawal is not clear. Nonetheless, this finding is similar to the elevated plasma EPA and DHA in horses fed FISH, and perhaps may be attributed to the mobilization of lipid reserves previously enriched in ALA

PAGE 112

112 from FLAX supplementation. Further study is warranted to identify lipid storage and mobilization kinetics in the horse and how they contribute to circulating plasma FA composition. The decline in plasma and RBC ALA is likely due to the shift from consumption of fresh bahiagrass during the supplementation period (May July ) to Coastal bermudagrass grass hay during the washout period (August September). The predominant FA present in bahiagrass is ALA, with the highest levels observed from April July in the region where this study was conducted (Warren and Kivipelto, 2007a) Although the ALA content of bahiagrass pasture is greater, Coastal bermudagras s hay, a warm season forage, also contains a high proportion of ALA (39% of total FA), LA ( 21 % of total FA) and the saturated FA C16:0 (24% of total FA), with the content of ALA decreasing as the maturity of the hay increases (Warren and Kivipelto, 2007b) Although the FA content of the specific hay fed to the horses in the current study was not measured, it is likely that the increase in plasma and RBC C16:0 observed in all horses during the washout period may be attributed to the presence of these FA in the hay, combined with that found in the basal grain (19% of total FA). In addition, the rise in plasma LA observed durin g the washout period may be attributable to the higher proportion of LA in hay as compared to pasture and the decrease in plasma ALA relates to the decreased ALA content of hay as compared to pasture. The major change in the n 6 FA profile of plasma was o bserved in horses fed FISH, as ARA was higher in these horses in response to 10 wk of supplementation. The FISH supplement contained modest amounts of ARA (1% of total FA), but ARA was absent in the FLAX and CON diets. An increase in plasma ARA was also ob served by King et al. (2008) in horses supplemented with similar amount s of encapsulated fish oil Plasma ARA in horses fed FISH

PAGE 113

113 declined 2 wk after the supplement was withdrawn, but remained slightly elevated over CON and FLAX through 5 wk post supplementation. No difference in RBC ARA was evident among treatment groups until 3 wk after supplementation ended, at which point the RBC ARA was higher in FISH than in CON and FLAX. Again, this may be related to the dynam ics of lipid mobilization in the horse. In contrast to ARA, horses consuming FISH had the lowest plasma and RBC LA after the supplementation period. A similar response was found in human subjects that consumed 3.6 g n 3/d (approximately 5.1 g n 3/100 kg BW /d) for 12 mo, as their plasma and RBC LA was lower than in then non supplemented control group (Katan et al., 1997) This is likely due to the i ncrease in proportions of EPA and DHA in the FISH group, resulting in a concomitant decrease in plasma and RBC LA content. The reason for the increase in plasma LA paired with a simultaneous decrease in RBC LA observed in all horses during the washout peri od is unclear. Because this response was observed in all horses, there may have been seasonal and/or dietary influences that affected all horses contributing to this effect. The nature of these influences remains to be elucidated. In conclusion, these data indicate that for the purposes of designing Latin square or cross over studies involving n 3 FA supplementation, a washout period of at least 8 wk is likely needed when using supplements containing EPA and DHA. In addition, the need for additional studies to characterize the kinetics of lipid storage and mobilization are warranted.

PAGE 114

114 Table 4 1. Fatty acid composition of plasma before (baseline) and after 10 wk of supplementation with milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementat ion (CON) and during the 8 wk washout period Supplementation Washout P v alues Fatty Acid 1 Treatment Baseline +10 wk 2 wk 3 wk 5 wk 8 wk SEM Trt Time Trt* time C18:2 n 6 CON 48.7 48.1 ab 54.1 53.9 56.8 55.1 0.6 FLAX 48.2 49.3 a 54.5 54.9 54.8 55.3 0.5 0.63 <0.01 <0.01 FISH 48.2 46.7 b 54.5 54.3 54.7 55.5 0.6 C18:3 n 3 CON 3.2 3.6 a 3.5 3.0 2.4 b 1.6 0.2 FLAX 3.6 3.7 a 3.5 2.7 3.3 a 2.0 0.1 0.05 <0.01 0.24 FISH 3.1 2.6 b 3.0 2.7 2.4 b 1.8 0.1 C20:4 n 6 CON 1.9 2.0 b 1.5 1.6 1.6 1.2 0.1 FLAX 2.0 2.1 b 1.4 1.6 1.5 1.3 0.1 0.23 <0.01 0.02 FISH 2.2 2.4 a 1.5 1.6 1.7 1.4 0.1 C20:5 n 3 CON ND ND b ND b ND b ND b ND ND FLAX ND ND b ND b ND b ND b ND ND <0.01 <0.01 <0.01 FISH ND 1.4 a 0.1 a 0.1 a 0.2 a ND 0.1 C22:6 n 3 CON ND ND b ND b ND b ND b ND ND FLAX ND ND b ND b ND b ND b ND ND <0.01 <0.01 <0.01 FISH ND 1.6 a 0.3 a 0.2 a 0.6 a ND 0.1 Sum n 6 CON 50.7 50.2 55.6 55.5 58.1 56.3 0.5 FLAX 50.3 51.4 55.9 56.5 56.4 b 56.6 0.5 0.77 <0.01 <0.01 FISH 50.4 49.1 56.1 55.8 56.4 56.9 0.6 Sum n 3 CON 3.2 3.6 b 3.5 3.0 2.4 b 1.6 0.2 FLAX 3.6 3.7 b 3.5 2.7 3.3 a 2.0 0.1 0.05 <0.01 <0.01 FISH 3.1 5.6 a 3.4 3.1 3.3 a 1.8 0.2 1 Fatty acids expressed as a percentage of total fatty acids. Within a treatment and fatty acid, values different than baseline (P<0.05) Within a treatment and fatty acid, values different than +10 wk (P<0.05) a,b,c Within a fat ty acid, values in the same column not sharing a common superscript are different (P<0.05) ND=not detected.

PAGE 115

115 Table 4 2. Fatty acid composition of red blood cell membranes before (baseline) and after 10 wk of supplementation with milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (CON) and during the 8 wk washout period Supplementation Washout P v alues Fatty Acid 1 Treatment Baseline +10 wk 2 wk 3 wk 5 wk 8 wk SEM Trt Time Trt* time C18:2 n 6 CON 42.4 39.8 a 43.6 27.1 18.2 4.8 2.5 FLAX 42.2 42.0 a 43.7 31.0 20.5 5.4 2.5 0.33 <0.01 0.47 FISH 43.2 37.0 *b 42.9 30.9 17.2 4.5 2.6 C18:3 n 3 CON 1.4 2.2 *a 2.6 1.0 0.2 ND 0.2 FLAX 1.3 2.3 *a 2.6 a 1.3 ND ND 0.2 0.10 <0.01 0. 31 FISH 1.2 1.5 b 2.1 0.8 ND ND 0.1 C20:4 n 6 CON 2.6 2.3 2.3 1.1 b 0.1 ND 0.2 FLAX 2.8 2.6 2.5 1.5 b 0.6 ND 0.2 0.20 <0.01 0.84 FISH 2.7 2.4 2.5 1.7 a 0.3 ND 0.2 C20:5 n 3 CON ND 0.2 b ND b ND b ND ND 0.03 FL AX ND ND b ND b ND b ND ND ND <0.01 <0.01 <0.01 FISH ND 1.7 a 0.9 a 1.1 a ND ND 0.1 C22:6 n 3 CON ND ND b ND b ND b ND ND ND FLAX ND 0.4 b ND b ND b ND ND 0.04 <0.01 <0.01 <0.01 FISH ND 1.6 a 1.3 a 0.3 a ND ND 0.1 Sum n 6 CON 45.0 42.1 45.9 28.2 18.4 4.8 2.7 FLAX 45.0 44.6 46.2 32.5 21.1 5.4 2.7 0.29 <0.01 0.49 FISH 45.8 39.4 45.5 32.6 17.5 4.5 2.8 Sum n 3 CON 1.4 2.3 b 2.6 b 1.0 b 0.2 ND 0.2 FLAX 1.3 2.8 b 2.6 b 1.3 b ND ND 0.2 <0.01 <0.01 <0.01 FISH 1.2 5.2 a 4.7 a 2.7 a ND ND 0.4 1 Fatty acids expressed as a percentage of total fatty acids. Within a treatment and fatty acid, values different than baseline (P<0.05) Within a treatment and fatty acid, values different than +10 wk (P<0.05) a,b,c Within a fatty acid, values in the same column not sharing a common superscript are different (P<0.05) ND=not detected.

PAGE 116

116 Figure 4 1 linolenic acid resp onse to 10 wk of supplementation and during an 8 wk washout period in horses receiving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplement ation (NON)

PAGE 117

117 Figure 4 2 Plasma and red blood cell eicosapentaenoic acid response to 10 wk of supplementation and during an 8 wk washout period in horses receiving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplement ation (NON)

PAGE 118

118 Figure 4 3 Plasma and red blood cell docosahexaenoic acid response to 10 wk of supplementation and during an 8 wk washout period in horses receiving milled flaxseed (FLAX), encapsulated fish oil (FISH), or no supplementation (NON)

PAGE 119

119 CHAPTER 5 E FFECT OF FISH OIL SU PPLEMENTATION ON INN ATE AND ACQUIRED IMM UNE FUNCTION IN YEARLING HORSES Abstract To determine if dietary omega 3 (n 3) fatty acid (FA) supplementation affects overall immune function in horses, 18 Thoroughbred yearling fillies (n=12) and colts (n=6) were randomly and equally assigned to one of two treatments for 56 d: encapsulated fish oil (FISH, n=9) providing 9 g n linolenic acid (ALA), 4 g eicosapentaenoic acid (EPA) 0.4 g docosapentaenoic acid, 4 g docosahexaenoic acid (DHA)/100 kg BW ) or no n 3 supplementation (CON, n=9). The basal diet consisted of a grain mix concentrate (4.5% crude fat) fed individually at 1.5% BW and Coastal bermudagrass hay provided ad libitum Whole oats (0.5 0.7 kg/d) were added to CON to make both diets is oenergetic. Treatment did not affect weight gain, which averaged 0.9 0.1 kg/d Supplementation with FISH for 56 d resulted in a higher proportion of EPA, DHA, and total n 3 FA in plasma (P<0.05), red blood cells (RBC; P<0.05), and white blood cells (WBC; P<0.05) compared to CON. Plasma linoleic acid was lower (P<0.05) and arachidonic acid (ARA) was higher (P<0.05) in horses fed FISH, but similar effects on these FA was not observed in RBC or WBC. Positive correlations were found between plasma and RBC ARA (P<0.05; r=0.47), EPA (P<0.05; r=0.98), and DHA (P<0.05; r=0.98), demonstrating successful incorporation of dietary FA into biological membranes. Supplementation with FISH had no effect on phagocytosis or phagocytosis induced oxidative burst of Staphyloco ccus aureus by neutrophils. Antigen specific serum IgGa subclass titers produced in response to a novel vaccine were not different between FISH or CON horses. Results from this study indicate that daily supplementation of horses with

PAGE 120

120 9 g n 3/100 kg BW f or 56 d does not alter neutrophil function or secondary humoral response in healthy yearling horses. Introduction The immune system functions through a complex variety of cells and mechanisms that coordinate to identify and eliminate foreign pathogens. Innate immunity provides protection in an immediate but non specific manner, eliminating foreign invaders through mechanisms such as phagocytosis and eicosanoid mediated inflammation (Tizard, 2 004) Many actions characteristic of innate immunity are essential for the subsequent activation and initiation of the adaptive, or acquired, immune response. Acquired immunity allows for the specific recognition of previously encountered pathogens, and t he maintenance of this specificity is This process is what provides additional protection during future encounters with the same antigen, and it is also what allows for successful vaccination (Tizard, 2004) The two branches of the immune system utilize different mechanisms for eliminating pathogens, but they are dependent upon one another for optimal function. Modulating immune function through nutrition has the potential to enhance health and performance, and recent research in humans and other species indicates that dietary omega 6 (n 6) and omega 3 (n 3) fatty acid (FA) supplementation affects inflammatory and other immune responses, primarily through t he modulation of cell membrane FA composition and subsequent inflammatory mediator production. Specifically, supplementation with eicosapentaenoic acid (C20:5 n 3; EPA) and docosahexaenoic acid (C22:5 n 3; DHA) has been shown to suppress inflammatory eicos anoid production (James et al., 2000) EPA and DHA compete with arachidonic acid (C20:4 n 6 ; A R A) for incorporation in to immune cell membranes, and eicosanoids produced from the precursors EPA and DHA are less pote nt than eicosanoids

PAGE 121

121 produced from A R A (Calder and Grimble, 2002) In addition, EPA and DHA have been shown to alter cell signaling capacity gene expression, and other mechanisms that play key roles in other immune functions such as neutrophil activity and antigen presentation (Calder, 2007) In the horse, research has almost ex clusively focused on the effect that n 3 FA have on i nflammatory mediator production whic h is a function of the innate branch of the immune system. Hall et al. (2004a) reported that fish oil supplementation to horses decreased PGE 2 production by stimulated bronchoalveolar lavage fluid cells. In additio n, supplement ation with linseed oil (rich in the n 3 FA linolenic acid) decreased endotoxin induced production of the inflammatory cytokine tumor necrosis factor TNF ) by macrophages (Morris et al., 1991) Currently, l ittle is known about how dietary n 3 FA suppl ementation affects o ther aspects of innate and acquired immunity. In a previous study in our laboratory intradermal phytohemagglutinin injection caused increased early local swelling response in horses supplemented with fish oil or flaxseed, compared to unsupplemented contro ls (Vineyard et al., 2006; Chapter 3) Part of the early inflammatory response involves the activation and extravasation of neutrophils to the site of insult. Th e early inflammatory response observed in our previo us study prompted further investigation into how n 3 supplementation affects innate to phagocytize and kill foreign microorganisms. S tudies in other species indicate that supplementation with n 3 FA may have an effect on acquired immunity by altering circulating antibody titers in vaccinated animals (Fritsche et al., 1991) However, horses supplemented with either corn or fish oil and sensitized with keyhole limpet hemocyanin (KLH) did not show differences in a ntibody titers to KLH 4 wk after

PAGE 122

122 vaccination (Hall et al., 2004a) It has been suggested that the humoral immune response to n 3 and n 6 FA supplementation may be antigen dependant (Sijben et al., 2001) Fish oil is a rich source of n 3 FA and supplementation with marine derived n 3 FA to horses has been shown to increase plasma n 3 FA concentration (Hall et al., 2004b; King et al., 2008) However, little is currently know about how fish oil supplementation affects cell membrane FA composition of both red blood cells (RBC) and white blood cells (WBC) in ng chain FA into cell membranes is important because the PUFA composition of cell membranes plays an important role in cell function and can influence immune function in a variety of ways (Calder, 2007) Therefore, the objective of this investigation was to test the hypothesis that encapsulated fish oil supplem entation will increase plasma, RBC, and WBC n 3 FA content and alter aspects of innate and acquired immune function in comparison to unsupplemented horses Materials and Methods Horses Eighteen Thoroughbred yearling fillies (n=12) and geldings (n=6) with a meanSE age of 15.1 0.2 mo and an initial bodyweight of 371.5 1.3 kg were utilized in this study During the investigation horses were housed individually in 3.7 x 3.7 m stalls for 12 h /d and fillies were housed in pairs and colts individually in 9. 8 x 21.3 m dry lot paddocks for 12 h / d at the Institute of Food and Agricultural Sciences Horse Te aching Unit in Gainesville, FL. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Florida

PAGE 123

123 Diet ary Treatments Horses were blocked by sex and age and randomly and equally assigned to one of two treatments for 56 d: encapsulated fish oil (FISH, n=9; JBS United, Sheridan, IN) providing 9 g n 3/100 kg BW daily or no n 3 supplementation (CON, n=9). The basal diet consisted of a non fat added grain mix concentrate ( Gest O Lac, OBS Feeds, Ocala, FL ) fed individually at 1.5% BW and Coastal bermudagrass ( Cynodon dactylon ) hay provided ad libitum (estimated to be 1.5% BW). Whole oats (0.5 0.7 kg/d) were added to the CON diet to make both diets isoenergetic. The nutrient and FA composition of the feeds and supplements are presented in Table 5 1. Sample Collection and Processing T o allow adequate ti me for sample processing in the laboratory h orses were divided into 3 groups of 6 horses, with equal treatment representation in each group and blood samples were obtained from one group per day over three consecutive days. Blood samples for FA analysis and neutrophil function were obtained prior to the start of supplementation (d 0) and at 28 and 56 d of supplementation. At each sampling interval, a pproximately 40 mL blood was collected from each horse by jugular venipuncture into evacuated tubes contain ing the anticoagulant sodium heparin (Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ) Tubes were continually mixed by gentle inversion until processing. Whole blood (25 mL) was diluted with 10 mL PBS slowly layered over lymphocyte separation medium (LSM; MP Biomedicals, Solon, OH) and centrifuged at 400 x g for 25 min at 22C (room temperature). After centrifugation, plasma was harvested and stored at 80C until later analysis. Peripheral blood mononuclear cells (PBMC) were removed from the interf ace, washed in PBS, and counted and re suspended in freezing media consisting of 90% fetal bovine

PAGE 124

124 serum (FBS) and 10% DMSO. Cells were gradually frozen at a 1C/ min cooling rate to 80C (Mr. Frosty, Nalgene Labware, Rochester, NY ) and stored in liquid ni trogen until later analysis. Red blood cells (RBC) were removed from below the LSM interface and stored at 80C until later analysis. Small aliquots (<0.5 m L ) of fresh whole blood were retained for the neutrophil assay. Blood samples for antibody producti on in response to vaccin ation were obtained immediately prior to vaccination (d 21 ) 2 wk later at the time of booster administration ( d 35 ) and 3 wk following the booster (d 56) Blood was collected into tubes containing no anticoagulant, and serum was h arvested after centrifugation at 2000 x g for 10 min and stored at 20C until later analysis. Feedstuff, Plasma, Red Blood Cell, and White Blood Cell Fatty Acid Analysis To analyze FA composition of the diets feed samples were lyophilized (FreeZone 6 Lit er Freeze Dryer System, Labconco Corp, Kansas City, Mo) and ground and representative samples consisting of 2 g hay 0.5 g grain mix, or 0.2 g flaxseed or encapsulated fish oil were utilized for FA extraction. For analysis of blood FA, 2 mL of plasma and 4 mL of RBC were frozen at 20 C in 4 mL Wheaton polypropylene Omni vials, lyophilized, sealed with snap caps and stored at 20 C. A mixed reference standard containing 33 FA methyl esters (FAME) (GLC 461, Nu Chek Prep, Elysian, MN) was reconstituted in 10 mL hexane. The FA in freeze dried plasma and RBC were methylated using the two step procedure with a 10 min incubation in sodium methoxide as described by Jenkins et al. (2001; see Appendix E) To maximize the amount of fat extracted, the FA in freeze dried PBMC (approximately 5 x 10 6 cells) an d in feedstuffs were extracted and methylated using the procedure using the procedure of Folch et al. (1957; see Appendix D) A CP 3800 Gas Chromatograph with a CP 8400 Autosampler and Injector, a split injection port, flame ionization detector (Varian, Inc, Walnut Creek, CA), and a 100 m CP SIL 88 fused

PAGE 125

125 silica capillary column (0.25 mm i.d. x 0.2 mm film thickness; Varian, Inc, Walnut Creek, CA), were used for the analysis of individual FA. An injection of 1.0 L was split 1:20 and the He carrier gas maintained at 1.0 mL/min. Column temperature was held at 120C for 1 min following injection, increased at the rate of 5C/min to 190C and held at that temperature for 30 min, then increased at the rate of 2C/min to 220C and held for 50 min. The injector temperature was set at 250C and the detector at 255C. Identification of 21 FAME ( C8:0, C1 0:0, C12:0, C14:0, C14:1, C16:0, C16:1 n 7 C17:0, C17:1, C18:0, C18:1n 9, C18:2n 6, C18:3n 3, C20:0, C20:1, C20:2, C20:4n 6, C20:5n 3, C22:0, C22:5n 3, and C22:6n 3) from the chromatograms were determined by comparing peak retention times with those from t he mixed reference standard. The inclusion of an internal standard (C19:0) was used to verify FA extraction efficiency. The retention times of each individual FA in the mixed FAME standard were verified by the use of reference standards (Nu Chek Prep, Elys ian, MN) containing a single FA population diluted to concentrations expected to be found in blood and feedstuffs The FA present in the extracted sample were quantified (g/L) by multiplying the g/L of an individual FA standard by the area of that indi vidual FA found in the sample, then dividing that by the area of the individual standard. Percent of each FA in the sample was calculated by dividing the g/L of each FA by the total g/L FA in the sample then multiplying that by 100. The sum of n 6 FA i n feedstuffs, plasma RBC, and W BC was calculated by adding the percentages of linoleic acid (LA; C18:2n 6) and arachidonic acid (ARA; C20:4n 6) present in each sample, and the sum of n 3 FA was calculated by the addition of ALA, EPA, DPA, DHA.

PAGE 126

126 Preparation of Bacterial Targets Staphylococcus aureus isolates provided by the Clinical Microbiology, Parasitology and Serology service at the University of Florida Veterinary Medical Center were grown in tryptic soy broth for 18 h at 37C to a final concentration o f approximately 5 x 10 9 cells/mL (see Appendix H) Bacteria in 10 mL of broth culture were heat killed at 56C for 60 min and harvested, washed in sterile PBS, and resuspended in 10 m L of propidium iodide stock solution (1 mg/mL). After mixing by continuou s rotation while protected from light exposure at 22C for 90 min, propidium iodide labeled bacteria was harvested by centrifugation and re suspended in 10 mL sterile PBS to a concentration of 1 x 10 9 cells/mL. Bacterial suspension was protected from ligh t exposure during storage at 4C and warmed to 22C prior to use. Neutrophil Function A method for simultaneously measuring phagocytosis and oxidative burst activity of neutrophils in whole blood using a modified dual color flow cytometry assay was adapted from a procedure developed for use in feline blood (Hanel et al., 2003; see Appendix I) In a preliminary study utilizing five mature Quarter Horse mares, dihydrorhodamine (DHR) loading dose, bacteria to neutrophil ratio, and incubation time were optimized to induce maximum neutrophil phagocyto sis and oxidative burst responses (Vineyard et al., 2007a; see Appendix I) Immediately after blood sample acquisition, white blood cell differential counts were performed to determine neutrophil concentration in each sample. Neutrophils in 100 L aliquots of heparinized blo od were loaded with 4 dihydrorhodamine (DHR ) and incubated at 37C for 10 min with constant rotation. The appropriate amount of propidium labeled Staphylococcus aureus was added to create a bacteria: neutrophil ratio of 30:1 and incubated for 30 min at 37C. After incubation samples were immediately placed on ice to halt phagocytic and oxidative burst

PAGE 127

127 activity and processed for flow cytometry using an Immunoprep reagent system (Coulter Corporation, Miami, FL) and an automated Q Prep Epics immunology workstation (Coulter Corp oration Miami, FL). The completion of hemolysis was achieved by the addition of 500 L of water to each tube, and extracellular fluorescence was quenched with 10 L of 0.4% trypan blue solution. The percentage of neutrophils undergoing phagocytosis and ph agocytosis induced oxidative burst was determined from the acquisition of 10,000 events/sample using a FACSort flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using CELL Quest software (Becton Dickinson, San Jose, CA). Antibody Response to V ac cination To determine the effect of n 3 FA supplementation on humoral immune response, horses were vaccinated with a multivalent vaccine containing inactivated infectious bovine rhinotracheitis, bovine viral diarrhea types 1 and 2, parainfluenza type 3, an d bovine respiratory synctial virus (Triangle 4+Type II BVD, Ft. Dodge Animal Health, Overland Park, KS) This bovine vaccine was utilized to insure that horses had no preexisting antibodies to the vaccine antigens, and has been shown to be immunogenic in horses (Slack et al., 2000) The primary vaccination was administered i.m. on d 21 and a booster vaccination was administered on d 35 of supplementation. Antigen specific serum IgGa subclass titers were determined by ELISA prior to the first vaccination (d 21), 2 wk later immediately prior to booster administration (d 35), and 3 wk after the booster (d 56) using the method as described by Slack et al. (2000) with slight modifications (See Appendix J) Plates were coated with the vaccine diluted in 0.05 M carbonate/bicarbonate buffer (1:500) and incubated overnight at 4C. After blocking with 1% teleostean gelatin in PBS for 90 min, s erum diluted in 1% teleostean gelatin in PBS ( 1:100 ) was added to the plate and incubated at 37 C for 90 min P eroxidase c onjugated goat anti equine

PAGE 128

128 IgGa polyclonal antibody (Serotec, Raleigh, NC) was added to the plate and incubated for an additional 90 min h at 37 C Color was developed by addition of 3,3',5,5' tetramethylbenzidine sburg, MD) and the reaction was stopped by addition of 1 M H 2 SO 4 Absorbance (optical density) of each well at a wavelength of 450 nm was determined spectrophotometrically. Statistical Analysis Differences in plasma RBC and WBC FA content and phagocytosi s and oxidative burst activity were analyzed using the MIXED procedure of SAS (Version 9.0, SAS Institute Inc., Cary, NC) with repeated measures. T reatment, sex, time, and treatment x time interaction were evaluated as fixed effects. Horse within treatment was a random effect. The PDIFF option of the LSMEANS statement of the MIXED procedure was used to compare treatment means. The CORR procedure of SAS was used to identify the relationship between plasma and serum FA in all horses. Serum IgGa subclass antib ody data was log transformed and analyzed with repeated measures using PROC MIXED Bodyweight measurements were analyzed using PROC GLM with repeated measures in SAS. Differences were considered significant at P<0.05. Results Horses gained an average of 0.9 0.1 kg/d over the trial period, and there were no differences in weight gain between the two dietary treatment groups. Throughout the study, horses fed the FISH treatment received 9 g n 3/100 kg BW daily. Plasma, R ed Blood Cell and W hite Blood Cell Fatty Acid Composition The FA composition of plasma, RBC, and WBC are presented in Table 5 2. After 28 d of supplementation, horses consuming FISH had a higher proportion of EPA, DHA, and sum n 3 FA in plasma (P< 0 .05) and RBC (P<0.05) compared to non suppl emented horses. These

PAGE 129

129 differences were sustained through 56 d of supplementation. Horses fed FISH also had lower plasma LA at 56 d (P<0.05) and higher plasma ARA at 28 d (P<0.05) and 56 d (P<0.05) than CON The long chain FA EPA and DHA were only detectabl e the in plasma and RBC of supplemented horses. After 56 d of supplementation, plasma and RBC A R A (P< 0 .01, r= 0 .46), EPA (P< 0 .01, r= 0 .98), and DHA (P< 0 .01, r= 0 .98) were positively correlated (Figures 5 1 and 5 2 ). Across treatments, WBC contained a greater percentage of A R A and l ess LA than both plasma and RBC. The fat extracted from d 0 WBC was lower than expected d ue to a low concentration of PBMC in d 0 samples therefore the values reported in Table 5 2 are not likely reflective of the actual FA composit ion of these cells. Supplementation with FISH resulted in greater WBC EPA DHA, and sum n 3 in WBC after 28 d of supplementation compared to CON (P<0.05), and this difference was sustained through 56 d In addition, the concentration of DHA and the sum of n 3 in WBC was greater (P<0.05) in horses fed FISH than CON after 56 d of supplementation. Over time, plasma ALA concentration decreased in all horses (P<0.05). Plasma LA and sum n 6 decreased in horses fed FISH (P<0.05) after 28 d of supplementation and r emained lower than baseline through 56 d In addition, an increase in plasma and RBC ARA, EPA, DHA, and sum n 3 (P<0.05) was detectable at 28 d in horses fed FISH and was sustained through 56 d In plasma of non supplemented horses, the sum of n 3 declined (P<0.05) after 28 d of supplementation and remained lower than baseline at 56 d In WBC of horses fed FISH, EPA, DHA, and sum n 3 was elevated after 28 d of supplementation and increased even further af t er 56 d of supplementation (P<0.05).

PAGE 130

130 Neutrophil F unction Neutrophil function did not differ between treatment s before (d 0) or after 28 d of supplementation. At d 56, the percentage of total neutrophils in 100 L of whole blood undergoing phagocytosis averaged 89.2 5.6% in non supplemented CON horses, which was similar to the mean of 89.0 6.2% observed in hor ses supplemented with FISH. In the same cell population, phagocytosis induced oxidative burst averaged 63.0 18.9% in CON, which did not differ from the mean of 58.8 12.1% for FISH Figure 5 3 depicts a representative scatter plot (A) showing the gated neutrophil population. The representative dot plot (B) depicts neutrophils that have undergone phagocytosis in both the upper left and right quadrants and those which have undergone phagocytosis induced oxidative burst in the upper right quadrant. Antibody Response to Vaccination Vaccination with an inactivated bovine viral vaccine induced a specific IgGa antibody response in all ho rses, and the primary response to vaccination was greater (P< 0 .05) than the secondary response (Table 5 3). However, FISH supplementation had no effect on antibody titer concentration. Discussion Results from this study demonstrate that daily supplement ation of horses with 9 g n 3 FA/100 kg BW (providing 4 g EPA + 4 g DHA/100 kg BW ) for 56 d can increase the concentration of EPA and DHA in plasma, RBC, and WBC cell membranes, but does not appear to alter neutrophil function or antibody production in resp onse to vaccination. The presence of dietary derived n 3 FA these long chain FA into the membranes of the cells where they will have the most biological impact. The lack of effect on neu trophil function and antibody production suggests that at the

PAGE 131

131 current level and duration of supplementation, fish oil neither enhances nor impairs these aspects of immune function in healthy yearling horses. Plasma, R ed Blood Cell, and W hite Blood Cell Fat ty Acids P lasma FA composition in the horse has been shown to be affected by fish oil supplementation. Hall et al. (2004b) reported increased EPA and DHA and decreased LA, ALA and n 6: n 3 ratio in the plasma of horses fed fish oil compa red to horses fed corn oil In a study by King et al. (2008) horses were fed an encapsulated fish oil product similar to that utilized in the current study After 28 d of supplementation, these horses displayed an increase in plasma ARA EPA, and DHA similar to that observed in the current investigation Previous studies in humans and other species have identified a correlation between plasma and red blood cell FA composition. Skeaff et al. (2006) reported that dietary induced changes in the linoleic a cid composition of both plasma and RBC are similar after 2 wk of supplementation in humans Also in humans, increases in plasma EPA and DHA were correlated with an increase in RBC EPA and DHA, detectable after 56 d of fish oil supplementation (Cao et al., 2006) Harris et al. (2007) reported an increase in the EPA and DHA content of plasma in human subjects consuming 0.5 g EPA+DHA /d that plateaued after 28 d of supplementation. However, RBC EPA+DHA content continued to rise throughout the 16 wk study. In the current study, EPA and DHA in plasma increased after 28 d of supplementation, but did not increase further after an additional 28 d of fish oil feeding. In contrast, the proportion of DHA in RBC and EPA and DHA in WBC continued to increase from 28 to 56 d of fish oil supplementation. T he average life span of an equine RBC is 140 150 d (Fraser et al., 1991) and the life span of WBC varies from w ee k s to y ears depending on the cell type (Janeway et al., 2005) The prolonged dynamics of FA incorporation into RBC and WBC membranes is likely due to the slower turnover and longer

PAGE 132

132 lifespan of RBC and WBC as compared to plasma phospholipids, which are more closely related to daily fluctuations in dietary FA intake (Katan et al., 1997; Bakan et al., 2006) Neutrophil F unction The neutrophil plays a key role in innate i lines of defense against foreign pathogens. Phagocytosis occurs upon binding of the pathogen to the pathogen i nitiates the elimination of the pathogen. Once engulfed, reactive oxygen and nitrogen species are produced by the neutrophil and released to create the lethal oxidative burst. In vitro studies have demonstrated that alterations in phagocyte membrane FA com position are associated with altered phagocytic capacity and suggest that PUFA supplementation may increase uptake of target material by phagocytes (Calder, 2007) Kew et al. (2004) reported that the proportions of total PUFA in PBMC membranes were positively correlated with neutrophil phagocytosis and oxidative burst activity but the spec ific PUFA content of neutrophil cell membranes were not measured in th at particular study Nonetheless th is relationship was attributed to the effect that PUFA can have on the physical nature of the cell membrane. This suggests that increasing membrane EPA and DHA content would enhance neutrophil fun ction. However, this effect has rarely been demonstrated in dietary intervention studies. One study in humans reported a 62% increase in phagocytic activity and a concomitant increase in the rate of reactive oxygen spe cies production after 2 mo of suppleme ntation with 3 g EPA+DHA/d (Gorjao et al., 2006) Thies et al. (1999) examined the capacity of neutrophils from pigs to phagocytize E. coli and found that feeding 5% fish oil significantly decreased the percentage of neutrophils engaged in phagocytosis. However, m ost investigations have shown n 3 supplementation in humans at levels ranging from 4.5 9.5 g ALA/d or 1 4.9 g EPA+DHA/d to have no effect on

PAGE 133

133 either phagocytosis or oxidative burst (Th ies et al., 2001a; Kew et al., 2003a; Kew et al., 2004; Miles et al., 2004a) In the current study, supplementation with FISH had no effect on phagocytosis or phagocytosis induced oxidative burst activity of neutrophils This finding agrees with Skjolaas Wilson et al. (2005) who found no difference in granulocyte phagoc ytic and oxidative burst functions in pregnant mares supplemented with either encapsulated fish oil ( providing 8.6 g EPA/d and 10.4 g DHA/d ) or corn oil for 60 d Similarly, in humans, supplementation with either 4.7 g EPA/d or 4.9 g DHA/d was reported to have no effect on the percentage of neutrophils undergoing phagocytosis when compared to a placebo supplement of olive oil (Kew et al., 2004) Antibody Response to V accination T he hallmark of the humoral immune response is the production of a ntibodies by B cells in res ponse to antigen stimulation. B the antigen and secrete antibodies, and this capability for memory is what allows for vaccination protocols to be successful. In poultry, the antibody response to vaccination with infectious bronchitis virus was not affected by supplementation with dietar y fish oil (Korver and Klas ing, 1997) In contrast, Fritsche et al. (1991) reported that fish oil supplementation increased antibody titers to sheep red blood cell immunization in pullets. However, n 3 FA supplementation has also been shown to decrease antibody ti ters to bovine serum albumin (Parmentier et al., 1997) The discrepancies in these observations are likely the result of different levels of supplementation and the use of different antigens Antibody responses to antigens that elicit a T H 1 type response may be more sensitive to n 3 FA supplementation, as a high level of dietary n 3 FA are expected to favor the T H 1 like response at the expense of T H 2 like responses (Sijben et al., 2001) It has been suggested that EPA supplementation influences the T H 1 / T H 2 balance via PGE 2 inhibition ;

PAGE 134

134 however, studies conducted to confirm this have been highly inconsistent and another mechanism is likely responsible for altered T H 1 / T H 2 bala nce in response to n 3 supplementation (Sijben and Calder, 2007) In the current study, a ntibody responses to a bovine specific vaccine against several bovine vir al strains were not different between non supplemented and fish oil supplemented horse s. Because these yearling horses had all previously been exposed to common equine vaccines utilized for herd health management, using a vaccine that none of the horses had previously been exposed to allowed for the assessment of the humoral response in na ve horses. In this study, supplementation commenced 3 wk prior to the primary vaccination, and perhaps the response may have been different if horses had been consuming supplemental n 3 FA for a longer period of time to allow for more n 3 incorporation int o WBC membranes. In addition, the lack of a true secondary antibody response observed in response to booster vaccination may indicate a limited immunogenicity of this vaccine in horses. Implications Despite alterations in cell membrane FA composition, t he results from this study indicate that daily supplementation of horses with 9 g n 3/100 kg BW (supplying 4 g EPA + 4 g DHA/100 kg BW ) for 56 d does not alter neutrophil function or antibody production in response to vaccination with a novel bovine vaccine. Although dietary n 3 FA have been shown to affect inflammatory mediator production in the horse, results of the current study provide no evidence that fish oil supplementation either enhances or impairs innate or acquired immunity in healthy horses. Additi onal study is needed to identify any immunomodulatory effects that n 3 FA supplementation may have in horses that are affected by chronic inflammatory conditions,

PAGE 135

135 because the response of these horses to dietary FA manipulation may differ from the response of healthy, clinically normal horses.

PAGE 136

136 Table 5 1 Nutrient and fatty acid composition of the grain mix concentrate and hay making up the basal diet, and th e oats and encap sulated fish oil supplement Nutrient 1 Concentrate Hay Oats Encapsulated Fish Oil DM, % 89.3 92.6 88.6 91.7 Crude Fat, % 4.5 2.0 5.4 22.3 CP, % 18.7 11.9 13.0 11.6 NDF, % 17.5 72.2 23.7 9.7 ADF, % 9.9 35.3 12.9 35.3 Ca, % 1.0 0.4 0.1 0 .4 P, % 0.7 0.2 0.4 0.2 Fatty acid 2 C18:2 n 6 50.2 23.0 45.3 9.2 C18:3 n 3 3.3 36.9 1.8 2.3 C20:4 n 6 0.1 ND ND 0.8 C20:5 n 3 ND ND ND 14.8 C22:5 n 3 ND ND ND 1.7 C22:6 n 3 ND ND ND 14.8 Other fatty acids 3 46.4 40.1 52.9 56.4 1 With the except ion of DM, all values are presented on 100% DM basis 2 Fatty acids expressed as a percentage of total fatty acids 3 C12:0, C14:0, C16:0, C16:1 n 7 C18:0, C18:1n 9 C20:0, C20:1, C20:2, C20:3, C22:1

PAGE 137

137 Table 5 2. P lasma, red blood cell, and white blood cell fatty acid composition in yearling horses receiving no n 3 fatty acid supplementation (CON) or encapsulated fish oil (FISH) Day 0 Day 28 Day 56 P values CON FISH CON FISH CON FISH SEM Trt Time Trt*Time Plasm a 1 C18:2 n 6 53.1 b 54.8 a ,x 53.6 53.0 y 53.9 a 52.0 b,y 0.3 0.62 0.22 0.009 C18:3 n 3 1.4 x 1.4 x 0.2 y 0.1 y 0.3 y 0.3 y 0.1 0.48 <0.01 0.89 C20:4 n 6 1.8 b ,x 2.0 a ,x 1.7 b ,x,y 2.5 a ,y 1.7 b ,y 2.3 a ,z 0.04 <0.01 <0.01 <0.01 C20:5 n 3 ND ND x ND b 3.1 a ,y ND b 3.1 a ,y 0.2 <0.01 <0.01 <0.01 C22:6 n 3 ND ND x ND b 2.2 a ,y ND b 2.3 a ,z 0.1 <0.01 <0.01 <0.01 Sum n 6 54.9 b 56.8 a ,x 55.4 55.4 x,y 55.6 54.3 y 0.3 0.69 0.31 0.04 Sum n 3 1.4 x 1.4 x 0.2 b ,y 5.5 a ,y 0. 3 b ,y 5.9 a ,y 0.3 <0.01 <0.01 <0.01 Red Blood Cells 1 C18:2 n 6 36.6 35.9 38 .3 35.4 39.9 36.4 0.7 0.18 0.40 0.60 C18:3 n 3 0.4 b 0.8 a ,x 0.5 0.5 y 0.3 0.3 y 0.1 0.17 0.03 0.14 C20:4 n 6 2.1 2.5 x 2.4 2.8 x,y 2.6 3.1 y 0.1 0.09 0.03 0.86 C20:5 n 3 ND ND x ND b 1.8 a ,y ND b 2.0 a ,y 0.1 <0.01 <0.01 <0.01 C22:6 n 3 ND ND x ND b 1.3 a ,y ND b 2.1 a z 0.1 <0.01 <0.01 <0.01 Sum n 6 38.7 38.4 40.7 38.2 42.5 39.6 0.7 0.33 0.31 0.68 Sum n 3 0.4 0.8 x 0.6 b 3.8 a ,y 0.3 b 4.9 a ,z 0.3 <0.01 <0.01 <0.01 White Blood Cells 1 ,2 C18:2 n 6 3.1 x 2.7 x 13.2 y 12.9 y 11.6 y 11.4 y 0.7 0.74 <0.01 0.99 C18:3 n 3 ND ND ND ND 0.1 ND 0.02 0.36 0.36 0.43 C20:4 n 6 ND x 0.4 x 7.8 y 6.8 y 6.8 y 6.4 y 0.5 0.59 <0.01 0.58 C20:5 n 3 ND ND x ND b 0.9 a ,y ND b 1.9 a ,z 0.1 <0.01 <0.01 <0.01 C22:6 n 3 ND ND x ND 0.6 y 0.1 b 2.5 a ,z 0.2 <0.01 <0.01 <0.01 Sum n 6 3.1 x 3.1 x 21.0 y 19.6 y 18.3 y 17.8 y 1.2 0.63 <0.01 0.88 Sum n 3 ND ND x ND 1.7 y 0.7 b 6.1 a ,z 0.4 0.01 <0.01 <0.01 1 Fatty acids expressed as a percentage of total fatty acids 2 Day 0 values are not likely reflective of actual white blood cell fatty acid composition due to a low concentration of c ells available for fat extraction a, b Within each day, means in the same row not sharing a common superscript differ (P<0.05) x,y,z Within a treatment, means in the same row not sharing a common superscript differ (P<0.05)

PAGE 138

138 Table 5 3. Antigen specific IgGa production (mean optical densitySE) by non supplemented (CON) and encapsulated fish oil supplemented (FISH) horses in response to a bovine vaccine Treatment 2 wk following primary vaccination 3 w k following booster vaccination 1 CON 2.150.4 a 1.290.4 b FISH 2.090.4 a 0.680.1 b 1 Booster vaccine administered 2 wk following primary vaccination Means differ from pre vaccination titers (P< 0 .05) a,b Means in the same row not sharing a common superscript differ (P<0.05)

PAGE 139

139 Fig ure 5 1. Relationship between plasma and red blood cell (RBC) arachidonic acid (ARA) concentration after 56 d of supplementation with either encapsulated fish oil or no supplementation Figure 5 2. Relationship between plasma a nd red blood cell (RBC) eicosapentaenoic acid (EPA ) and docosahexaenoic acid (DHA) concentration after 56 d of supplementation with either encapsulated fish oil or no supplementation

PAGE 140

140 Figu re 5 3 Representative scatter (A) and dot (B) plots generated by flow cytometric evaluation of neutrophil function A B

PAGE 141

141 CHAPTER 6 EFFECT OF HIGH FAT D IETS AND FAT SOURCE ON IMMUNE FUNCTION I N YEARLING HORSES Abstract Recent research in humans indicates omega 3 (n 3) and omega 6 (n 6) fatty acids (FA) may each affect immune function di fferently. To determine if the fat source used in a high fat diet affects immune function in horses, 24 Quarter Horse and Thoroughbred yearlings were randomly and equally assigned to one of three treatments for 42 d: a fish oil (1/3) and olive oil (2/3) bl end (FISH, n=8), corn oil (CORN, n=8), and no supplemental fat (NON, n=8). Horses had free choice access to bahiagrass pasture and a grain mix top dressed with 6% FISH or CORN to create a 10% fat concentrate fed at 1.25% BW/d. The g rain mix was fed to NON horses at a rate of 1.37% BW/d t o make diets isocaloric FISH contained 8.6 g linoleic acid (LA), 5.1 g eicosapentaenoic acid (EPA), 0.5 g docosapentaenoic acid, and 2.4 g docosahexaenoic acid (DHA)/100 g FA and was fed to supply 7.2 g n 3/100 kg BW/d. COR N contained 57.7 g LA/100 g FA, supplying 43.2 g n 6/100 kg BW/d. Blood samples were obtained at 0 and 42 d for determination of plasma and red blood cell membrane (RBC) FA composition, lymphocyte proliferation (LP), PGE 2 production by peripheral blood mon onuclear cells (PBMC), and neutrophil phagocytic and oxidative burst activity. Horses were administered a tetanus booster on d 21, and antibody titers were analyzed at d 42. Data were analyzed using the MIXED procedure of SAS, and contrasts were utilized t o compare NON vs. fat added treatments. Treatment did not affect BW gain, which averaged 0.70.1 kg/d. Plasma and RBC from horses fed FISH had higher (P<0.05) EPA, DHA, and total n 3 and lower (P<0.05) LA and total n 6 compared to CORN and NON. Plasma and RBC LA was highest in CORN horses (P<0.05) and RBC arachidonic acid was lower in CORN horses compared to NON (P<0.05). Neutrophil

PAGE 142

142 function and lymphocyte proliferation were not affected by treatment. PGE 2 production was lower for FISH and CORN than NON (P< 0.05). Using baseline titers as a covariate, horses fed fat added diets had higher tetanus antibody titers at d 42 than NON (P<0.05). Results demonstrate that fat source in a 10% total fat concentrate affects the FA profile of plasma and cell membranes, bu t immune response did not differ between horses supplemented with n 3 or n 6 FA. Rather, total dietary fat, instead of fat source, appeared to alter immune function. Introduction Feeding high fat diets to horses has become a common practice, as it provides a safe way to increase the energy density of the diet for horses with high energy demands. The sources of fat used in most commercially available fat added feeds are rich in omega 6 (n 6) fatty acids (FA). However, research suggests that n 6 FA may affect immune function differently than omega 3 (n 3) FA (James et al., 2000) and therefore the inclusion of n 3 FA in the diets of horses being fed a fat added diet may be of benefit. Recent research in both animals an d humans indicates that dietary n 6 and n 3 FA supplementation affects immune response primarily through the modulation of inflammatory mediator production and cell membrane FA composition (Calder and Grimble, 2002) In the horse, research has almost exclusively focused on the effects of n 3 FA on inflammatory mediator production (Morris et al., 1991; Hall et al., 2004a) However, less is understood about how dietary n 3 and n 6 FA supplementation may affect other aspects of immune function in the horse. Previous studies in our laboratory indicate d that supplementing n 3 FA to a low fat diet has no marked effect on neutrophil function or antibody production compared to horses receiving no supplemental FA (Vineyard et al., 2007b; Chapter 5) The inclusion of n 6 or n 3 FA as part of a high fat diet ha s not yet been investigated for their impact on immune function in the horse.

PAGE 143

143 The objective of the current study was to identify the effects that a fat added diet rich in either n 6 or n 3 FA has on aspects of both innate and acquired immunity in the horse. Because of claims about the pro inflammatory nature of n 6 FA and their metaboli tes in humans (Sargent, 1997) it was hypothesized that n 6 FA supplementation could result in altered immune function through impaired neutrophil function, increased inflammatory mediator production, and/or impai red response to vaccination compared to either n 3 FA supplementation or a non fat added diet. Materials and Methods Horses Twenty four Quarter Horse (n=21) and Thoroughbred (n=3) yearlings (12 fillies, 10 geldings, 2 colts) with a meanSE age of 16.3 0. 2 mo and an initial bodyweight of 441.18.2 kg were utilized in this study. During the trial, fillies and geldings/colts were housed separately in two adjacent 8 ha pastures at the Institute of Food and Agricultural Sciences Hors e Research Center in Ocala, FL. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Florida. Dietary Treatments Horses were blocked by sex and age and randomly and equally assigned to one of three dietary treatments for 42 d: an oil blend consisting of 1/3 fish oil (Omega Protein, Houston, TX) and 2/3 olive oil (FISH, n=8); corn oil (CORN, n=8); or no supplemental fat (NON, n=8). Olive oil contains very low amounts of polyunsaturated FA; therefore, blending it with fish oil allowed the provision of a high level of dietary fat while maintaining the total daily n 3 FA intake at a level used previously to assess immune function in horses (Vineyard et al., 2006; Chapter 3; Vineyard et al., 2007b; Chapter 5) The basal grain mix concentrate ( Gest O Lac, OBS Feeds,

PAGE 144

144 Ocala, FL ) was to p dressed with 6% FISH or CORN to create a 10% total fat concentrate and fed individually at a rate of 1.25% BW/d. This amount of CORN provided 43.2 g n 6/100 kg BW/d and FISH provided 7.2 g n 3/100 kg BW/d. To make the diets isocaloric, horses receiving n o supplemental fat (NON) were fed the same grain mix at a rate of 1.37% BW/d. Bodyweight gain resulting from growth was recorded every 2 wk and adjustments were made to the amount of feed and oil offered. Throughout the study (July August), all horses ha d free choice access to bahiagrass ( Paspalum notatum ) pasture. The nutrient and FA composition of the feeds and supplements are presented in Table 6 1. Sample Collection a nd Processing To allow adequate time for sample processing in the laboratory, horses were divided into four groups of six horses with equal treatment representation in each group. Blood samples were obtained from one group per day over four consecutive day s prior to the start of the study (d 0) and at the end of the supplementation period (d 42) for analysis of plasma and red blood cell membrane FA composition, neutrophil function and lymphocyte proliferation. At each sampling period, approximately 30 ml of b lood was collected from each horse by jugular venipuncture into evacuated tubes containing the anticoagulant sodium heparin (Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ) Tubes were continually mixed by gentle inversion until processing. Whole b lood in 25 mL aliquots was diluted with 10 mL PBS, slowly layered over lymphocyte separation medium (LSM; MP Biomedicals, Solon, OH), and centrifuged at 400 x g for 25 min at 22C (room temperature). After centrifugation, plasma was harvested and stored at 80C until later analysis. Peripheral blood mononuclear cells (PBMC) were removed from the interface, washed in PBS, and counted and re suspended in freezing media consisting of 90% fetal bovine serum (FBS) and 10% DMSO. Cells were gradually frozen at a 1C/minute cooling rate to

PAGE 145

145 80C (Mr. Frosty, Nalgene Labware, Rochester, NY) and stored in liquid nitrogen until later analysis. Red blood cells (RBC) were removed from below the LSM interface and stored at 80C until later analysis. Small aliquots (<0. 5 mL) of fresh whole blood were retained for the neutrophil assay. Blood samples for antibody production in response to a tetanus booster vaccine were obtained immediately prior to booster administration (d 21) and 3 wk following the booster (d 42). Blood was collected into evacuated tubes containing no anticoagulant, and serum was harvested after centrifugation at 2000 x g for 10 min and stored at 20C until later analysis. Feedstuff, Plasma and Red Blood Cell Fatty Acid Analysis To analyze FA composition of the basal diet feed samples were lyophilized (FreeZone 6 Liter Freeze Dryer System, Labconco Corp, Kansas City, Mo) and ground and representative samples consisting of 2 g grass or 0.5 g grain mix were utilized for FA extraction. For FA analysis of o ils, 0.25 g of corn, olive, or fish oil was used. For analysis of blood FA, 2 mL of plasma and 4 mL of RBC were frozen at 20 C in 4 mL Wheaton polypropylene Omni vials, lyophilized, sealed with snap caps and stored at 20 C. A mixed reference standard con taining 33 FA methyl esters (FAME) (GLC 461, Nu Chek Prep, Elysian, MN) was reconstituted in 10 mL hexane. Fatty acids in freeze dried plasma and RBC were methylated using the two step procedure with a 10 min incubation in sodium methoxide as described by Jenkins et al. (2001) FA in freeze dried feedstuffs and oils were extrac ted and methylated using the procedure of Folch et al. (1957) A CP 3800 Gas Chromatograph with a CP 8400 Autosampler and I njector, a split injection port, flame ionization detector (Varian, Inc, Walnut Creek, CA), and a 100 m CP SIL 88 fused silica capillary column (0.25 mm i.d. x 0.2 mm film thickness; Varian, Inc, Walnut Creek, CA), were used for the analysis of individual FA. An injection of 1.0 l was split 1:20 and the He

PAGE 146

146 carrier gas maintained at 1.0 mL/min. Column temperature was held at 120 o C for 1 min following injection, increased at the rate of 5 o C/min to 190 o C and held at that temperature for 30 min, then increased at the rate of 2 o C/min to 220 o C and held for 50 min. The injector temperature was set at 250 o C and the detector at 255 o C. Identification of 23 FAME ( C8:0, C10:0, C12:0, C14:0, C16:0, C16:1 n 7 C17:0, C17:1, C18:0, C18:1n 9, C18:2n 6, C18:3n 3, C20:0, C20 :1, C20:2, C20:3n 3, C20:3n 6, C20:4n 6, C20:5n 3, C22:0, C22:2, C22:5n 3, and C22:6n 3) from the chromatograms were determined by comparing peak retention times with those from the mixed reference standard. An internal standard (C19:0) was included in eac h sample to verify FA extraction efficiency. Retention times of each individual FA in the mixed FAME standard were verified by use of reference standards (Nu Chek Prep, Elysian, MN) containing a single FA population diluted to concentrations expected to be found in blood and feedstuffs Fatty acids present in the extracted sample were quantified (g/L) by multiplying the g/L of an individual FA standard by the area of that individual FA found in the sample, then dividing that by the area of the individua l standard. Percent of each FA in the sample was calculated by dividing the g/L of each FA by the total g/L FA in the sample then multiplying that by 100. The sum of n 6 FA in feedstuffs, plasma or RBC was calculated by adding the percentages of linol enic acid (LA; C18:2n 6) and arachidonic acid (ARA; C20:4n 6) present in each sample, and the sum of n 3 FA was calculated l i nolenic acid (ALA; C18:3n 3), eicosapentaenoic acid (EPA; C20:5n 3), docosapentaenoic acid (DPA; C22:5n 3), an d docosahexaenoic acid (DHA; C22:6n 3). Preparation of Bacterial Targets Prior to performing the neutrophil function analysis, bacteria were fluorescently labeled to be utilized in the assay. Staphylococcus aureus isolates provided by the Clinical Microbio logy,

PAGE 147

147 Parasitology and Serology service at the University of Florida Veterinary Medical Center were grown in tryptic soy broth for 18 h at 37C to a final concentration of approximately 5 x 10 9 cells/mL. Bacteria in 10 mL of broth culture were heat killed at 56C for 60 min and harvested, washed in sterile PBS, and resuspended in 10 mL of propidium iodide stock solution (1 mg/mL). Cultures were protected from light exposure and mixed by continuous rotation at 22C for 90 min. Propidium iodide labeled bacter ia were then harvested by centrifugation and re suspended in 10 mL sterile PBS to a concentration of 1 x 10 9 cells/mL. Bacterial suspension was protected from light exposure during storage at 4C and warmed to 22C prior to use. Neutrophil Function A meth od for simultaneously measuring phagocytosis and oxidative burst activity of neutrophils in whole blood using a modified dual color flow cytometry assay was adapted from a procedure developed for use in feline blood (Hanel et al., 2003; see Appendix I) In a preliminary study utilizing five matu re Quarter Horse mares, dihydrorhodamine (DHR) loading dose, bacteria to neutrophil ratio, and incubation time were optimized to induce maximum neutrophil phagocytosis and oxidative burst responses (Vineyard et al., 2007a; see Appendix I) Immediately after blood sample acqu isition, white blood cell differential counts were performed to deter mine neutrophil concentration in each sample. Neutrophils in 100 L aliquots of rotation. The appropriate amount of propidium labeled Staphylococcus aureus was added to create a bacteria: neutrophil ratio of 30:1 and incubated for 30 min at 37C. After incubation, samples were immediately placed on ice to halt phagocytic and oxidative burst activity and processed for flow cytometry using an Immunoprep reagent system ( Coulter Corp., Miami, FL) and an automated Q Prep Epics immunology workstation (Coulter Corp., Miami, FL). The

PAGE 148

148 completion of hemolysis was achieved by the addition of 500 L of distilled water to each tube, and extracellular fluorescence was quenched with 10 L of 0.4% trypan blue solution. The percentage of neutrophils undergoing phagocytosis and phagocytosis induced oxidative burst were determined from the acquisition of 10,000 events/sample using a FACSort flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using CELLQuest software (Becton Dickinson, San Jose, CA). Prostaglandin E 2 Analysis Frozen PBMC from samples obtained at d 42 were thawed gradually and immediately ted with 10% FBS, 2 mM glutamine, 25 mM HEPES, and penicillin streptomycin (100 IU/ml and 100 g/ml, respectively). Cells were counted and viability was determined to be greater than 80% using trypan blue exclusion. Cells were challenged with lipopolysacch aride (LPS) at a final concentration of 1 10 6 cells/well with 10 ng LPS/well (final volume = 1 ml/well) and incubated at 37C with 5% CO 2 for 24 h. Prostaglandin E 2 production (PGE 2 ) in culture supernatant was analyzed colorimetrically using a commercial ly available kit (Correlate EIA, Assay Designs, Ann Arbor, MI), and optical density of each well was analyzed spectrophotometrically using a plate reader set to read at a wavelength of 405 nm (PowerWave XS, BioTek Instruments Inc., Winooski, VT). Lymphocy te Proliferation Frozen PBMC were thawed gradually and immediately washed and resuspended in ( DMEM ) supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, and penicillin streptomycin (100 IU/mL and 100 g/mL, respectivel y). Cell viability was determined to be greater than 80% using trypan blue exclusion. Proliferative

PAGE 149

149 response to mitogen stimulation was assessed with a nonradioactive colorimetric assay, which has been shown in many species (including horses) to closely co rrelate with the conventional radioactive [ 3 H]thymidine incorporation assay (Ahmed et al., 1994; Witonsky et al., 2003) Aliquots of 100 L of the cell suspension (1 10 6 cells/mL) were stimulated in triplicate wi th 100 L of either Concanavalin A (Con A, 2 g/mL) phytohemagglutinin (PHA, 25 g/mL), or culture media (no mitogen control). Cells were incubated at 37C in 6% CO 2 for a total of 72 h. Alamar Blue (20 L) was added to each well 18 h prior to the end of incubation. Fluorescence was determined with a fluorometer (Synergy HT; BioTek Instruments Inc., Winooksi, VT) at wavelengths of 530 nm excitation, 590 nm emission. The change in fluorescence was calculated by subtracting the mean fluorescence of the non stimulated control w ells f rom the mean fluorescence of the stimulated w ells. Tetanus Antibody Titers All horses had previously been vaccinated for tetanus as weanlings. A tetanus booster vaccination (Tetanus Toxoid, Fort Dodge Animal Health, Fort Dodge, IA ) was administered i.m on d 21 of this study. Serum samples were obtained immediately prior to booster administration and 3 wk later on d 42. Tetanus specific IgG titers were determined by ELISA (Scintilla Development, Bath, PA). Results were calculated a gainst a standard curve derived from calibrator samples, and results were multiplied by the dilution factor to ac hieve the quantitative result reported as IU/mL. Any samples with values greater than the high calibrator were diluted again and rerun in the a ssay so that the abs orbance value fell within the standard curve. Statistical Analysis Differences in plasma and RBC FA composition, neutrophil phagocytosis and oxidative burst activity, and lymphocyte proliferation were analyzed using PROC MIXED with repe ated

PAGE 150

150 measures in SAS (Version 9.0, SAS Institute Inc., Cary, NC). The effects of treatment, sex, time, and treatment x time interaction were evaluated as fixed effects. Horse within treatment was included in the model as a random effect. The PDIFF option o f the LSMEANS statement of PROC MIXED was used to compare treatment means. PGE 2 production by PBMC was analyzed similar to the variables described above, but without repeated measures. Differences in tetanus antibody titers were also analyzed using the MIX ED procedure of SAS, with baseline titer serving as a covariate. In addition, preplanned contrasts were utilized to compare antibody titers between NON vs. fat supplemented (FISH and CORN) treatments. Bodyweight measurements were analyzed using the GLM pro cedure of SAS with repeated measures. Differences were considered significant at P<0.05. Results On average, horses gained 0.70.1 kg/d over the 42 d trial period, and there were no differences in BW gain among the three dietary treatment groups. Through out the study, horses consuming FISH were supplemented with 7.2 g n 3/100 kg BW/d and horses consuming CORN were supplemented with 43.2 g n 6/100 kg BW/d. As horses gained weight, the average daily intake of ALA, EPA, DPA, and DHA provided by the FISH supp lement increased from 5.5, 17.2, 1.6, and 8.0 g/d at the beginning of the study to 5.8, 18.4, 1.8, and 8.6 g/d at the conclusion of the study, respectively. For horses consuming CORN, supplemental LA increased from an average of 184.5 g at the beginning of the study to 200.7 g at the conclusion. Plasma and Red Blood Cell Fatty Acid Composition The FA composition of plasma and RBC is presented in Table 6 2. No differences in plasma or RBC FA composition were detected among treatments prior to the start of su pplementation. After 42 d of supplementation, horses consuming FISH had a higher proportion

PAGE 151

151 of EPA, DHA, and sum n 3 FA in plasma (P<0.05) and RBC (P<0.05) compared to CORN and NON. Horses fed FISH also had lower plasma (P<0.05) and RBC (P<0.05) LA and sum n 6 at d 42 than CORN and NON. Horses consuming CORN had higher (P<0.05) plasma LA and sum n 6 compared to NON and FISH after 42 d of supplementation. In addition, CORN supplementation resulted in lower sum n 3 in RBC than the other treatments (P<0.05). N on supplemented horses had higher RBC ALA than CORN and FISH (P<0.05) and higher RBC ARA than CORN (P<0.05). Overall effects of time and treatment x time were detected for many of the n 3 and n 6 FA in plasma and RBC (Table 6 2). From d 0 to d 42, plasma and RBC LA increased in CORN (P<0.05) and decreased in FISH (P<0.05), but did not change in NON. Plasma ALA increased in response to supplementation with FISH or CORN (P<0.05), but RBC ALA content was unaffected by supplementation. In contrast, ALA remaine d unchanged in the plasma of non supplemented horses, but increased in RBC (P<0.05) from d 0 to d 42. Plasma and RBC ARA content increased (P<0.05) during the study period in all treatments. Only supplementation with FISH resulted in an increase in plasma (P<0.05) and RBC (P<0.05) EPA and DHA. The sum of n 6 FA in plasma increased from day 0 to d 42 in CORN and FISH (P<0.05), but remained unchanged in non supplemented horses. In RBC, however, the sum of n 6 increased in NON and CORN (P<0.05), but not FISH. The sum of n 3 FA increased in the plasma of horses fed FISH (P<0.05) and decreased in plasma of horses fed CORN (P<0.05), but did not change in non supplemented horses. The sum of n 3 FA in RBC increased (P<0.05) in horses fed FISH and in non supplemented horses, but did not change in horses fed CORN.

PAGE 152

152 Neutrophil Function Neutrophil function did not differ among treatments before or after 42 d of supplementat ion. At d 42, the percentage of neutrophils in 100 L of whole blood undergoing phagocytosis averaged 97.50.7% in non supplemented horses, which was similar to the mean of 96.71.1% observed in horses fed CORN and 97.10.7% observed in horses supplemented with FISH. In the same cell population, phagocytosis induced oxidative burst was similar across treatments, averaging 29.74.3, 25.54.4, and 29.63.9% in NON, CORN, and FISH, respectively. The percent of neutrophils undergoing phagocytosis and phagocytos is induced oxidative burst increased (P<0.05) from d 0 to d 42 in all treatment groups (data not shown). Figure 6 1 depicts a representative scatter plot (A) showing the gated neutrophil population. The representative dot plot (B) depicts neutrophils that have undergone phagocytosis in both the upper left and right quadrants and those which have undergone phagocytosis induced oxidative burst in the upper right quadrant. Prostaglandin E 2 Production Treatment with LPS increased PGE 2 production by PBMC in all treatment groups (P<0.05). After 42 d of supplementation, PBMC from horses consuming FISH and CORN produced less PGE 2 upon LPS st imulation than horses receiving no suppl emental fat (P<0.05) (Figure 6 2 ). Lymphocyte Proliferation Although PBMC positively responded to ConA and PHA stimulation, proliferative responses were not different between dietary treatments after 42 d of supplementation (F igure 6 3 ). There were no differences in proliferative response between treatments at d 0, but

PAGE 153

153 proliferation in response to ConA and PHA decreased (P<0.05) from d 0 to d 42 (data not shown) Tetanus Antibody Titers At d 42 (21 d after the tetanus booster was administered), tetanus antibody titers were higher (P<0.05) in horses fed fat (CORN and FISH) than in horses not su pplem ented with fat (NON) (Figure 6 4 ). Mean tetanus antibody titers for NON, CORN and FISH were 3.40.1, 3.80.1, and 3.70.1 IU/mL, respectively. There was no difference in titer levels between CORN and FISH or between FISH and NON, but horses consuming CORN had higher (P<0.05) tetanus titers than NON (Figure 6 5 ) Discussion Feeding horses a fat added diet for 42 d that supplied 12% of total daily DE from corn oil or a fish/olive oil blend did not appear to negatively affect immune function and, in fact, was shown to have a positive impact on some measures of immun ity No differences in neutrophil function or lymphocyte proliferation were found among treatments, but h orses fed a high fat diet had increased antibody titers in response to a tetanus booster vaccination and decreased PGE 2 production by stimulated PBMC. These impacts on immune function occurred regardless of whether the supplemental fat source contained pre dominantly n 3 (fish/olive oil blend) or n 6 (corn oil) fatty acids. Furthermore, similar responses occurred despite the impact that dietary fat source had on plasma and RBC fatty acid composition. Supplementing with corn oil increased n 6 and decreased n 3 fatty acids in plasma, while supplementing with fish oil had the opposite effect. In addition, feeding corn oil increased n 6 fatty acids in RBC, whereas feeding fish oil increased n 3 fatty acids in RBC.

PAGE 154

154 Plasma and Red Blood Cell Fatty Acids The plasma responses to corn oil and fish oil supplementation observed in this study agree with results of other studies performed in horses. Hall et al. (2004b) reported that 12 wk of corn oil supplementation increased LA and the sum of n 6 FA in plasma, whereas fish oil supplementation increased EPA and DHA and the sum of n 3 in plasma. Similarly, King et al. (2005; 2008) fed horses an encapsulated fish oil supplement for 28 d and observed an increase in EPA, DHA, and the sum of n 3 FA present in both plasma and RBC. In the current study, ALA and the sum of n 3 FA in RBC increased in horses fed FISH and in non supplemented horses. These increases are likely due to the rising ALA content of pasture during t he study period, as well as the additional n 3 FA provided by the fish/olive oil blend in the FISH treatment. Forage can serve as a significant source of ALA in equine diets (Warren and Kivipelto, 2007a) and the bahiagrass available to horses in the current study contained 2.3% total fat with ALA comprising 58% of the total FA content. By comparis on, the proportion of n 3 FA in RBC of horses fed corn oil did not change during the study period, although they too were eating ALA rich forage. Instead, the proportion of n 6 FA increased in the plasma and RBC of horses fed corn oil. The high dietary n 6 FA intake from corn oil may have competitively inhibited dietary n 3 FA for membrane incorporation (James et al., 2000) In addition, RBC membranes may possess some form of homeostatic regulatory mechanism that ma intains a minimum threshold level of n 3 FA, preventing further decreases even in the face of increased n 6 FA incorporation into the cell membrane. The proportion of ARA found in plasma and RBC increased during the study period in all horses. Small amount s of ARA were measured only in the fish oil supplement, while large quantities of the ARA precursor, LA, were found in corn oil. However, because the increase in

PAGE 155

155 plasma and RBC ARA was observed across treatments, including non supplemented horses, this sug gests season or possibly the process of growth and development may influence ARA biosynthesis in the horse. Further research is warranted to understand non dietary influences on plasma and RBC FA composition and on how LA, EPA, and DHA affect the presence of ARA in the growing horse. Lymphocyte Proliferation The proliferative capacity of lymphocytes from horses in this study was not affect by dietary treatment. This is in contrast to studies in humans, where supplementation with fish oil supplying up to 2 0.7 g EPA+DHA /100 kg BW has been shown to decrease lymphocyte proliferation (Calder and Grimble, 2002) In addition, high levels of fish oil fed to laboratory animals suppressed spleen lymphocyte proliferation compared to diets rich in other fat sources (Miles and Calder, 1998) For the most part, these findings suggested fish oil possessed potential immuno suppr essive and anti inflammatory effects, which were considered favorable and beneficial to health (Calder, 2001) On the other hand, a drastic reduction in the function and activity of lymphocytes could result in comp romised host defenses. Therefore, other investigators set out to identify the effects of moderate levels of n 3 FA supplementation on lymphocyte proliferation. Kew et al. (2003a) reported that supplementation with EPA+DHA /100 kg BW did not affect lymphocyte proliferation in healthy human subjects (Kew et al., 2003a) Similarly, supplementation providing 4.6 6.6 g EPA+DHA /100 kg BW showed no effect on lymphocyte pro liferation in humans (Endres et al., 1993; Yaqoob et al., 2000) In the current study, FISH horses consumed 5.6 g EPA+DHA /100 kg BW and at this level of supplementation, PBMC response to mitogen stimulation was not affected. Similarly,

PAGE 156

156 supplementation with 57.7 g n 6/100 kg BW from corn oil neither enhanced nor suppressed lymphocyte proliferation. Neutrophil Function Phagocytosis of foreign pathogens is a fundamental part of innate immune function, and dietary induc ed alterations in membrane fatty acid composition have been associated with altered phagocytic capacity (Calder and Grimble, 2002; Calder, 2007) Increased cell membrane polyunsaturated FA content has been shown to increase the uptake of target material by the neutrophil in vitro due to the altered physical nature of the membrane (Calder et al., 1990) In the current study, treatment had no effect on phagocytosis or phagocyt osis induced oxidative burst activity of neutrophils. This finding agrees with results of a previous study in yearling horses consuming a low fat diet that was supplemented with encapsulated fish oil for 56 d (Vineyard et al., 2007b) In addition, Skjolaas Wilson (2005) found no difference in granulocyte phagocytic and oxidative burst functions in pregnant mares supplemented with 19 g EPA+DHA/d or corn oil f or 60 d. The majority of studies in humans have been unable to demonstrate that n 3 FA supplementation has any significant effect on neutrophil function in vivo (Kew et al., 2003a; Miles et al., 2004a; Kew et al., 2 004) It has been suggested that polyunsaturated FA supplementation may affect the extent of phagocytic activity, rather than the percentage of neutrophils undergoing phagocytosis. The contradiction between observations made in vitro and in vivo may be du e, in part, to the methodological approach of measuring neutrophil function. Measuring the percentage of neutrophils that engage in phagocytosis and oxidative burst, which was assessed in the current study, may not be as sensitive as a measurement of the e xtent of phagocytic or oxidative burst activity (i.e. the amount of target material the neutrophil has engulfed and destroyed) (Calder, 2007)

PAGE 157

157 PGE 2 Production The eicosanoid PGE 2 is a potent lipid mediator of inflammation that can increase pain sensitivity and initiates vasodilation. It is synthesized from ARA present in the cell membrane (primarily of macrophages) in response to a variety of stimuli. The positive relationship between 2 has been well documented (Calder, 2007) In addition, it has been demonstrated that increased incorporation of EPA and DHA in to cell membranes results in decreased PGE 2 production (Calder and Grimble, 2002) This is partly because o f decreased availability of ARA in the cell membrane due to competition with EPA and DHA and partly due to the inhibitory effect EPA has on ARA metabolism to PGE 2 In the cyclooxygenase enzymatic pathway, ARA is the precursor to PGE 2 As the n 3 homologue of ARA, EPA can inhibit ARA metabolism by competing for the enzymes necessary to convert ARA to PGE 2 (James et al., 2000) In the current study, horses consuming either FISH or CORN produced less PGE 2 than horses r eceiving no supplemental fat. Horses consuming no supplemental fat had the highest cell membrane ARA concentration, which could contribute to greater PGE 2 production. In horses fed FISH, the higher EPA and DHA in cell membranes may be responsible for a red uced PGE 2 production compared to NON, as it is well documented that supplementation with n 3 fatty acids decreases PGE 2 production by PBMC (Calder and Grimble, 2002) Despite the greater concentration of LA in RBC, horses fed CORN also had lower ARA in RBC than NON. T here is evidence demonstrating that high LA intake exerts an inhibitory effect on the enzymes n ecessary for conversion of LA to ARA (Huang et al., 1996) In mice, high total fat intake suppressed PGE 2 produ ction with no additional effect of increased dietary n 3 FA intake (Broughton an d Wade, 2002) In contrast to these findings, Hall et al. (2004a) reported lower PGE 2 production by

PAGE 158

158 LPS stimulated bronchoalveolar lavage fluid cells in horses supplemented with fish oil compared to horses supplem ented with corn oil. However, their study did not include a non fat supplemented control treatment and the horses were supplemented with only 3% fat. Perhaps in addition to the specific FA composition of supplemental fat, the total amount of fat consumed p lays a role in the capacity of PBMC to produce PGE 2 Tetanus Antibody Titers The hallmark of the humoral immune response is the production of antibodies in response crete antibodies, and this capability for memory is what allows for vaccination protocols to be successful. One general way to assess humoral immune function is by measuring specific antibody production against a previously encountered antigen. In the curr ent study, all horses had previously received a primary series of tetanus vaccinations as weanlings. The horses were fed their respective dietary treatments for 42 d, and at the midpoint of the trial (d 21) baseline tetanus titers were measured and horses were administered a tetanus booster vaccine. At the end of the trial period (d 42), tetanus antibody titers were assessed in all horses. Because baseline titers were different among treatment groups prior to booster administration, baseline titer was used as a covariate for the analysis. When measured 3 wk after booster vaccination, tetanus antibody titers were higher in horses fed fat added diets (CORN and FISH) than in horses fed a non fat added diet (NON). This agrees with Fritsche et al. (1991) who reported an increased antibody response to sheep red blood cell vaccination in chicks supplemented with 7% fish oil. In another study, chickens supplemented with 70 g sunflower oil (69% LA)/ kg diet mounted increased antibody response to mal eyl bovine serum albumin immunization compared to chickens not supplemented with fat (Parmentier et al., 2002)

PAGE 159

159 A review of the literature reveals relatively few definitive studies on the effects of n 3 and n 6 fa tty acids on antibody production. In the studies that have been performed, results have been contradictory. In contrast to what was observed in poultry, Hall et al. (2004a) observed no effect of fish oil supplement ation on antibody production in horses sensitized to keyhole limpet hemocyanin. Similarly, Lessard et al. (2003) reported that antibody response to ovalbumin sensitization was not affected by dietary n 3 FA supplementation in cattle. It appears that antibody response may be depe ndent upon the type of antigen used to promote antibody production and the dietary supplementation level of total dietary fat (Fritsche et al., 1991) In addition, alterations in membrane fatty acid composition of lymphocytes involved in antibody product ion will affect several aspects of immune cell function, including cell signaling and (Calder and Grimble, 2002) More work is necessary to elucidate the specific mechanisms that are responsible for alterations in antibody production due to dietary fat supplementation. I mplications Results from this study show that feeding supplemental fat alters plasma and RBC fatty acid composition and has an effect on immune response in yearling horses, including decreased production of PGE 2 by PBMC and increased antibody response to t etanus vaccination. The responses do not appear to be different between corn oil and a fish/olive oil blend when provided at a rate to supply 12% of total daily digestible energy as fat. Both n 6 and n 3 fatty acids play a role in proper function of the im mune system in clinically normal horses, and perhaps the n 3: n 6 ratio of the diet may influence immune response as well. When estimated FA intake from pasture is included, the diets utilized in this study provided a relatively similar intake ratio of n 6 : n 3, despite the additional n 6 and n 3 FA provided by the corn oil and fish/olive oil blend.

PAGE 160

160 The n 6: n 3 ratio of the FISH diet was 1.4, NON was 1.7, and CORN was 3.7. These ratios are considered low in comparison to the recommended dietary n 6: n 3 ra tio of 6:1 for optimal cardio protective effects in humans (Wijendran and Hayes, 2004) In horses with limited access to fresh forage, supplementation with high amounts of n 6 FA may further increase the n 6: n 3 ratio, but more research is necessary to determine if a high n 6: n 3 ratio in the diet also affects immune function in horses. The horses utilized in this study were healthy yearlings and did not suffer from any apparent inflammatory conditio ns or depressed immune function. It is unclear if effects in humans, it has been suggested that the presence of an inflammatory condition might increase the sensi tivity to the immunomodulatory effects of dietary n 3 supplementation and that condition specific clinical end points may be more sensitive markers of to these effects than immune markers (Sijben and Calder, 2007) Further study is warranted to iden tify the effects of feeding fat to horses with autoimmune or inflammatory disease and the potential for attenuating disease symptoms and/or progression by altering the dietary n 6: n 3 ratio through increased n 3 intake. However, in healthy horses, the add ition of fat to the diet from sources rich in either n 6 or n 3 FA appears to have potential benefi ts on immune function.

PAGE 161

161 Table 6 1 Nutrient and fatty acid composition of the grain mix concentrate and Bahiagrass pasture maki ng up the basal diet, and the fish/olive oil blend and corn oil supplement s Nutrient 1 Concentrate Pasture Fish/Olive oil blend Corn oil DM, % 88.8 23.8 100 100 Crude Fat, % 4.1 2.3 100 100 CP, % 16.8 13.2 0 0 NDF, % 20.1 65.2 0 0 ADF, % 11.0 38.3 0 0 Ca, % 0.8 0.4 0 0 P, % 0.5 0.3 0 0 Fatty acid 2 C16:0 16.1 18.8 17.1 12.9 C18:0 2.3 2.7 2.7 1.7 C18:1 27.4 2.4 48.0 26.1 C18:2 n 6 49.7 17.6 8.6 57.7 C18:3 n 3 4.6 57.7 1.6 1.3 C20:4 n 6 0 0 0.3 0 C20:5 n 3 0 0 5.1 0 C22:5 n 3 0 0 0.5 0 C22: 6 n 3 0 0 2.4 0 Other fatty acids 3 0.7 0.8 13.2 0.4 1 With the exception of DM, all values are presented on 100% DM basis 2 Fatty acids expressed as g/100 g fatty acids. 3 C14:0, C16:1 n 7 C17:0, C20:0, C20:1, C22:0, C22:1, and C22:4

PAGE 162

162 Table 6 2 Plasma and red blood cell fatty acid c omposition before (d 0) and after (d 42) supplementation with corn oil (CORN), fish/olive oil blend (FISH), or no supplementation (NON) Day 0 Day 42 P v al ues Fatty Acid 1 NON CORN FISH NON CORN FISH SEM Trt Time Trt* Time Plasma C18:2 n 6 51.5 51.5 49.6 52.1 b 57.1 a 46.8 *c 0.5 <0.01 0.04 <0.01 C18:3 n 3 3.5 3.9 4.0 2.5 a 1.8 *ab 0.9 *b 0.3 0.54 <0.01 0.06 C20:4 n 6 0.6 0.6 0.6 1.4 1.2 1.5 0.1 0.43 < 0.01 0.61 C20:5 n 3 0 0 0 0 b 0 b 2.5 a 0.1 <0.01 <0.01 <0.01 C22:6 n 3 0 0 0 0 b 0 b 1.7 a 0.1 <0.01 <0.01 <0.01 Sum n 6 52.2 52.1 50.3 53.5 b 58.2 a 48.2 *c 0.5 <0.01 <0.01 <0.01 Sum n 3 3.5 3.9 4.0 2.7 b 1.9 b 5.7 a 0.3 <0.01 0.23 <0.01 Red Blood Cell s C18:2 n 6 38.5 37.2 39.4 40.2 a 41.5 *a 36.0 *b 0.5 0.36 0.31 <0.01 C18:3 n 3 1.4 1.7 1.5 2.5 *a 1.0 b 1.1 b 0.1 0.16 0.95 <0.01 C20:4 n 6 1.5 1.4 1.8 2.8 *a 2.1 *b 2.5 *ab 0.1 0.08 <0.01 <0.01 C20:5 n 3 0 0 0 0.1 b 0 b 2.1 a 0.1 <0.01 <0.01 <0.01 C22:6 n 3 0 0 0 0.02 b 0 b 1.3 a 0.1 <0.01 <0.01 <0.01 Sum n 6 39.7 38.5 41.3 43.6 *a 44.2 *a 38.8 b 0.6 0.47 0.01 <0.01 Sum n 3 1.9 1.7 1.5 3.1 b 1.4 c 5.1 a 0.2 <0.01 <0.01 <0.01 1 Fatty acids expressed as a percentage of total fatty acids Within a treatment, t he fatty acid concentration at d 42 differs from d 0 (P<0.05) a,b,c Within a day means not sharing a common superscript are different (P<0.05)

PAGE 163

163 Figure 6 1 Representative scatter (A) and dot (B) plots generated by flow cytometr ic evaluation of neutrophil function Figure 6 2 Production of PGE 2 by stimulated PBMC from horses receiving no supplement (NON), a fish/olive oil blend (FISH), or corn oil (CORN) for 42 d A B

PAGE 164

164 Figure 6 3 Proliferative responses of PBMC in horses receiving no supplementation (NON), or supple mented with corn oil (CORN) or a fish/olive oil blend (FISH) for 42 d. Figure 6 4 Tetanus specific IgG titers at d 42 in horses supplemented with corn oil (CORN), fish oil (FISH), or no supplementation (NON)

PAGE 165

165 Figure 6 5 Tetanus specific IgG titers at d 42 in response horses supplemented with corn oil (CORN), fish oil (FISH), or no supplementation (NON)

PAGE 166

166 CHAPTER 7 SUMMARY AND CONCLUSI ONS Taken together, the results from the studies in this dissertation highlight several key considera tions for n 3 FA supplementation in the horse. Although fed to supply a similar level of n 3 fatty acids, feeding encapsulated fish oil had a greater impact on plasma and RBC n 3 composition than milled flaxseed when horses were allowed free choice pasture access during the growing season However, it is possible that under conditions of limited pasture access combined with a high grain diet, flaxseed supplementation could impact plasma and RBC ALA composition. Fish oils contain EPA and DHA, which are not f ound in flaxseed, and these are the PUFA which have the ability to alter e icosanoid production during inflammatory processes. In addition, other mechanisms, such as cell signaling, membrane fluidity, and gene expression, are affected by an increased concen tration of EPA and DHA in cell membranes and can ultimately to convert ALA to EPA and DHA appears to be very limited, but more research is warranted to determine how different dietary concentrations of ALA may affect the efficiency of its elongation and desaturation to longer chain PUFA. Supplementation with both fish oil and flaxseed resulted in a more pronounced early inflammatory response to PHA injection but these findings need to be confirmed in future studies in order to verify repeatability and to determine specifically which cellular mechanisms are affected at the local site of injection. It appears that plasma ARA, EPA, and DHA are positively correlated with that in RBC after 56 d of fish oil supplementation, and that alterations generated by dietary n 3 supplementation can be detected in plasma before RBC. The delayed kinetics of n 3 FA incorporation into RBC can also be observed after supplementation has been discontinued, as

PAGE 167

167 EPA and DHA rem ain elevated in RBC for a longer period of time than in plasma. It takes a minimum of 8 wk for complete washout of dietary n 3 FA, as 5 wk after cessation of supplementation, plasma EPA and DHA experience a secondary rise, most likely due to mobilization o f stored lipids. The PUFA composition of WBC is slightly different than RBC, with a lower concentration of LA and a higher concentration of ARA in WBC regardless of n 3 FA supplementation. Nonetheless, fish oil supplementation does increase the n 3 FA con centration of WBC, and changes can be detected after 28 d of supplementation. When n 3 FA are supplemented as part of a low fat diet, the effect on overall immune function appears to be minimal. No differences were observed among non supplemented horses a nd those supplemented with 6 g n 3/100 kg BW for PGE 2 production by PBMC, or in horses supplemented with 9 g n 3/100 kg BW for neutrophil function or antibody production in response to a novel vaccine. However, when both n 3 and n 6 FA are supplemented as part of a high fat diet, some aspects of immune function were affected. When horses were supplemented with either 7.2 g n 3 FA/100 kg BW or 57.7 g n 6/100 kg BW daily, PGE 2 production by PBMC decreased and antibody response to tetanus booster vaccination i ncreased. These findings can be interpreted as favorable, but they were unexpected for horses supplemented with n 6 FA, especially the decline in PGE 2 However, reports that LA can inhibit ARA eicosanoid metabolism helps to explain this finding. In additio n, the enhanced antibody response observed in fat supplemented horses is in contrast to the findings from the previous study that demonstrated no effect of n 3 FA supplementation on antibody production. In addition to the higher level of dietary fat, it is likely that the type of antigen plays a role in the nature of the

PAGE 168

168 effect that n 3 FA supplementation has on humoral immune response, and further study is warranted to identify the relationship between antibody production and fat supplementation. Overall, it can be concluded that supplementation of horses with fish oil is an effective way to increase the n 3 FA concentration of plasma and cell membranes. In all of the studies conducted, horses readily consumed the fish oil products when acclimation was achi eved through a gradual increase over a period of approximately 5 7 d. In addition, feeding a high fat diet (12% digestible energy provided by fat) rich in either n 3 or n 6 FA does not appear to negatively affect immune function and perhaps may even supp ort immune function. The results from these studies do not provide strong evidence to support commercial claims that n 3 FA supplementation will enhance immune function or decrease inflammation, at least when supplied at a rate of 6 9 g n 3/100 kg BW to he althy horses. The aim of these studies, however, was not to specifically target the anti inflammatory effects of n 3 FA, but rather to gain a broader view of how aspects of innate and acquired immune function are affected by n 3 FA supplementation. The res ults do indicate potential to positively impact at least some aspects of immune function through n 3 FA supplementation. More studies are needed to investigate the effects of feeding fat to horses on immune function, especially those suffering from autoimm une or inflammatory disease s, and to further explore the potential for attenuating disease symptoms and progression by altering dietary n 6:n 3 ratio through increased n 3 intake

PAGE 169

169 APPENDIX A PROCEDURE FOR PERIPH ERAL BLOOD MONONUCLE AR CELL ISOLATION (CHAP TER 3) NOTE: rinse pipette with PBS before drawing cells into clean pipette (cells are sticky); Label all conicals in preparation 1) Centrifuge 5 vaccutainer tubes at 2500 rpm (1000 x g) at 22 C (RT ; room temperature ) f or 10 min 2) Pipette 3 mL 0.9% PBS in tw o 15 mL conicals. 3) Pipette buffy coat ( ~1 .5 mL ) from 3 blood tubes into 1 PBS conical and pipette the buffy coat from the remaining 2 tubes into another PBS conical. Mix. 4) In each conical, bring to 10 mL volume with PBS. 5) Pipette 5 mL LSM (RT) in two 15 mL conicals. Slowly layer diluted blood over LSM. 6) Centrifuge at 1240 rpm (400 x g) for 30 min at 22 C (RT). 7) Remove plasma, then remove interface between plasma and LSM from each tube. Minimize amt. of LSM removed. 8) Place in 1 new 15ml tube. 9) Add 5 10 mL (3 vol umes) PBS to wash cells. Mix. 10) Centrifuge at 750 rpm (160 x g) for 10 min. 11) Decant and discard supernatant. 12) Repeat washing with 6 mL PBS. 13) Centrifuge at 750 rpm (160 x g) for 10 min. 14) Decant and discard supernatant. 15) Repeat washing with 6 mL PBS. 16) Centrifuge at 750 rpm (160 x g) for 10 min. 17) Resuspend cells in 1 ml PBS (mix gently) 18) Count cells (live and total).

PAGE 170

170 After counting, b ring cells to desired concentration using freezing media (90% fetal bovine serum, 10% DMSO). Freeze in 1 mL aliquots in tubes rated for fr eezing in liquid nitrogen. Place cells in Mr. Frosty container filled with 100% isopropyl alcohol in to 80C freezer; after 24 h place in nitrogen storage tank for long term storage.

PAGE 171

171 APPENDIX B PROCEDURE FOR RED BL OOD CELL ISOLATION ( CHAPTER 3) 1) Hematocri ts were previously det ermined and all plasma removed before samples were brought to Nu trition Lab. Only RBC cells fro m heparin tube s were brought to our lab for processing. 2) d pipette 3 mL s labeled with the horses number and # 1, # 2 and # 3. 3) Pipette at least 5 mL of cold saline into each of the 3 tubes containing the 3 mL and mix well 4) Place tubes in t he large centrifuge in the balance room. The centrifuge must be balanced, so place the equally filled tubes directly across for each other. Use water in a tube to balance any extra blood tube. 5) Close and lock the lid on the centrifuge. To turn the cen trifuge on, just turn the large black dial on the right of the centrifuge clockwise to 40 (yellow tape with arrow makes the spot) to centrifuge at 4000 RPM. 6) Spin blood for 15 min. Turn centrifuge off by turning dial counterclockwise back to 0. 7) Wait for the centrifuge to stop spinning before you open the lid and attempt to remove the vacutainers. 8) Place vacutainers in a rack and remove the tops. 9) Now use the aspirator on the lab bench to remove most of the salin e from the top of the tube #1, # 2 and #3. Use the aspirator wit h care and try NOT to aspirate the Keep the tip of the pasteur pipette on the edge of glass vial when aspirating. This helps eliminate RBC loss. 10) Add 5 mL of cold saline to the but use at least 5 mL .) Mix well until all RBC have been diluted into the saline. 11) Centrifuge the 3 tubes at 4000 RPM for 10 minutes. 12) Discard saline supernatant from centrifuged RBC samples w Use aspirator. 13) Add 5 mL but use at least 5 mL .) Mix well until all RBC have been diluted into the saline. 14) Centrifuge the 3 tubes at 4000 RPM for 10 minutes.

PAGE 172

172 15) Use aspirator. 16) Add 5 mL but use at least 5 mL .) Mix well until all RBC have been diluted into the saline. 17) Centrifuge the 3 tubes at 4000 RPM for 10 minutes. 18) Discard supernatant from centrifuged RBC samples. Use aspirator. At this point, you must also CAREFULLY ul not to 19) Now, add EXACTLY 2 mL of cold saline #2 and # 3. 20) After mixing each tube well transfer 2 mL of the RBC suspension from tube # 1, # 2 and #3 and put in labeled 4ml freeze r tubes. 21) Make sure the caps are firmly in place, then place tubes at an angle to provide additional surface area (for when we later freeze dry these samples). Freeze samples at this angle in 20 C freezer before placing in storage boxes in 8 0 C freezer.

PAGE 173

173 APPENDIX C PROCEDURE FOR PERIPH ERAL BLOOD MONONUCLE AR CELL ISOLATION (CHAPTERS 5 AND 6) NOTE: When bringing tubes from farm to lab, invert every 10 15 min to keep blood well mixed. R inse pipette with PBS before drawing cells into clean pipette (cells are sticky) Label all conicals (50 mL ) in preparation. 1) Dilute 25 mL whole blood in 10 mL PBS. 2) SLOWLY layer 35 mL diluted blood over 15 mL LSM. **maintain sharp interface** 3) Centrifuge at 1500rpm (400 x g) for 25 min at room temperature ( RT ) (21 C). 4) Rem ove plasma for FA analysis. 5) Aspirate remaining plasma to within 2 3mm above PBMCs. 6) Remove PBMCs and approx. LSM below, placing in new 50 mL tube. **do not disturb RBCs; Reserve RBCs for FA analysis.** 7) Bring to 40 mL volume with PBS. Mix. 8) Centrifuge at 7 40rpm (100 x g) for 10 min at RT. 9) Decant and discard supernatant, being careful not to disturb cell pellet. 10) Add 10 mL PBS. Mix. 11) Centrifuge at 740rpm (100 x g) for 10 min at RT. 12) Decant and discard supernatant. 13) Resuspend cells in 1 mL PBS (mix gently). 14) Count cells (live and total). Bring cells to desired concentration using freezing media (90% fetal bovine serum, 10% DMSO). Freeze in 1 mL aliquots in tubes rated for freezing in liquid nitrogen. Place cells in Mr. Frosty container filled with 100% isopropyl al cohol in to 80C freezer; after 24 h place in nitrogen storage tank for long term storage

PAGE 174

174 APPENDIX D PROCEDURE FOR EXTRAC TION AND METHYLATION OF FATTY ACIDS FOLCH METHOD Always wear eye protection, gloves and apron and work under a fume hood during L CFA extraction 1) Place weighted or measured sample into a 40 mL screw cap vial with Teflon lined cap and record sample weight or record sample volume. Approximate weight or volume of samples : 2 mL plasma, freeze dried 2 mL freeze dried 5 x10 6 cells (WBC minimum) 2 3 grams of hay or grass 0.25 0.5 grams of ration/concentrate 0.1 0.3 grams fish oil supplement or flax 2) Pipette 50 L of C19:0 (1 g / L concentration) into each sample ( the internal standard should be at room temperature before pipetting ). less C19:0 because you do not want the C19:0 to be the largest peak in the sample. 3) Run a C19:0 standard with each set of samples extracted. For this C19:0 std, wait until after filtration step to add C19:0 to the indi vidual C19:0 standard vial. 4) After adding internal std to samples, add Folch 1 (2:1 chloroform: methanol). To C19:0 standard run, add only Folch 1. 20 mL Folch 1 for grasses, hay, ration, fish oil and flax 20 mL 10 mL 5) Vortex (2 minutes). (Do not vortex WBCs) 6) Let sample stand at room temperature over night (16 hours). 7) The next morning, fill both water baths, the 37C (use deionized water here) and the 90 o C and let them be preheating.

PAGE 175

175 8) Vortex sample and filter them through #40 Whatman filter paper ( use the 150mm filter paper ) into a previously weighted 40 mL screw cap vial with teflon lined cap. 9) Add another 10 to 20 mL of Folch1 to original sample vial, (use 20 mL for grasses) vortex, and filt er into previously weighted 40 mL screw cap vial to recover any LCFA remaining in original sample vial. 10) Pipette Folch 1 on filter paper, using pasteur pipette, to rinse any remaining FA off of filter. 11) Add 25 100 L of 10% BHT to the Folch1 solution coll ected from the filtration. Gently rotate vial to mix, and dry sample under nitrogen gas flow in a 37 degrees centigrade water bath. *** BHT sometimes will overlap C14:0, so if you need to see C14:0, you may not want to use the BHT*** 12) At this point add the C:19 std to filtered Folch 1 for the c19:0 std. Use a 0.333 g / L concentration of C:19 13) For 37C water bath, make sure valve is open on #1 valve of the evaporation unit before turning on nitrogen tank. 14) While tubes are drying, label 8 mL septa vials and GC vials. 15) P 70" (1=samples id, P=plasma, 70=day) of course, always make a 16) The dry tube now contains fat collected from the sample. Allow tube to cool and dry the outside of t he vial completely with a paper towel When tube is cool, weigh tube containing fat to determine the amount of fat in the original sample. 17) Wrap teflon tape around the threads of each vial to prevent evaporation of methanol (cap must be tight and leakproo f for methylation to take place). 18) Add 2 mL of 4% H2SO4 in methanol to each 40 mL vial containing the dried fat. Make sure the screw cap in tightly closed on the vial. 19) Heat vials in 90 degree centigrade water bath for 15 minutes. 20) Take teflon tape off o f vials. 21) Allow sample to cool. Make sure samples are completely cool before adding hexane. 22) Get some double distilled water Get fresh double distilled water each day that you are using it.

PAGE 176

176 23) Add hexane to sample. Use 1 or 3 mL hexane for hay or rati on samples, depending on fat content of sample. A sample with 1.0gm fat can use 3 mL of hexane. Use 1.0 mL hexane for 2 mL of freeze dried plasma and 0.5 mL hexane for RBCs. 24) Vortex samples and transfer sample to an 8 mL vial with a teflon septa screw cap. 25) With a needle and syringe, add 2 mL of double distilled water to the 8 mL vial. Vortex. Let stand, inverted, for 30 minutes at room temperature. 26) Remove bottom layer (water and chemicals), the layer closest to the septa using a needle and syringe. Be careful not to remove any hexane!!! Leave a little water rather than remove hexane!!!!! 27) With a needle and syringe, add 2 mL of double distilled water to the 8 mL vial. Vortex. Let stand, inverted for 30 minutes at room temperature. 28) Remove bottom l ayer (water and chemicals), the layer closest to the septa using a needle and syringe. Be careful not to remove any hexane!!! Leave a little water rather than remove hexane!!!!! 29) With a needle and syringe, add 2 mL of double distilled water to the 8 mL v ial. Vortex. Let stand, inverted, for 30 minutes at room temperature. 30) Remove bottom layer (water and chemicals), the layer closest to the septa using a needle and syringe. Be careful not to remove any hexane!!! Leave a little water rather than remove he xane!!!!! 31) With a needle and syringe, add 2 mL of double distilled water to the 8 mL vial. Vortex. Let stand, upright... not inverted this time...for 30 minutes at room temperature. If hexane in sample is not separating from water or is cloudy, you can l eave sample in fridge overnight so it will separate. You can centrifuge these vials to help separate off the hexane 32) If you are having trouble getting the hexane off without getting water as well, first, transfer hexane to slender, 1 mL autosampler vials to help insure you do not get any water in final gc vial. Then transfer hexane layer to GC vial for analysis. You need at least 0.5 mL of hexane in a 2 mL glass GC vial! If you have less than 0.5 mL of hexane you will need to use the 200 L polypropylen e tubes.

PAGE 177

177 33) Mark level of hexane on GC vials with a sharpie. We do this in case the sample evaporates in the freezer. If it does evaporate we can reconstitute to the previous level. 34) Hex ane can be filtered through #1 W hatman filter containing sodium sulf ate anhydrous into sample vial and crimp seal vial cap, if you have enough hexane (1 or 2 mL ) and 35) Sample is ready to be run on GC REAGENTS 10% BHT ( antioxidant ) : 20gm BHT in 200 mL total volume with methanol 4% H 2 SO 4 for methylation : 8.33 mL H 2 SO 4 IN 200 mL total volume w/ methanol. This causes a violent reaction when reagents are mixed. Use a magnetic stirrer and mix these chemicals under the hood. Wear eye protection, gloves and a lab apr on! : 1 part methanol to 2 parts chloroform Double distilled water: Get fresh daily. C19:0 internal std : 1 g / L non methylated

PAGE 178

178 APPENDIX E PROCEDURE FOR FATTY ACID EXTRACTION AND METHYLATION JENKINS METHOD ( CLEMSON 2 STEP ) Reagents 1) Internal standard : 1mg/ mL C19:0 in methanol The concentration of the internal standard (IS) should be adjusted according to the type of sample being run. For example, the feed samples and pure fat samples may require 2 mg/ mL IS concentration, but sa mples containing less total fatty acids (such as feces or blood) required lower IS concentration such as 1 or 0.5 mg/ mL 2) 0.5M Sodium Methoxide Solution in Methanol Sodium Methoxide Solution CH 3 ONa (F.W.=54.02) Sigma cat. # 403607 800 mL ACS reag ent, 0.5 M in methanol 3) 5% Methanolic HCL Acetyl chloride CH 3 COCL F.W.=78.50 (Fisher cat. #A27 250) Reagent grade 98% 10 mL acetyl chloride (Fisher cat. #A27 250) added to100 mL cold methanol. Caution !!!! This is an explosive reaction!!!! The acetyl chloride must be added to the methanol very slowly while stirring. It is helpful to do this reaction with the methanol on ice. NOTE: The shelf life of this mixture is one week 4) 6% Potassium Carbonate 5) Potassium Carbonate, anhydrous : Fisher cat. #P208 5 00 Dissolve 60 grams of Potassium Carbonate (K 2 CO 3 ) in 1 liter distilled water. 6) 0.5 mL 1.0 mL Hexane

PAGE 179

179 Procedure 1) Weigh the sample into a screw top capped (Teflon lined caps) culture tube. Weigh to 4 decimal places. 2) Sample weight depends on the type of sample being analyzed. For example, most pure fats require a sample weight of 20 25 mg, while feed samples without added fat and feces samples require 500 mg. Feed samples with added fat require about 250 mg. 3) NOTE: The lip of the tube must NOT h ave any chips so that the tube can be completely sealed. If a tube is chipped, throw it away. 4) Add 50 L of prepared internal standard. 5) Add 2 mL of Sodium Methoxide, 0.5 M Solution in Methanol. Cap tightly and vortex lightly to mix. 6) Incubate in 50 0 C water bath for 10 minutes. 7) Remove from water bath and allow to cool for 5 minutes. 8) Uncap and add 3 mL of 5% Methanolic HCL. Recap and vortex. 9) Incubate in a n 80 0 C water bath for 10 minutes. 10) Remove from water bath and allow to cool for 10 minutes. 11) Ad d 1 mL of Hexane and 7.5 mL 6% K 2 CO 3 Recap and vortex. 12) Centrifuge at 1200 rpm for 5 minutes to separate layers. 13) Decant off Hexane and samples are ready for GC analysis.

PAGE 180

180 APPENDIX F PROCEDURE FOR LYMPHO CYTE PROLIFERATION Cell culture media DMEM 100 U/m L Pen/Strep 1 mL/100 mL l glutamine 1 mL/100 mL HEPES 10% Fetal Bovine Serum To make 1 L (1000 mL) of media: Pen/Strep = 10 mL l glutamine = 10 mL HEPES = 10 mL 10% FBS = 100 mL FBS DMEM = 1000 (10+10+10+100) = 870 mL All components should be passed th rough a sterile filter into a sterilized bottle. add L glutamine to media weekly fresh media should be made up every 3 weeks 1) Thaw frozen cell suspen sion using the following method: a) Remove from liquid nitrogen and quick thaw in 37C water bath for 90 sec (swirl as thawing) b) Place in 15 mL conical c) Add 3 drops of warm media; swirl d) Repeat until at an approximate volume of 2 mL e) Add 6 drops; swirl; until an approximate volume of 5 mL f) Add 1 mL at a time; swirl; until a vol ume of 10 mL is reached g) Centrifuge at 22 C 1000 x g for 10 min h) Decant supernatant i) Re suspend in 1 mL media Alternate Method: a) Slightly thaw sample in hand until pellet loosens b) Place pellet in 10 mL warm culture media c) Slowly invert until pellet dissolves d) Leave overnight in 37C incubator

PAGE 181

181 2) Count cells using hemacytometer 3) Spin cell suspension to create pellet 4) Re suspend cells in culture media to concentration of 1 x 10 6 cells/ mL 5) Prepare mitogen suspensions: ConA at 2 g/ mL PHA at 25 g/ mL 6) Add 100l of cell suspension (1x10 6 cells/ mL ) to each we ll in triplicate for blank, ConA, and PHA for each sample 7) Add 100l of media to blank wells 8) Add 100l of ConA suspension to ConA wells 9) Add 100l of PHA to PHA wells 10) Incubate at 37C with 6% CO 2 for 72 hours 11) At 54 hours (18 h prior to endpoint), add 20 l of alamar blue to each well and incubate for another 18 hours. 12) At 72 hours, read on fluorometer at excitation 530 nm and emission 590 nm Samples are averaged. The blank is subtracted from each control and unknown average and the sample is reported as change in fluorescence.

PAGE 182

182 APPENDIX G PROCEDURE FOR PGE 2 ANALYSIS Separate PBMC (Chapter 3 PGE 2 samples): 1) Separate PBMC as per the Accuspin procedure provided with the kit upon purchase 2) 3) Resu spend pellet in 1 mL of RPMI with 10% FBS, 1% antibiotic/antimycotic 4) Count cells (via hemacytometer) Separate PBMC (Chapter 6 PGE 2 samples): See Appendix C Challenge with LPS (Can be modified to your experimental design) : Use PGE 2 kit Co rrelate EIA, Assay Designs, Ann Arbor, MI Challenge 1x10 6 cells/mL with 10 ng/mL of LPS U se 24 well plates and prepare cells suspension so that there are 2x10 6 cells/mL. A dd 0.5 mL of the cell suspension to the appropriate wells, followed by 0.5 mL of 20ng/mL of LPS or 0.5 mL of culture media for control wells. This leads to a final concentrati on of 1x10 6 cells/well with 10 ng of LPS per well (final volume is 1 mL/well). 1) Resuspend cells to achieve desired cell concentration 2) Prepare control wells = cells c ultured with RPMI culture media only 3) Prepare LPS wells = cells cultured with 10 ng/mL LPS. 4) Incubate 37C, 5% CO 2 for 24 hours. 5) Collect supernatant from each well 6) Store at 20 to 70C until performing an ELISA (PGE 2 evaluation Assay Des igns kit )

PAGE 183

183 APPENDIX H PROCEDURE FOR PREPAR ATION OF BACTERIAL T ARGETS 1) Obtain a preparation of Staphylococcus aureus bacteria grown in tryptic so y broth at final volume of 10 mL and final concentration of 5x10 9 cells/mL 2) Kill the bacteria by heating the b roth culture at 56 C for 30 60 min. Harvest the heat killed bacteria by centrifugation at 2000 rpm for 15 minutes. 3) Decant supernatant and re suspend the bacterial pellet in 10 mL sterile PBS. Vortex. Centrifuge at 2000 rpm for 15 min. 4) Decant the supernat ant and re suspend the bacterial pellet in 10 mL of 300 g/ mL propidium iodide (PI) solution (7 mL PBS + 3 mL of PI stock solution (1 mg/ mL )). 5) Cover tube in aluminum foil to protect against light and mix by continuous rotation at 22 C overnight. 6) Harves t the PI labelled bacteria by centrifugation at 2000 rpm for 15 min. Decant the supernatant and re suspend the bacteria in 10 mL sterile PBS. Good for up to one year

PAGE 184

184 APPENDIX I PROCEDURE OPTIMIZATION, AND VALIDATION DATA FOR NEUTROPHIL FUNCTION ASSAY NOTE: Prior to analysis, perform WBC differential analysis to determine neutrophil concentration in whole blood sample. Procedure 1) Label 3 tubes for each horse: neg control (DHR only), pos control (DHR + PMA), and SA (DHR + Staph aureus) 2) working solution of DHR from 500 uM stock solution: 100 L DHR stock + 900 L PBS = 1000 L of 50 uM DHR 3) Add 100 L of heparinized whole blood to each tube. 4) Add 10 L of 50 M DHR into the tubes (final DHR concentration/tube = 4 M). 5) Incubate tubes at 37 o C for 10 minutes with constant rotation to load the DHR into the neutrophils. 6) Prepare 5 g / mL working solution of PMA from 1 mg/ mL stock solution: 5 L PMA stock + 995 L PBS = 1000 L of 5 g/ mL PMA solution Store working solution on ice pending use 7) Add 10 L of the PMA working solution to the pos control. The final concentration of PMA per tube = 50 ng 8) Add the appropriate amount of bacterial suspension (10 6 cells/ L ) to the tubes for a bacterial:neutrophil ratio of 30:1 9) Incubate all tubes at 37 o C for 30 minutes with constant rotation. 10) Immediately place tubes on ice to stop phagocytosis and oxidative burst activity. 11) Process the tubes for flow cytometry using the automated Q Prep Epics immunology workstation set on the 35 second cycle. 12) Add 5 00 L of cold distilled water to each tube for completion of hemolysis. 13) Add 10 L of 0.4% trypan blue to each tube to quench extracellular fluorescence.

PAGE 185

185 Optimization and Validation Staphylococcus aureus bacteria was grown in tryptic soy broth for 18h at 37C and heat killed, labeled with propidium iodide (PI), and resuspended in PBS to a concentration of 1x109 cells/ mL Dihydrorhodamine (DHR), a non fluorescent substrate which is converted to fluorescent rhodamine during OB, was dissolved in dimethyl sul fo xide to a stock concentration bacteria, was dissolved in ethanol to a stock concentration of 1 mg/ mL Samples were processed for flow cytometry utilizing a Q prep au tomated lysing system (Coulter Corp., Miami, FL). A FACSort flow cytometer (Becton Dickinson, San Jose, CA) was utilized to measure fluorescent intensity. Data were collected from 10,000 cells/sample and analyzed using CellQuest software (Becton Dickinson) To determine optimal assay conditions, whole blood was obtained via jugular venipuncture into heparinized tubes from four mature Thoroughbred geldings. DHR loading dose in 100 aliquots of whole blood was evaluated at 1, 1.25, 2, 2.5, 4, 5, 8, and 10 optimization of maximal OB upon PMA (10 mL ) stimulation. Percentage of PMN that underwent OB plateaued at 4 as evaluated at 0, 2.5, 5, and 10 mL Percentage of PMN that underwent OB plateaued at 5 mL of PMA. Bacteria:PMN ratios were evaluated at 10, 20, 30, 40, 50, and 60:1. Maximum phagocytosis and OB reached a plateau at 30:1. Incubation time of whole bl ood with bacteria was evaluated for 10, 20, 30, 40, and 50 min. Maximum phagocytosis and OB achieved a plateau at 30 min. To determine day to day variability of PMN function measurement with the optimized settings, blood samples from five mature Quarter Ho rse mares were obtained and analyzed for simultaneous phagocytosis and OB act ivity. The inter assay coefficient of variation (CV) was

PAGE 186

186 assessed by testing 3 samples obtained on 3 consecutive days for each of the five mares. PMN in labeled S. a ureus at a 30:1 bacteria:PMN ratio. DHR loaded PMN served as the negative control and DHR loaded PMN stimulated with PMA served as the positive control. Figure I 1 DHR optimization F igure I 2 PMA c oncentration optimization

PAGE 187

187 Figure I 3 B acteria:neutrophil ratio optimizatio n Figure I 4 Incubation time optimization

PAGE 188

188 Figure I 5 Validation of assay settings in 5 hor ses over 3 consecutive days for percent of neutrophils undergoing phagocytosis Figure I 6. Validation of assay settings in 5 horses over 3 consecutive days for percent of neutrophils undergoing phagocytosis induced oxidative bu rst Using the optimized assay, the mean percentage of PMN that underwent phagocytosis and phagocytosis induced OB over the 3 day period was 876.3% and 53.85.8%, respectively. The mean CV was 6% for phagocytosis and 21% for phagocytosis induced OB.

PAGE 189

189 A PPENDIX J PROCEDURE FOR SERUM I G G SUBCLASS TITER DET ERMINATION BY ELISA A. Plate Coating 1. Dilute whole vaccine 1:500 in 0.05 M carbonate/bicarbonate buffer (pH 9.4) by adding vaccine to 9.980 mL carbonate/bicarbonate buffer. 2. diluted vaccine to each well. Cover plate with plate sealer and incubate overnight at 4C. 3. Aspirate dilute PBS solution containing 0.05% Tween 20. Aspirate wash solution. 4. Block wells with 1% t ) for 1 h at 3 7C. 5. Aspirate blocking buffer and proceed to assay as described below: B. ELISA 1. Dilute serum samples 1:100 in 1% teleostea of dilute serum to triplicate wells of the plate On each plate, include a ll serum samples (pre vacci ne, d14, d 35) from one animal, a positive control (serum obtained from a vaccinated animal), and a negative control (serum obtained from a non vaccinated animal). 2. Incubate plate at 37C for 90 min 3. Aspirat PBS solution cont aining 0.05% Tween 20. Aspirate wash solution. 4. Dilute detection antibody 1:10,000 IgG subclass antibody to 11.99988 mL buffer. 5. dilute detection antibody to each well. Cover plate and Incubate at 37C for 90 min 6. Aspi rat PBS solution containing 0.05% Tween 20. Aspirate wash solution. 7. TMB substrate to develop color. Cover and incubate in the dark for 20 min at room temperature. 8. St Stop Solution to each well. Allow to equilibrate for 5 min. 9. Read absorbance (OD) of each well at wavelength 450 nm

PAGE 190

190 LIST OF REFERENCES Ahmed, S. A., R. M. Gogal, Jr., and J. E. Walsh. 1994. A new rapid and simple non radioactive assay to monitor an d determine the proliferation of lymphocytes: An alternative to [3h]thymidine incorporation assay. J. Immunol. Methods 170: 211 224. Arab, L. 2003. Biomarkers of fat and fatty acid intake. J. Nutr. 133 Suppl 3: 925S 932S. Aymond, W. M., and M. E. Van Elswy k. 1995. Yolk thiobarbituric acid reactive substances and n 3 fatty acids in response to whole and ground flaxseed. Poult. Sci. 74: 1388 1394. Bagga, D., L. Wang, R. Farias Eisner, J. A. Glaspy, and S. T. Reddy. 2003. Differential effects of prostaglandin derived from omega 6 and omega 3 polyunsaturated fatty acids on cox 2 expression and il 6 secretion. Proc. Natl. Acad. Sci. USA 100: 1751 1756. Bakan, E., A. Yildirim, N. Kurtul, M. F. Polat, H. Dursun, and K. Cayir. 2006. Effects of type 2 diabetes mellit us on plasma fatty acid composition and cholesterol content of erythrocyte and leukocyte membranes. Acta Diabetol. 43: 109 113. Berbert, A. A., C. R. Kondo, C. L. Almendra, T. Matsuo, and I. Dichi. 2005. Supplementation of fish oil and olive oil in patient s with rheumatoid arthritis. Nutrition 21: 131 136. Brinsko, S. P., D. D. Varner, C. C. Love, T. L. Blanchard, B. C. Day, and M. E. Wilson. 2005. Effect of feeding a dha enriched nutriceutical on the quality of fresh, cooled and frozen stallion semen. Ther iogenology 63: 1519 1527. Brouard, C., and M. Pascaud. 1990. Effects of moderate dietary supplementations with n 3 fatty acids on macrophage and lymphocyte phospholipids and macrophage eicosanoid synthesis in the rat. Biochim. Biophys. Acta 1047: 19 28. Br oughton, K. S., C. S. Johnson, B. K. Pace, M. Liebman, and K. M. Kleppinger. 1997. Reduced asthma symptoms with n 3 fatty acid ingestion are related to 5 series leukotriene production. Am. J. Clin. Nutr. 65: 1011 1017. Broughton, K. S., and J. W. Wade. 200 2. Total fat and (n 3):(n 6) fat ratios influence eicosanoid production in mice. J. Nutr. 132: 88 94. Burdge, G. C. 2006. Metabolism of alpha linolenic acid in humans. Prostaglandins Leukot. Essent. Fatty Acids 75: 161 168. Burdge, G. C., and P. C. Calder. 2005. Conversion of alpha linolenic acid to longer chain polyunsaturated fatty acids in human adults. Reprod. Nutr. Dev. 45: 581 597. Burdge, G. C., A. E. Jones, and S. A. Wootton. 2002. Eicosapentaenoic and docosapentaenoic acids are the principal produc ts of alpha linolenic acid metabolism in young men*. Br. J. Nutr. 88: 355 363. Burdge, G. C., and S. A. Wootton. 2002. Conversion of alpha linolenic acid to eicosapentaenoic, docosapentaenoic and docosahexaenoic acids in young women. Br. J. Nutr. 88: 411 4 20.

PAGE 191

191 Bush, J. A., D. E. Freeman, K. H. Kline, N. R. Merchen, and G. C. Fahey, Jr. 2001. Dietary fat supplementation effects on in vitro nutrient disappearance and in vivo nutrient intake and total tract digestibility by horses. J. Anim. Sci. 79: 232 239. Ca lder, P. C. 2001. Polyunsaturated fatty acids, inflammation, and immunity. Lipids 36: 1007 1024. Calder, P. C. 2003. N 3 polyunsaturated fatty acids and inflammation: From molecular biology to the clinic. Lipids 38: 343 352. Calder, P. C. 2006. Polyunsatur ated fatty acids and inflammation. Prostaglandins Leukot. Essent. Fatty Acids 75: 197 202. Calder, P. C. 2007. Immunomodulation by omega 3 fatty acids. Prostaglandins Leukot. Essent. Fatty Acids 77: 327 335. Calder, P. C., J. A. Bond, D. J. Harvey, S. Gord on, and E. A. Newsholme. 1990. Uptake and incorporation of saturated and unsaturated fatty acids into macrophage lipids and their effect upon macrophage adhesion and phagocytosis. Biochem. J. 269: 807 814. Calder, P. C., and R. F. Grimble. 2002. Polyunsatu rated fatty acids, inflammation and immunity. Eur. J. Clin. Nutr. 56 (Suppl. 3): S14 19. Calder, P. C., P. Yaqoob, F. Thies, F. A. Wallace, and E. A. Miles. 2002. Fatty acids and lymphocyte functions. Br. J. Nutr. 87 (Suppl. 1): S31 48. Cao, J., K. A. Schw ichtenberg, N. Q. Hanson, and M. Y. Tsai. 2006. Incorporation and clearance of omega 3 fatty acids in erythrocyte membranes and plasma phospholipids. Clin. Chem. 52: 2265 2272. Cassil, B., S. Hayes, J. Ringler, and L. Lawrence. 2006. The effect of sedation on serum cortisol concentration in mares during weaning. J. Anim. Sci. 84 (Suppl. 1): 329 330. Caughey, G. E., E. Mantzioris, R. A. Gibson, L. G. Cleland, and M. J. James. 1996. The effect on human tumor necrosis factor alpha and interleukin 1 beta produc tion of diets enriched in n 3 fatty acids from vegetable oil or fish oil. Am. J. Clin. Nutr. 63: 116 122. Chandrasekharan, N. V., and D. L. Simmons. 2004. The cyclooxygenases. Genome Biol. 5: 241. Christensen, M. S., and C. E. Hoy. 1996. Effects of dietary triacylglycerol structure on triacylglycerols of resultant chylomicrons from fish oil and seal oil fed rats. Lipids 31: 341 344. Cleland, L. G., J. K. French, W. H. Betts, G. A. Murphy, and M. J. Elliott. 1988. Clinical and biochemical effects of dietary fish oil supplements in rheumatoid arthritis. J. Rheumatol. 15: 1471 1475. Cleland, L. G., and M. J. James. 2000. Fish oil and rheumatoid arthritis: Antiinflammatory and collateral health benefits. J. Rheumatol. 27: 2305 2307.

PAGE 192

192 Conquer, J. A., J. B. Martin I. Tummon, L. Watson, and F. Tekpetey. 1999. Fatty acid analysis of blood serum, seminal plasma, and spermatozoa of normozoospermic vs. Asthenozoospermic males. Lipids 34: 793 799. Contreras, M. A., and S. I. Rapoport. 2002. Recent studies on interaction s between n 3 and n 6 polyunsaturated fatty acids in brain and other tissues. Curr. Opin. Lipidol. 13: 267 272. Cooper, A. L., L. Gibbons, M. A. Horan, R. A. Little, and N. J. Rothwell. 1993. Effect of dietary fish oil supplementation on fever and cytokine production in human volunteers. Clin. Nutr. 12: 321 328. Cotter, S. M. 2001. Hematology. Teton NewMedia, Jackson Hole, WY. Cunha, T. J. 1991. Horse feeding and nutrition. 2nd ed. Academic Press, Inc., San Diego, CA. D'Ambola, J. B., E. E. Aeberhard, N. Tr ang, S. Gaffar, C. T. Barrett, and M. P. Sherman. 1991. Effect of dietary (n 3) and (n 6) fatty acids on in vivo pulmonary bacterial clearance by neonatal rabbits. J. Nutr. 121: 1262 1269. De Moffarts, B., K. Portier, N. Kirschvink, J. Coudert, N. Fellmann E. van Erck, C. Letellier, C. Motta, J. Pincemail, T. Art, and P. Lekeux. 2007. Effects of exercise and oral antioxidant supplementation enriched in (n 3) fatty acids on blood oxidant markers and erythrocyte membrane fluidity in horses. Vet. J. 174: 113 121. Drevon, C. A. 1992. Marine oils and their effects. Nutr. Rev. 50: 38 45. Dunnett, C. E., D. J. Marlin, and R. C. Harris. 2002. Effect of dietary lipid on response to exercise: Relationship to metabolic adaptation. Equine Vet. J. Suppl.: 75 80. Duvaux Ponter, C., M. Tournie, L. Detrimont, F. Clement, C. Ficheux, and A. A. Ponter. 2004. Effect of a supplement rich in linolenic acid added to the diet of mares on fatty acid composition of mammary secretions and the qcquisition of passive immuneity in the f oal. Anim. Sci. 78: 399 407. Ellis, A. D., and J. Hill. 2005. Nutritional physiology of the horse. Nottingham University Press, Nottingham, UK. Emken, E. A., R. O. Adlof, and R. M. Gulley. 1994. Dietary linoleic acid influences desaturation and acylation o f deuterium labeled linoleic and linolenic acids in young adult males. Biochim. Biophys. Acta 1213: 277 288. Endres, S., R. Ghorbani, V. E. Kelley, K. Georgilis, G. Lonnemann, J. W. van der Meer, J. G. Cannon, T. S. Rogers, M. S. Klempner, and P. C. Weber. 1989. The effect of dietary supplementation with n 3 polyunsaturated fatty acids on the synthesis of interleukin 1 and tumor necrosis factor by mononuclear cells. N. Engl. J. Med. 320: 265 271. Endres, S., S. N. Meydani, R. Ghorbani, R. Schindler, and C. A. Dinarello. 1993. Dietary supplementation with n 3 fatty acids suppresses interleukin 2 production and mononuclear cell proliferation. J. Leukoc. Biol. 54: 599 603.

PAGE 193

193 Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: 497 509. Folsom, R. W., M. A. Littlefield Chabaud, D. D. French, S. S. Pourciau, L. Mistric, and D. W. Horohov. 2001. Exercise alters the immune response to equine influenza virus and increases susceptibility to infection. Equine Vet. J. 33: 664 669. Fraser, C. M., J. A. Bergeron, A. Mays, and S. E. Aiello (Editors). 1991. The merck veterinary manual. Merck and Co., Inc., Rathway, N. J. Friberg, C. A., and D. Logas. 1999. Treatment of c ulicoides hypersensitive horses with high dose n 3 fatty acids: A double blinded crossover study. Vet. Derm. 10: 117 122. Fritsche, K. L., D. W. Alexander, N. A. Cassity, and S. C. Huang. 1993. Maternally supplied fish oil alters piglet immune cell fatty a cid profile and eicosanoid production. Lipids 28: 677 682. Fritsche, K. L., N. A. Cassity, and S. C. Huang. 1991. Effect of dietary fat source on antibody production and lymphocyte proliferation in chickens. Poult. Sci. 70: 611 617. Galbraith, H., T. B. Mi ller, A. M. Paton, and J. K. Thompson. 1971. Antibacterial activity of long chain fatty acids and the reversal with calcium, magnesium, ergocalciferol and cholesterol. J. Appl. Bacteriol. 34: 803 813. Gallai, V., P. Sarchielli, A. Trequattrini, M. Francesc hini, A. Floridi, C. Firenze, A. Alberti, D. Di Benedetto, and E. Stragliotto. 1995. Cytokine secretion and eicosanoid production in the peripheral blood mononuclear cells of ms patients undergoing dietary supplementation with n 3 polyunsaturated fatty aci ds. J. Neuroimmunol. 56: 143 153. Goodrich, L. R., and A. J. Nixon. 2006. Medical treatment of osteoarthritis in the horse a review. Vet. J. 171: 51 69. Gorjao, R., R. Verlengia, T. M. Lima, F. G. Soriano, M. F. Boaventura, C. C. Kanunfre, C. M. Peres, S C. Sampaio, R. Otton, A. Folador, E. F. Martins, T. C. Curi, E. P. Portiolli, P. Newsholme, and R. Curi. 2006. Effect of docosahexaenoic acid rich fish oil supplementation on human leukocyte function. Clin. Nutr. 25: 923 938. Gurr, M. I., J. L. Harwood, and K. N. Frayn. 2002. Lipid biochemistry. 5th ed. Blackwell Science Ltd., Oxford, UK. Hall, J. A., L. R. Henry, S. Jha, M. M. Skinner, D. E. Jewell, and R. C. Wander. 2005. Dietary (n 3) fatty acids alter plasma fatty acids and leukotriene b synthesis by stimulated neutrophils from healthy geriatric beagles. Prostaglandins Leukot. Essent. Fatty Acids 73: 335 341. Hall, J. A., R. J. Van Saun, S. J. Tornquist, J. L. Gradin, E. G. Pearson, and R. C. Wander. 2004a. Effect of type of dietary polyunsaturated fat ty acid supplement (corn oil or fish oil) on immune responses in healthy horses. J. Vet. Intern. Med. 18: 880 886.

PAGE 194

194 Hall, J. A., R. J. Van Saun, and R. C. Wander. 2004b. Dietary (n 3) fatty acids from menhaden fish oil alter plasma fatty acids and leukotrie ne b synthesis in healthy horses. J. Vet. Intern. Med. 18: 871 879. Hanel, R. M., P. C. Crawford, J. Hernandez, N. A. Benson, and J. K. Levy. 2003. Neutrophil function and plasma opsonic capacity in colostrum fed and colostrum deprived neonatal kittens. Am J. Vet. Res. 64: 538 543. Hansen, R. A., G. K. Ogilvie, D. J. Davenport, K. L. Gross, J. A. Walton, K. L. Richardson, C. H. Mallinckrodt, M. S. Hand, and M. J. Fettman. 1998. Duration of effects of dietary fish oil supplementation on serum eicosapentaeno ic acid and docosahexaenoic acid concentrations in dogs. Am. J. Vet. Res. 59: 864 868. Hansen, R. A., C. J. Savage, K. Reidlinger, J. L. Traub Dargatz, G. K. Ogilvie, D. Mitchell, and M. J. Fettman. 2002. Effects of dietary flaxseed oil supplementation on equine plasma fatty acid concentrations and whole blood platelet aggregation. J. Vet. Intern. Med. 16: 457 463. Harris, M. A., L. H. Baumgard, M. J. Arns, and S. K. Webel. 2005. Stallion spermatozoa membrane phospholipid dynamics following dietary n 3 supp lementation. Anim. Reprod. Sci. 89: 234 237. Harris, P. A. 1998. Developments in equine nutrition: Comparing the beginning and end of this century. J. Nutr. 128: 2698S 2703S. Harris, P. A., J. D. Pagan, K. G. Crandell, and N. Davidson. 1999. Effect of feed ing thoroughbred horses a high unsaturated or saturated vegetable oil supplemented diet for 6 months following a 10 month fat acclimation. Equine Vet. J. Suppl. 30: 468 474. Harris, W. S., J. V. Pottala, S. A. Sands, and P. G. Jones. 2007. Comparison of th e effects of fish and fish oil capsules on the n 3 fatty acid content of blood cells and plasma phospholipids. Am. J. Clin. Nutr. 86: 1621 1625. Henry, M. M., J. N. Moore, E. B. Feldman, J. K. Fischer, and B. Russell. 1990. Effect of dietary alpha linoleni c acid on equine monocyte procoagulant activity and eicosanoid synthesis. Circ. Shock 32: 173 188. Henry, M. M., J. N. Moore, and J. K. Fischer. 1991. Influence of an omega 3 fatty acid enriched ration on in vivo responses of horses to endotoxin. Am. J. Ve t. Res. 52: 523 527. Hernandez, A., J. A. Yager, B. N. Wilkie, K. E. Leslie, and B. A. Mallard. 2005. Evaluation of bovine cutaneous delayed type hypersensitivity (dth) to various test antigens and a mitogen using several adjuvants. Vet. Immunol. Immunopat hol. 104: 45 58. Hoffman, R. M., D. S. Kronfeld, J. H. Herbein, W. S. Swecker, W. L. Cooper, and P. A. Harris. 1998. Dietary carbohydrates and fat influence milk composition and fatty acid profile of mare's milk. J. Nutr. 128: 2708S 2711S.

PAGE 195

195 Hoffman, R. M., D. S. Kronfeld, J. L. Holland, and K. M. Greiwe Crandell. 1995. Preweaning diet and stall weaning method influences on stress response in foals. J. Anim. Sci. 73: 2922 2930. Holland, J. L., D. S. Kronfeld, and T. N. Meacham. 1996. Behavior of horses is affected by soy lecithin and corn oil in the diet. J. Anim. Sci. 74: 1252 1255. Holman, R. T. 1986. Nutritional and biochemical evidences of acyl interaction with respect to ess ential polyunsaturated fatty acids. Prog. Lipid Res. 25: 29 39. Holmes, M. A., H. G. Townsend, A. K. Kohler, S. Hussey, C. Breathnach, C. Barnett, R. Holland, and D. P. Lunn. 2006. Immune responses to commercial equine vaccines against equine herpesvirus 1 equine influenza virus, eastern equine encephalomyelitis, and tetanus. Vet. Immunol. Immunopathol. 111: 67 80. Howard, A. D., G. D. Potter, E. M. Michael, P. G. Gibbs, D. M. Hood, and B. D. Scott. 2003. Heart rates, cortisol, and serum cholesterol in exe rcising horses fed diets supplemented with omega 3 fatty acids. In: Proc. 18th Equine Nutr. Physiol. Soc., East Lansing, MI. p 41 46. Huang, Y. S., D. E. Mills, R. C. Cantrill, and J. P. Poisson. 1996. In vivo and in vitro metabolism of linoleic and gamma linolenic acids. In: Y. S. Huang and D. E. Mills (eds.) Gamma linolenic acid metabolism and its roles in nutrition and medicine. p 84 105. AOCS Press, Champaign, IL. Hussein, N., E. Ah Sing, P. Wilkinson, C. Leach, B. A. Griffin, and D. J. Millward. 2005. Long chain conversion of [13c]linoleic acid and alpha linolenic acid in response to marked changes in their dietary intake in men. J. Lipid Res. 46: 269 280. James, M. J., R. A. Gibson, and L. G. Cleland. 2000. Dietary polyunsaturated fatty acids and infla mmatory mediator production. Am. J. Clin. Nutr. 71: 343S 348S. Janeway, C. A., P. Travers, M. Walport, and M. J. Shlomchik. 2005. Immunobiology : The immune system in health and disease. 6th ed. Garland Science Publishing New Youk, NY. Jansen, W. L., S. N. Geelen, J. van der Kuilen, and A. C. Beynen. 2002. Dietary soyabean oil depresses the apparent digestibility of fibre in trotters when substituted for an iso energetic amount of corn starch or glucose. Equine Vet. J. 34: 302 305. Jansen, W. L., M. M. S. v Oldruitenborgh Oosterbaan, J. W. Cone, H. T. De Vries, J. M. Hallebeek, R. Hovenier, J. Van der Kuilen, C. M. Huurdeman, D. C. G. M. Verstappen, M. C. Gresnigt, and A. C. Beynen. 2007. Studies on the mechanism by which a high intake of soybean oil depres ses the apparent digestibility of fibre in horses. Anim. Feed Sci. Technol. 138: 298 308. Jansen, W. L., J. van der Kuilen, S. N. Geelen, and A. C. Beynen. 2000. The effect of replacing nonstructural carbohydrates with soybean oil on the digestibility of f ibre in trotting horses. Equine Vet. J. 32: 27 30.

PAGE 196

196 Jenkins, T. C., E. J. Thies, and E. E. Mosley. 2001. Direct methylation procedure for converting fatty amides to fatty acid methyl esters in feed and digesta samples. J. Agric. Food Chem. 49: 2142 2145. Jo e, B., and B. R. Lokesh. 1994. Role of capsaicin, curcumin and dietary n 3 fatty acids in lowering the generation of reactive oxygen species in rat peritoneal macrophages. Biochim. Biophys. Acta 1224: 255 263. Kampen, A. H., T. Tollersrud, S. Larsen, J. A. Roth, D. E. Frank, and A. Lund. 2004. Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes. Vet. Immunol. Immunopathol. 97: 105 114. Kapoor, R., and Y. S. Huang. 2006. Gamm a linolenic acid: An antiinflammatory omega 6 fatty acid. Curr. Pharm. Biotechnol. 7: 531 534. Kasama, T., Y. Miwa, T. Isozaki, T. Odai, M. Adachi, and S. L. Kunkel. 2005. Neutrophil derived cytokines: Potential therapeutic targets in inflammation. Curr. D rug Targets Inflamm. Allergy 4: 273 279. Katan, M. B., J. P. Deslypere, A. P. van Birgelen, M. Penders, and M. Zegwaard. 1997. Kinetics of the incorporation of dietary fatty acids into serum cholesteryl esters, erythrocyte membranes, and adipose tissue: An 18 month controlled study. J. Lipid Res. 38: 2012 2022. Kearns, R. J., M. G. Hayek, J. J. Turek, M. Meydani, J. R. Burr, R. J. Greene, C. A. Marshall, S. M. Adams, R. C. Borgert, and G. A. Reinhart. 1999. Effect of age, breed and dietary omega 6 (n 6): Om ega 3 (n 3) fatty acid ratio on immune function, eicosanoid production, and lipid peroxidation in young and aged dogs. Vet. Immunol. Immunopathol. 69: 165 183. Kelley, D. S., P. C. Taylor, G. J. Nelson, and B. E. Mackey. 1998. Arachidonic acid supplementat ion enhances synthesis of eicosanoids without suppressing immune functions in young healthy men. Lipids 33: 125 130. Kelley, D. S., P. C. Taylor, G. J. Nelson, P. C. Schmidt, A. Ferretti, K. L. Erickson, R. Yu, R. K. Chandra, and B. E. Mackey. 1999. Docosa hexaenoic acid ingestion inhibits natural killer cell activity and production of inflammatory mediators in young healthy men. Lipids 34: 317 324. Kew, S., T. Banerjee, A. M. Minihane, Y. E. Finnegan, R. Muggli, R. Albers, C. M. Williams, and P. C. Calder. 2003a. Lack of effect of foods enriched with plant or marine derived n 3 fatty acids on human immune function. Am. J. Clin. Nutr. 77: 1287 1295. Kew, S., T. Banerjee, A. M. Minihane, Y. E. Finnegan, C. M. Williams, and P. C. Calder. 2003b. Relation betwee n the fatty acid composition of peripheral blood mononuclear cells and measures of immune cell function in healthy, free living subjects aged 25 72 y. Am. J. Clin. Nutr. 77: 1278 1286.

PAGE 197

197 Kew, S., E. S. Gibbons, F. Thies, G. P. McNeill, P. T. Quinlan, and P. C. Calder. 2003c. The effect of feeding structured triacylglycerols enriched in eicosapentaenoic or docosahexaenoic acids on murine splenocyte fatty acid composition and leucocyte phagocytosis. Br. J. Nutr. 90: 1071 1080. Kew, S., M. D. Mesa, S. Tricon, R. Buckley, A. M. Minihane, and P. Yaqoob. 2004. Effects of oils rich in eicosapentaenoic and docosahexaenoic acids on immune cell composition and function in healthy humans. Am. J. Clin. Nutr. 79: 674 681. Khol Parisini, A., R. van den Hoven, S. Leinker, H. W. Hulan, and J. Zentek. 2007. Effects of feeding sunflower oil or seal blubber oil to horses with recurrent airway obstruction. Can. J. Vet. Res. 71: 59 65. King, S. S., A. A. Abughazaleh, S. K. Webel, and K. L. Jones. 2008. Circulating fatty acid profil es in response to three levels of dietary omega 3 fatty acid supplementation in horses. J. Anim. Sci. 86: 1114 1123. King, S. S., K. L. Jones, G. A. Apgar, and A. Abughazaleh. 2005. Fatty acid profiles from red blood cells in response to three levels of di etary omega 3 fatty acid supplementation. In: Proc. 19th Equine Sci. Soc. Symp. (Abstr.), Tucson, AZ. p 362 363. Korver, D. R., and K. C. Klasing. 1997. Dietary fish oil alters specific and inflammatory immune responses in chicks. J. Nutr. 127: 2039 2046. Kronfeld, D. S. 1996. Dietary fat affects heat production and other variables of equine performance, under hot and humid conditions. Equine Vet. J. Suppl.: 24 34. Kronfeld, D. S., J. L. Holland, G. A. Rich, T. N. Meacham, J. P. Fontenot, D. J. Sklan, and P A. Harris. 2004. Fat digestibility in equus caballus follows increasing first order kinetics. J. Anim. Sci. 82: 1773 1780. Kruglik, V. L., J. M. Kouba, C. M. Hill, K. A. Skjolaas Wilson, C. Armendariz, J. E. Minton, and S. K. Webel. 2005. Effect of feedi ng protected n 3 polyunsaturated fatty acids on plasma and milk fatty acid levels and igg concentrations in mares and foals. In: Proc. 19th Equine Sci. Soc. Symp. (Abstr.), Tucson, AZ. p 135 136. Lau, C. S., K. D. Morley, and J. J. Belch. 1993. Effects of fish oil supplementation on non steroidal anti inflammatory drug requirement in patients with mild rheumatoid arthritis -a double blind placebo controlled study. Br. J. Rheumatol. 32: 982 989. Lauritzen, L., H. S. Hansen, M. H. Jorgensen, and K. F. Michael sen. 2001. The essentiality of long chain n 3 fatty acids in relation to development and function of the brain and retina. Prog. Lipid. Res. 40: 1 94. LeBlanc, C. J., D. W. Horohov, J. E. Bauer, G. Hosgood, and G. E. Mauldin. 2008. Effects of dietary suppl ementation with fish oil on in vivo production of inflammatory mediators in clinically normal dogs. Am. J. Vet. Res. 69: 486 493.

PAGE 198

198 Lessard, M., N. Gagnon, D. L. Godson, and H. V. Petit. 2004. Influence of parturition and diets enriched in n 3 or n 6 polyuns aturated fatty acids on immune response of dairy cows during the transition period. J. Dairy Sci. 87: 2197 2210. Lessard, M., N. Gagnon, and H. V. Petit. 2003. Immune response of postpartum dairy cows fed flaxseed. J. Dairy Sci. 86: 2647 2657. Levy, B. D., C. B. Clish, B. Schmidt, K. Gronert, and C. N. Serhan. 2001. Lipid mediator class switching during acute inflammation: Signals in resolution. Nat. Immunol. 2: 612 619. Liu, Y. L., D. F. Li, L. M. Gong, G. F. Yi, A. M. Gaines, and J. A. Carroll. 2003. Effe cts of fish oil supplementation on the performance and the immunological, adrenal, and somatotropic responses of weaned pigs after an escherichia coli lipopolysaccharide challenge. J. Anim. Sci. 81: 2758 2765. Lorenzo Figueras, M., S. M. Morisset, J. Moris set, J. Laine, and A. M. Merritt. 2007. Digestive enzyme concentrations and activities in healthy pancreatic tissue of horses. Am. J. Vet. Res. 68: 1070 1072. Lumsden, J. H., R. Rowe, and K. Mullen. 1980. Hematology and biochemistry reference values for th e light horse. Can. J. Comp. Med. 44: 32 42. Manhart, D. R., B. D. Scott, E. M. Eller, C. M. Honnas, D. M. Hood, J. A. Coverdale, and P. G. Gibbs. 2007. Effect of pufas on markers of inflammation in arthritic horses. In: Proc. 20th Equine Sci. Soc. Symp. ( Abstr.), Hunt Valley, MD. p 11 12. McCann, M. E., J. N. Moore, J. B. Carrick, and M. H. Barton. 2000. Effect of intravenous infusion of omega 3 and omega 6 lipid emulsions on equine monocyte fatty acid composition and inflammatory mediator production in vi tro. Shock 14: 222 228. Meydani, S. N., S. Endres, M. M. Woods, B. R. Goldin, C. Soo, A. Morrill Labrode, C. A. Dinarello, and S. L. Gorbach. 1991. Oral (n 3) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: Compariso n between young and older women. J. Nutr. 121: 547 555. Meydani, S. N., A. H. Lichtenstein, S. Cornwall, M. Meydani, B. R. Goldin, H. Rasmussen, C. A. Dinarello, and E. J. Schaefer. 1993. Immunologic effects of national cholesterol education panel step 2 d iets with and without fish derived n 3 fatty acid enrichment. J. Clin. Invest. 92: 105 113. Mickleborough, T. D., M. R. Lindley, A. A. Ionescu, and A. D. Fly. 2006. Protective effect of fish oil supplementation on exercise induced bronchoconstriction in as thma. Chest 129: 39 49. Miles, E. A., T. Banerjee, and P. C. Calder. 2004a. The influence of different combinations of gamma linolenic, stearidonic and eicosapentaenoic acids on the fatty acid composition of blood lipids and mononuclear cells in human volu nteers. Prostaglandins Leukot. Essent. Fatty Acids 70: 529 538.

PAGE 199

199 Miles, E. A., T. Banerjee, M. M. Dooper, L. M'Rabet, Y. M. Graus, and P. C. Calder. 2004b. The influence of different combinations of gamma linolenic acid, stearidonic acid and epa on immune f unction in healthy young male subjects. Br. J. Nutr. 91: 893 903. Miles, E. A., T. Banerjee, S. J. Wells, and P. C. Calder. 2006. Limited effect of eicosapentaenoic acid on t lymphocyte and natural killer cell numbers and functions in healthy young males. Nutrition 22: 512 519. Miles, E. A., and P. C. Calder. 1998. Modulation of immune function by dietary fatty acids. Proc. Nutr. Soc. 57: 277 292. Morris, D. D., M. M. Henry, J. N. Moore, and J. K. Fischer. 1991. Effect of dietary alpha linolenic acid on end otoxin induced production of tumor necrosis factor by peritoneal macrophages in horses. Am. J. Vet. Res. 52: 528 532. Mossmann, T. R., and S. Sad. 1996. The expanding universe of t cell subsets: Th1, th2, and more. Immunology Tod. 17: 138 146. Munsterman, A. S., A. L. Bertone, T. A. Zachos, and S. E. Weisbrode. 2005. Effects of the omega 3 fatty acid, alpha linolenic acid, on lipopolysaccharide challenged synovial explants from horses. Am. J. Vet. Res. 66: 1503 1508. Nesse, L. L., G. I. Johansen, and A. K. Blom. 2002. Effects of racing on lymphocyte proliferation in horses. Am. J. Vet. Res. 63: 528 530. NRC. 2007. Nutrient requirements for horses. The National Academies Press, Washington, D.C. O'Connor, C. I., L. M. Lawrence, and S. H. Hayes. 2007. Dietary f ish oil supplementation affects serum fatty acid concentrations in horses. J. Anim. Sci. 85: 2183 2189. O'Connor, C. I., L. M. Lawrence, A. C. Lawrence, K. M. Janicki, L. K. Warren, and S. Hayes. 2004. The effect of dietary fish oil supplementation on exer cising horses. J. Anim. Sci. 82: 2978 2984. O'Neill, W., S. McKee, and A. F. Clarke. 2002. Flaxseed (linum usitatissimum) supplementation associated with reduced skin test lesional area in horses with culicoides hypersensitivity. Can. J. Vet. Res. 66: 272 277. Oomah, B. D., G. Mazza, and E. O. Kenaschuk. 1992. Cyanogenic compounds in flaxseed. J. Agr. Food Chem. 40: 1346 1348. Parmentier, H. K., A. Awati, M. G. Nieuwland, J. W. Schrama, and J. W. Sijben. 2002. Different sources of dietary n 6 polyunsaturate d fatty acids and their effects on antibody responses in chickens. Br. Poult. Sci. 43: 533 544. Parmentier, H. K., M. G. Nieuwland, M. W. Barwegen, R. P. Kwakkel, and J. W. Schrama. 1997. Dietary unsaturated fatty acids affect antibody responses and growth of chickens divergently selected for humoral responses to sheep red blood cells. Poult. Sci. 76: 1164 1171.

PAGE 200

200 Poland, T. A., J. M. Kouba, C. M. Hill, C. Armendariz, J. E. Minton, and S. K. Webel. 2006. Effects of fatty acid supplementaion on plamsa fatty ac id concentrations and characteristics of the first postpartum estrous in mares. J. Anim. Sci. (Suppl. 1) (Abstr.) 84: 393. Radi, Z. A., and N. K. Khan. 2006. Effects of cyclooxygenase inhibition on the gastrointestinal tract. Exp. Toxicol. Pathol. 58: 163 173. Rees, D., E. A. Miles, T. Banerjee, S. J. Wells, C. E. Roynette, K. W. Wahle, and P. C. Calder. 2006. Dose related effects of eicosapentaenoic acid on innate immune function in healthy humans: A comparison of young and older men. Am. J. Clin. Nutr. 83 : 331 342. Ribeiro, W. P., S. J. Valberg, J. D. Pagan, and B. E. Gustavsson. 2004. The effect of varying dietary starch and fat content on serum creatine kinase activity and substrate availability in equine polysaccharide storage myopathy. J Vet Intern Med 18: 887 894. Robinson, N. E., F. J. Derksen, M. A. Olszewski, and V. A. Buechner Maxwell. 1996. The pathogenesis of chronic obstructive pulmonary disease of horses. Br. Vet. J. 152: 283 306. Robson, P. J., T. D. Alston, and K. H. Myburgh. 2003. Prolonged suppression of the innate immune system in the horse following an 80 km endurance race. Equine Vet. J. 35: 133 137. Rossdale, P. D., R. Hopes, N. J. Digby, and K. offord. 1985. Epidemiological study of wastage among racehorses 1982 and 1983. Vet. Rec. 116: 66 69. Salgar, S. K., M. J. Paape, B. Alston Mills, and R. H. Miller. 1991. Flow cytometric study of oxidative burst activity in bovine neutrophils. Am. J. Vet. Res. 52: 1201 1207. Sampath, H., and J. M. Ntambi. 2005. Polyunsaturated fatty acid regulation of genes of lipid metabolism. Annu. Rev. Nutr. 25: 317 340. Sargent, J. R. 1997. Fish oils and human diet. Br. J. Nutr. 78 Suppl 1: S5 13. Siciliano, P. D., S. K. Webel, L. S. Brown, L. K. Warren, T. E. Engle, and P. D. Burns. 2003. Effect of n 3 polyunsa turated fatty acid source on plasma fatty acid profiles of horses. J. Anim. Sci. (Suppl. 1) 81: 72. Sijben, J. W., and P. C. Calder. 2007. Differential immunomodulation with long chain n 3 pufa in health and chronic disease. Proc. Nutr. Soc. 66: 237 259. S ijben, J. W., M. G. Nieuwland, B. Kemp, H. K. Parmentier, and J. W. Schrama. 2001. Interactions and antigen dependence of dietary n 3 and n 6 polyunsaturated fatty acids on antibody responsiveness in growing layer hens. Poult. Sci. 80: 885 893. Skeaff, C. M., L. Hodson, and J. E. McKenzie. 2006. Dietary induced changes in fatty acid composition of human plasma, platelet, and erythrocyte lipids follow a similar time course. J. Nutr. 136: 565 569.

PAGE 201

201 Skjolaas Wilson, K. A., V. L. Kruglik, C. M. Hill, J. M. Kouba E. G. Davis, S. K. Webel, and J. E. Minton. 2005. Effects of marine derived dietary omega 3 fatty acid (n 3) supplementation on isolated peripheral blood polymorphonuclear cells of pregnant mares and their foals. In: Proc. 19th Equine Sci. Soc. Symp. (Ab str.), Tucson, AZ. p 305. Slack, J., J. M. Risdahl, S. J. Valberg, M. J. Murphy, B. R. Schram, and D. P. Lunn. 2000. Effects of dexamethasone on development of immunoglobulin g subclass responses following vaccination of horses. Am. J. Vet. Res. 61: 1530 1 533. Smits, E., C. Burvenich, and R. Heyneman. 1997. Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood. Vet. Immunol. Immunopathol. 56: 259 269. Spearman, K. R., E. A Ott, J. Kivipelto, and L. K. Warren. 2005. Effect of fatty acid supplementation of the mare on milk and foal plasma composition and foal growth. In: Proc. 19th Equine Sci. Soc. (Abstr.), Tucson, AZ. p 3 4. Spears, J. K., C. M. Grieshop, and G. C. Fahey, Jr. 2004. Evaluation of stabilized rice bran as an ingredient in dry extruded dog diets. J. Anim. Sci. 82: 1122 1135. Sprecher, H. 2000. Metabolism of highly unsaturated n 3 and n 6 fatty acids. Biochim. Biophys. Acta 1486: 219 231. Sprecher, H., D. L. Lut hria, B. S. Mohammed, and S. P. Baykousheva. 1995. Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids. J. Lipid Res. 36: 2471 2477. Stelzleni, E. L., L. K. Warren, and J. Kivipelto. 2006. Effect of dietary n 3 fatty acid suppl ementation on plasma and milk composition and immune status of mares and foals. J. Anim. Sci. (Suppl. 1) 84: 392. Stull, C. L., and A. V. Rodiek. 2000. Physiological responses of horses to 24 hours of transportation using a commercial van during summer con ditions. J Anim Sci 78: 1458 1466. Thewke, D., M. Kramer, and M. S. Sinensky. 2000. Transcriptional homeostatic control of membrane lipid composition. Biochem. Biophys. Res. Commun. 273: 1 4. Thies, F., E. A. Miles, G. Nebe von Caron, J. R. Powell, T. L. H urst, E. A. Newsholme, and P. C. Calder. 2001a. Influence of dietary supplementation with long chain n 3 or n 6 polyunsaturated fatty acids on blood inflammatory cell populations and functions and on plasma soluble adhesion molecules in healthy adults. Lip ids 36: 1183 1193. Thies, F., G. Nebe von Caron, J. R. Powell, P. Yaqoob, E. A. Newsholme, and P. C. Calder. 2001b. Dietary supplementation with gamma linolenic acid or fish oil decreases t lymphocyte proliferation in healthy older humans. J. Nutr. 131: 19 18 1927. Thies, F., L. D. Peterson, J. R. Powell, G. Nebe von Caron, T. L. Hurst, K. R. Matthews, E. A. Newsholme, and P. C. Calder. 1999. Manipulation of the type of fat consumed by growing pigs affects plasma and mononuclear cell fatty acid compositions and lymphocyte and phagocyte functions. J. Anim. Sci. 77: 137 147.

PAGE 202

202 Tizard, I. R. 2004. Veterinary immunology : An introduction. 7th ed. Elsevier (USA), Philadelphia, PA. Trebble, T., N. K. Arden, M. A. Stroud, S. A. Wootton, G. C. Burdge, E. A. Miles, A. B Ballinger, R. L. Thompson, and P. C. Calder. 2003a. Inhibition of tumour necrosis factor alpha and interleukin 6 production by mononuclear cells following dietary fish oil supplementation in healthy men and response to antioxidant co supplementation. Br. J. Nutr. 90: 405 412. Trebble, T. M., S. A. Wootton, E. A. Miles, M. Mullee, N. K. Arden, A. B. Ballinger, M. A. Stroud, G. C. Burdge, and P. C. Calder. 2003b. Prostaglandin e2 production and t cell function after fish oil supplementation: Response to ant ioxidant cosupplementation. Am. J. Clin. Nutr. 78: 376 382. Vineyard, K. R., P. C. Crawford, and L. K. Warren. 2007a. Simultaneous measurement of neutrophil phagocytosis and oxidative burst activity by flow cytometric evaluation in equine whole blood. In: Proc. 20 th Equine Sci. Soc. Symp. (Abstr.), Hunt Valley, MD. p 272 273. Vineyard, K. R., L. K. Warren, and J. Kivipelto. 2007b. Effect of fish oil supplementation on neutrophil function and antibody production in yearling horses. In: Proc. 20 th Equine Sci. Soc. Symp. (Abstr.), Hunt Valley, MD. p 13 14. Vineyard, K. R., L. K. Warren, K. A. Skjolaas, J. E. Minton, and J. Kivipelto. 2006. Effects of dietary fish oil and flaxseed on plasma fatty acid composition and immune function in yearling horses. J. Anim. Sci. (Suppl. 1) 84: 393. Wallace, F. A., E. A. Miles, and P. C. Calder. 2000. Activation state alters the effect of dietary fatty acids on pro inflammatory mediator production by murine macrophages. Cytokine 12: 1374 1379. Wallace, F. A., E. A. Miles, and P. C. Calder. 2003. Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects. Br. J. Nutr. 89: 679 689. Wallace, F. A., E. A. Miles, C. Evans, T. E. Stock, P. Yaqoob, and P. C. Calder. 2001. Dietary fatty acids influence the production of th1 but not th2 type cytokines. J. Leukoc. Biol. 69: 449 457. Wang, Y. W., C. J. Field, and J. S. Sim. 2000. Dietary polyunsaturated fatty acids alter lymphocyte subset proportion and proliferation, se rum immunoglobulin g concentration, and immune tissue development in chicks. Poult. Sci. 79: 1741 1748. Warren, L. K., and J. Kivipelto. 2007a. Effect of season, forage maturity and grazing on the fatty acid composition of bahiagrass pasture. J. Anim. Sci. (Suppl. 1) 85: 141. Warren, L. K., and J. Kivipelto. 2007b. Fatty acid content of grass and legume hays commonly fed to horses. J. Anim. Sci. (Suppl. 1) 85: 140 141.

PAGE 203

203 Watson, T. D., L. Burns, S. Love, C. J. Packard, and J. Shepherd. 1991. The isolation, ch aracterisation and quantification of the equine plasma lipoproteins. Equine Vet. J. 23: 353 359. Wijendran, V., and K. C. Hayes. 2004. Dietary n 6 and n 3 fatty acid balance and cardiovascular health. Annu. Rev. Nutr. 24: 597 615. Williams, C. A., D. S. Kr onfeld, W. B. Staniar, and P. A. Harris. 2001. Plasma glucose and insulin responses of thoroughbred mares fed a meal high in starch and sugar or fat and fiber. J. Anim. Sci. 79: 2196 2201. Williams, C. M., and G. Burdge. 2006. Long chain n 3 pufa: Plant v. Marine sources. Proc. Nutr. Soc. 65: 42 50. Wilson, K. R., G. D. Potter, E. M. Michael, P. G. Gibbs, D. M. Hood, and B. D. SCott. 2003. Alteration in the inflammattory response in athletic horses fed diets containing omega 3 polyunsaturated fatty acids. I n: Proc. 18th Equine Nutr. Physiol. Soc. Symp. p 20 25. Wistuba, T. J., E. B. Kegley, J. K. Apple, and M. E. Davis. 2005. Influence of fish oil supplementation on growth and immune system characteristics of cattle. J. Anim. Sci. 83: 1097 1101. Witonsky, S. R. M. Gogal, Jr., V. Buechner Maxwell, and S. A. Ahmed. 2003. Immunologic analysis of blood samples obtained from horses and stored for twenty four hours. Am. J. Vet. Res. 64: 1003 1009. Wood, J. L., J. R. Newton, N. Chanter, and J. A. Mumford. 2005. Inf lammatory airway disease, nasal discharge and respiratory infections in young british racehorses. Equine Vet. J. 37: 236 242. Woodward, A. D., B. D. Nielsen, C. I. O'Connor, S. K. Webel, and M. W. Orth. 2005. Dietary long chain polyunsaturated fatty acids increase plasma eicosapentaenoic acid and docosahexaenoic acid concentrations and trot stride length in horses. In: Proc. 19th Equine Sci. Soc. Symp. (Abstr.), Tucson, AZ. p 101 105. Yaqoob, P., and P. Calder. 1995. Effects of dietary lipid manipulation up on inflammatory mediator production by murine macrophages. Cell Immunol. 163: 120 128. Yaqoob, P., and P. C. Calder. 2007. Fatty acids and immune function: New insights into mechanisms. Br. J. Nutr. 98 Suppl 1: S41 45. Yaqoob, P., H. S. Pala, M. Cortina Bo rja, E. A. Newsholme, and P. C. Calder. 2000. Encapsulated fish oil enriched in alpha tocopherol alters plasma phospholipid and mononuclear cell fatty acid compositions but not mononuclear cell functions. Eur. J. Clin. Invest. 30: 260 274. Zeyner, A., J. B essert, and J. M. Gropp. 2002. Effect of feeding exercised horses on high starch or high fat diets for 390 days. Equine Vet. J. Suppl.: 50 57.

PAGE 204

204 Zeyner, A., C. Hoffmeister, A. Einspanier, J. Gottschalk, O. Lengwenat, and M. Illies. 2006. Glycaemic and insuli naemic response of quarter horses to concentrates high in fat and low in soluble carbohydrates. Equine Vet. J. Suppl.: 643 647. Zhang, P., R. Smith, R. S. Chapkin, and D. N. McMurray. 2005. Dietary (n 3) polyunsaturated fatty acids modulate murine th1/th2 balance toward the th2 pole by suppression of th1 development. J. Nutr. 135: 1745 1751. Zhao, G., T. D. Etherton, K. R. Martin, P. J. Gillies, S. G. West, and P. M. Kris Etherton. 2007. Dietary alpha linolenic acid inhibits proinflammatory cytokine product ion by peripheral blood mononuclear cells in hypercholesterolemic subjects. Am. J. Clin. Nutr. 85: 385 391.

PAGE 205

205 BIOGRAPHICAL SKETCH Kelly Robertson Vineyard is an Alabama native, growing up primarily in Tuscaloosa. When she was 10 years old, she convinced her parents to allow her to take horseback riding lessons even though they had no prior horse experience whatsoever. Throughout her teenage years, Kelly mucked stalls and performed barn chores to earn riding privileges, and she can remember being especial ly curious about how the barn managers knew exactly what to feed each individual horse. In high school, Kelly stayed active in several activities, including the 4 H horse club, classical piano lessons, church youth choir, and as the pianist for a performin g jazz choir. After high school, Kelly attended Auburn University on an academic scholarship and graduated cum laude Kelly became a founding member of the Auburn Equestrian t eam, worked at the veterinary hospital in both the equine reproduction service and pharmacy, spent three summers working as the wrangler on a large cow calf and stocker cattle ranch in northern Colorado, and took a semester off in the spring of 1998 to bac kpack across Europe. After graduating college she spent a year training horses and became a certified instructor with the North American Riding for the Handicapped Association She then chose to attend the University of Florida in 2001 to study equine nu trition under Dr. Edgar Ott after being offered an oligosaccharide supplementation on immune function in broodmares and foals. Her interest in nutrition and immune function continued int o her Ph.D. work under Dr. Lori Warren as s he investigated the effects of omega 3 fatty acid supplementation on immune function in yearling horses During her tenure as a graduate student, Kelly instructed multiple undergraduate courses in the areas of ho rse handling, broodmare management, and foal care. She also travelled across the state giving

PAGE 206

206 lectures on a variety of horse management topics. Kelly served on the College of Agriculture and Life Sciences Academic Development Committee and Associate Dean S earch Committee and she was the Professional Development Chair for the Animal Science Graduate Student Association In 2006, she received the Omega Protein Innovative Research Award and in 2007 she was awarded the Ph.D. S tudent o f the Y ear honor in the D epartment of Animal Sciences. Kelly is also a current member of the Gamma Sigma Delta Honor Society of Agriculture and has recently joined the Board of Directors for Florida Agri Women. Also during her graduate program, Kelly was lucky to have moved across the street from a wonderful man, Mort Vineyard, whom she married in 2006. They purchased a small farm and built a home, where they now live with their 2 dogs, 2 cats, and a few horses. Kelly continues to ride and train her 8 year old horse Togey, whom she bought as a yearling when she started graduate school, in the sport of dressage. U pon completion of her Ph.D., Kelly plans to pursue a career in the equine feed industry, and she does not discount the possibility of returning to academia one day in the fu ture.