ASSESSMENT OF OXIDATIVE STRESS AND MUSCLE DAMAGE IN EXERCISING HORSES IN RESPONSE TO LEVEL AND FORM OF VITAMIN E by MADISON FAGAN (Under the Direction of KYLEE JO DUBERSTEIN) ABSTRACT Vitamin E is an essential antioxidant noted for reducing oxidative stress. The goal was to determine (1) if supplemental vitamin E is beneficial to exercising horses and (2) if there is a benefit of natural vs synthetic supplements. After a 2wk washout 18 horses were divided into groups and fed a control diet plus: (1) 1000 IU synthetic α-tocopherol (SYN-L), or (2) 4000 IU/d synthetic α-tocopherol (SYN-H), or (3) 4000 IU/d RRR-α- tocopherol (NAT). Horses began a 6wk exercise protocol, with standard exercise tests (SET) performed pre and post the protocol. Venous blood samples were collected. NAT horses had higher α-tocopherol (P<0.05). Plasma MDA levels were lower in NAT vs SYN-L horses post SET2 (P=0.02). Serum AST was significantly lower post SET2 in NAT horses vs SYN-L or SYN-H (P<0.05). In conclusion, feeding higher levels of the more bioavailable natural vitamin E source had a beneficial effect of reducing oxidative stress. INDEX WORDS: Vitamin E, Equine, Exercise, Alpha-tocopherol, Oxidative stress, Nutrition ASSESSMENT OF OXIDATIVE STRESS AND MUSCLE DAMAGE IN EXERCISING HORSES IN RESPONSE TO LEVEL AND FORM OF VITAMIN E by MADISON FAGAN B.S., North Carolina State University, 2015 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2018 © 2018 Madison Fagan All Rights Reserved ASSESSMENT OF OXIDATIVE STRESS AND MUSCLE DAMAGE IN EXERCISING HORSES IN RESPONSE TO LEVEL AND FORM OF VITAMIN E by MADISON FAGAN Major Professor: Kylee Jo Duberstein Committee: Robert Pazdro Jarrod Call Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia August 2018 This paper is dedicated to my family for their constant encouragement. iv ACKNOWLEDGEMENTS I would like to first acknowledge my major professor, Dr. Kylee Duberstien. Thank you for allowing me to be involved in this project. I would not have been nearly as successful in this program without your constant support and advice. I am thankful for your continued mentorship and guidance throughout this process. Thank you for always being available to help with anything, project related or not. I look forward to continuing to work with you! I would also like to acknowledge all of the undergraduates that worked long, hard hours during this project. Thank you for committing your weekends and free time to cleaning stalls, exercising horses and being so dedicated to the success of this project. Each of you played an important role in carrying out a very labor intensive project and I appreciate each of your contributions. Last but not least, thank you to my husband, Zac, for your support during this project. Thank you for your willingness to help clean stalls early in the morning every weekend and your openness to learning everything about equine care. Thank you for taking the time to help me study and for always suggesting ice cream after the most stressful days! v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .................................................................................................v LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF COMMONLY USED ABBREVIATIONS………………………………...…. x CHAPTER 1 Introduction ........................................................................................................1 2 Literature Review...............................................................................................4 Vitamin E .....................................................................................................4 Free Radicals ..............................................................................................14 Antioxidants ...............................................................................................17 Oxidative Stress .........................................................................................22 Muscle Damage .........................................................................................28 Current Recommendations.........................................................................33 3 Assessment of Oxidative Stress and Muscle Damage in Exercising Horses in Response to Level and form of Vitamin E.......................................................35 Abstract ......................................................................................................36 Introduction ................................................................................................37 Materials and Methods ...............................................................................39 Results ........................................................................................................44 vi Discussion ..................................................................................................46 Tables and Figures .....................................................................................50 Literature Cited ..........................................................................................56 4 Conclusions ......................................................................................................61 REFERENCES ..................................................................................................................63 APPENDICES A TBARS Procedure ...........................................................................................73 B Protein Carbonylation Procedure .....................................................................75 C Diet and Treatment Groups ..............................................................................77 vii LIST OF TABLES Page Table 1: Weight, age and sex distribution for treatment groups. .......................................50 Table 2: Six-week exercise protocol ..................................................................................51 Table 3: Standard Exercise Test preformed before and after six weeks of exercise condition by each treatment group .........................................................................51 viii LIST OF FIGURES Page Figure 2.1: Tocopherol and tocotrienol isoforms differ based on the methylation around the chromonal ring ...................................................................................................5 Figure 2.2: Stereoisomers of -tocopherol based on the R or S orientation of methyl groups around 3 chiral carbons ................................................................................6 Figure 2.3: : -tocopherol inhibits chain propagation of lipid peroxidation .....................20 Figure 2.4: The p-orbital orientation of electrons on the aromatic ring and small of - tocopherol enhances stabilization of the molecule as it neutralizes free radicals ..22 Figure 2.5: Various pathways that contribute to protein carbonylation, leading to oxidative stress .......................................................................................................26 Figure 3.1: Timeline of project ...........................................................................................51 Figure 3.2: Average serum -tocopherol concentrations ..................................................52 Figure 3.3: Average change in aspartate aminotransferase concentrations .......................52 Figure 3.4: Average change in creatine kinase concentrations ..........................................53 Figure 3.5: Average plasma protein carbonylations ..........................................................53 Figure 3.6: Average total glutathione concentrations ........................................................54 Figure 3.7: Average plasma malondialdehyde concentrations as measured by TBARS ...54 Figure 3.8a: Percent change of total stride duration for front legs ....................................55 Figure 3.8b: Percent change of total stride duration for hind legs .....................................55 ix LIST OF COMMONLY USED ABBRVEATIONS SET Standard Exercise Test -TPP Alpha-tocopherol transport protein IU International Unites •- O2 Superoxide Radical OH• Hydroxyl Radical H2O2 Hydrogen Peroxide ROS Reactive Oxygen Species ATP Adenosine Triphosphate ETC Electron Transport Chain TBARS Thiobarbituric Acid Reactive Substances MDA Malondialdehyde DI Deionized TEP 1,1,3,3-Tetraethoxypropane CK Creatine Kinase AST Aspartate aminotransferase SOD Superoxide dismutase GPx Glutathione Peroxidase GSH Reduced Glutathione GSSG Oxidized glutathione x PUFA Polyunsaturated Fatty Acid DOMS Delayed Onset Muscle Soreness xi CHAPTER 1 INTRODUCTION Reactive oxygen species (ROS) are unstable, highly reactive molecules that contain an unpaired electron in their outer orbital and are capable of damaging proteins, lipids and DNA. Oxidative stress occurs if the production of free radicals outweighs the level of antioxidants needed to stabilize them (Devasagayam et al., 2004). The occurrence of oxidative stress is much more likely as exercise intensity increases and the electron transport chain becomes overwhelmed. Oxidative stress has been implicated as a cause of muscle damage and decreased athletic performance. This has lead researchers