EFFECT OF ACUTE AND CHRONIC EXERCISE ON IMMUNOENDOCRINE RESPONSES IN PROFESSIONAL RUGBY UNION by BRIAN CUNNIFFE A submission presented in partial fulfilment of the requirements of the University of Glamorgan/Prifysgol Morgannwg for the degree of Doctor of Philosophy This research programme was carried out in collaboration with Wales Rugby Union and University of Wales Institute Cardiff October 2009 Statement of Originality This work has not been submitted for any degree or doctoral candidate at any university. To the best of my knowledge and belief, the dissertation contains no material previously written or published by another person except where due reference is made in the thesis itself. Signature: Brian Cunniffe TABLE OF CONTENTS ABSTRACT i ACKNOWLEDGEMENTS iii PUBLICATIONS/COMMUNICATIONS v LIST OF TABLES vii LIST OF FIGURES ix LIST OF ABBREVIATIONS xii Chapter One GENERAL INTRODUCTION Page 1.1 Introduction 1 1.2 Reasons for present investigation: Current state of play 2 1.3 Intended process of Investigation 6 Chapter Two LITERATURE REVIEW 2.1 Rugby Union: The game 8 2.1.1 Player characteristics and physiological demands 8 2.1.2 Evolutionary trends within the game 10 2.2 Exercise stress defined 11 2.2.1 Stress and Rugby union 12 2.3 Exercise induced immunoendocrine disturbances 13 2.3.1 Circulating cells and functional role 13 2.3.2 Effects of exercise on circulating cell number 16 2.3.3 Effects of exercise on cellular function 18 2.3.4 Hormonal response to exercise 19 2.4 Immune suppression: Possible causes? 20 2.4.1 Exercise and blood neutrophil function 21 2.4.2 Exercise and Plasma Glutamine/Glutamate 22 2.4.3 Tissue trauma and associated immune response 26 2.4.4 Cytokine release and Overtraining 27 2.5 Tissue inflammation and acute phase response 29 2.5.1 Interleukin 6 (IL-6) 29 2.5.2 C-reactive Protein (CRP) 30 2.5.3 Exercise-induced muscle damage: Creatine Kinase 31 2.6 Brain-immune system interaction: Psychological stress 33 2.7 Exercise stress: clinical outcomes 34 2.7.1 Overreaching, Overtraining and Burnout 34 2.7.2 Changes in athlete mood 36 2.7.3 Increased predisposition to Illness 38 2.8 Exercise and illness 38 2.8.1 How much is too much? 38 2.8.2 Effects of upper respiratory illnesses (URIs) on performance 41 2.8.3 Symptomology and clinical course 42 2.9 Mucosal immunity: role in host defence 43 2.9.1 Exercise, viral entry and mucosal protection 43 2.9.2 Constituents of human saliva: role in host protection 44 2.9.3 Saliva release and flow 44 2.10 Salivary Iga 45 2.10.1 Modulation 46 2.10.2 Role of s-IgA in oral protection 47 2.10.3 Effects of exercise on s-IgA 47 2.10.4 Role of s-IgA in illness development 50 2.10.5 Reasons for observed change 51 2.11 Salivary Lysozyme 52 2.11.1 Modulation 53 2.11.2 Role of lysozyme in oral protection 53 2.11.3 Effects of exercise on salivary lysozyme 53 2.11.4 Role of saliva lysozyme in illness development 54 2.11.5 Reasons for observed change 54 2.12 Psychological stress: muscosal immunity and illness 55 2.13 Hormones in saliva 56 2.13.1 Saliva Cortisol 56 2.13.2 Saliva Testosterone 57 2.13.3 Testosterone:Cortisol ratio 58 2.13.4 Transportation of analytes into saliva 59 2.13.5 Other methodological issues 60 2.14 Circadian variation in selected immunoendocrine markers 60 2.15 Psychometric assessment: RESTQ-sport 62 2.16 Summary 64 Chapter Three GENERAL METHODS [Acute studies] 3.1 Study entry and ethical approval 65 3.2 Preliminary measurements 65 3.2.1 Determination of Peak Oxygen Uptake ( & OV 2peak ) 65 3.3 Blood sampling and analysis 66 3.3.1 Blood collection and treatment 66 3.3.2 Determination of Total and differential leukocyte counts 67 3.3.3 Determination of LPS-stimulated neutrophil degranulation 69 3.3.4 Determination of serum Cortisol and Testosterone 70 3.3.5 Determination of high sensitive C-Reactive Protein (hs-CRP) 70 3.3.6 Determination of interleukin-6 (IL-6) 71 3.3.7 Determination of Creatine Kinase (CK) 71 Chapter Four IMMUNOENDOCRINE RESPONSES TO ACUTE AND REPEATED EXERCISE IN PROFESSIONAL RUGBY UNION Study One Time-Course Of Changes In Immuneoendocrine Markers Following An International Rugby Game 4.1 Introduction 72 4.2 Methods 74 4.3 Results 76 4.4 Discussion 82 Study Two Evaluation Of Immunoendocrine Responses Throughout A Three Week International Rugby Series 4.5 Introduction 87 4.6 Methods 88 4.7 Results 91 4.8 Discussion 96 Chapter Five GENERAL METHODS [Longitudinal studies] 5.1 Study entry and ethical approval 101 5.2 Preliminary measures 101 5.2.1 Determination of Peak Oxygen Uptake ( & OV 2peak ) 101 5.2.2 Body mass and stature 101 5.2.3 Skinfold Assessment 101 5.3 Saliva sampling and analysis 102 5.3.1 Collection of saliva samples 102 5.3.2 Determination of saliva flow 103 5.4 Saliva analysis 103 5.4.1 Determination of s-IgA 103 5.4.2 Determination of s-Lys 104 5.4.3 Determination of Saliva Cortisol and Testosterone 104 5.4.4 Blood Contamination Assay (Transferrin) 105 5.4.5 Saliva Osmolality 105 5.5 Blood sampling and analysis 106 5.5.1 Determination of CK and hs-CRP 106 5.5.2 Determination of Plasma Glutamine and Glutamate 106 5.5.3 Urine Osmolality 109 5.6 Physical activity and Training Load assessment 109 Chapter Six EFFECTS OF CHRONIC EXERCISE ON IMMUNOENDOCHRINE MARKERS AND ILLNESS INCIDENCE IN PROFESSIONAL RUGBY Study Three Investigation Of Player Attitudes To Contemporary Demands In Professional Rugby Union 6.1 Introduction 111 6.2 Methods 113 6.3 Results 113 6.4 Discussion 118 Study Four Changes In Mucosal Immunity And Infectious Incidence Within Elite Rugby Union Players Over A Competitive Season 6.5 Introduction 121 6.6 Methods 124 6.7 Results 130 6.8 Discussion 139 Study Five Biochemical And Endocrine Responses To Training Load During A Competitive Elite Rugby Union Season 6.9 Introduction 144 6.10 Methods 145 6.11 Results 153 6.12 Discussion 159 Study Six Training Related Psychological Stress In Elite Rugby Union Players Over A Competitive Season 6.13 Introduction 164 6.14 Methods 165 6.15 Results 167 6.16 Discussion 173 Chapter Seven GENERAL DISCUSSION 7.1 General discussion 177 7.2 Strengths and limitations 181 7.3 Main findings from thesis 182 7.4 Practical suggestions and future research 184 Chapter Eight SUPPORTING CASE STUDIES Case Study One: Determination Of An Appropriate Saliva Collection Method 188 For Analysis Of Human Salivary Lysozyme Case Study Two: Cross Comparison Of Varying Methods Used For Calculation 196 Of Training Load In Rugby Union Case Study Three: Illness Monitoring In Team Sports Using A Web-Based 204 Training Diary Case Study Four: An Evaluation Of The Physiological Demands Of 216 Elite Rugby Union REFERENCES 225 APPENDICES A. Informed consent forms 273 B. Study information sheets 275 C. Health screen questionnaires 280 D. Food record diaries [24 h and 4 day] 283 E. Nutritional supplement questionnaire 288 F. Sample training load calculation 290 G. RESTQ Sport Questionnaire 293 ABSTRACT Prolonged and intense exercise is known to modulate and suppress certain aspects of the immunoendocrine system. Such effects are thought to be largely mediated by the release of stress hormones and regulatory cytokines which originate from a variety of stress related paradigms in sport. These include acute physical exertion, chronic and repetitive exercise as well as other psychological and psychosocial aspects of training and competing in an elite environment. It may be of particular interest to study the effects of regular competition and training on immunoendocrine markers in rugby union players. At the professional level, rugby is an intense and physically demanding game where a significant amount of tissue trauma occurs as a result of the many game collisions. The aims of the studies outlined in this thesis were to determine the effects of acute, repeated and chronic exercise exposure on immunoendocrine markers and illness incidence in professional rugby union. Additional case studies were also undertaken to supplement main study findings. The first part of the thesis documented the effects of acute and repeated exercise on immunoendocrine markers in a cohort of international rugby union players. Data in study 1 showed that large disturbances in immunoendocine and hormone levels occur in players (n = 10) following game play. The magnitude of this response appeared dependent on game physicality (number of rucks/mauls, tackles) and the number of collisions players received during match play. Findings also showed suppression in host immunity, and in particular, innate immune function (neutrophil degranulation) which was not resolved 38 h (-29%) into the recovery period. In study 2, bloods were taken from players (n = 8) across a 21-day international rugby series. Data revealed that players entered the international camp with residual muscle damage (creatine kinase; CK) and inflammation (hs-CRP) following previous club involvement in European cup rugby. Further increases in stress related markers (cortisol, IL-6, CK, CRP) were not evident throughout the players time at the international training base. Conversely, a progressive increase in anabolic-catabolic balance (T/C ratio) was observed across time. In comparison to values on camp-entry (day 1), increases in T/C ratio were evident on day-5 (9.8%), day-7 (13%), day-19 (35%) and day-21 (45%) (P < 0.05).