Effects of the Soy Phytoestrogen Genistein
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EFFECTS OF THE SOY PHYTOESTROGEN GENISTEIN ON THE REPRODUCTIVE DEVELOPMENT OF IMMATURE FEMALE BROILER CHICKENS Except where reference is made to the work of others, the work described in this thesis is my own or was done in collaboration with my advisory committee. This thesis does not include proprietary or classified information. ________________________________________ Lindsay Marie Stevenson Certificate of Approval: ________________________ _______________________ Joseph B. Hess Wallace D. Berry, Chair Professor Associate Professor Poultry Science Poultry Science ________________________ _______________________ John P. Blake George T. Flowers Professor Interim Dean Poultry Science Graduate Schoool THE EFFECTS OF THE SOY PHYTOESTROGEN GENISTEIN ON THE REPRODUCTIVE DEVELOPENT OF IMMATURE FEMALE BROILER CHICKENS Lindsay Marie Stevenson A Thesis Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Master of Science Auburn, Alabama May 10, 2007 EFFECTS OF THE SOY PHYTOESTROGEN GENISTEIN ON THE REPRODUCTIVE DEVELOPMENT OF IMMATURE FEMALE BROILER CHICKENS Lindsay Marie Stevenson Permission is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. ______________________________ Signature of Author ______________________________ Date of Graduation iii VITA Lindsay Marie Stevenson, daughter of John Alvin and Colleen Marie Stevenson, was born May 30, 1982 in Toledo, Ohio. She graduated from Lake High School in Millbury, Ohio in June 2000. She attended The Ohio State University in Columbus, Ohio majoring in Animal Sciences with a minor in Agricultural Business. She graduated in June 2004 with a Bachelor of Science in Agriculture degree. In August 2004, she entered Graduate School at Auburn University under the direction of Wallace Berry, Ph.D. iv THESIS ABSTRACT THE EFFECTS OF THE SOY PHYTOESTROGEN GENISTEIN ON THE REPRODUCTIVE DEVELOPMENT OF IMMATURE FEMALE BROILER CHICKENS Lindsay Marie Stevenson Master of Science, May 10, 2007 (Bachelor of Science, The Ohio State University, 2004) 152 Typed Pages Directed by Wallace D. Berry Developmentally inappropriate exposures to estrogenic compounds are known to alter morphology and function of the reproductive tract in various species. Chickens are continually exposed to the relatively potent estrogenic soy isoflavones through the diet. It has been shown that the primary soy isoflavone genistein induces proliferation of the chick oviduct. However, information is lacking as to the specific reproductive tract developmental effects of genistein exposure in chicks. Three experiments were done to compare specific oviduct morphological and functional responses to genistein exposure with responses elicited by a classical estrogen, diethylstilbestrol (DES) in female broiler chicks. To avoid the effects of dietary soy v isoflavones, the experimental diets were formulated with dried egg white, rather than the usual soybean meal, as a protein source. These experiments examined the effects of genistein in the on the morphology and growth of the oviduct and functional responses to estrogen consisting of plasma vitellogenin content and oviductal ovalbumin synthesis. From the three experiments, it was determined that genistein acts as a weak estrogen in the immature female chick. It has effects on the growth and morphology of the oviduct. Genistein can also induce the synthesis of estrogen-dependent secretions in the chick including vitellogenin and ovalbumin. vi ACKNOWLEDGEMENTS The author would like to express sincere appreciation to her graduate committee, Drs. Joseph Hess and John Blake for their input and support. Special thanks are extended to Dr. Wallace Berry for his instruction and guidance throughout the project. Thanks are also extended to the faculty and staff of the Poultry Science Department especially to Ms. Suzanne Oates for her help with laboratory procedures and preparations. The author would also like to thank her friends at Auburn and her fellow graduate students, Alexis, Candace, Gina, Leslie and Scott, for all of their support and assistance. Thanks are also due to Dr. Fred Hoerr and the staff at the Alabama Veterinary Diagnostics Laboratory for embedding and sectioning oviduct sections. This manuscript is dedicated to the author’s parents, John and Colleen Stevenson, and siblings, Nichole and Matthew, for always being there. vii Journal style used: Poultry Science Computer software used: Microsoft Office 2000® iPhoto 4.0.3© SAS 9.1® viii TABLE OF CONTENTS LIST OF TABLES xii LIST OF FIGURES xv CHAPTER I. INTRODUCTION 1 CHAPTER II. LITERATURE REVIEW 3 Estrogens 3 Endogenous Estrogens 3 Exogenous Estrogens 4 Estrogen Action 7 Estrogen Receptors 8 Estrogen Receptor Alpha 10 Estrogen Receptor Beta 11 Estrogen Receptor Gamma 12 Functions in Animals 12 Phytoestrogens 14 Sources of Phytoestrogens 14 Classes of Phytoestrogens 17 Coumestans 17 Lignans 17 Isoflavones 18 Soybean Phytoestrogens 19 ix General Effects on Animals 22 Genistein 24 Metablism 25 Analytical Methods to Determine Genistein Levels 26 Effects of Genistein 28 Molecular Effects 28 General Effects on Animals 30 Effects on Birds and Chickens 31 CHAPTER III. STATEMENT OF RESEARCH OBJECTIVES 37 CHAPTER IV. MANUSCRIPT 1 38 ESTROGENIC EFFECTS OF SHORT-TERM ORAL EXPOSURE TO THE SOY ISOFLAVONE GENISTEIN IN THE IMMATURE BROILER CHICKEN Abstract 38 Introduction 39 Materials and Methods 42 Results and Discussion 46 CHAPTER V. MANUSCRIPT 2 69 EFFECTS OF SHORT-TERM ORAL EXPOSURE TO THE SOY ISOFLAVONE GENISTEIN IN THE IMMATURE FEMALE BROILER CHICKEN Abstract 69 Introduction 70 Materials and Methods 73 Results and Discussion 77 CHAPTER VI. MANUSCRIPT 3 87 x EFFECTS OF SHORT-TERM INJECTED EXPOSURE TO THE SOY ISOFLAVONE GENISTEIN ON THE IMMATURE FEMALE BROILER CHICKEN Abstract 87 Introduction 88 Materials and Methods 91 Results and Discussion 95 CHAPTER VII. SUMMARY OF RESULTS 105 BIBLIOGRAPHY 107 APPENDICES 128 Appendix 1. Immunostaining of Paraffin Sections with Microwave 129 Antigen Retrieval Appendix 2. Bio-Rad Protein Assay 131 Appendix 3. Direct Enzyme Linked Immunosorbant Assay 132 Appendix 4. Sandwich Enzyme Linked Immunosorbant Assay 134 Appendix 5. Determining Total Isoflavones in Plasma with HPLC 136 xi LIST OF TABLES Table 1. Natural and synthetic xenoestrogens. 5 Table 2. Levels of isoflavones and lignans in various food sources. 15 Table 3. Various forms of isoflavones. 19 Table 4. Ingredient percentages and calculated analysis of broiler starter 51 diet with a low level of isoflavones. Table 5. Starting and final body weight. 52 Table 6. Oviduct weight and relative weight of the oviduct at 16 days 52 of age. Table 7. Wet femur weight and relative wet femur weight at 16 days 52 of age. Table 8. Dry femur weight and relative dry femur weight at 16 days 53 of age after 48 hours at 50°C. Table 9. Ashed femur weight and relative ashed femur weight at 16 53 days of age after 18 hours at 600°C. Table 10. Amount of protein and vitellogenin contained in the plasma 53 of birds at 16 days of age. Table 11. Ingredient percentages and calculated analysis of broiler starter 81 diet with a low level of isoflavones (control diet). Table 12. Ingredient percentages and calculated analysis of a standard 82 broiler starter diet (standard diet). Table 13. Average body weight per bird on days 0 and 5. 83 Table 14. Average body weight per bird on days 10 and 15. 83 xii Table 15. Average body weight gain per bird per period for days 0 to 5 83 and 5 to 10. Table 16. Average body weight gain per bird per period for days 10 to 15 84 and 0 to 15. Table 17. Liver weight and relative liver weight at 15 days of age. 84 Table 18. Oviduct weight and relative oviduct weight at 15 days of age. 84 Table 19. Wet femur weight and relative wet femur weight at 15 days 85 of age. Table 20. Dry femur weight and relative dry femur weight at 15 days 85 of age after 48 hours at 50°C. Table 21. Ashed femur weight and relative ashed femur weight at 15 days 85 of age after 18 hours at 600°C. Table 22. Amount of protein and vitellogenin contained in the plasma 86 of birds at 15 days of age. Table 23. Amount of genistein contained in the plasma of birds at 15 days 86 of age. Table 24. Ingredient percentages and calculated analysis of broiler starter 99 diet with a low level of isoflavones (control diet). Table 25. Ingredient percentages and calculated analysis of a standard 100 broiler starter diet (standard diet). Table 26. Average body weight per bird on days 0 and 5. 101 Table 27. Average body weight per bird on days 10 and 15. 101 Table 28. Average body weight gain per bird per period for days 0 to 5 101 and 5 to 10. Table 29. Average body weight gain per bird per period for days 10 to 15 102 and 0 to 15. Table 30. Liver weight and relative liver weight at 15 days of age. 102 Table 31. Oviduct weight and relative oviduct weight at 15 days of age. 102 Table 32. Wet femur weight and relative wet femur weight at 15 days 103 of age. xiii Table 33. Dry femur weight and relative dry femur weight at 15 days 103 of age after 48 hours at 50°C. Table 34. Ashed femur weight and relative ashed femur weight at 15 103 days of age after 18 hours at 600°C. Table 35. Amount of protein and vitellogenin contained in the plasma 104 of birds at 15 days of age. Table 36. Amount of genistein contained in the plasma of birds at 15 days 104 of age. xiv LIST OF FIGURES Figure 1. Structure of endogenous estrogens. 3 Figure 2. Structure of the synthetic estrogen diethylstilbestrol. 6 Figure 3. Structures of coumestans. 17 Figure 4. Structures of isoflavones. 19 Figure 5. Hemotoxylin and eosine stained paraffin embedded oviduct 54 sections from chicks at 16 days of age.