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Development of a Model Cryopreserved Germplasm Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2003 From gamete collection to database development: development of a model cryopreserved germplasm repository for aquatic species with emphasis on sturgeon William Rittenhouse Wayman Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Wayman, William Rittenhouse, "From gamete collection to database development: development of a model cryopreserved germplasm repository for aquatic species with emphasis on sturgeon" (2003). LSU Doctoral Dissertations. 2637. https://digitalcommons.lsu.edu/gradschool_dissertations/2637 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. FROM GAMETE COLLECTION TO DATABASE DEVELOPMENT: DEVELOPMENT OF A MODEL CRYOPRESERVED GERMPLASM REPOSITORY FOR AQUATIC SPECIES WITH EMPHASIS ON STURGEON A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The School of Renewable Natural Resources By William Rittenhouse Wayman B.S., Auburn University, 1991 M.S., Louisiana State University, 1996 August 2003 Acknowledgements I thank Dr. Terrence Tiersch for serving as my major advisor. Besides helping in the fulfillment of the obligations to obtain my doctoral degree, he has helped in obtaining a full-time position with the U. S. Fish and Wildlife Service, and in beginning my professional career. For this I am truly thankful. I thank Mr. Vincent Mudrak for offering me a position at the Warm Springs Fish Technology Center, and for providing the resources of the U. S. Fish and Wildlife Service during this dissertation. I thank the rest of my Advisory Committee, Dr. John Caprio, Dr. John Hawke, Dr. Jill Jenkins, and Dr. Robert Romaire, for the assistance they have provided during my studies. I thank the U. S. Fish and Wildlife Service for the initial funding of these efforts through a research work order, and for allowing the continuation of the work through the position at Warm Springs. I thank Sheldon Hawkins, James Henne, Malcolm Mohead, Wayne Tucker, and Kent Ware of the Bears Bluff National Fish Hatchery for help in the spawning and cryopreservation work with shortnose sturgeon. I thank Dr. Edward Baker of the Michigan Department of Natural Resources, his crew (Dean Burdett, Dan Daugherty, Karen Koval, and Jessica Mistak), Brenda and Gil Archambo of the Black Lake Chapter of Sturgeon for Tomorrow, and Kregg Smith for their assistance in the lake sturgeon efforts. I especially thank Rob Holm for his assistance and friendship in research of pallid sturgeon and shovelnose sturgeon, and in the development of a repository for pallid sturgeon sperm. I thank the staff and volunteers at the Garrison Dam National Fish Hatchery and the Charles M. Russell National Wildlife Refuge for assistance and housing during the cryopreservation work on pallid sturgeon and shovelnose sturgeon. I thank Karen Kilpatrick, Jan Dean and the staff at the Natchitoches National Fish Hatchery for their assistance in the collection of blood samples from shovelnose sturgeon and pallid sturgeon. ii I thank the staff of the Warm Springs Regional Fisheries Center (Robert Bakal, Joe Bodine, Josh Bradley, Irene Duke, Carlos Echevarria, Lawrence Ford, Norm Heil, Brian Hickson, Harvey Ingram, Haile Macurdy, and Rosla Plant) for making me feel comfortable at work and helping me to accomplish this project. I especially thank Greg Looney for the help, advice, and assistance that he has given during this research, many times while being away from his family for extended periods. I thank the Student Conservation Association interns at Warm Springs, Elizabeth Osier, Catherine Bradley, and Jaclyn Zelko, for assistance, and for putting up with me for the 6 months that I was their supervisor. I thank the staff and students of the Aquaculture Research Station for all the assistance they provided over the years. I miss being around there to enjoy the friendships that were developed over the past decade. I owe John Buchanan immensely for his critical review of this dissertation, his advice on coping with stress, his guidance on research directions, and especially his friendship. I express extreme gratitude to my wife, Candice, for her unwavering support during this period, and especially during the last 6 months when she was largely responsible for the care of our newborn child, William, Jr. I express thanks to my parents for their monetary and emotional support throughout my life and to my sisters for their competitive nature and support, without them none of this would have been possible. I am sure that I am leaving out others who have helped me during the last 7 years, and for this I am sorry, but please be assured that I appreciate your help. iii Table of Contents Acknowledgements................................................................................................................ii List of Tables .........................................................................................................................vi List of Figures ........................................................................................................................viii Abstract ..................................................................................................................................xiii Chapter 1 Foreword .................................................................................................................1 Literature Cited ..............................................................................................7 2 Introduction............................................................................................................. 9 Literature Cited .............................................................................................. 28 3 Development of Methods to Verify the Field Identifications of Species ............... 35 Introduction.................................................................................................... 35 Materials and Methods................................................................................... 37 Results............................................................................................................ 41 Discussion...................................................................................................... 42 Literature Cited .............................................................................................. 45 4 Bacterial Contamination of Sperm Samples ........................................................... 48 Introduction.................................................................................................... 48 Materials and Methods................................................................................... 51 Results............................................................................................................ 55 Discussion...................................................................................................... 59 Literature Cited .............................................................................................. 65 5 Development of Refrigerated Storage Methods...................................................... 69 Introduction.................................................................................................... 69 Materials and Methods................................................................................... 70 Results............................................................................................................ 75 Discussion...................................................................................................... 96 Literature Cited .............................................................................................. 103 6 Development of Cryopreservation Techniques.......................................................108 Introduction....................................................................................................108 Materials and Methods...................................................................................109 Results............................................................................................................114 Discussion......................................................................................................127 Literature Cited ..............................................................................................131 iv 7 Development of Methods for the Objective Determination of Sperm Quality.......133 Introduction....................................................................................................133 Materials and Methods...................................................................................134 Results............................................................................................................138 Discussion......................................................................................................156 Literature Cited ..............................................................................................159
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