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A GENERAL APPROACH for VITRIFICATION of FISH SPERM a Dissertation Submitted to the Graduate Faculty of the Louisiana State Unive

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A GENERAL APPROACH FOR VITRIFICATION OF FISH SPERM 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 Rafael Cuevas Uribe B.S., Universidad de Guadalajara, 2003 M.S., Kentucky State University, 2006 December 2011 In memory of Herbert Stein and Raymundo Ramirez Delgadillo who taught me science and motivated me to pursue a Ph.D. “My duty is to communicate to those who are to become professional thinkers, the future Doctors of Philosophy, the pure love of Truth.” Father Basile J. Luyet ii Acknowledgements There are many people I wish to thank. “Thanks” is a simple word but it can possess a transcendental meaning. This Ph.D. came not just from my effort, but from the support of many others. It took me 5.5 years to finish. This required a lot of patience from my major professor Dr. Tiersch, who was a guide and mentor in my professional and personal life. He always is going to be a good friend and a good example to follow. Most of my learning in cryobiology came by the teaching of Dr. S. P. Leibo who I regard as the “living encyclopedia of cryobiology”. I thank Dr. Leibo for sharing his knowledge, and thank the other members of my Advisory Committee, Dr. Romaire and Dr. Green for their time and for reading this long dissertation. Thanks also to Dr. Walter for serving on my Committee from Texas State University. Also thanks to Dr. Sibel Bargu for accepting at the last minute to serve as Dean‟s Representative for my Committee. Thanks to all of the members of Dr. Tiersch‟s laboratory, especially Dr. Yang, Noel Novelo, E Hu, Doug Kuenz, and Mike Christensen for their friendship and team work. Thanks is extended to the labor force in the laboratory, especially undergraduate student workers Meredith Hunt, Jennifer Atilano, Adam Duethman, Courtney Staudermann, Ereene Tan, and Tu Dao. Thanks also to all the students and staff at the Aquaculture Research Station, especially Cenelia Cuartas for her sincere friendship. Other scientists that assisted me in this project were Dr. Ana Viveiros from Brazil, Dr. Jonathan Daly and Kim Daly from Australia, Markita Savage and Leona Hazlewood from Texas State University, Dr. Ed Chesney and Jeremy Miller from the Louisiana Universities Marine Consortium, and Dr. Harry Daniels, Adriane Gill, and Robin Sanderson from North Carolina State University. Thanks to all the scientists from my previous universities who taught me science during those years, especially my previous advisors Dr. Gomelsky and Dr. Mims. iii The field of cryobiology, as seen in other scientific fields, started with contributions from Priest-Scientists or men of God and missionaries to science. Father Lazzaro Spallanzani (l‟Abate Spallanzani) was the first to freeze and thaw sperm in 1776. In fact, the „Father of Cryobiology‟ is Father Basile J. Luyet, from the Order of Missionaries of St. Francis de Sales. St. Francis once said that science, knowledge and wisdom was an eighth sacrament, a way by which divine life was to be dispensed to humankind. During my Ph.D. journey, I realized that the common denominator of these great scientists was a synthesis of their spiritual and professional lives. I have discovered the meaning of “Religioso y estudiante, Religioso por delante” (religious and student, but primarily religious) (St. John of the Cross), and of “Ora et labora” (St. Benedict), and working with fish has been “Piscis assus Christus est Passus” (St. Augustine). “The solution of the problem of life is life itself. Life is not attained by reason and analysis but first of all by living” (Thomas Merton). When Father Luyet was asked how he reconciled being a scientist and priest, he responded “Truth does not contradict truth.” With this I want to give thanks to God for letting me learn to surrender and accept His Will. Thanks to my family for their love and support. Thanks to my parents who taught me values and principles which are part of my morality. Thanks to my brothers Jorge and Antonio, and my sisters Aurora, Marines, and Fatima for companionship and unconditional help during this journey. Thanks to all of my nieces and nephews, who although too young to understand what a Ph.D. is, have taught me that it is important to enjoy the simple things in life. Thanks to my uncles Antonio, Javier, and Rafael for their support. Thanks to my friends Roberto Rodriguez (who came to my dissertation defense), Ivan, and Molina. Finally, thanks to my sweetheart Elibe Noriega (milagrito de amor), for all her love and support during the writing of this dissertation. iv These words were dedicated to me by my father on the day of my dissertation defense: “Effort and perseverance take us to success. An objective without efforts is never reached. Efforts without an objective, it‟s a waste. Rafo: Today, you achieve your Ph.D. objective, which has demanded your last five years, in addition to your master‟s and your bachelor degree. As the mountain climber, you reached the top. For this reason you are very happy. Congratulations, a lot of congratulations. But as any activity in life, when you reach an objective, immediately you think of another bigger than your last. This is your case, it‟s your post-doctorate, post-Ph.D. You will continue the way walking and climbing the mountains, which are in front of you: your personal life and your professional life. Remember that work and studies are part of our lives, they are not everything. Finally remember, give thanks to God, Who gives you these moments. With God, all our objectives can be achieved; without Him, we are nothing and we cannot do anything. Rafo: Congratulations.” Jorge Luis Cuevas Miguel, August 5, 2011 v Table of Contents Dedication ............................................................................................................................... ii Acknowledgements ................................................................................................................. iii List of Tables .......................................................................................................................... viii List of Figures ......................................................................................................................... xi List of Abbreviations .............................................................................................................. xvi Abstract ................................................................................................................................... xvii Chapter 1. Foreword ............................................................................................................... 1 Biomedical Research Fish Models..................................................................................... 3 Genetically Improved Lines ............................................................................................... 4 Endangered Fish Species ................................................................................................... 4 References .......................................................................................................................... 6 Chapter 2. Introduction ........................................................................................................... 11 Cell Dehydration in Cryopreservation ............................................................................... 11 Equilibrium vs. Non-equilibrium Cryopreservation .......................................................... 14 Source of Variation in Vitrification ................................................................................... 18 Advantages of Vitrification ................................................................................................ 18 Sperm Vitrification ............................................................................................................ 19 Vitrification of Fish Eggs and Embryos ............................................................................ 22 New Strategies for Application of Cell Vitrification ......................................................... 24 References .......................................................................................................................... 25 Chapter 3. Production of Channel Catfish with Sperm Cryopreserved by Rapid Non-Equilibrium Cooling ........................................................................... 37 Materials and Methods ....................................................................................................... 41 Results ................................................................................................................................ 50 Discussion .......................................................................................................................... 56 Conclusions ........................................................................................................................ 64 References .......................................................................................................................... 65 Chapter 4. Production of F1 Offspring with Vitrified Sperm from a Live-bearing Fish, the Green Swordtail Xiphophorus hellerii ............................................................. 75 Materials and Methods ......................................................................................................
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