Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2017 On-site Sperm Cryopreservation for Aquatic Repository Development and User Group Linkage William Martin Childress Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Environmental Sciences Commons Recommended Citation Childress, William Martin, "On-site Sperm Cryopreservation for Aquatic Repository Development and User Group Linkage" (2017). LSU Master's Theses. 4577. https://digitalcommons.lsu.edu/gradschool_theses/4577 This Thesis 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 Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. ON-SITE SPERM CRYOPRESERVATION FOR AQUATIC REPOSITORY DEVELOPMENT AND USER GROUP LINKAGE A Thesis 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 Master of Science in The School of Renewable Natural Resources by William M. Childress B.S., University of Southern Mississippi, 2011 August 2017 Acknowledgements There are many people that have guided me and provided support along the way during this thesis. I would like to thank my major professor, Dr. Terrence Tiersch, for his guidance, mentorship, and the many opportunities to travel, conduct research, and grow both professionally and personally. I thank Dr. Edward Chesney for serving on my graduate committee and providing me with my foundation in aquaculture and research. I would not be where I am today without his guidance and teaching during my time working for him at Louisiana Universities Marine Consortium. I would also like to thank Dr. Rex Caffey for serving on my graduate committee, teaching me about economics, and strengthening the cost analysis portion of my project. I would like to thank all the members of the Aquatic Germplasm and Genetic Resources Center. I thank Amy Guitreau, Yue Liu, Mike Christensen, Leticia Torres, and Noel Novelo for their support and involvement during this project. I thank all student workers, including Thomas Lee, Austin Daspit, and Megan Chesnut for their hard work. Thanks to Jane Niu for helping to create the GIS map. I would like to thank my entire family for their support and encouragement. Thanks to my mom and dad who instilled my love for the water and shaped me into who I am today. Thanks to my girlfriend, Kandis, for support from my first day of this project to the last and always encouraging me. Lastly, thanks to all my friends and the friends I have made along with way through this journey. ii Table of Contents Acknowledgements ......................................................................................................................... ii List of Tables ...................................................................................................................................v List of Figures ............................................................................................................................... vii Abstract ............................................................................................................................................x Chapter 1. Thesis Overview .............................................................................................................1 References .................................................................................................................................5 Chapter 2. Introduction ....................................................................................................................7 Equilibrium and Non-equilibrium Cryopreservation................................................................9 Sample Collection ...................................................................................................................12 Sperm Activation ....................................................................................................................13 Estimating Motility and Cell Concentration ...........................................................................14 Evaluation of Sperm Quality ..................................................................................................16 Packaging ................................................................................................................................18 Freezing Devices ....................................................................................................................21 Storage and Transportation .....................................................................................................22 Thawing and Fertilization .......................................................................................................22 References ...............................................................................................................................24 Chapter 3. Design and Cost Analysis of a Self-contained Mobile Laboratory for Aquatic Species Cryopreservation...............................................................................................................30 Methods ..................................................................................................................................33 Results.....................................................................................................................................45 Discussion ...............................................................................................................................55 Conclusions.............................................................................................................................60 References ...............................................................................................................................61 iii Chapter 4. Use of a Mobile Laboratory for Cryopreservation of Blue Catfish (Ictalurus furcatus), Xiphophorus, and Red Snapper (Lutjanus campechanus) at Remote Locations ...........................64 Methods ..................................................................................................................................66 Results.....................................................................................................................................72 Discussion ...............................................................................................................................76 Conclusions.............................................................................................................................78 References ...............................................................................................................................78 Chapter 5. Summary and Conclusions ...........................................................................................81 Mobile Laboratory Design ......................................................................................................82 Field Testing ...........................................................................................................................83 Integration of Mobile Laboratories into Existing Programs ...................................................84 References ...............................................................................................................................87 Appendix A. Standard Operating Procedures ................................................................................89 Appendix B. Mobile Laboratory Client and Site Assessment Form .............................................98 Appendix C. General Supply Checklist .......................................................................................100 Appendix D. Step-by-step Guide to Loading and Unloading the Mobile Laboratory.................101 Appendix E. Equipment and Supply List for Cryopreservation ..................................................104 Appendix F. Cooling Curves .......................................................................................................107 Vita ...............................................................................................................................................110 iv List of Tables 1.1. Oral presentations and published abstracts at meetings of the Louisiana Chapter of the American Fisheries Society (LA-AFS), the Gulf Coast Conservation Biology Symposium (GCCBS), the World Aquaculture Society (WAS), and the Joint Meeting of Herpetologists and Ichthyologists based on thesis research. ...................4 2.1. Comparison of user groups, relative body size, habitat, reproductive mode, sperm activation model, and collection method for the model species used in this thesis. .............................................................................................................................9 3.1. Hypothetical assumptions for cryopreservation of Blue Catfish for the aquaculture production of hybrid catfish (Channel Catfish female X Blue Catfish male) sac fry .............39 3.2. Hypothetical assumptions for cryopreservation of multiple species in the genus Xiphophorus for repository development ...............................................................................40 3.3. Spreadsheet model assumptions