Aus der Klinik für kleine Haustiere der Tierärztlichen Hochschule Hannover und dem Research Department des Saint Louis Zoo, St. Louis, Missouri, USA Cryopreservation of Mexican gray wolf (Canis lupus baileyi) semen - evaluation of different times and rates of pre-freeze cooling and Equex pastasupplementation - in comparison with semen of the domestic dog and generic gray wolf (Canis lupus) INAUGURAL-DISSERTATION Zur Erlangung des Grades einer DOKTORIN DER VETERINÄRMEDIZIN (Dr. med. vet.) durch die Tierärztliche Hochschule Hannover Vorgelegt von Claudia Zindl aus Singen/Hohentwiel Hannover 2006 Bibliographische Information der Deutschen Bibliothek Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliographie; Detaillierte bibliographische Daten sind im Internet über http://dnb.ddb.de abrufbar. 1. Auflage 2006 © 2006 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH, Gießen Printed in Germany ISBN 3-938026-89-8 Verlag: DVG Service GmbH Frankfurter Straße 89 35392 Gießen 0641/24466 [email protected] www.dvg.net 2 Aus der Klinik für kleine Haustiere der Tierärztlichen Hochschule Hannover und dem Research Department des Saint Louis Zoo, St. Louis, Missouri, USA Cryopreservation of Mexican gray wolf (Canis lupus baileyi) semen - evaluation of different times and rates of pre-freeze cooling and Equex pastasupplementation - in comparison with semen of the domestic dog and generic gray wolf (Canis lupus) INAUGURAL-DISSERTATION Zur Erlangung des Grades einer DOKTORIN DER VETERINÄRMEDIZIN (Dr. med.vet.) durch die Tierärztliche Hochschule Hannover Vorgelegt von Claudia Zindl aus Singen/Hohentwiel Hannover 2006 3 Wissenschaftliche Betreuung: Univ.-Prof’in Dr. med. vet. Anne-Rose Günzel-Apel Cheryl S. Asa, PhD (St. Louis Zoo, MO, USA) 1. Gutachterin: Univ.-Prof’in Dr. med. vet. Anne-Rose Günzel-Apel 2. Gutachter: Apl. Prof. Dr. med. vet. Michael Böer Tag der mündlichen Prüfung: 29. Mai 2006 Die Dissertation wurde gefördert durch ein Kurzstipendium des Deutschen Akademischen Austauschdienstes 4 Meinen Eltern 11 12 Contents 1 Introduction……………………………………………………………………..…..11 2 Literature review………………………………………………………………..….13 2.1 Carnivore Conservation…………………………………………………..….13 2.1.1 Mexican wolf conservation…………………………………………………..13 2.1.2 Role of reproduction sciences in carnivore conservation – semen banking…15 2.1.3 Seasonality and canid reproduction………………………………………….17 2.2 Semen collection by electroejaculation………………………………………18 2.3 Temperature decrease <37 °C as part of the cryopreservation process………19 2.4 Molecular basis of cooling cells ………………….…………………………..21 2.4.1 Membrane structure and thermotropic behaviour…………………………….22 2.4.2 Thermotropic behaviour and membrane function…………………………….23 2.4.2.1 Sperm damage caused by low temperature per se…….………………………24 2.4.2.2 Sperm damage caused by cold shock…………………………………………26 2.5 Cooling, equilibration and chilling in canine species…………………….…...29 2.5.1 Cooling period………………………………………………………………...29 2.5.2 Equilibration period…………………………………………………………...37 2.5.3 Chilling period………………………………………………………………...38 2.6 Extender additives…………………………………………………………….39 2.6.1 Extender additives containing sodium dodecyl sulphate……………………...39 2.7 Hypoosmotic swelling test …………………………………………………...41 2.8 Computer assisted semen analysis…………………………………………….43 2.9 Electron microscopy……...…………………………………………………...45 3 Materials and Methods…………………………………….………………………..48 3.1 Preliminary cooling and temperature trial ……………………………………48 3.2 Semen donors…………………………………………………………………48 3.3 Determination of testicular size………………………...…………………….50 3.4 Semen collection…………………………………………...…………………50 3.5 Experimental design…………………………………………………………..52 13 3.6 Semen handling……………………………………………...……………….54 3.7 Semen evaluation …………..………………………………………………...56 3.8 Statistical Analysis……………………………………………………………63 4 Results………………………………………………………………………………..64 4.1 Preliminary temperature trial………………………………….………………64 4.2 Cooling patterns of diluted semen…………………………….………………65 4.3 Morphology of testes………………………………………….………………67 4.4 Characteristics of dog and wolf ejaculates……………………………………67 4.5 Cooled semen…………………………………………………………………68 4.5.1 Influence of different cooling times and rates on sperm plasma membrane integrity……………………………………………………………………….68 4.5.2 Influence of different cooling times and rates on morphology and acrosome integrity……………………………………………………………………….68 4.5.3 Electron microscopic evaluation of sperm morphology…..………………….70 4.6 Frozen-thawed semen…………………………………………………………72 4.6.1 Differences of collected fresh, cooled and frozen-thawed semen….…………72 4.6.2 Influence of different cooling times and Equex pasta on post-thaw motility...73 4.6.4.1 Microscopic evaluation…………………………………………………….....73 4.6.4.2 Computer assisted semen analysis of Mexican wolf semen………………….77 4.6.3 Influence of different cooling times and Equex pasta on post-thaw plasma membrane integrity……………………………………………………………86 4.6.4 Influence of different cooling times and Equex pasta on post-thaw sperm morphology and acrosome integrity………………………………………......94 5 Discussion…………………………………………………………………………….98 5.1 Species differences of ejaculate and fresh semen characteristics…..…………98 5.2 Testes measurements……………………………………………..….………..98 5.3 Influence of the cooling process on semen……………………………………99 5.3.1 General observations……………………………………………………….....99 5.3.2 Specific influence of cooling rates, cooling times and final temperature 14 attained……………………………………………………………………….100 5.3.3 Electron microscopic findings after the cooling period……………………..102 5.4 Influence of the freezing-thawing process on semen………………………..104 5.4.1 General observations………………………………………………………...104 5.4.2 Influence of applied cooling rates and times on frozen-thawed semen……...105 5.5 Influence of Equex pasta on frozen-thawed generic gray and Mexican wolf semen…………………………………………………………………...108 5.6 Influence of cooling times and rates and Equex pasta on motility parameters of frozen-thawed Mexican wolf semen evaluated by CASA………….…..……111 6 Summary....................................................................................................................114 7 Zusammenfassung.....................................................................................................116 8 Literature references………………………………………………………………118 9 Appendix……………………………………………………………………………165 9.1 Recipes for media……………………………………………………………165 9.2 Defined set-up of the Hamilton Thorn IVOS-10……………………………168 9.2 Tables………………………………………………………………………..169 15 Abbreviations ALH lateral head displacement ATP adenosine triphophate ANOVA analysis of variance AZA American Zoo and Aquarium Association BCF beat cross frequency BEEM ballistic electron emission microscopy BHT butylated hydroxytoluene BSA bovine serum albumine Ca2+ calcium ions Canid TAG Taxon Advisory Group of canid species for AZA conservation programs cAMP circular adenosin triphophate CASA computer assisted semen analysis chap chapter 6-CFDA 6-carboxyfluoresceindiacetate CITES Convention on International Trade in Endangered Species of Wild Fauna and Flora CO2 carbon dioxide ext extender EZA European Zoo Association e.g. exempli gratia h hour 16 HEPES N-(2-hydroxyethyl)piperazinN-2-ethane HOST hypoosmotic swelling test HT Hamilton Thorn IVOS-10 HSP90 90 kDa heat-shock protein i.e. id est IMP intramembraneous particle IUCN The World Conservation Union kg kilogram LIN linearity LN2 liquid nitrogen max maximum mg milligram min minimum mins minutes μg microgram μl microliter mOsm milliosmol n number of dogs/generic gray wolves/Mexican wolves OEP Orvus Es paste p probability of error pH negative decadic logarithm of the hydrogen ion concentration PI propidium iodide PUFA polyunsaturated fatty acids RT room temperature 17 SDS sodium dodecyl (lauryl) sulphate SEM statistic error of the mean SMI sperm motility index SQA sperm quality analysis SSP® Species Survival Plan STR straightness TRIS Tris(hydroxymethyl)-aminomethane USFWS United States Fish and Wildlife Service VAP average path velocity VCL curvilinear velocity VSL straightline velocity WAZA World Association of Zoos and Aquariums WCSRC Wildlife Canid Survival and Research Center wb water bath 18 Introduction 1 Introduction Recovery of the Mexican gray wolf* (Canis lupus baileyi), a subspecies of the generic gray wolf (Canis lupus), depends on careful genetic management of the captive population. Because of their monogamous mating system, transfer of gametes using cryopreservation and artificial insemination is preferable to breaking pair-bonds and transfer of animals. As there are only few Mexican wolves available for evaluating the requisite technology, such as sperm cryopreservation, it has been necessary to use the generic gray wolf as a model and to base the techniques on those developed for the domestic dog, the wolf’s closest relative. As cooling is one of the critical steps in semen freezing, the effect of various cooling rates on semen quality was compared in both generic gray and Mexican wolves. Spermatozoa of several species require a rest of several hours during cooling, before freezing, to develop maximal resistance to the effects of freezing. The first studies on cooling and equilibration of canine spermatozoa stated that none of the temperatures or time factors significantly affected the survival rate (MARTIN 1963a, b). A number of studies have dealt with the effects of slow and rapid cooling of canine semen (e.g. OLAR et al. 1989; HAY et al. 1997b). Some investigations (OSINOWO and SALAMON 1976; OETTLÉ 1986a)