CORE Metadata, citation and similar papers at core.ac.uk Provided by Helsingin yliopiston digitaalinen arkisto KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 162 KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 162 Photo: Tuomas Selänne, SS HELI LINDEBERG Embryo Technology in the Farmed European Polecat (Mustela putorius) UNIVERSITY OF KUOPIO KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 162 KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 162 HELI LINDEBERG Embryo Technology in the Farmed European Polecat (Mustela putorius) Doctoral dissertation To be presented, with the permission of the Faculty of Veterinary Medicine, University of Helsinki, for public criticism in the Auditorium Maximum, Hämeentie 57, Helsinki, on December 5, 2003 at 12 noon Institute of Applied Biotechnology University of Kuopio UNIVERSITY OF KUOPIO KUOPIO 2003 Distributor: Kuopio University Library P.O. Box 1627 FIN-70211 KUOPIO, FINLAND TEL. +358 17 163 430 FAX +358 17 163 410 Series editors: Professor Lauri Kärenlampi, Ph.D. Department of Ecology and Environmental Science Professor Jari Kaipio, Ph.D. Department of Applied Physics Author’s address: Institute of Applied Biotechnology University of Kuopio P.O. Box 1627, FIN-70211 Kuopio, Finland [email protected] Supervisors: Professor Emerita Maija Valtonen, DVM, Ph.D. Institute of Applied Biotechnology University of Kuopio Professor Terttu Katila, DVM, Ph.D. Department of Clinical Veterinary Sciences University of Helsinki, Saari Unit Saarentaus, Finland Sergei Amstislavsky, Senior Researcher, Ph.D. Institute of Cytology and Genetics Russian Academy of Sciences, Siberian Branch Novosibirsk, Russia Reviewers: Professor Gábor Vajta, MD, Ph.D., DVSc Section of Reproductive Biology Danish Institute of Agricultural Sciences Research Centre Foulum Tjele, Denmark Professor Emeritus Keith J. Betteridge, MVSc, Ph.D., FRCVS Department of Biomedical Sciences Ontario Veterinary College University of Guelph Guelph, Canada Opponent: Professor Wenche Farstad, DVM, Ph.D., MDNV Department of Production Animal Clinical Sciences Norwegian School of Veterinary Science Oslo, Norway ISBN 951-781-300-7 ISSN 1235-0486 Kopijyvä Kuopio 2003 Finland Lindeberg, Heli. Embryo technology in the farmed European polecat (Mustela putorius). Kuopio University Publications C. Natural and Environmental Sciences 162. 2003. 110 p. ISBN 951-781-300-7 ISSN 1235-0486 ABSTRACT The present research project started the development of assisted reproductive technology in endangered mammals in Finland. It concentrated on the endangered European mink (Mustela lutreola) which is assumed to have become extinct in Finland. The farmed European polecat (Mustela putorius) served as a model animal for the European mink. Studies on embryo technology of the farmed European polecat focused on the development of embryo recovery, cryopreservation and transfer techniques, which could further be applied to the conservation of the endangered European mink. Oestrous donors were mated to fertile males once daily on two consecutive days. The recipients were mated to vasectomized males to induce ovulation. A total of 582 embryos were recovered from 66 donors either post mortem or surgically under anaesthesia 3 to 13 days after the first mating. The embryos were subjected to one of the following treatments 1) fresh embryo transfer to recipients 2) in vitro culture 3) conventional slow freezing, thawing and transfer to recipients 4) vitrification, warming, in vitro culture and transfer to recipients. During in vitro culture, 1- to 16-cell stage embryos developed to blastocysts but did not expand. The embryos placed in culture as morulae or blastocysts expanded in vitro during the first 24-h period in the same manner as their in vivo counterparts. In vitro culture of polecat embryos using the described technology is applicable for temporary storage of embryos waiting to be transferred or cryopreserved. The numbers of transferred embryos per recipient were 10.8, 10.0 and 12.5 for fresh, frozen-thawed and vitrified-warmed embryos, respectively. The percentages of live kits per transferred embryos (= survival rate) were 42, 11 and 16, and the numbers of live kits per recipient were 4.5, 1.1 and 2.0 for fresh, frozen-thawed and vitrified-warmed embryos, respectively, with an overall survival rate of 30% (89 live kits/302 transferred embryos). Transfer of cryopreserved embryos resulted in a low kit yield, but nonetheless did produce the first mustelids ever from frozen-thawed and vitrified-warmed embryos. Universal Decimal Classification: 619, 502.74, 636.93, 591.16 CAB Thesaurus: Mustelidae; polecats; embryo transfer; cryopreservation; in vitro culture; embryonic development ACKNOWLEDGEMENTS I became a researcher in an ex situ project of my principal supervisor, Professor Maija Valtonen, DVM, Ph.D., in 1997 at the Department of Applied Zoology and Veterinary Medicine (now Institute of Applied Biotechnology) of the University of Kuopio where we worked with blue foxes, polecats and reindeer. Three years of intense project work and lots of data provided me with a fine batch of publications and finally a thesis. The mustelid part of the ex situ project has been a huge success: it was an excellent idea of Professor Valtonen to create a project like this, at a time when the importance of ex situ conservation work and assisted reproductive technology of endangered species was not yet well established in Finland. The Finnish Biodiversity Research Programme (FIBRE) funded our ex situ project during the period 1997- 1999. The Institute of Applied Biotechnology and the University of Kuopio are warmly thanked for providing the facilities and the animals at the Juankoski Research Station. The writing was financially supported by grants from the Finnish Cultural Foundation, the Finnish Veterinary Foundation and the Finnish Cultural Foundation of Northern Savo (Alma ja Jussi Jalkanen Fund). Our ex situ project was the first in the world concentrating on blue fox and reindeer which were the model species of Scandinavian arctic white fox and wild forest reindeer, and the second when it comes to mustelids. I wish to express my deepest gratitude to Professor Maija Valtonen, who besides being my mentor, was always easy to turn to, had time to discuss with me and gave me valuable support during the hard times of my personal life. Thank you, Professor Terttu Katila, DVM, Ph.D., my second supervisor from the Faculty of Veterinary Medicine, Department of Clinical Veterinary Sciences, Animal Reproduction for guiding me through the difficulties in writing this thesis and for allowing me to change from cryopreservation of stallion semen which I started in 1994…eventually to preservation of mustelids. I wish to express my gratitude to Professors Gábor Vajta, MD, Ph.D., and Keith Betteridge, MVSc, Ph.D., for reviewing my thesis. Thank you for your excellent comments and improvement of the thesis. Professor Gábor, you have provided me with many of your laboratory protocols which I have greatly appreciated. I warmly thank my co-workers who have been indispensable during the course of the studies. Jussi Aalto, DVM, developed the surgical flank method and kindly allowed me to describe it in this thesis. Jussi, you were the driving force in deciding the practical parts of the experiments, in pulling me down from skies when my spirit was lost in the clouds. Sergei Amstislavsky, Ph.D., my third supervisor and our Russian friend actively participated in the experiments of this thesis in 1998-99. Subsequently our collaboration has focused on the conservation of the European mink. Sergei, I owe you my thanks for sharing the interesting world of science and mustelids. Your cultural and intellectual facts have been a source of refreshment in this project. Thanks for sharing those with us! Jaana Peippo, Ph.D., has been the driving force in setting up the in vitro production of polecat embryos for our experiments. Jaana, your ever-lasting enthusiasm in science is a source of inspiration for all of us. We were lucky to have such talented graduate students, Mikko Järvinen, M.Sc., Katja Piltti, M.Sc., and Hanna Korhonen, M.Sc., working in our project. We all counted a LOT of embryos, over and over again, during this project. Thank you Mikko, Katja and Hanna for being so thorough and skilful! This project would not have been completed without the animals. I express my gratitude to Anitta Helin and Matti Tengvall who took care of the polecats at the Juankoski Research Station of the University of Kuopio. Elina Reinikainen organized facilities and equipment for this project. Risto Savolainen, Seppo Kukkonen, Helena Könönen are thanked for their technical assistance during the experiments of this thesis. To the staff of the Juankoski Research Station, Maija Miskala, Rose-Marie Nybondas, Marja-Leena Rönkä and many short time employees, thank you for feeding the polecats. My colleagues, Liisa Jalkanen, DVM, Merja Voutilainen, DVM, and Vesa Rainio, DVM, Ph.D., are acknowledged for the excellent network of help they have provided me during all the years. Professor Maria Halmekytö, Ph.D., present leader of the embryo technology group of Animal Biotechnology and group researcher, Kirsi Kananen-Anttila, Ph.D., are indebted for providing me with new research challenges after this thesis. All other professors, researchers and staff members at the Institute of Applied Biotechnology are thanked for friendship and many nice discussions. Thanks