Embryo Technology in the Farmed European Polecat (Mustela Putorius)

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KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 162 KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 162

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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 are due to Jaakko Mäkelä for telling me about the early stages of polecat fur farming in Finland and Dr. Raija Sauna-aho, DVM, for kindly providing me with information about feeding minks and polecats during the breeding season. Professor Riitta-Mari Tulamo, DVM, Ph.D., your advice has always been useful. Thank you for remembering Juhani with me.

Special thanks to several individuals who have contributed to this thesis: Professor Erkki Koskinen, DVM, Ph.D., and Antti-Pekka Kangas, M.Sc., for statistical analysis, Teija Peura, Ph.D., for providing Katja Piltti with her OPS protocol, Jo Gayle Howard, DVM, Ph.D., for providing me with her protocol of semen collection and cryopreservation in the ferret, Ziyi Li, Ph.D., for providing me with his unpublished results of ferret embryos hatching in vitro and for many interesting email discussions about ferret reproduction, Professors Yrjö Gröhn, DVM, Ph.D., and R.H. Foote, Ph.D., for helping me gather valuable references for the thesis, William Swanson, Ph.D., for his communications about cat in vivo embryos, Tiit Maran, M.Sc., for providing me with his literature on the European mink, Lauri Ann Willingham-Rocky, M.Sc., and Steve Metcalfe, M.Sc., for providing me with their Master of Science theses, Gabriela Kania, M.Sc., for her experience in zona hardening, Outi Lohi, Ph.D., Anniina Laurema, MD, for good references and Pekka Hyvönen, Ph.D., for supporting my mental health. I also wish to thank Kimmo Kivinen and Tuija Karhu for technical support and Ewen MacDonald, D. Pharm., for revising the language of this thesis.

I owe thanks to people other than my family members who have helped me taking care of the children while I was busy doing research; Annika Olev, Jenni Sutinen, Paula Rokkila, Hilkka Pitkänen and family Rissanen. Heikki Konttinen is appreciated for helping in economy and taxation.

My parents, Irma and Mauno Lindeberg, you have constantly supported me during all my life. My two sisters, Mai and Satu, and Satu’s husband, Mikko Nuutinen, now it is your turn to do research in our family. Thanks for sharing my life. I love you all.

The Hirvonen family has provided me with a second home. Juhani’s parents, his late mother, Katri and father, Erkki, Juhani’s sisters, Riitta Leinonen, Leena Heljälä, Marjatta Rissanen and brothers, Olavi Hirvonen and Veijo Hirvonen, thank you for keeping us inside the family. I wish my dissertation would give us a little consolation for the fact that Juhani passed away just before his own dissertation for which I will dedicate this book to the families left behind.

My late husband, Juhani Hirvonen, DVM, I miss you inconceivably. My heart grieves that you are not here to share this with me. Our children, Jussi and Helena Hirvonen, I would not have survived without you.

Kuopio 27th October, 2003

Heli Lindeberg

Photo: Pentti Vänskä

To my families To Jussi and Helena To the animals behind

ABBREVIATIONS

AI artificial insemination ATP adenosine triphosphate B blastocyst BSA bovine serum albumin CL corpus luteum CM compact morula CO2 carbon dioxide CPA cryoprotective agent CZB Czatot-Ziomek-Bavister DMSO dimethylsulphoxide DPBS Dulbecco’s phosphate buffered saline EB early blastocyst ExB expanded blastocyst eCG equine chorionic gonadotrophin EG ethylene glycol FBS fetal bovine serum FSH follicle stimulating hormone FCS fetal calf serum GLY glycerol GnRH gonadotrophin releasing hormone GS goat serum HB hatched blastocyst hCG human chorionic gonadotrophin ICM inner cell mass i.c. intracardiae i.m. intramuscular i.v. intravenous IU international unit IVC in vitro culture IVF in vitro fertilization IVM in vitro maturation IVP in vitro production LH luteinizing hormone LN2 liquid nitrogen M morula MII metaphase two mPBS modified phosphate buffered saline NCS newborn calf serum NBSC newborn calf serum NCSU North Carolina State University OPS open pulled straw PB1 modified phosphate buffered saline PBS phosphate buffered saline PVA polyvinylalcohol PVP polyvinylpyrrolidone ® registered trademark SCN suprachiasmatic nuclei SOF synthetic oviductal fluid T temperature TCM tissue culture medium  trademark UTJ utero-tubal junction w/wo with/without XB expanded blastocyst

ORIGINAL ARTICLES

This thesis is based on the following original articles which are referred to in the text by their Roman numerals I-IV:

I. Lindeberg H, Järvinen M. Early embryonic development and in vitro

culture of in vivo produced embryos in the farmed European polecat

(Mustela putorius). Theriogenology 2003;60:965-975.

II. Lindeberg H, Amstislavsky S, Järvinen M, Aalto J, Valtonen M.

Surgical transfer of in vivo produced farmed European polecat (Mustela

putorius) embryos. Theriogenology 2002;57:2167-2177.

III. Lindeberg H, Aalto J, Amstislavsky S, Piltti K, Järvinen M, Valtonen

M. Surgical recovery and successful surgical transfer of conventionally

frozen-thawed in vivo produced embryos in the farmed European

polecat (Mustela putorius). Theriogenology 2003;60:1515-1525.

IV. Piltti K, Lindeberg H, Aalto J, Korhonen H. Live cubs born after

transfer of OPS vitrified-warmed embryos in the farmed European

polecat (Mustela putorius). Theriogenology 2004. In press.

This thesis also contains previously unpublished data. Unpublished data from the experiment presented in IV are based on the material and methods of the original publication and marked with supercript u (i.e. IVu).

CONTENTS

1 Introduction 15

2 Review of literature 16 2.1 Reproductive physiology of Mustelidae 16 2.2. Reproductive physiology of the domestic ferret 17 2.2.1 Photoperiods and seasonality 17 2.2.2 Oestrous cycle and vaginal cytology 19 2.2.3 Ovulation 20 2.2.4 Oocyte maturation and fertilization 21 2.2.5 Early embryonic development 22 2.2.6 Oviductal passage 23 2.2.7 Early embryonic coats 24 2.2.8 Implantation 26 2.2.9 Gestation and function of corpora lutea 28 2.2.10 Parturition, rebreeding and induction of oestrus 29 2.3 Assisted reproductive technology 29 2.3.1 Artificial insemination 29 2.3.2 Embryo recovery and transfer 31 2.3.3 In vitro embryo technology 35 2.4 Cryopreservation of in vivo embryos 37 2.4.1 Conventional slow freezing 38 2.4.2 Vitrification 40

3 Aims of the study 46

4 Materials and methods 47 4.1 Animals 47 4.2 Vasectomy of the males 49 4.3 Test matings 49 4.4 Detection of oestrus and mating of the females 50 4.5 Post mortem embryo recovery 51 4.6 Surgical embryo recovery 51 4.6.1 The linea alba method (III) 51 4.6.2 The flank method (IVu) 52 4.7 Evaluation of recovered embryos 55 4.8 Measurement of the diameters of the embryos 56 4.9 In vitro culture of fresh embryos 56 4.10 Processing of fresh embryos before transfer 57 4.11 Conventional slow freezing of embryos 58 4.12 Open pulled straw vitrification, warming, culture and transportation of embryos 58 4.13 Surgical embryo transfer 60 4.14 Post mortem examinations 60 4.15 Postoperative care of the surgically flushed donors and recipients 61 4.16 Statistics 61

5 Results 62 5.1 Early embryonic development and oviductal passage 62 5.2 Embryo recovery and evaluation 63 5.3 In vitro culture of in vivo embryos 66 5.4 Treatment of fresh and cryopreserved embryos before transfer 68 5.5 Transfer of fresh, frozen-thawed and vitrified-warmed embryos 69

6 Discussion 74 6.1 Early embryonic development 74 6.2 Evaluation of embryos 75 6.3 Embryo recovery techniques 77 6.4 In vitro culture 80 6.5 Conventional slow freezing 82 6.6 Vitrification 86 6.7 Surgical transfer of fresh, frozen-thawed and vitrified-warmed embryos 88

7 Conclusions 91

8 References 93

Appendix: Original publications

1 INTRODUCTION

Biodiversity, i.e. the total variety of genetic strains, species and ecosystems (Glosser 1993) is continuously threatened, and prevention of loss in biodiversity demands intervention from mankind (Woodford 1990, Garde et al. 1998, Holt and Pickard 1999). Conservation efforts consist of both in situ and ex situ programs. In situ programs protect and manage animal populations within their natural, native habitat. Ex situ programs remove individuals, gametes or embryos from wild populations for controlled breeding and management in captivity (Rall et al. 1991). However, breeding in captivity is subject to two major problems. First, animals undergo genetic adaptation during captivity, i.e. the traits that improve their fitness to survive in artificial conditions will be selected. Secondly, reproduction in small populations results in inbreeding (Bainbridge and Jabbour 1998). Assisted reproductive technology (artificial insemination, embryo transfer and cryopreservation of gametes and embryos) can be used to reduce these threats. Some of the population can be conserved as frozen embryos or oocytes using genetic resource banking (Wildt et al. 1992, 1997, Loskutoff 1998, Amstislavsky et al. 1996, 1997a, 2000). Though this is already reality for some mouse strains it is not, so far, the case for wildlife. In captivity, many individuals experience difficulties in reproduction due to aggressive behaviour, debility, diseases or injuries (Lasley et al. 1994). Modern biotechnology may assist these animals to mate or carry their offspring to term provided that enough information exists on the reproductive physiology of these species (Goodrowe 2001, Wildt et al. 2001).

The present research project was the first study on the development of assisted reproductive technology for three mammalian species which are endangered or under observation in Finland, one of which was the European mink (Mustela lutreola). The farmed European polecat (Mustela putorius) served as a model species for the European mink. The European mink is classified as an endangered species (EN A1ace) on the IUCN Red List (IUCN 2002), and it is almost extinct in the wild in many parts of Europe (Maran and Henttonen 1995, Maran 1996, Maran et al. 1998, Sidorovich 2000). The European mink and the European polecat are close relatives and have similar reproductive physiology so that hybrids can be obtained both in captivity (Ternovsky and Ternovskaya 1994, Amstislavsky and Ternovskaya 2000)

16 and in the wild (Davison et al. 2000). The technology developed in the present study has recently been applied to the European mink (Amstislavsky et al., in press).

2 REVIEW OF LITERATURE

2.1 Reproductive physiology of Mustelidae

The family of Mustelidae (weasels, badgers, skunks and otters) consists of 23 genera and 65 species (Howard 2001). It is subdivided into 5 recognized subfamilies: 1) Mustelinae (weasels) 2) Mellivorinae (honey badgers) 3) Melinae (badgers) 4) Mephitinae (skunks) and 5) Lutrinae (otters). The subfamily Mustelinae includes weasels, ermines, stoats, minks and polecats (Howard 2001). In the genus Mustela, the group of polecats consists of domestic ferret (Mustela putorius furo), European polecat (Mustela putorius), European mink (Mustela lutreola), steppe polecat (Mustela eversmanni) and black-footed ferret (Mustela nigripes). The American mink (Mustela vison) is the most aberrant in the genus Mustela (Davison et al. 2000). The domestic ferret is considered as the domesticated form of the European polecat, and is widely used as a laboratory research animal all over the world (Thomson 1951, Fox 1998). Phylogenetically the European polecat and the Siberian polecat (Mustela sibirica) are probably the closest relatives of the European mink (Maran 1996, Maran et al. 1998). Table 1 describes the reproductive similarities of the farmed European polecat and the European mink collected from several reference sources (Moshonkin 1981, Ternovsky and Ternovskaya 1994, Maran and Robinson 1996, Fox and Bell 1998, Davison et al. 2000). These similarities justify the use of the polecat/ferret as a model species for the European mink. The European polecat and the domestic ferret produce intraspecies hybrids that are fertile. Hybrid females between the European polecat and the European mink are also fertile but males are infertile (Ternovsky and Ternovskaya 1994).

Mustelids are polytocous carnivorous species demonstrating a variety of unique reproductive characteristics. The reproductive physiology of some species has been quite thoroughly studied while less is known for other species (Mead 1989, Ternovsky and Ternovskaya 1994). This literature review of reproductive physiology

17 concentrates on results obtained with the domestic ferret. When necessary, results concerning other mustelids and other carnivores are also included.

Table 1. Reproductive features of the domestic ferret (Mustela putorius furo) and the European mink (Mustela lutreola).

Mustela putorius furo Mustela lutreola Chromosome number 2n = 40 2n = 38 Breeding season March-August March-June Onset of oestrus Enlarged pink vulva Enlarged pink vulva up to 50x normal size up to 25x normal size Length of oestrus Continuous until intromission 1-10 days Oestrous cycles/season 1-3 1-3 Type of ovulation Induced Induced Implantation 12 days after mating Not studied Gestation length 6 weeks, 42 ± 1 days 41.6 ± 0.8 (39-44) days Pseudopregnancy 5.5 to 6 weeks Not studied Average litter size 8.6 ± 0.6 (1-17) 4.3 ± 0.1 (1-9)

2.2 Reproductive physiology of domestic ferret

2.2.1 Photoperiods and seasonality

The domestic ferret is considered to be a seasonally polyoestrous species, but females exhibit a constant oestrus between late March and early August if they are not bred (Mead 1989). Marshall (1904) classified the ferret as a mono-oestrous species. Offspring born the previous summer reach puberty by the following spring at the age of 8-12 months (Fox and Bell 1998). Initiation of gonadal activity is totally dependent on the light-dark cycle, which stimulates or inhibits reproduction through transmission of information about the day length to the brain (Bissonnette 1932, Turek and Van Cauter 1988). Information about day length is transmitted from the retinal cells of the eye, via the optic nerves, to the suprachiasmatic nuclei (SCN), located in the anterior hypothalamus. The output from the SCN is transmitted via the paraventricular nucleus and the superior cervical ganglia to the pineal gland, which secretes the pineal hormone, melatonin, the concentration of which in serum is high at night and low during the day (Forsberg 1992). A decrease in the time when there is a high

18 circulating melatonin concentration stimulates gonadal activity of ferrets, which breed when the days are long.

The ferret’s gonadal response to a given photoperiod depends both on the duration of the photoperiod and on the previous photoperiodic experience. Ferrets, like other “long-day” seasonal breeders, need alternating periods of lengthening days, during which they are sensitive to light, and shortening days during which they are insensitive to light, so that the annual cycle recurs normally (Herbert 1989). The state of temporary unresponsiveness to photic stimuli is referred to as the “photorefractory” condition (Elliott and Goldman 1981) and it is required to induce a return to the photosensitive state (Forsberg 1992). Ferrets exposed to artificially changing long and short day light conditions (long days: 14 hours light and 8 hours darkness; short days: 8 hours light and 16 hours darkness) start showing oestrus about 3 weeks after the change from short days to long days (Fox and Bell 1998) and cease showing oestrus about the same time after a change from long days to short days. The repeated change from long days to short days after every six months causes one period of gonadal activity per year similar to a natural breeding season when the animals are exposed to natural outdoor light conditions. A change in light conditions every four or two months causes two or three periods of gonadal activity a year, respectively (Herbert 1989). Through the use of reverse light cycles, ferrets can be induced to breed anytime during the year (Harvey and MacFarlane 1958).

As the melatonin secretion decreases, the pulsatile secretion of gonadotrophin releasing hormone (GnRH) increases, which initiates the release of LH and FSH (Baum et al. 1986). The pituitary gonadotrophins initiate follicular development and, as the follicular production of oestradiol starts, the females enter oestrus. After electrical stimulation of hypothalamus of anaesthetized female ferrets, there were low basal concentrations of FSH and LH for 5 oestrous females, two of which ovulated, one had luteinized follicles and 2 failed to ovulate after stimulation. This suggests that a small rise in the plasma LH levels following hypothalamic stimulation in oestrous females was sufficient to cause ovulation (Donovan and Ter Haar 1977a).

2.2.2 Oestrous cycle and vaginal cytology

Increasing tumescence in the pink coloured vulva is a sign of prooestrus in the ferret. The vulva enlarges up to 50 times its normal size during a 2- to 3 -week period at the beginning of the breeding season (Hammond and Marshall 1930), which extends from March to August (Marshall 1904). No change in the turgidity of the vulva occurs up to 36 hours after copulation (Hammond and Walton 1934) but 3 or 4 days after mating the vulva starts regressing and regains its normal size in 2-3 weeks. If the vulva does not recede, ovulation has probably not taken place and the female may need to be remated (Lagerqvist 1992). Oestrus can persist for up to 5 months, but once ovulation is induced, either pregnancy or pseudopregnancy ensues (Hammond and Marshall 1930).

In conjunction with the vulval swelling during oestrus, there is a thickening of the uterine endometrium and follicles develop in the ovaries. Primordial follicles develop to primary follicles during all stages of the reproductive life span of mammals, including prenatal, prepubertal, anoestrous periods and pregnancy (Murphy 1989). Pituitary gonadotrophins are required for follicular maturation (Murphy 1979). The pattern of follicular development during a prolonged oestrus is unknown but it is assumed to be continuous (Robinson 1918). In the absence of copulation, which results in a prolonged oestrus, a cohort of follicles develops, degenerates and is replaced by a new cohort of follicles. Follicular development and atresia overlap so that there is a recent cohort of follicles available for ovulation whenever copulation might occur (Murphy 1989). After copulation, some preovulatory follicles may persist in the ovaries and luteinize but they do not rupture (Robinson 1918). The interval required for development from primordial to preovulatory follicle has not been reported for any mustelid. It has been proposed that the time required for a wave of follicles to develop prior to induced ovulation in ferrets is approximately 6 days (Murphy 1989).

Oestradiol secreted by the follicles controls vulval swelling, uterine development, sexual receptivity and changes in vaginal cells (Hamilton and Gould 1940). Ferrets with vulval diameters greater than 1 cm have mated successfully (Murphy 1989). Vaginal cytology is commonly used to detect oestrus in some domestic carnivores,

20 especially dogs (Olson et al. 1984), and the technique has been described for domestic ferrets (Hamilton and Gould 1940, Williams et al. 1992), mink (Hansson 1947) and silver foxes (Jalkanen et al. 1988). In ferrets, prooestrus was characterized by an increasing percentage of superficial epithelial cells together with enlargement of the vulva. During oestrus, usually > 90% of epithelial cells were superficial cells and after several days these cells were fully keratinised. Neutrophils were common during all stages of the oestrous cycle (Williams et al. 1992). Following copulation, which may last from 15 min to 3 h (Hammond and Walton 1934) the average time being 1 hour (Fox and Bell 1998), the percentage of superficial cells in the vagina declines together with vulval swelling. During mating, the male grips the female’s neck and makes repeated pelvic thrusts until intromission is achieved (Carroll et al. 1985).

2.2.3 Ovulation

Ovulation is non-spontaneous, i.e. it is induced by pressure on the cervix associated with copulation (Carroll et al. 1985) or by hCG treatment (Mead et al. 1988a). During oestrus, large amounts of oestradiol are secreted from antral follicles on the ovaries. Extended coital stimuli under high oestradiol concentration are required to prolong and potentiate the release of GnRH to elicit sufficient LH for ovulation (Donovan and Ter Haar 1977b). The increase in the LH concentration after copulation is modest (only 3- to 4-fold elevation) and, during oestrus, LH does not exhibit the usual mammalian pattern of pulsatile release (Carroll et al. 1985, Tritt 1986). After sufficient LH release, the preovulatory follicles mature and an average of 12 oocytes (5 to 13) per female, are ovulated 30-40 h after copulation (Hammond and Walton 1934, Chang and Yanagimachi 1963, Carroll et al. 1985) into the ovarian bursa. The ferret ovary is encapsulated in a fatty ovarian bursa, so that the ovulated oocytes are not shed into the abdominal cavity (Marshall 1904). The oocytes remain capable of being fertilized for a period of 30-36 hours after ovulation (Hammond and Walton 1934, Chang and Yanagimachi 1963), but matings during a period of 18-30 hours after ovulation produce small litters (1-3 kits) (Hammond and Walton 1934). If the number of penetrated oocytes is considered as evidence of fertilization, the ferret oocytes are most capable of being fertilized up to 12 hours after ovulation, i.e. 42-52 hours after copulation (Chang and Yanagimachi 1963). Spermatozoa were found in the ovarian bursa 6 hours after copulation in the ferret (Hammond and Walton 1934)

21 or 1 hour after copulation in the stoat (Amstislavsky, personal communication), but they require 3.5-11.5 hours of capacitation time in the female reproductive tract to become fertile (Chang and Yanagimachi 1963). The spermatozoa apparently retain their capability to induce fertilization for up to 126 hours after copulation (Chang 1965b), but the rate of fertilization depends on the short lifespan of oocytes. The exact site where fertilization takes place is not known (Murphy 1989), though the middle third of the oviduct (Robinson 1918) has been suggested. According to Chang and Yanagimachi (1963), ferrets may sometimes ovulate spontaneously when they are handled. Generally, preintromission events do not lead to any increase in LH and thus do not induce ovulation (Carroll et al. 1985).

2.2.4 Oocyte maturation and fertilization

Ferret oocytes ovulate at the metaphase of the second meiotic division (MII) (Mainland 1931, Piltti et al. 2003) embedded in three layers of corona radiata cells (Chang 1950), which detach after fertilization. A positive correlation between the naked appearance of oocytes and fertilization has been reported (Chang 1965b). Metaphase II is the resting phase in the second meiosis, and the second meiosis can proceed only after penetration of the oocyte by a spermatozoon or after parthenogenetic activation (Valtonen and Jalkanen 1993, Farstad et al. 2001). A nuclear maturation process is initiated, and the formation of the first polar body and the appearance of the second maturation spindle occur, after copulation (Hamilton 1934). Without coital stimuli, oocytes remain immature in the germinal vesicle stage until they degenerate. Parthenogenetic cleavage of oocytes, i.e. the formation and division of blastomeres in the absence of fertilization, is common up to 2-6 cells and may occur in 43-60% of the unfertilized oocytes, most likely as a result of oocyte ageing (Chang 1950, 1957). The majority of unfertilized oocytes are embedded in the corona radiata even 4 to 5 days after sterile mating (Chang and Yanagimachi 1963). If the oocyte is fertilized, the entire spermatozoon (head together with tail) passes through the zona pellucida (Hamilton 1934). In the cytoplasm, the head of the fertilizing spermatozoon first enlarges, its chromatin condenses and the head detaches from the tail (Chang 1965b, Chang and Yanagimachi 1963). The sperm head forms the male pronucleus, and the female chromatin forms the smaller female pronucleus by 12-18 h after oocyte penetration by a spermatozoon (Chang 1965b). The first

22 pronuclei are discovered 40¾ to 43½ hours after copulation (Mainland 1930, Hamilton 1934, Chang and Yanagimachi 1963, Piltti et al. 2003). The second polar body is extruded soon after oocyte penetration by a spermatozoon, as in most other mammals (Hamilton 1934). In the ferret, the morphological differences between the first and the second polar body are not distinctive (Chang 1965a). The number of polar bodies that may change their location in the distinct perivitelline space (Hamilton 1934) varies from two to four (Mainland 1931, Chang and Yanagimachi 1963) indicating that in both fertilized and unfertilized oocytes, the first polar body may frequently either divide (Chang and Yanagimachi 1963) or become fragmented. The thickness of the zona pellucida varies from 2 to 20 µm (Mainland 1932, Chang 1950). The size of an unfertilized oocyte without a corona radiata has been reported to be between 140 and 160 µm (Hamilton 1934, Chang 1950). The oocyte is spherical or ovoid; its nucleus is large, round and eccentric with one or, rarely, two nucleoli (Robinson 1918). It is rich in lipid particles which in one-half of the oocyte are less dense than in the other half (Hamilton 1934).

2.2.5 Early embryonic development

Timing of early embryonic development and morphology of the ferret embryos has been thoroughly described from fertilized oocytes to expanded blastocysts on day 11 after mating (Hamilton 1934). Robinson (1918) described the development and location (oviduct/uterus) of embryos from fertilized oocytes to blastocysts on Day 6 after mating. The first cleavage produces two blastomeres which are similar in appearance but unequal in size. The first two blastomeres divide asynchronously, so that further stages of uneven numbers of blastomeres are produced (Hamilton 1934). Asynchronous divisions are a common feature for carnivore embryos (Amstislavsky et al. 1993b, Lindeberg et al. 1993) but not for embryos of ruminants (Betteridge and Fléchon 1988). A rough estimation of the intervals between the first and second, and the second and third cleavages was 10-16 hours for each stage (Chang 1965b). The assessment of oocytes and embryos is complicated in carnivores, including the ferret, by the dark cytoplasm which may obscure the identification of the pronuclei in oocytes and individual blastomeres in preimplantation embryos (Wildt and Goodrowe 1989). Fat appears as yellowish refractile droplets in living embryos and completely fills the vitellus in oocytes, obscuring the nucleus (Hamilton 1934). Asynchronous

23 division of the cells and differences in size are detected up to the 16-cell stage but no morphological differences can be detected between the central (inner cell mass) cells and the surrounding (trophoectoderm) cells at the morula stage, in which the cells are grouped closely together and a distinct perivitelline space is present. Fat is equally distributed among the cells (Hamilton 1934).

The degree of development of embryos of the same age varies considerably (Rider and Heap 1986). At the same time after mating, embryos from one ferret can be at the blastocyst stage, whereas those of another ferret may still be at the morula stage (Chang 1969). Blastocysts may be further developed than those in which a longer time interval has elapsed after mating. Furthermore, blastocysts at different stages of development are found in the same ferret (Hamilton 1934). Blastocysts appear to be almost spherical and completely fill the zona pellucida, which has become thinned by the presence of the expanding blastocyst. The inner cell mass appears as a dark mass at one pole of the blastocyst. The flattened cells of the trophoectoderm and the inner cell mass are dotted with fatty granules (Robinson 1925, Hamilton 1934). Uterine blastocysts expand from a size of 200 µm in diameter to more than 2 mm in diameter during their preimplantation development (Daniel 1970, Enders and Schlafke 1972). Progesterone has been reported to support blastocyst expansion up to a diameter of 1 mm but, for further blastocyst expansion, additional ovarian factors are required (McRae 1992). Without the presence of maternal progesterone neither cleavage of embryos in the oviducts and in the uterus (Rider and Heap 1986) nor expansion of blastocysts in the uterus takes place (Buchanan 1969, McRae 1992). As in the ferret, the elimination of progesterone from pregnant animals has been reported to have deleterious effects on embryonic development and implantation in the mouse (Wang et al. 1989) and in the rat (Singh et al. 1996). However, in the pig, progesterone seems to act indirectly through the mother’s reproductive tract rather than directly on embryos (Ying et al. 2000).

2.2.6 Oviductal passage

Ferret embryos enter the uterus over a period of several days starting on Day 5 after mating. The embryos start entering the uterus on Day 4, if females are treated with hCG (Chang 1969), and almost all embryos are found in the uterus by Day 7 as

24 described in Table 2; studies of Robinson (1918), Hammond and Walton (1934), who analysed Robinson’s (1918) data and Hamilton (1934). The remaining reports included in Table 2 deal with embryos that have been recovered for purposes other than to reveal the length of time the embryos remain in the oviducts or stage of development they achieve before entering the uterus. Ferret preimplantation embryos experience a prolonged period of oviductal residence - a phenomenon which has also been demonstrated in the cat (Swanson et al. 1994), the blue fox (Valtonen et al. 1985, Valtonen and Jalkanen 1993), the silver fox (Jalkanen 1992), the dog (Holst and Phemister 1971, Renton et al. 1991) and the horse (Betteridge et al. 1982).

2.2.7 Early embryonic coats

By Day 12 after mating, implanting ferret blastocysts are converted into bilaminar vesicles through the outgrowth of the primitive endoderm, called hypoblast, which then underlies the trophoectoderm. The embryonic coat is reduced to only a little over 2 µm in thickness (Enders 1971). At this stage, blastocyst coats may indeed be different from the original zona pellucida in many (if not all) species as suggested by Denker (2000) who speculated that this is most likely to be the case in those species, like the ferret, with a central type of implantation and, related to this, with a high degree of blastocyst expansion. A more appropriate designation for embryos from this stage onward would be “a chorionic vesicle” (Wimsatt 1975).

Formation of a mucin-like capsular glycoprotein coat has been observed by the horse blastocyst (Betteridge et al. 1982) and by the rabbit blastocyst (see Denker 2000 for review). Uterine secretion material has been reported to attach to the zona pellucida in the cat (Roth et al. 1994, Swanson et al. 1994) and uterine protein to the capsule in the horse (Stewart et al. 1995). In carnivores, the actual mechanism of hatching has not been described in the literature but preimplantation in vivo blastocysts experience zonal thinning and expansion. By some unknown mechanism, the volume of the coat increases considerably during this process in order to compensate for the effects of stretching (Enders 1971, Denker 2000). In the ferret, Enders (1971) favored the idea that swelling due to hydration was the mechanism behind the increasing volume of the coat rather than any addition of material. In species with a capsular coat (the horse and the rabbit), dissolution of the zona pellucida is an enzymatic process and is still

Table 2. Developmental stages of domestic ferret embryos recovered on different days after natural mating or after hCG-induced ovulation. Day 0 = first day of mating or day of hCG administration. Numbers indicate cell numbers, the percentage of uterine embryos is in parenthesis.

Days after first mating or treatment with hCG Treatment 1 2 3 4 5 6 7 8 of donors Reference 1 - 2 1 - 10 1 - 20 1 - M 1 - B M - B Mating Robinson 1918 (0) (0) (0) (0) (25) (71) 1 1 - 8 4 - 16 5 - 32 9 - M 16 - B Mating Hamilton 1934 at UTJ 1 - 2 1 - 8 4 - 32 8 - M 4 - B Mating Hammond and Walton 1934 at UTJ 1 - 4 sterile mating + Chang and Yanagimachi 1963 (0) AI 6 - 42 h later B B NR Chang 1965a (NR) (NR) 1 - 6 6 - 8 Mating or AI + 90 IU Chang 1965b (0) (0) HCG 0 - 96 h later 1 - M M - B B 90 IU hCG Chang 1968 (25) (99) (100) 1 - 2 4 - 8 4 - B M - B M - B 90 IU hCG Chang 1969 (0) (15) (42) (99) (96) 1 - 8 M - XB Mating Marston and Kelly 1969 (0) (100) in ovary 1 - 16 M B Mating Daniel 1970 (0) (0) (100) (100) M - B Mating on two McRae 1994 (100) consecutive days B - NR NR - NR NR - NR 100 IU hCG + Kidder et al. 1999a (100) (100) (100) Mating M - NR NR - XB hCG + 2 matings Kidder et al. 1999b (100) (100) 12 h apart 8 - 16 eCG + hCG + Li et al. 2001 (NR) sterile mating UTJ = utero-tubal junction, NR = not reported, M = morula, B = blastocyst, AI = artificial insemination, XB = expanded blastocyst, eCG/hCG = equine/human chorionic gonadotrophin

26 under way in the rabbit during capsular coat formation (Denker 2000) but in the horse the zona is shed from the outside of the then well-developed capsule at a later stage (Betteridge 1989). In most ungulates the blastocyst hatches from the zona pellucida before it expands considerably. For this reason, there is no need for addition of much uterine derived material to the zona. Hatching, i.e. general dissolution of the embryonic coat, does not occur prior to implantation in the ferret (Enders and Schlafke 1972). In several places, parts of the coat remain between the trophoblast and the uterine epithelium (Gulamhusein and Beck 1973). The greatest thinning of the coat takes place at the lateral walls of the antimesometrial portions of the swelling, which are the first areas of penetration of trophoblast into the uterine epithelium (Enders and Schlafke 1972).

2.2.8 Implantation

Three types of pregnancy have been identified in the Mustelidae (Mead 1989, Ternovsky and Ternovskaya 1994). All polecat species and the European mink have a short period of pregnancy of constant duration (37-44 days). Species like the stoat and the sable exhibit an obligatory diapause at the blastocyst stage, and a long gestation period (7-10 months) (Amstislavsky and Ternovskaya 2000). In the American mink, the gestation period is short but variable (range 45-61 days) and may or may not include implantation delay (Mead 1989).

Implantation in the ferret is central with rapid invasion of the uterine epithelium by the trophoblast over a broad area that eventually becomes a zonary band of endotheliochorial placenta (Strahl and Ballman 1915, cited in Enders and Schlafke 1972). The endotheliochorial placenta has three fetal (endothelium, connective tissue, trophoblast) but only two maternal layers (connective tissue, endothelium); since the maternal epithelium is lost. The fetal trophoblast invades the endometrial epithelium, but does not destroy the endothelium of the maternal capillaries (Mossman 1987, Valtonen 1992). Between Days 12 and 13 after mating, the embryos have become implanted in the endometrium (Enders and Schlafke 1972, Mead et al. 1988b).

Prior to implantation, the trophoblast differentiates rapidly and gives rise to patches of syncytial trophoblast (Gulamhusein and Beck 1973). The first feature of penetration is

27 the projection of a thin fold of syncytial trophoblast between adjacent epithelial cells. As implantation progresses, more of the blastocyst is involved in implantation, and by Day 14, a continuous sheet is either penetrating or overlying the area of the wall of the uterus which constitutes for two-thirds of the circumference of the future zonary band placenta (Enders and Schlafke 1972). At the cervical and ovarian ends of the chorionic vesicle and in the mesometrial region, the trophoectoderm is non-invasive (Gulamhusein and Beck 1975). In these non-attached regions, the trophoectodermic cells absorb uterine milk (Gulamhusein and Beck 1973). In the immediate vicinity of implanting chorionic vesicles, a highly localized increase in the permeability of uterine blood vessels is associated with the final stage of attachment to the uterine epithelium, this being first detectable on the morning of Day 12 after mating (Mead et al. 1988b). Prostaglandins are proposed to play an important role in the process of implantation but the process is unrelated to decidual formation, as the ferret is an adeciduate species (Mead et al. 1988b); i.e. ferret endometrium is not known to be capable of decidual transformation during implantation (Beck 1974) in contrast to the situation of rodents and humans that have a primary decidualization reaction before blastocyst(s) start penetrating the uterine epithelium (Johnson and Everitt 2000). Following implantation, a wave of epithelial hypertrophy sweeps progressively from the uterine lumen towards the bases of the glands and epithelial cells become extraordinarily enlarged with nuclei as large as 90-100 µm in diameter. Most of the luminal cells lose their integrity and form masses containing whole or fragmented nuclei. This degenerate tissue is termed a symplasma since, although technically it is a syncytium, it is not active tissue (Amoroso 1952, Buchanan 1966). This degenerated tissue probably contributes to the histiotrophe since it is ingested by the syncytiotrophoblast (Gulamhusein and Beck 1975).

In the ferret, the placental labyrinth has fully developed at Day 18, when the greatly hypertrophied maternal capillaries are completely surrounded by a layer of syncytiotrophoblast (Gulamhusein and Beck 1975). At the same time, accumulations of maternal blood which vary considerably in size and location and constitute the “haemophagous organ” (Creed and Biggers 1964) appear in the antimesometrial region between the placental discs. This organ is fully formed by Day 28, and it maintains its size almost to term (Gulamhusein and Beck 1975). It is thought to act as an alternative source of iron for the fetuses (Baker and Morgan 1973).

2.2.9 Gestation and function of corpora lutea

Gestation length is 41 days (39-42 days) in the domestic ferret (Hammond and Marshall 1930, Ternovsky and Ternovskaya 1994, Fox and Bell 1998). If fertilization does not occur, pseudopregnancy lasting 40 to 42 days ensues, the functional life of corpora lutea (CL) being similar to that in normal pregnancy (Hammond and Marshall 1930, Chang and Yanagimachi 1963). Ferret CL start secreting progesterone immediately after ovulation, and the progesterone concentrations rise continuously to peak at about Day 12-14 during the period of implantation (Daniel 1976), then decrease steadily after about Day 15 and level off by day 24 of pregnancy (Heap and Hammond 1974). Ferret CL consist of small (<25 µm) and large (>25 µm) luteal cells, with the smaller cells predominating on Day 6. A shift toward larger sizes is observed by Days 13 and 24 of pregnancy, and concurrently the percentage of smaller cells declines (Joseph and Mead 1988). Progesterone concentrations decline continuously between Day 24 and parturition at Day 42 (Blatchley and Donovan 1976). Mustelids and other carnivores display a protracted decline in circulating progesterone, and progesterone levels reach basal concentrations ≥1 week after parturition (Møller 1973). The conceptuses have no effect on the duration of the luteal phase, because pregnancy and pseudopregnancy are indistinguishable (Hammond and Marshall 1930, Heap and Hammond 1974, Agu et al. 1986). Removal of the uterus during the luteal phase has no effect on the life span of the CL (Deanesly 1967). Factors causing luteal regression in the ferret are not known.

Functional corpora lutea (CL) require anterior pituitary support for the maintenance of pregnancy and pseudopregnancy (Hill and Parkes 1932, Donovan 1967, Blatchley and Donovan 1976). A critical period exists between Days 6 and 8 after mating, during which other luteal factor(s) are required to facilitate the actions of progesterone on the uterus and on the embryos (Wu and Chang 1973, Foresman and Mead 1978). One of these factors, prolactin, induces implantation in hypophysectomized ferrets and sustains the luteal progesterone production during the first half of gestation (Murphy 1979). High densities of uterine prolactin binding sites are exhibited in ovariectomized progesterone- and oestradiol-treated ferrets suggesting that ovarian

29 steroids have a role in the development of uterine prolactin binding sites, but the mechanism of the interaction is still unknown (Rose et al. 1993). The functional ferret CL secrete many proteins, but proteins specific for implantation have not been isolated or identified so far (Mead et al. 1988c, Huang et al. 1993). It seems that oestrogen is not essential for implantation in the ferret (Foresman and Mead 1978, Mead and McRae 1982). Experimentally induced maternal pregnancy reactions (for instance traumatization with an intrauterine thread which results in the formation and subsequent necrosis of symplasmal nests of endometrial epithelial cells and hypertrophy of the maternal capillary endothelium) show that endometrium is sensitive for implantation between Days 9 and 14 (Gulamhusein and Beck 1977).

2.2.10 Parturition, rebreeding and induction of oestrus

The domestic ferret gives birth to an average of 8 kits (1-18 kits), which weigh 6-12 g at birth (Ternovsky and Ternovskaya 1994, Fox and Bell 1998). Females will return to oestrus within two weeks after weaning if they are exposed to a stimulatory photoperiod. If the kits are removed at birth, the mothers return to oestrus 8 weeks after mating, as do pseudopregnant animals and females that have resorbed their fetuses (Marston and Kelly 1969). If females give birth to only a small number of kits, 1-5, they may return to oestrus while nursing (Fox and Bell 1998). Induction of oestrus in anoestrous ferrets has been accomplished by treatment with crude pituitary extracts (Hill and Parkes 1930). Most anoestrous females maintained on a non- stimulatory light cycle (10 h light and 14 h dark) showed oestrous signs and were bred in 6-13 days after treatment with 0.25 mg of FSH, administered twice daily (Mead and Neirinckx 1989).

2.3 Assisted reproductive technology

2.3.1 Artificial insemination

In artificial insemination, semen is collected from a male and is deposited into the uterus of an oestrous female, a procedure which has been accomplished widely, including in the domestic ferret (Mustela putorius furo). The first attempts were

30 performed by Hammond and Walton (1934) who inseminated 19 females into the vagina and mated them with vasectomised males several times 0-7 days after insemination. However, none of the females became pregnant. Kidder et al. (1998) inseminated 26 hormonally treated domestic ferrets into the vagina and none conceived, but intrauterine insemination of 48 hormonally treated ferrets resulted in 23 fetuses at 20-day post ovulation (48%, 23/48). An additional 5 + 5 domestic ferrets were transcervically inseminated either into the uterine horn or into the uterine body. All females inseminated into the uterine horns, but only 2/5 (40%) inseminated into the uterine body, became pregnant. These results demonstrate that AI should take place in the uterine horns.

Chang (1965b) inseminated 40 female ferrets surgically into the uterine horns with fresh epididymal semen 0 to 120 hours before treatment with 90 IU of hCG in order to study the viability of spermatozoa in the female genital tract. Eight, 5 and 9 kits were born following semen deposition 24, 36 and 60 hours before hCG treatment, respectively. Chang and Yanagimachi (1963) deposited semen into the ovarian capsule at different times from the estimated time of ovulation in hormonally ovulated ferrets. The number of oocytes fertilized by fresh epididymal semen injected into the ovarian capsule varied from 0% (24 hours before ovulation) to 58-64% (6-0 hours before ovulation), and 6 hours after ovulation, the percentage of fertilized oocytes decreased to 30%. Of the 48 transcervically inseminated females in the study of Kidder et al. (1998), 24 were inseminated 24 hours post hCG and 19 fetuses were detected at 20 d post ovulation (79%, 19/24). The remaining 24 females were inseminated immediately after hCG and 4 became pregnant (17%, 4/24). These results indicate that conception is possible following AI over a wide time range around ovulation; 90-100 hours before ovulation (assuming that ovulation takes place 30 hours after hCG; Chang 1965b) until 6 hours after ovulation. The optimal time for insemination is 6 hours before ovulation; i.e. females should be inseminated 24 hours after hCG treatment or mating to a vasectomized male.

In the family of Mustelidae, the conservation programme for the black-footed ferret (Mustela nigripes) in USA has been a good example of a successful rescue mission of a species destined to extinction (Seal et al. 1989, Howard 2001, Wildt et al. 2001). Although based on natural mating, laparoscopic artificial insemination with fresh and

31 frozen-thawed semen has been used to increase the number of captive individuals (Howard 2001). To develop this technology, related mustelid species, i.e. the domestic ferret, polecat and steppe polecat (Mustela eversmanni), have been used as model animals. The first attempts to cryopreserve domestic ferret semen were described by Atherton et al. (1989). They tested several extenders and obtained the highest post-thaw motility (45-48%) when semen was frozen in a commercial horse semen extender (E-Z Mixin) in straws and by adding 3.0-4.5% glycerol and 2 mM ATP, but no inseminations were performed. Budworth et al. (1989) froze domestic ferret semen in Egg-yolk-TEST extender with either 2% or 4% glycerol. One of two surgically inseminated hormonally treated ferrets became pregnant and delivered kits. The first in-depth study for determining the optimum cryopreservation method for ferret spermatozoa combined with test-inseminations was conducted by Howard et al. (1991). A 70% (7 pregnant/10 inseminated females) pregnancy rate and 31 kits (mean litter size 4.4; range 1-9 kits) were produced after laparoscopic intrauterine insemination using frozen-thawed electroejaculated semen. The developed technology was thereafter used to produce kits in the black-footed ferret. A total of 76 black- footed ferret individuals have been produced with the help of artificial insemination in the conservation programme (Howard 2001). Howard et al. (1996) laparoscopically inseminated 7 steppe polecats (Mustela eversmanni) and 6 black-footed ferrets using fresh or frozen-thawed semen from steppe polecats and black-footed ferrets. Of 7 + 6 females, 6 + 4 became pregnant (77%, 10/13) with mean litter sizes of 5.3 and 2.3, respectively. In another study, Howard et al. (1997) laparoscopically inseminated 17 black-footed ferrets and 3 steppe polecats using fresh and frozen-thawed black-footed ferret semen. Of 17 + 3 females, 13 + 2 delivered (75%, 15/20) altogether 35 + 10 offspring (2.1 + 5.0 kits/female, respectively). The pregnancy results by Howard et al. (1996, 1997) were reported in a way that prevents comparison between fresh and frozen-thawed semen.

2.3.2 Embryo recovery and transfer

Another assisted reproductive technique, embryo transfer, is a process in which preimplantation embryos are collected from one female (the donor) and transferred to other females (recipients) for development to term. This technology is already well developed and widely used commercially, more in cattle than in other domestic

32 species. Among carnivores, this technology has progressed fastest in the Felidae (see Pope 2000) due to financial interest in conserving endangered felids. This has provided better opportunities for the pursuit of these experimental techniques. The first live offspring born after transfer of fresh in vivo embryos in the domestic cat (Felis catus) were reported by Schriver and Kraemer (1978) and Kraemer et al. (1979). In this first successful transfer study, 47 embryos were transferred to 9 recipients and 4 developed into live kittens (success rate 8.5%, 4/47; 0.4 live kittens/recipient). In the Canidae, the first successful surgical embryo transfer of the domestic dog was reported by Kinney et al. (1979) and Kraemer et al. (1980). In these studies, a total of 37 surgically recovered in vivo embryos were transferred to recipients in natural oestrus within 4 days of the onset of oestrus in the donors. Seven recipients delivered 4 offspring (success rate 11%, 4/37; 0.6 live pups/recipient). Subsequently surgical embryo transfer of fresh in vivo embryos has resulted in live offspring, as reported by Takeishi et al. (1980), Tsutsui et al. (1989) in the domestic dog and by Jalkanen and Lindeberg (1998) in the farmed silver fox (Vulpes vulpes) but with low survival rates. Due to the increasing use of IVF technology in the domestic dog, transfer of embryos in earlier developmental stages has become necessary. Transfer of zygote to 8-cell stage embryos to the uterine tubes of recipient bitches resulted in a survival rate of 34% (10 live pups/29 transferred embryos) (Tsutsui et al. 2001). All these studies show that very often, and for unknown reasons, the transfer of embryos in polytocous species typically results in lower numbers of offspring born than would be predicted from the number of embryos transferred or may result in no term pregnancy at all (Wildt and Goodrowe 1989).

The domestic ferret was the first carnivorous species in which surgical transfer of fresh in vivo embryos resulted in live offspring (Chang 1968). In embryo transfer programs for the domestic ferret, embryos have mainly been recovered post mortem using a variety of flushing media (Table 3). In Chang’s (1968, 1969) studies, hormonal treatment with 90 IU of hCG was used to induce ovulation in donors and recipients. Hormonal treatment together with a low number of embryos transferred to each recipient may have lowered the success rates and kit yield (Table 4). Transfer of fresh embryos has been reported in the American mink (Chang 1968, Zhelezova and Golubitsa 1978, Adams 1982) with success rates (live offspring/transferred embryos) ranging between 25% and 33%.

Table 3. Summary of embryo recoveries in experiments resulting in live offspring after transfer of in vivo embryos in the domestic ferret. Day 0 = first day of mating or day of hCG treatment.

Number Day of No. of Recovered of Treatment of oestrous donors embryo Flushing medium recovered embryos/ Reference donors recovery embryos donor NR Mating 6 - 8 TCM199 + 10-20% ferret serum 51 NR Chang 1968 62 90 IU hCG i.p., surgical AI one day later 2 - 8 TCM199 + 20-50% ferret serum 636 10.3 Chang 1969 4 Mating once daily on two consecutive days 6 DPBS + 15% FCS 39 9.7 McRae 1994 37 100 IU hCG + coincident mating and second 6 - 7 25 mM Hepes + 1% PVA + 324 8.8 Kidder et al. 1999bc mating 12 hours later 0.1% antibiotic-antimycotic 6a 50-150 IU eCG, 50-200 IU hCG 72 hours 2 DPBS + 2% NCS 116 19.3 Li et al. 2001 6b later w/wo coincident mating 2 DPBS + 2% NCS 106 17.7 Li et al. 2001 aDonors were mated. bDonors were not mated. cNon-surgical recovery, all others were done post mortem. hCG = human chorionic gonadotrophin, AI = artificial insemination, eCG = equine chorionic gonadotrophin TCM199 = tissue culture medium 199, DPBS = Dulbecco’s phosphate buffered saline, NR = not reported FCS = fetal calf serum, PVA = polyvinylalcohol, NCS = newborn calf serum, w/wo = with/without

Table 4. Summary of transfer results of fresh in vivo embryos in the domestic ferret.

No. of No. of Number hCG trans- transferred Survival Live of or ferred embryos/ Live rateb kits/ Reference recipients mating embryos recipient kits (%) recipient 11 hCG 51 4.6 17 33 1.5 Chang 1968 23 hCG 133 5.8 36 27 1.6 Chang 1969 4 hCG 27 6.8 14c 52 3.5 McRae 1994 31 hCG 251a 8.1 61 24 2.0 Kidder et al. 1999b 3 Mating 54 18 27 50 9 Li et al. 2001 atranscervical transfer, others were done surgically blive fetuses or kits/transferred embryos cat 32-34 days of gestation

It is difficult to traverse the cervix in small mammals, a fact which has fundamentally prevented the development of non-surgical embryo collection and transfer technologies in these animals. Kidder et al. (1999b) developed a non-surgical method for both recovery and transfer of embryos. The embryo recovery and transfer equipment used consisted of an endoscope with a halogen light source connected to a video camera for visualizing the cervix. The average time for traversing a cervix was 7 min (range 2 to 20 min). The majority of the catheterisations (85%) were successful, failures were attributed to tissue folds obscuring the cervical os and, most often, a failure to guide the stylette through the cervical canal (Kidder et al. 1999b). The nonsurgical embryo collection and transfer technologies were comparable to surgical ones (Table 4).

Human chorionic gonadotrophin has been used in embryo transfer programs to induce ovulation in the donor ferrets. Li et al. (2001) have reported a regimen for superovulation treatment for ferret donors. Optimum superovulation (19.3±0.6 oocytes and embryos per female) was achieved after treatment with 100 IU of eCG and 150 IU of hCG. The ovulation rate was doubled from that induced by mating (8.9±2.5 oocytes and embryos per female). Superovulation has also been reported in the stoat (Mustela erminea) (Amstislavsky et al. 1993b, 1997b).

2.3.3 In vitro embryo technology

In vitro embryo production includes collection of oocytes from ovaries post mortem or laparoscopically/transvaginally under anaesthesia/sedation from ovaries of live animals, maturation, fertilization and culture in vitro to stages appropriate for transfer to a recipient animal or cryopreservation. This technology is now a routine procedure in cattle but not so far in other species other than humans. Mean calving rate after single embryo transfer of fresh bovine IVP embryos has been reported to be 30% (range 12-50%) (Peterson and Lee 2003). The lambing rate of in vitro produced fresh sheep and goat embryos after transfer experiments varies between 40 and 50% (Dattena et al. 2000, Cognié et al. 2003). In the pig, the efficiency of IVP embryos, in terms of yield of transferable embryos, is still less than desired, and rarely exceeds 40% (Day 2000).

Among carnivores, the technology is further advanced in felids than in canids (Farstad 2000a). In the domestic cat and two wild cats, Indian desert cat (Felis silvestris ornata) and tiger (Panthera tigris), IVF embryos have developed to term after transfer (Donoghue et al. 1990, Pope et al. 1993, Pope 2000). Live offspring have been successfully born after transfer of fresh (Pope et al. 1997) and frozen-thawed (Gómez et al. 2003) IVM-IVF-embryos in the domestic cat. Development of cat embryos in vitro is comparable to their development in vivo up to the morula stage (Roth et al. 1994). In the study of Roth et al. (1994), up to 70% of in vivo derived embryos at earlier developmental stages developed to blastocysts in vitro, whereas none of the in vitro derived embryos reached the blastocyst stage. However, the blastocyst production rate has been improved by modifications of the culture conditions (Kanda et al. 1998, Pope et al. 1999).

Live offspring have not been born after transfer of IVF embryos in the domestic dog (Canis familiaris). A 22-day pregnancy has been achieved after transfer of in vivo matured oocytes fertilized in vitro and cultured to the 2-cell stage before transfer (England et al. 2001). In vitro maturation of oocytes has been the major problem in canine IVP (Rodrigues and Rodrigues 2002), but intense research in the maturation process and development of the technology for canids is underway (Farstad 1993, Metcalfe 1999, Farstad et al. 2001, Luvoni et al. 2001, Willingham-Rocky 2002).

Canine oocytes are spontaneously ovulated at the beginning of the first meiotic division as primary oocytes and they then mature in the oviducts (Valtonen and Jalkanen 1993, Farstad 2000b). Culture of in vivo derived embryos has been reported in the domestic dog (Renton et al. 1991) and in the silver fox (Lindeberg et al. 1993). In these experiments, few embryos developed further during in vitro culture.

In the ferret, in vivo matured oocytes have successfully been fertilized in vitro and 10% of the zygotes developed to blastocysts during in vitro culture (Lindeberg et al. 2001). In the same study, in vitro matured oocytes developed only to the morula stage. The following year, electroejaculated semen was processed by swim-up only whereas it was centrifuged in Sperm-TALP during the first year. The blastocyst production rate for oocytes matured in vivo was lower than during the first year due to their poor fertilization rate. Seven blastocysts were produced from 163 in vivo matured oocytes (4.3%). One fourth of the in vitro matured oocytes reached metaphase II, and three out of 84 in vitro matured and fertilized oocytes cleaved up to the 4-cell stage (Lindeberg et al., unpublished results).

To study the timing of nuclear maturation of in vivo oocytes, 14 European polecat females were mated with vasectomized males and killed at different intervals after the mating (Piltti et al. 2003). The oocytes were recovered from the preovulatory follicles and stained for determination of the stage of nuclear maturation. By 33 to 39 hours after mating, 80% of the oocytes had reached metaphase II. Li et al. (2002) harvested immature ferret oocytes from small follicles (diameter 0.5-2.0 mm) after superovulation treatment with 100 IU/kg of eCG, followed by 100-150 IU/kg of hCG 72 hours later. The percentage of matured oocytes obtained after a 24-48-h maturation period in three different IVM media varied from 28 to 70% (Table 5).

In vitro culture of ferret embryos recovered post mortem at different stages of development has been reported by Whittingham (1975) and Li et al. (2001) (Table 6) though Whittingham (1975) did not report the treatment of the donors. Li et al. (2001) superovulated their donors with 50-150 IU eCG followed by 50-200 IU hCG 72 h later with the donors being mated immediately after hCG treatment. The first study establishing protocols for the reconstruction of ferret embryos by nuclear transfer using G0/G1 -phase donor fetal fibroblasts has recently been reported by Li et al.

(2003). Three of the 478 reconstructed embryos developed into 3-week old fetuses after embryo transfer. One of these fetuses had identical size and structure to that of in vivo conceived 3-week fetuses, while the remaining two had blunted growth.

Table 5. Results of in vitro maturation (IVM) of ferret oocytes in three different IVM media.

% of matured % of matured In vitro maturation medium oocytes after 24 h oocytes after 48 h TCM-199 + 10% FBS 28 56 TCM-199 + 10% FBS + 10 IU/ml eCG + 5 IU/ml hCG 70 66 TCM-199 + 10% FBS + 10 IU/ml eCG + 5 IU/ml hCG + 2 µg/ml 17β-oestradiol 37 70 TCM = tissue culture medium, FBS = fetal bovine serum eCG = equine chorionic gonadotrophin, hCG = human chorionic gonadotrophin

Table 6. Results of in vitro culture of in vivo produced embryos in the domestic ferret.

Stage of Duration of Blastocysts/ development in vitro Culture cultured % of Reference at recovery culture (d) medium embryos blastocysts 1-cell 5 NR 8/15 53 Whittingham 1975 2-8-cell 4 NR 20/28 71 Whittingham 1975 8-cell 3 NR 127/162 78 Whittingham 1975 TCM-199 + 1-2-cell 5 10% FBS 16/34 47 Li et al. 2001 1-2-cell 5 CZB 20/31 65 Li et al. 2001 1-2-cell 5 NCSU-23 6/35 17 Li et al. 2001 NR = not reported, TCM = tissue culture medium, FBS = fetal bovine serum CZB = Czatot-Ziomek-Bavister, NCSU = North Carolina State University

2.4 Cryopreservation of in vivo embryos

There are two basic approaches to cryopreserve cells, the conventional slow freezing method and the newer vitrification technique, both of which have been used successfully to preserve a wide variety of cell types, including spermatozoa, oocytes and embryos. Conventional slow freezing is equilibrium cooling by which cells are

38 suspended in a relatively low concentration of a cryoprotectant (usually 1.5 M solution) for 10-15 min and cooled at low rates of 0.3-2 °C/min until being plunged in liquid nitrogen.

Vitrification is non-equilibrium cooling by which cells are exposed to high concentrations of a mixture of cryoprotectants (4 to 8 M solutions) usually briefly for 30 sec-1 min and cooled at high rates ranging from 500 °C/min to more than 20 000 °C/min. Standard cooling rates from 500 to 3000 °C/min can be achieved when a straw is used as a sample container. Ultra-rapid cooling rates from 9000 to more than 20 000 °C/min can be achieved when specimens in containers come directly in contact with LN2 (Rall 2001, Leibo and Songsasen 2002).

To date, the first reports of live-born offspring from cryopreserved embryos have been published for 24 different mammalian species. Conventional slow freezing was used to cryopreserve embryos of 22 species and vitrification embryos of two species (Pope et al. 2000, Rall 2001, Swanson 2001).

2.4.1 Conventional slow freezing

During slow cooling, the individual cells are assumed to lose water rapidly enough by exosmosis to concentrate the intracellular solutes sufficiently to eliminate supercooling and maintain the chemical potential of intracellular water in equilibrium with that of extracellular water (Mazur 1963). The result is that the cell dehydrates and only minor ice crystals will be formed intracellularly. If the cells are cooled too rapidly, they are not able to lose water fast enough to maintain equilibrium; they become increasingly supercooled and eventually attain equilibrium by freezing intracellularly (Mazur 1988). Most cells also require the presence of a cryoprotectant for successful cryopreservation. The presence of a cryoprotectant is required to decrease the concentration of electrolytes during freezing and to decrease the extent of osmotic shrinkage at a given low temperature (Mazur 1984). Cryoprotectans have also other, only partially understood, effects. The cyoprotectant must be a non-toxic, highly water-soluble chemical capable of preventing or reducing freezing injury. One class of cryoprotectants must permeate the cells to protect (e.g. glycerol, ethylene

39 glycol, dimethyl sulphoxide, methanol, propylene glycol, and dimethylacetamide) whereas the other class is composed of nonpermeating solutes. This class includes sugars and higher molecular weight compounds such as polyvinylpyrrolidone, polyethylene glycols, dextrans, and hydroxyethyl starch (Fahy 1988, Gao and Critser 2000).

Whittingham et al. (1972) and Wilmut (1972) reported the birth of the first live offspring after transfer of frozen-thawed embryos in the mouse. Now 31 years later, transfer of cryopreserved embryos has become routine in countries with major cattle breeding industries, and pregnancy rates of 40-70% are reported (Hasler 2001). Use of ethylene glycol (EG) as a cryoprotectant is widely accepted because of its ease of use under farm conditions (Massip 2001). EG is a low molecular weight (62.07) organic compound which was initially used as a cryoprotectant for mouse and rat embryos (Miyamoto and Ishibashi 1977). It freely permeates most cells (Leibo 1981) and does not need to be stepwise diluted after thawing (Bracke and Niemann 1995).

Sheep and goat embryos can be frozen with good results (Dobrinsky 2002). In contrast, large equine blastocysts do not tolerate cryopreservation and transfer of frozen-thawed morulae and smaller blastocysts do not yield survival rates high enough to permit wide commercial application (Squires et al. 1999). Porcine embryo cryopreservation is still at the experimental stage with increasing number of studies reporting live offspring after transfer of frozen-thawed embryos which have been treated with cytoskeletal stabilizers, such as cytochalasins, prior to cryopreservation (Dobrinsky 2002). In the carnivores, offspring of the domestic cat and ocelot (Felis pardalis) have been born after transfer of frozen-thawed embryos (Dresser et al. 1988, Swanson 2001). Cryopreserved in vitro produced African wildcat (Felis silvestris) embryos have developed to term in domestic cat recipients (Pope et al. 2000). However, embryo cryopreservation has not been successful in the canids so far (Farstad 2000a,b, Lindeberg et al. 2000). In the mustelids, Amstislavsky et al. (1993a, 1996, 2000) have studied cryopreservation and in vitro development of frozen-thawed ermine and polecat embryos, but no one had reported the birth of live offspring after transfer of frozen-thawed embryos until the work in this thesis was published.

2.4.2 Vitrification

Vitrification is a glass-like solidification of solutions, brought about by an extreme elevation in viscosity during cooling, first suggested as an alternative to freeze- preservation of life forms by Luyet (Luyet 1937). Unlike the conventional method known as solidification of a liquid into a crystalline or partially crystalline state, vitrification does not produce ice crystals during the rapid cooling process or storage (Luyet 1937, Rall and Fahy 1985a,b). The total absence of ice during vitrification has been confirmed (Valdez et al. 1990). This lack of extracellular and intracellular ice formation during vitrification is attributable to the high concentrations of cryoprotectans needed to achieve vitrification. Some ice formation from the previously vitreous liquid, i.e. devitrification, has been considered as harmless during warming as many systems, including embryos, are capable of surviving this brief devitrification during rapid warming provided the ice formed does not recrystallize (Rall 1987, Fahy 1988).

The composition of the vitrification medium is the key to successful vitrification (Fahy et al. 1984, Rall and Wood 1994). First, the embryos must be suspended in a solution of cryoprotectants that is sufficiently concentrated to avoid crystallization and solidify as a glass at practicable cooling rates. Second, embryos must tolerate exposure and dehydration in this solution. The embryos are liable to be injured by the toxicity of the high concentration of the cryoprotectant(s), this being the greatest obstacle to successful vitrification (Kasai 1995). Sucrose or trehalose as a frequently used replacement for sucrose and occasionally certain macromolecules (BSA, Ficoll, polyethylene glycol) are combined with cryoprotectants to maintain a high osmotic pressure in the extracellular medium. Macromolecules acting as nonpermeating cryoprotectans may help to replace some of the volume of the permeating cryoprotectants and therefore concentrations of all cryoprotectants can be reduced (Rall et al. 1987, Gao and Critser 2000). Macromolecules facilitate vitrification by contributing to the enhancement of vitrification of the solutes, stabilize proteins and membranes, and preventing devitrification, i.e. formation of ice both in the solution and the embryos, during warming (Fahy et al. 1984, Kasai et al. 1990). Sucrose or trehalose is required during cooling for protection against the toxic effects of

41 cryoprotectants. During warming, the presence of sucrose or trehalose reduces osmotic swelling in the embryos due to removal of cryoprotectants from the embryos.

In an ideal situation, sufficient amounts of cryoprotectants permeate the cells. The influx and efflux of cryoprotectants are temperature dependent processes; lower temperatures allow embryos to be incubated for a longer time (Rall and Fahy 1985a,b). Diffusion is better at higher temperatures, but at elevated incubation temperatures, cryoprotectants enter the embryos faster and may easily damage or even kill the embryos. Full permeation is not necessary, as it causes injury due to chemical toxicity or osmotic stress during removal (Kasai 1995). During the lowering of the temperature, the liquid solution becomes supercooled due to the high concentration of its solutes; it stays liquefied and becomes more viscous down to very low temperatures, eventually to the point that it can no longer flow on a measurable timescale. Then, the extremely viscous solution and the embryos within it become glass without ice formation (MacFarlane 1987). The warming process is fast, heating rates of 400-1000 °C/min are required to suppress devitrification (Fahy et al. 1984).

Vitrification in 0.25-ml straws was described for the first time in the cryopreservation of mouse embryos (Rall and Fahy 1985a,b). Mouse embryos were exposed to a vitrification solution (VS1) containing three permeating agents, 20.5% w/v dimethyl sulphoxide (DMSO), 15.5% w/v acetamide, 10% w/v propylene glycol and, 6% w/v polyethylene glycol (a macromolecule) in a modified Dulbecco’s phosphate buffered saline (DPBS) at pH 8.0. After an initial stepwise equilibration of approximately 35 min starting at room temperature and ending at 4 °C, the embryos were plunged in straws into liquid nitrogen. The requirement for low temperature and the composition of relatively toxic ingredients including, DMSO, acetamide and propylene glycol have reduced the use of the first described vitrification medium under field conditions. Subsequently, many different compositions of in-straw vitrification media have been reported. Live offspring or pregnancies have been produced in a variety of species using different protocols with various cryoprotectants. Table 7 summarizes studies in which live offspring or pregnancies have been produced after transfer of in vivo embryos flushed from uteri, vitrified and warmed in 0.25-ml straws using ethylene glycol as one component or as the sole cryoprotectant.

Table 7. A summary of transfers of embryos flushed from uteri, vitrified and warmed in 0.25- ml straws using EG as one component or as the sole cryoprotectant in different species.

In-straw (0.25 ml) No.of No. of Survival Species vitrification medium transferred live rate Reference embryos offspring(%) Mouse 40% EG, 30% Ficoll, 0.5M sucrose in PB1 at 20 °C 167 57 34 Kasai et al. 1990 Mouse 25% EG, 25% DMSO 0.4% BSA in PBSa 342 82 24 Ishimori et al. 1992b Mouse 40% EG, 18% Ficoll, 0.3M sucrose in PB1 at 20 °C 419 112 27 Miyake et al. 1993 Mouse 40% EG, 30% Ficoll, 0.5M sucrose in PB1 at 20 °C 188 84 44.7 Zhu et al. 1993 Rabbit 40% EG, 18% Ficoll, 0.3M sucrose in PBS at 20 °C 120 60 50 Kasai et al. 1992 Bovine 25% EG, 25% DMSO, 0.4% BSA in PBSa 14 7b 50 Ishimori et al. 1992a Bovine 25% EG, 25% DMSO, 5.56 M glucose, 4mg/ml BSA, 23 9 39 Ishimori et al. 1993 0.33 mM pyruvate in PBS at 22-24 °C Equine 40% EG, 18% Ficoll, 0.3M sucrose in PBSa 5 2b 40 Hochi et al. 1994 Ovine 3.4M GLY, 4.6M EG, 0.5M sucrose, 20% FCS in PBS 19 12 63 Naitana et al. 1997 at 15-20 °C 3.4M GLY, 4.6M EG, 0.5M sucrose, 0.1% PVA in PBS 17 10 59 Naitana et al. 1997 at 15-20 °C Ovine 40% EG, 18% Ficoll, 0.3M Martinez and sucrose at room temp. 65 26 40 Matkovic 1998 Ovine 25% GLY, 25% EG, 20% NBCS in PBS at room temp 50 25 50 Baril et al. 2001 aVitrification temperature not reported, bNumber of pregnancies at Day 60. NR = not reported, EG = ethylene glycol, PB1 = modified phosphate buffered saline, PBS = phosphate buffered saline, DMSO = dimethyl sulphoxide, GLY = glycerol, BSA = bovine serum albumin, PVA = polyvinylalcohol, NBCS = newborn calf serum

One of the recent methods of ultra-rapid vitrification for cryopreserving oocytes and embryos with increased cooling and warming rates is the open pulled straw (OPS) vitrification technique (Vajta et al. 1997). In this method, straws are previously heat- pulled to the half of their diameter and thickness of the wall and embryos are loaded with a capillary effect by touching a 1-2 µl droplet containing embryos with the narrow end of a modified straw. Other technologies use different containers such as electron microscope grids (Martino et al. 1996), cryoloops (Lane et al. 1999a,b), steel

43 cubes that are covered with aluminium foil (Dinnyés et al. 2000), glass micropipettes (Hochi et al. 2001, Kong et al. 2000) or nylon 60 µm meshes (Matsumoto et al. 2001). It is also possible to drop the specimen in a droplet of vitrification medium directly into liquid nitrogen (Říha et al. 1991, Misumi et al. 2003). Immersing the OPS or other open containers into liquid nitrogen (LN2) allows direct contact between the vitrification medium and LN2 which leaves the possibility that the cells may become contaminated if LN2 contains microbes (Bielanski et al. 2003). This is not the case with the methods using either filtered LN2, or sealed 0.25-ml open pulled straws (Vajta et al. 1998b, López-Béjar and López-Gatius 2002).

In OPS, all equilibration procedures are conducted on heated stages. If the temperature of the stages is set to 39 °C, an incubation temperature around + 37 °C is produced. The cooling rate for an open pulled straw between 0 degrees and -195 degrees has been estimated as 16,700 °C/min compared to 2,436 °C/min for a 0.25-ml straw (Vajta et al. 1998a). At high temperature, the cryoprotectants are assumed to permeate the embryos well, providing good protection against cryoinjury. OPS vitrification allows for the use of a lower cryoprotectant concentration than can be achieved with in-straw vitrification. Rapid loading and expelling from OPS straws minimize both toxic and osmotic injuries (Vajta 1997).

In vitro produced bovine embryos have also been vitrified by OPS and the first live offspring were born in 1998 (Vajta et al. 1998a). Mouse and rat embryos have been successfully OPS vitrified-warmed and in vitro cultured but so far not transferred into recipients (Kong et al. 2000, Isachenko et al. 2003b). The OPS method seems to be suitable also for oocyte cryopreservation. In the first report of successful OPS vitrification of bovine oocytes, 25% of oocytes developed to blastocysts in vitro after fertilization and culture of 7 days (Vajta et al. 1998a). Table 8 summarizes the successful transfers of OPS vitrified in vivo embryos in different species.

Table 8. Live offspring of different species born after transfer of in vivo produced embryos flushed from uteri and vitrified in open pulled straws using ethylene glycol as one component or as the sole cryoprotectant.

Species Vitrification Days from No. of No. of Survival medium mating to embryos live rate Reference recovery transferredoffspring (%) Swine 8 M EG, 7% 5 180 5 2.8 Beebe et al. PVP in mPBS 2000 at 25 °C Swine 8 M EG, 7% 6 147 5 3.4 Beebe et al. PVP in PBSa 2002 Swine 3.2 M EG, 2.5 M DMSO, 0.6 M 5-6 400 38 9.5 Berthelot et al. sucrose in TCM/ 2000 PBS at 39 °C Swine 20% EG, 20% DMSO, 0.4 M 5-6 200 26 13 Berthelot et al. sucrose in TCM 2001 At 22-24 °C Goat 20% EG, 20% DMSO, 20% GS 7 28 18 64 El-Gayar et al. in TCM-199 2001 at 39 °C Rabbit 25% GLY, 25% EG, 20% FBS, 2-3 238 123 51.7 López-Béjar 5.6 mM glucose and In PBS at López-Gatius room temp. 2002 Rabbit 25% EG, 0.25 M sucrose, 20% 2-3 238 100 42.0 López-Béjar FBS, 5.6 mM and glucose in PBS López-Gatius at 22-25 °C 2002 Sheep 20% EG, 20% DMSO, 0.3M 6.5-7.5 50 31 62 Isachenko trehalose, 15% et al. 2003a FCS in TCM-199 at room temp. aIncubation temperature not reported, EG = ethylene glycol, DMSO = dimethyl sulphoxide, PVP = polyvinylpyrrolidone, mPBS = modified phosphate buffered saline, GLY = glycerol, PBS = phosphate buffered saline, FBS = fetal bovine serum, FCS = fetal calf serum, TCM = tissue culture medium, GS = goat serum, NR = not reported

Zona damage in in-straw vitrification has been reported to occur at a frequency of 1.6% to 3.6 % of embryos (Kasai et al. 1996) or 31% of embryos (Vajta et al. 1997) when the embryos are rapidly cooled and warmed in 0.25-ml straws. Due to the high rate of cooling and warming, there is less danger of zona or embryo fractures in open pulled straws (Vajta 1997).

To date, there have been no published reports of the transfer of carnivore embryos after OPS vitrification. Crichton et al. (2000) OPS vitrified 2- to 4-cell stage IVF embryos of Siberian tiger (Panthera tigris altaica) in 3 M EG, 2.3 M DMSO and 0.5 M sucrose prepared in commercial HEPES-TL solution containing 10% fetal calf serum. Warming took place rapidly at room temperature. Of the vitrified-warmed embryos, 46% survived vitrification based on further development in vitro during a 24-h post-warming period. The embryos were not transferred into recipients. In the domestic cat, pregnancies have been achieved following OPS vitrification and subsequent transfer of the in vitro cultured embryos produced by both intracytoplasmic sperm injection and in vitro fertilization of in vitro matured oocytes (Pushett et al., submitted, cited in Vajta et al. 2001).

3 AIMS OF THE STUDY

The primary aim of the present study was to:

Develop methods for embryo recovery, embryo transfer and cryopreservation of embryos for conservation purposes of the endangered European mink by using the farmed European polecat as a model species.

The following issues were chosen as specific objectives of the work presented in this thesis:

To investigate early embryonic development in the farmed European polecat. (I)

To study the ability of in vivo derived embryos of the farmed European polecat to continue their development in the in vitro medium developed for cattle and to compare the development of polecat embryos in vivo with their development in vitro. (I)

To investigate the effects of the developmental stage, and the number of transferred embryos, weight of recipient, and synchronization between donors and recipients on embryo survival after surgical transfer of fresh in vivo embryos into recipients in the farmed European polecat. (II)

To develop a surgical embryo recovery technique for polecat donors. (III, IVu)

To study the ability of in vivo embryos of the farmed European polecat to withstand conventional embryo freezing and the newly developed open pulled straw vitrification technique and the ability of frozen/vitrified embryos to develop into full term offspring after thawing/warming and surgical embryo transfer. (III, IV)

4 MATERIALS AND METHODS

4.1 Animals

The animals included in the present study were farmed polecats, animals which originated from a population of 70 wild European polecats captured for fur production purposes in Finland during the 1970's. However, difficulties were experienced in breeding the wild animals in captivity. The industry was interested in producing lighter-coloured skins than the skins of the original wild European polecats; therefore, the wild animals were mixed with 200 imported lighter-coloured ferrets in the late 1970's. This captive population forms the basis for all farmed polecats in Finland.

A total number of 121 females and 50 males were used in the experiments described in original articles I-IV (Table 9). Of the 66 donors, 59 were yearlings, 4 were two- year-olds, 2 were three-year-olds and one was a 5-year-old. All 28 recipients were yearlings. The experimental design is shown in Figure 1.

Table 9. Total numbers of animals used in the experiments of original articles I-IV.

Original Breeding Number Number of Number of Number of Number of article season of donors fertile males vasectomised males test females recipients I + II 1998 34 16 6 10 16 III 1999 18 13 5 10 8 IV 2001 14 7 3 7 4 Total 66 36 14 27 28

All animals were kept in individual wire mesh cages measuring 70 x 30 x 38 cm (length x width x height). Nesting boxes measuring 40 x 29 x 32 cm, respectively, were accessible to the females but external to the main cage area. The animals were housed in their cages in an unheated barn, which had windows at the level of the roofs of the cages. During the breeding season, the animals were exposed to the outdoor temperature (mean monthly temperatures 2 °C in April, 9 °C in May) and light conditions (15 h light and 9 h dark in April, 17 h light and 7 h dark in May) and given wet feed prepared in the local kitchen and water ad libitum. The recommendations given by the Finnish Fur Breeders Association for feeding mustelids during the

48 breeding season include energy 1150-1250 kcal/kg complete wet feed, 40-50% protein, 30-32% fat and 15-25% carbohydrates, supplemented with vitamins and minerals. All experiments were carried out at the Fur Animal Research Station of the Institute of Applied Biotechnology, University of Kuopio, Juankoski, Finland, 27°35´E, 62°53´N. All experiments were approved by the Animal Ethics Committee of the University of Kuopio.

Post mortem Surgical embryo recovery recovery

I, II III, IV Donors

Fresh embryos -Early embryonic Cryopreservation development (I) IV III I II Vitrified- Frozen- In vitro culture warmed thawed embryos embryos IV

Surgical transfer

Recipients

Birth of pups

Figure 1. The experimental design.

4.2 Vasectomy of the males

Vasectomized males were used to induce ovulation in the recipient females by mating. A group of 11 polecat males were vasectomized surgically 1 month before the breeding seasons of 1998 (n=6) and 1999 (n=5). Anaesthesia was induced by intramuscular injection of 0.22 mg/kg medetomidine hydrochloride (Domitor 1 mg/ml, Orion-Farmos, Turku, Finland) and 11.0 mg/kg ketamine hydrochloride (Ketalar 50 mg/ml, Parke-Davis, S.A., Barcelona, Spain). Skin was incised 5 cm above the testicles 0.5 cm lateral to the linea alba, muscles and fat were separated bluntly, and the spermatic cord lifted. The cord was incised longitudinally and the vas deferens was separated from the vein and the nerve with a concave needle. The duct was ligated with 3-0 silk (FS-1, Ethicon, Germany) in 2 locations, 0.5 cm apart, and a piece of duct was excised between the ligatures. The peritoneum and muscle layers were sutured with absorbable material (Vicryl, Ethicon, Germany) and the skin with stainless steel wire (Monofilament wire, B. Braun Melsungen, Germany). The males were treated with antibiotic 57.5 mg benzylpenicillinbenzazine/75 mg benzylpenicillin- procaine (Duplocillin LA, Intervet, Gist-Brocades NV Delft, Holland) postoperatively. Both sperm ducts were operated in the same manner.

4.3 Test matings

Test females were mated to vasectomized males to confirm the sterility of the males. Matings and treatments of the mated test females were the same as for the donors and recipients.

In 1998, four vasectomized males mated 2 test females each and the 2 remaining males mated 1 female each. After a pseudopregnancy period of 42 d, the test females were killed and their uteri collected and examined for no signs of implantation. (II). In 1999, the 5 vasectomized males were test mated to 1 to 3 females each and none of the females whelped. (III). In 2001, the 3 vasectomized males were proven sterile with 7 test matings during the same breeding season. (IV).

4.4 Detection of oestrus and mating of the females

Females were visually examined 3 times a week for swelling of the vulva to confirm the beginning of prooestrus. Prooestrus was defined as the period of increase in size and tumescence of the vulva, and oestrus as the period of maximum vulval size. Matings were initiated after maximal vulval swelling, which appeared at between 1 and 2 weeks after the beginning of the prooestrus. The females to be mated were placed into the cages of the males, and the matings were supervised to confirm the beginning of intromission. The majority of the females were mated once daily on 2 consecutive days. If possible, the second mating of each female was with the same male. The females and males tied for a period of 0.5-2 h after which the females were returned back to their cages. To confirm mating, 0.5 ml of warm 3.2% Na-citrate was introduced into the vagina with a Pasteur pipette, aspirated back into the pipette and transferred to a slide for microscopic examination using a 400x magnification. The mating was considered successful if spermatozoa were detected. If no spermatozoa were detected, the female was remated on the next day, and this remating was considered as the first successful mating if spermatozoa were found in the vagina. Spermatozoa were not detected in the vaginal samples of the recipients mated with the vasectomized males.

Exceptions to this mating schedule, in the study reported in Article II, included one donor which was mated twice with a 5-day difference, three donors which were mated on 3 consecutive days and one donor which was mated only once. Two donors and 5 recipients had a prominently swollen vulva 1 week after 1 or 2 successful matings and were remated. These 2 rematings (2nd and 3rd or 3rd and 4th mating) were considered as the first and second successful matings. These donors and recipients entered into the embryo recovery programme after an appropriate period of time had elapsed since the rematings. In the study of surgical transfer and cryopreservation (III), one recipient was mated on 3 consecutive days. For the OPS study (IV), six donors were left in the same cage with a male overnight. Of the recipients, three were left in the same cage with a male overnight.

4.5 Post mortem embryo recovery

The donors were killed by electrocution 3 to 14 days after the first mating (I, II). Uteri and oviducts were removed, inspected and flushed as described in earlier studies (Valtonen et al. 1985, Lindeberg et al. 1993). The ovaries were removed from the oviducts and the oviducts transected at the uterotubal junction. Both uterine horns were transected 5 mm from the bifurcation and the body of the uterus was transected at the cervix. Each uterine horn was flushed with 20 ml, and the oviducts and the uterine body were flushed into five separate Petri dishes with 10 ml of Dulbecco’s PBS (Gibco, Life Technologies Ltd., Paisley, Scotland) supplemented with glucose (1 g/l) and BSA (1 g/l, Gibco, Life Technologies Ltd., Paisley, Scotland). The time elapse between the killing of the females and the start of embryo recovery was less than 5 minutes.

4.6 Surgical embryo recovery

4.6.1 The linea alba method (III)

The embryos were recovered surgically under general anaesthesia 8 to 9 days after the first mating. The donors were weighed, treated with 0.25 mg/kg medetomidine hydrochloride (i.m., Domitor 1 mg/ml, Orion-Farmos, Turku, Finland) and 13.0 mg/kg ketamine hydrochloride (i.m., Ketalar 50 mg/ml, Parke-Davis, S.A., Barcelona, Spain). Induction of anaesthesia occurred in 2 minutes. The eyes were covered with gel drops and hair coat was shaved at the linea alba, washed with iodine (Betadine 75 mg/ml, Leiras Ltd, Turku, Finland) and disinfected with 70% ethyl alcohol (Etax Aa, Primalco, Rajamäki, Finland). Females were placed onto a clean thick towel on the operating table and the skin was incised at the linea alba. Fat was opened bluntly, the peritoneum was incised and the uterine horns were lifted with fingers. A cannula (1.3 x 38 mm, Venflon® 2, I.V. Cannula, Viggo Products, Helsingborg, Sweden) containing 3 openings (approximate inner diameter 0.7-0.8 x 1.0 mm) made with a surgical blade was inserted into the tip of each uterine horn and pushed gently towards the corpus uteri. Both cannulae were then connected to plastic tubes and 20 ml flushing medium (EmcareTM, Immuno-Chemicals Ltd., Auckland, New Zealand,) supplemented with 2 g/l

52 of BSA (20% bovine serum albumin, Immuno-Chemicals Ltd.) at room temperature was introduced gently into the uterine horn and allowed to flow through the corpus uteri into the opposite horn. At the time of the flushing (8-9 days after mating) the liquid was held in the uterus by the closed cervix. From the opposite uterine horn the medium was allowed to flow out via the other cannula and into a Petri dish. After flushing, the peritoneum and muscle layers were sutured with absorbable material (Vicryl, Ethicon) and the skin with stainless steel (Monofilament wire, B. Braun Melsungen, Germany). The skin was protected with antibiotic powder (Bacibact®, Orion-Farmos, Turku, Finland) and aerosol wound bandaid (Hansaplast®, Beiersdorf AG, Hamburg, Germany). The females were treated with antibiotic, 57.5 mg bentsylpenicillinbentsatine/75 mg bentsylpenicillinprocaine (Duplocillin LA i.m., Intervet, Gist-Brocades NV Delft, Holland) postoperatively.

If few or no embryos were recovered from uteri during the surgical flushing, ovariohysterectomy was performed and the oviducts were removed for flushing. In such cases, the anaesthetized animals were killed (i.c., 2 ml of T-61 vet. inject., Intervet International, Holland).

4.6.2 The flank method (IVu)

The embryos were recovered surgically under general anaesthesia 6 to 8 days after the first mating. The donors were weighed, treated with 0.5 mg/kg medetomidine hydrochloride (i.m., Domitor 1 mg/ml, Orion-Farmos, Turku, Finland) and 25 mg/kg ketamine hydrochloride (i.m., Ketalar 50 mg/ml, Parke-Davis, S.A., Barcelona, Spain). Induction of anaesthesia occurred in 2 minutes. After shaving, both lumbar flanks were washed with iodine (Betadine 75 mg/ml, Leiras Ltd, Turku, Finland) and disinfected with 70% ethyl alcohol (Etax Aa, Primalco, Rajamäki, Finland). The eyes were covered with gel drops and the donors were treated with antibiotic, 57.5 mg benzylpenicillinbenzazine/75 mg benzylpenicillin-procaine (i.m., Duplocillin LA, Intervet, Gist-Brocades NV Delft, Holland) preoperatively.

Figure 2a. The fat covering the ovary is attached to the skin. 2b. The cannula is inserted into the uterine horn. 2c. The cannula is attached to the same stitch as the fat. Photos: Mikko Järvinen.

Figure 3a. The cannula is properly inserted. 3b. The plastic tube is connected. Photos: Mikko Järvinen.

The donor was placed in lateral recumbency onto a clean thick towel on the operation table. The skin was incised at the lumbar flank (1.5 cm), and the muscle layers separated bluntly. The peritoneum was cut with scissors and further opened bluntly with forceps. The fat covering the ovary was lifted to expose the tip of the uterine horn. A 2-0 vicryl suture was placed into the fat covering the ovary (Figure 2a) and the uterine horn was held by this suture without touching the horn while gently pushing an i.v. cannula (22G, 0.8/25mm, Vasculon® 2, BOC Ohmeda AB, Helsingborg, Sweden) from the tip of the horn towards the corpus uteri (Figure 2b). The cannula had 3 openings (approximate inner diameter 0.4-0.5 x 1.0 mm) made with a surgical blade. The cannula was then fastened to the fat by threading one piece

54 of the vicryl suture through the hole in the wing of the hub of the cannula (Figure 2c). The cannula inserted into the uterine horn and fastened into the fat was then sutured into the caudal edge of the flank incision by using the same vicryl suture, leaving only the hub of the cannula outside of the closed incision (Figure 3a). A plastic tube was connected to the cannula (Figure 3b), the operated flank was covered with a gauze, and the female was gently turned over to expose the unoperated flank for surgery.

The unoperated flank was processed as described earlier for the other flank and a total of 20 ml of prewarmed (+37 ºC) flushing medium (Complete Embryo Flushing Solution, Emcare™, Immuno-Chemicals Ltd., Auckland, New Zealand) was slowly flushed through the plastic tube into the uterus and out into a Petri dish through the opposite side tube without touching either the female or the uterus (Figure 4).

Figure 4. Flushing of the uterine horns using the flank method. In this photograph, medium has been flushed through the uterus into a Petri dish and the upper cannula has been removed from the uterine horn. Photo: Mikko Järvinen

After flushing, the tube and the cannula from the uterine horn together with the stitch were removed and the uterine horn was replaced into the abdominal cavity. The peritoneum and muscle layers were sutured with absorbable material (Vicryl, Ethicon) and the skin was sutured with stainless steel (Monofilament wire, B. Braun Melsungen, Germany). The skin was protected with antibiotic powder (Bacibact®, Orion-Farmos, Turku, Finland) and aerosol wound bandaid (Hansaplast®, Beiersdorf AG, Hamburg, Germany). The female was gently turned over to expose the previously operated flank and the same removal and closure procedures were repeated.

If few or no embryos were recovered from uteri during the surgical flushing, ovariohysterectomy was performed and the oviducts were removed for flushing before killing the anaesthetized animals (i.c., 2 ml of T-61 vet. inject., Intervet International, Holland).

4.7 Evaluation of recovered embryos

The recovered embryos were evaluated on an unheated stage of a stereomicroscope using a 64x magnification for the stage of development and photographed (I, II, III, IV). In case of expanded blastocysts, a smaller magnification was used so that all blastocysts of a same female were photographed in the same image. According to the stage of development, the embryos were divided into 6 groups; 1- to 16-cell stage embryos, morulae, early blastocysts, blastocysts, expanded blastocysts and hatched blastocysts. One to 16-cell stage embryos had clearly distinguishable individual blastomeres, which were counted to classify the individual embryos although, in the results, these embryos are presented as a group. Morulae consisted of embryos that had an uncountable number of small blastomeres but no signs of impending cavitation. Unexpanded blastocysts with a blastocoel cavity smaller than half the size of an embryo were considered as early blastocysts. Other unexpanded blastocysts with a cavity equal to or larger than half the size of an embryo were considered as blastocysts. Expanded blastocysts had a larger diameter than early blastocysts and blastocysts, and were either fully cavitated or had a collapsed cavity at recovery. Hatched blastocysts had no visible embryonic coat at recovery.

4.8 Measurement of the diameters of the embryos

The mean diameter of the embryos including the embryonic coat was measured retrospectively from the slides with a ruler (Absolute Digimatic Caliper, model CD- 15C, Mitutoyo (U.K.) Ltd., Andover, England) considering 1 mm length of the ruler as 100 µm of true diameter (Jalkanen and Lindeberg 1998) (I, II). The measurements were corrected by dividing with a coefficient of 1.33 which was counted from the magnifications of the objectives of the stereomicroscope. Day 7 and Day 8 (Day 0 = first day of mating) non-expanded blastocysts were grouped together with 1- to 16- cell stage embryos and morulae. Expanded blastocysts were grouped according to the days after first mating. Blastocysts with a diameter greater than 230 µm were considered as expanded blastocysts. In 2002, the diameters of the embryos were measured again using a computer program (DP-Soft, Version 3.0 for Windows 95 and Windows NT, Olympus, Hamburg, Germany) and the original measurements were checked. All slides were scanned onto files into the DP-Soft program and the different magnifications for the program were set using a 1000 µm objective micrometer (model OB-M, Olympus, Japan) which had been photographed using the different magnifications of the stereomicroscope used to photograph the original embryos. As the difference between diameters measured using these two methods was considered small (ranged from 0 to 41 µm), the original numerical values have been presented in the results.

4.9 In vitro culture of fresh embryos

Fourteen females were humanely killed according to the following schedule (days after first mating): 3 d (n=1 female), 4 d (n=1), 5 d (n=1), 6 d (n=1), 7 d (n=3), 9 d (n=1), 10 d (n=1), 11 d (n=1), 12 d (n=1), 13 d (n=2) and 14 d (n=1) in order to clarify when embryos enter the uterus, to have all stages of development for in vitro culture and to investigate the earliest date of implantation (I). Embryos of the same female were cultured in the same well of a four-well multidish (Cat. No. 176740, Nunc™, Roskilde, Denmark) and embryos of the females killed on the same day were cultured in the same four-well multidish. The number of embryos cultured in the same well varied from 5 to 12.

The culture medium was prepared into a tissue culture flask (Cat. No. 163371, Nunc™) and filtered (0.2µm filter). An aliquot of 500 µl was pipetted into each well and the dish was equilibrated for 15 minutes in the incubator (Automated CO2 Incubator, Model 3194, Forma Scientific Inc., Marietta, OH, USA) before transferring the embryos. The medium consisted of TCM-199 + glutamine (Medium 199 W/Glutamax I, Gibco, Life Technologies™, Paisley, Scotland) supplemented with 10% FBS (Foetal bovine serum, Lot 40G2379F, Gibco, Life Technologies™, Germany), 0.25 mM pyruvate (Pyruvic Acid, Sigma Chemicals Co, St. Louis, MO, USA) and 40 µg/ml gentamycin (Gentamycin Solution, Sigma Chemicals Co, St. Louis, MO, USA). The embryos were washed twice in the flushing medium and once in the culture medium before being pipetted into the wells. Dishes with embryos were incubated at 39.0 °C in a humidified atmosphere of 5% CO2/95% air until all embryos in the well ceased to show any signs of development. Embryos showing signs of degeneration during the culture period (i.e. the trophectodermal structure of the embryos disappeared and the inside of the zona pellucida turned into a black mass) were not removed from the wells.

During the culture period, the embryos were studied on an unheated stage under a stereomicroscope using a 64x magnification for stage of development and photographed once a day except on weekends when they were viewed only once. Daily, or during the weekends on Saturday or Sunday, the embryos were transferred into a new well containing 500 µl of fresh culture medium (equilibrated for 15 minutes before use). Hatching of the embryos was not assisted, only visually observed. Cell numbers were not counted.

4.10 Processing of fresh embryos before transfer

The embryos were washed 3 times in the flushing medium (II). They were transferred into 4-well multidishes (Nunc™) containing 500 µl of culture medium used for in vitro culture and incubated at 39.0 °C in a humidified atmosphere of 5% CO2/95% air, or they remained in the flushing medium protected from light at room temperature (+20- 24 °C) until transfer. If embryos were to be transferred within an hour, they remained at room temperature (+20-24 °C) but if transfer was delayed by >1 hour, the embryos

were stored in the incubator (Automated CO2 Incubator, Model 3194, Forma Scientific, Inc., Marietta, Ohio, USA).

4.11 Conventional slow freezing of embryos

From each donor, all different types of blastocyst (early blastocysts, blastocysts, expanded blastocysts), if present in the group of the recovered embryos, were chosen for cryopreservation (III). The embryos were washed once in embryo holding solution (EmcareTM, ECHM-100, Immuno-Chemicals Ltd.) and transferred into 1.5 M ethylene glycol (EG) (EmcareTM, ECEG-100, Immuno-Chemicals Ltd.). The embryos were equilibrated in EG for 15 min and within this time they were aspirated into 0.25 ml plastic straws (IMV, L'Aigle, France) with a 1-ml syringe. The straws were sealed with plastic plugs and loaded into a freezing machine (Freeze Control CL856, Biogenics, Napa, CA, USA). The number of embryos per straw ranged from 1 to 11. The freezing machine was programmed to decrease the temperature by 3.0 °C/min from +15 to -6 °C.

The straws were seeded at -6 °C with forceps precooled in liquid nitrogen (LN2). After a 10 min hold, cooling continued at 0.3 °C/min to -32 °C and the straws were then plunged into LN2. Straws were stored in LN2 for an average of 6 d before thawing. The straws were thawed in air at a rate of 400-500 °C/min, and the contents were emptied into a Petri dish. The embryos were either transferred immediately after thawing, or they were placed into 0.5 M sucrose medium for 6-7 min and then into fresh flushing medium. After thawing, embryos whose trophectoderm and inner cell mass had been shattered into small pieces inside the zona pellucida were discarded and the remaining embryos were transferred. The time elapsing between thawing and transfer was approximately 25 min for directly transferred embryos and 45 min for sucrose treated embryos.

4.12 Open pulled straw vitrification, warming, culture and transportation of embryos

Recovered embryos were evaluated under a stereomicroscope with 64x magnification and washed twice in modified Hepes SOF (Walker et al. 1996) without any of the following: BSA, myoinositol, glutamine, MEM and BME amino acids solutions,

59 warmed to +37 °C before vitrification (IV). Morulae and blastocysts were vitrified using the modified open pulled straw method (Vajta et al. 1997). One to 16-cell stage embryos were discarded. All manipulations were performed on +37 °C heated stages. Blastocysts were incubated in 1.34 M ethylene glycol and 1.05 M DMSO dissolved in holding medium (Hepes SOF and 20% fetal bovine serum) for 3 min. After 3 min, the embryos were transferred into a 20 µl droplet of 2.95 M ethylene glycol and 2.32 M DMSO dissolved in holding medium containing 0.9 M sucrose. Finally, the embryos were transferred into a 2 µl droplet and loaded with a capillary effect by touching the droplet containing the embryos with the narrow end of an open pulled straw and submerged in liquid nitrogen within 25-40 sec. Morulae were incubated in the first medium for 4 min and in the second medium for 30-45 sec.

Embryos were warmed by immersing the straw within 3 sec at a 30-45° angle in a warm holding medium containing 0.3 M sucrose. After 1 min of warming, embryos were transferred for 5 min into the holding medium containing 0.15 M sucrose and then into the holding medium without sucrose for another 5 min. After warming, embryos whose trophectoderm, inner cell mass and zona pellucida had turned into a black mass, or embryos that had shattered into small pieces, were discarded and the remaining embryos were washed once in SOF medium supplemented with 6 mg/ml BSA (Sigma), 0.5 µg/ml myoinositol (Sigma), 29 µg/ml glutamine (Gibco), 1% MEM non-essential amino acid solution (Sigma) and 3% BMEM essential amino acid solution (Sigma), and cultured in vitro (IVC) in the same medium at 38.5 °C in a humified atmosphere of 90% N2, 5% CO2 and 5% O2 for 3 h to 3 d, depending on the stage of the embryos at the time of collection. After in vitro culture, morulae which had developed into blastocysts, or blastocysts and expanded blastocysts which had collapsed but were able to cavitate again during the culture period, were transferred into a tube containing 500 µl of Hepes SOF and transported to the experimental farm in a CO2 incubator (Cell-trans, model 4016, Labotect, Göttingen, Germany). The duration of transport and processing of the embryos was approximately 2 h. The remaining in vitro cultured embryos were either discarded or stained for cell numbers.

4.13 Surgical embryo transfer

Recipients were weighed, anaesthetized and prepared as described earlier for embryo recovery and placed onto a clean thick towel in right lateral recumbency (II, III, IV). The left uterine horn was approached from an incision made in the left lumbar flank. The fat layer covering the ovary was gently grasped with forceps and suspended with a suture to exteriorize the left uterine horn. The transfer instrument was a thin sharpened haematocrit capillary, which was connected to a plastic tube containing either a mouthpiece (II, III, Amstislavsky et al. 1991) or a syringe (IV). Embryos were aspirated into the instrument with a 15-20 µl of medium (II: flushing medium or culture medium; III: flushing medium; IV: Hepes SOF medium) and gently transferred into the ovarian third of the uterine horn without insufflating air. Occasionally some localized uterine bleeding was observed after transfer. The flank incision was sutured as described earlier. The recipients received antibiotic, 57.5 mg bentsylpenicillinbentsatine/75 mg bentsylpenicillinprocaine (Duplocillin LA i.m., Intervet, Gist-Brocades NV Delft, Holland) postoperatively during the breeding seasons 1998 and 1999 and preoperatively during the breeding season 2001.

4.14 Post mortem examinations

After flushing the oviducts and the uteri, the ovaries were sliced in order to count the number of corpora lutea (CL). All recipients not whelping were killed and their uteri examined for signs of implantation. (II).

The ferret/polecat ovary is encapsulated in an ovarian fatty bursa which prevented the counting of CL of surgically flushed donors. Thus, to determine ovulation number, the original donors were killed and examined for CL 18 to 25 d after the original flushing. Recipients not whelping were killed and their uteri examined for implantation scars within 2 weeks after the expected delivery. (III).

4.15 Postoperative care of the surgically flushed donors and recipients

During a 24-h post-operative period, the females were observed while caged in a warm room next to the operating room (III, IV). The females were kept warm by wrapping them in a blanket and placing them between 2 hot-water bottles in the recovery cage. The females recovered from anaesthesia within 2-3 h after the operation.

During the 24-h after flushing, one donor died of hypothermia (III), while two donors (III) and another two donors (IV) needed intensive care. Intensive care comprised oral and subcutaneous fluid therapy (5 ml of water and 20 ml of Natrosteril, Orion Ltd, Espoo, Finland, respectively), pain medication (flunixine meglumine; Finadyne® 50 mg/ml vet. inject, Orion Ltd, Espoo, Finland) and prolonged antibiotic therapy (Duplocillin LA).

All recipients received the same management as other pregnant females on the research farm (II, III, IV). They were not examined for pregnancy to avoid possible fetal loss. The number of kits was counted and their sexes determined 5 d after squeaking was initially registered around the time of the expected delivery except in 1998 when the sexes were determined after weaning of the kits.

4.16 Statistics

The following statistical methods were used:

Descriptive statistics (all articles) Independent Samples t-test (II) χ2-test (IV)

The computer software Excel 2000 (Microsoft Co., Redmont, WA, USA) was used for the statistical analyses.

5 RESULTS

5.1 Early embryonic development and oviductal passage

A total of 582 embryos were recovered from 62 females 3 to 13 days after the first mating (Table 10). Until Day 4 after the first mating, only 1- to 16-cell stage embryos were recovered, the first morulae were recovered on Day 5 and the first blastocysts on Day 6 after the first mating. On Day 7, both morulae and blastocysts were recovered. Between Days 8 and 10, mainly expanded blastocysts were recovered.

Four females were humanely killed 11, 12, 13 and 14 days after the first mating and they had 10, 8, 10 and 14 swellings, respectively, in their uteri indicating that implantation had already occurred. On Days 11 and 12, flushing resulted in the recovery of remnants of 10 and 8 trophectoderms and 9 and 8 empty embryonic coats, respectively. On Days 13 and 14, no flushing medium could be passed through the uterine horns.

Table 10. Embryo recovery results of the farmed European polecat (Mustela putorius) donors and proportion of embryos in the oviducts (Ov) and the uterus (Ut) in relation to days after first mating.

Days after first mating 3 4 5 6 7 8 9 10 13 Ov/Ut Ov/Ut Ov/Ut Ov/Ut Ov/Ut Ov/Ut Ov/Ut Ov/Ut Ov/Ut Total 1-16-cell 11/0 7/0 10/0 8/3 3/6 1/32 0/10 40/51 M, CM 5/0 11/25 21/22 0/14 4/1 41/62 EB,B,XB 5/26 0/62 10/190 0/72 0/12 0/11 15/373 Total 11/0 7/0 15/0 24/54 24/90 11/236 4/83 0/12 0/11 96/486 Females 1 1 2 10 12 26 8 1 1 62 % in Ut 0 0 0 77 79 96 95 100 100 M = morulae, CM = compact morulae, EB = early blastocyst, B = blastocyst, XB = expanded blastocyst

Sixteen percent (96/582) of all embryos were recovered from the oviducts and 84% (486/582) from the uteri. Until Day 5 after the first mating, embryos were recovered only from the oviducts. Between Days 6 and 9, embryos were recovered both from the oviducts and the uteri. From Day 10 onwards, the embryos were recovered only from the uteri.

5.2 Embryo recovery and evaluation

A total of 582 in vivo embryos were recovered and utilized in this study (Figure 5). Summary of embryo numbers from the original articles is presented in Table 11.

1-16-cell stage Morulae Blastocysts embryos n=103 n=388 n=91

A total of 582 recovered embryos

Fresh transfers Cryopreserved n=172 n=130

In vitro cultured Used for other n=85 experiments n=101

Discarded n=94

Figure 5. Utilization of the recovered embryos.

The average number of corpora lutea (CL) per donor was 14 ± 4 (± SD) resulting in a total recovery rate of 66% (440 embryos/667 CL, n=47 donors) (Table 12). The average number of recovered embryos per donor was 9.4 ± 3.6 (± SD) (1-19 embryos recovered from uteri and oviducts/donor, n=62 donors and 582 embryos).

Table 11. Total numbers of the embryos in the experiments of the original articles I-IV.

No. of No. of No. of No. of No. of Original No. of flushed frozen IVC transferred No. of born kits article donors embryos embryos embryos embryos recipients I 14 95 0 85 0 0 0 II 20 200 0 0 172 16 72 III 18 172 93 0 80 8 9 IV 14 115 98 69 50 4 8 Total 66 582 191 154 302 28 89 IVC = in vitro culture

Table 12. Summary of results of embryo recoveries from uteri and oviducts in the farmed European polecat.

All donors Donors with CL No. of Recovered No. of Original No. of recovered embryos/ No. of recovered No. of Recovery article donors embryos donor donors embryos CL rate (%) I, II 30 295 9.8 30 295 403 73 III 18 172 9.6 12 116 192 60 IV 14 115 8.2 5 29 72 40 Total 62 582 9.4 47 440 667 66 CL = corpora lutea

After evaluation, most of the recovered embryos were considered as normal and developing (89%, 520/582) and only a minority of embryos (11%, 62/582) were classified as degenerated or retarded in development according to the days after first mating. All normal and developing embryos represented the right age compared to the days after first mating and were of excellent or good quality (Code 1, IETS). Retarded embryos, i.e. they were of an earlier stage of development than expected by the days after first mating, or degenerated embryos, i.e. the trophectoderm and the inner cell mass had turned into a black mass (blastocysts) or individual blastomeres were of largely unequal size or some of the blastomeres or all of them had turned into a black mass (1- to 16-cell stages and morulae), were of poor quality (Code 4, IETS).

The 295 embryos recovered post mortem, but not the 287 embryos recovered surgically, were evaluated for shape and diameter. Two thirds of the recovered 295 embryos (I, II) appeared ovoid, one third were spherical. Of these 295 embryos, the mean diameter of the recovered 1- to 16-cell stage embryos (n=59), morulae (n=73), Day 7 (n=36) and Day 8 (n=25) blastocysts (not expanded) including the embryonic coat was 193 ± 16 µm (± SD). The mean diameters of the recovered Day 7 (n=9), Day 8 (n=24), Day 9 (n=46), Day 10 (n=12) and Day 13 (n=11) expanded blastocysts including the embryonic coat were 261 ± 23, 332 ± 119, 432 ± 160, 624 ± 86 and 1328 ± 257 µm (± SD), respectively (Figure 6).

Results on embryo recovery using different embryo recovery methods are presented in Table 13. Results have been calculated using 355 morulae and blastocysts that were recovered from uteri on Days 6-9 after the first mating from 47 donors whose CL numbers were counted. All embryos were considered as transferable.

Table 13. Post mortem and surgical embryo recovery results from 355 transferable uterine embryos recovered on Days 6-9 after the first mating in the farmed European polecat (Mustela putorius). Day 0 = first day of mating.

Number of recovered transferable uterine embryos per donor (n)/ Number embryo recovery rate (flushed transferable embryos/corpora lutea) of Article On D6 n on D7 n on D8 n on D9 n on D6-9 n embryos I + II 4/29% 2 6/54% 10 8/66% 6 7/53% 6 7/54% 24 170 III 8/50% 1013/87% 2 8/56% 12 107 IV 4/32% 5 8/ - 2 9/ - 4 7/ - 11 78 Total 7 12 20 8 47 355

When embryos were recovered with linea alba method (III), post mortem examination of the uteri of the donors revealed adhesions caused by the flushing procedure. Uterine horns had adhesions either along the insertion line of the cannulae or both horns had stuck together.

In order to study the consequences of the flank method (IV) of surgical embryo recovery, on the reproductive capacity of the females, 8 operated donors (2 Day 6, 2 Day 7 and 4 Day 8 donors) were mated to fertile males during the second oestrus of

66 the same breeding season. Of these 8 females, 6 delivered a total of 31 kits (10 female and 21 male kits) after a gestation period of 41-42 days. One of the two donors not delivering was found to have a tumor in her right ovary at necropsy.

5.3 In vitro culture of in vivo embryos

Of the 85 cultured embryos, which were placed in culture at different stages of development, 55 (65%) developed further (i.e. cleaved at least once, expanded or hatched) during the culture period, while 30 embryos (35%) did not develop (no cleavage, no expansion, no hatching) (Table 14).

Table 14. In vitro culture results of in vivo produced farmed European polecat (Mustela putorius) embryos placed in culture at different stages of development.

Stage of Days Number Interval (d) between appearance of first and last development after first of M/CM B/ExB HB at recovery mating embryos during IVC (Number of developed embryos) 1-2-cell 3 10 3-4 (9) 4-5 (5) 9-11 (2) 4-16-cell 4 or 5 12 1-2 (10) 2-4 (10) 7 (1) M, CM 6 or 7 25 0-1 (24) 1-2 (21) 6 (2) EB, B 7 4 1-2 (4) ExB 9,10 or 13 32 2-5 (8) HB 13 2 Total 85 M = morula, CM = compact morula, EB = early blastocyst, B = blastocyst, ExB = expanded blastocyst, HB = hatched blastocyst, IVC = in vitro culture

With respect to the 1- to 16-cell stage embryos recovered between Days 3 and 5 after the first mating, the blastocyst development rate was 68% (15 blastocysts/22 cultured 1- to 16-cell stage embryos). These embryos had been cultured for 48 h before the first blastocysts and 120 h (5 d) before the last blastocysts were detected. For morulae and compact morulae recovered on Days 6 and 7 after the first mating, the blastocyst development rate was 84% (21 blastocysts/25 morulae and compact morulae). These embryos had been cultured for 24 h before the first blastocysts and 48 h before the last blastocysts were detected. Thirty-eight embryos were collected as early blastocysts, blastocysts, expanded or hatched blastocysts and 12 of them developed further (either

67 expanded after being cultured for 24 h (n=4) or hatched after being cultured for 48 to 120 h (n=8).

Of the 47 one to 16-cell stage embryos, morulae and compact morulae which were cultured in vitro and recovered between Days 3 and 7 after the first mating, 36 turned into blastocysts in vitro; 5 on Day 6, 17 on Day 7, 13 on Day 8 and 1 on Day 9 after the first mating. Of the remaining 11 one to 16-cell stage embryos, morulae and compact morulae, 7 ceased to develop at the compact morula stage and 4 did not develop beyond the stage they were at when recovered. The timing of blastocyst formation in vitro did not differ from the timing of blastocyst formation in vivo.

The in vitro cultured 1- to 16-cell stage embryos which developed into blastocysts in vitro were not capable of expanding like their in vivo counterparts (Figure 6). However, for the first 24 h in vitro, expansion of the embryos placed in culture as morulae or blastocysts on Day 6 and Day 7 was comparable to expansion in vivo. The embryos which were recovered on Day 9 and Day 10 as expanded blastocysts, and which hatched in vitro (n=5), increased their original diameter during the first 24 or 48 h in culture and then decreased their diameter towards the end of the culture period. The remaining embryos which were recovered as expanded or hatched blastocysts between Days 9 and 13 after the first mating (n=29, diameters not shown in Figure 6) showed either no change in their diameter or an initial decrease followed by a plateau or a continuing slow decrease in their diameter during the culture period.

The hatching rate was 15% (13 hatched/85 cultured embryos). Of the 13 hatched embryos, 10 were placed in culture as morulae or blastocysts and 3 at 1- to 16-cell stages. With respect to the 10 embryos placed in culture as morulae or blastocysts, hatching was observed to occur through lysis, leaving no remnants of the coverings in the medium. For the remaining 3 embryos placed in culture at 1- to 16-cell stages, 1 was observed to hatch through a hole in the zona pellucida, leaving an empty zona in the medium, and 2 still had the hatching process ongoing at the end of the culture period, this being apparent via a thinning of the zona over an entire pole with no breakthrough, or as protrusions through the zona. The remaining 70 non-hatched embryos and the 2 hatched blastocysts at recovery were degenerated at the end of the culture period.

uncultured in vivo 1550 embryos (n=295) cultured: 1350 Day 3, 1-2-cell stages (n=5) 1150 Days 4-5, 4-16-cell 950 stages (n=10)

750 Days 6-7, morulae and blastocysts 550 (n=25) Mean diameter of embryos (µm) 350 Day 9, blastocysts (n=3) 150 34567891011121314 Day 10, blastocysts (n=2) Days after first mating

Figure 6. Changes in mean diameter (± SD) of cultured in vivo produced embryos in the farmed European polecat (Mustela putorius): Days 3-5, one to 16-cell stage embryos which developed to blastocysts (n=15); Days 6-7, morulae and blastocysts which expanded (n=25); and Days 9-10, expanded blastocysts which hatched (n=5). Mean diameter (± SD) of 295 uncultured in vivo embryos at recovery is shown for reference. Day 0 = first day of mating.

5.4 Treatment of fresh and cryopreserved embryos before transfer

The time spent by fresh embryos outside the reproductive tract during flushing, evaluation, and transferring procedures was approximately 76 min (29-85 min; n=16 transfers). In unsuccessful transfers (n=5), the fresh embryos remained outside the reproductive tract in the flushing medium at room temperature on average for 65 min. For frozen-thawed embryos, the time spent outside the reproductive tract during flushing, freezing and thawing excluding storage time until transfer was approximately 164 min (69-256 min; n=8 transfers). The difference in duration of procedures between successful (n=3) and unsuccessful (n=5) transfers was less than 10 min. The times for processing embryos were not recorded for vitrified-warmed embryos.

Of 80 transferred frozen-thawed embryos, 38 were treated with sucrose to remove the EG before transfer into 4 recipients, while the remaining 42 embryos were transferred immediately after thawing into the remaining 4 recipients. One litter of 2 kits was born after transfer of embryos following sucrose treatment and 2 litters were born after direct transfer of embryos without removal of EG. Embryonic coat damage was observed in 34% (27/80) of the transferred frozen-thawed embryos.

The vitrified-warmed embryos were cultured in vitro for 3-72 hours after warming. Embryos, which developed from morulae into blastocysts, and blastocysts and expanded blastocysts which had collapsed but cavitated again during in vitro culture were chosen for embryo transfer. After in vitro culture, the average progress in development and re-expansion rate of the embryos was 51% (50/98) (Table 15). The development and re-expansion rate of morulae and blastocysts was 64% (45 transferred/70 vitrified) and for expanded blastocysts 18% (5 transferred/28 vitrified). In vitro survival was higher for Day 6 and Day 7 embryos (62%, 38 transferred/61 vitrified) than for Day 8 embryos (32%, 12 transferred/37 vitrified) (P<0.1).

Table 15. In vitro survival rate of vitrified and in vitro cultured embryos of farmed European polecat (Mustela putorius) according to the days after mating at recovery. The viability of the embryos was evaluated by their further development and re-expansion during culture. Day 0 = first day of mating.

Days after Number Recovered embryos Transferable mating at of Discarded embryos after Recovery donors M (%) B (%) ExB (%) embryos (%) vitrification (%) 6 8 16 (31) 28 (54) - 8 (15) 25/44 (57)a 7 2 - 17 (100) - - 13/17 (76)b 8 4 8 (17) 1 (2) 28 (61) 9 (20) 12/37 (32)c Total 14 24 (21) 46 (40) 28 (24) 17 (15) 50/98 (51) M = morula, B = blastocyst, ExB = expanded blastocyst abP<0.1, bcP<0.01, acP<0.1, ab-cP<0.1

5.5 Transfer of fresh, frozen-thawed and vitrified-warmed embryos

A total of 172 fresh, 80 frozen-thawed and 50 vitrified-warmed embryos were transferred into 28 recipients (Table 16). All recipients, which received ≥ 12 fresh, frozen-thawed or vitrified-warmed embryos, delivered kits (Table 17). There was an

70 average number of 10 ± 2 (± SD) transferred fresh embryos per recipient, 7-15 embryos per recipient. In the successful transfers, the average number of transferred fresh embryos was 11 ± 2, 8-15 embryos per recipient. In unsuccessful transfers, the average number of transferred fresh embryos was 9 ± 1, 7-11 embryos per recipient. The average weight of the 16 recipients receiving fresh embryos was 990 ± 129g (751-1174 g). A total of 11 of these recipients (69%) produced offspring. The average weight of the recipients delivering kits (n=11) was 1044 ± 106g, those not delivering (n=5) were lighter, 870 ± 92g (P<0.01). Altogether, 72 kits were born, representing a survival rate of 42% (72 kits/172 transferred fresh embryos). The average litter size was 4.5 ± 3.4 (0-9 kits; n=16 recipients).

Table 16. Characteristics of embryo transfers in the farmed European polecat (Mustela putorius) in the original articles II-IV.

Type of Number of Treatment Number No. of trans- No. Live transferred transferred before of ferred embryos/ of live kits/ embryos embryos transfer recipients recipient kits, (%) recipient fresh (II) 172 none 16 10.8 72 (42) 4.5 frozen (III) 80 w/wo sucrose 8 10 9 (11) 1.1 vitrified (IV) 50 3-72 h IVC 4 12.5 8 (16) 2.0 Total 302 28 10.8 89 (30) 3.2 w/wo = with/without, IVC = in vitro culture, % = live kits/transferred embryos

The average number of transferred cryopreserved embryos per recipient was 10 ± 3 (±SD), 7-18 embryos per recipient. In successful transfers, the average number of transferred cryopreserved embryos was 13 ± 2, 11-18 embryos per recipient. In unsuccessful transfers, the average number of transferred cryopreserved embryos was 8 ± 1, 7-11 embryos per recipient. The average weight of the 12 recipients receiving cryopreserved embryos was 1112 ± 89 g (991-1246 g). A total of 5 of these recipients (42%) produced offspring. The average weight of the recipients delivering kits (n=5) was 1074 ± 82 g, those not delivering (n=7) were heavier, 1134 ± 92 g. Altogether, 17 kits were born, representing a survival rate of 13% (17 kits/130 transferred cryopreserved embryos). The average litter size was 1.4 ± 2.0 (0-6 kits; n=12 recipients).

Table 17. Effect of number of embryos transferred on born offspring.

Number of Number of recipients giving birth after embryo transferred transfer/ total number of recipients embryos Fresh embryos Cryopreserveda embryos 7 0/1 0/2 8 1/2 9 2/3 0/3 10 1/2 0/1 11 0/1 2/3 12 or more 7/7 3/3 Total 11/16 5/12 aFrozen-thawed or vitrified-warmed

The overall survival rate indicated as number of kits per transferred embryo was 30% (89 kits/302 transferred embryos) in this study. The survival rate was 25% (89 kits/172 fresh and 191 cryopreserved embryos) if counted as number of kits per overall total of 363 fresh and cryopreserved embryos.

The number of donors per single recipient ranged from one to four. Of the 28 recipients, 12 received embryos from one donor, 8 from two donors, 3 from 3 donors and 5 from 4 donors. The average numbers of kits born per recipient in these groups were 2.6 ± 3.4, 4.0 ± 3.3, 4.3 ± 4.5 and 2.6 ± 2.8, respectively.

None of the females used to test the sterility of vasectomized males delivered kits and those whose uteri were examined post mortem revealed no implantation scars in their uteri, thus confirming the sterility of the vasectomized males. The recipients which did not whelp and received fresh (Figure 7a) or frozen-thawed embryos (Figure 7b) showed no implantation sites in their uteri. The recipients, which did not whelp and received vitrified-warmed embryos, were not examined. Two recipients died before the expected date of delivery.

Eleven recipients, which received fresh embryos, delivered a total of 72 live and normal kits 42 to 45 d after the first mating (i.e. 35 to 39 d after embryo transfer) (Table 18). Five recipients, which received cryopreserved embryos, delivered a total

72 of 17 live and normal kits 43 to 46 d after the first mating (i.e. 35 to 39 d after embryo transfer). One litter is shown in Figure 8.

Table 18. Sexes of the embryo transfer (ET) kits and the kits of the breeding females.

Sexes of the No. of Sexes of the Original born ET kits Breeding breeding born kits article Female Male NE Total season females Female Male Total II 24 41 7 72 1998 39 125 146 271 III 3 6 9 1999 29 99 82 181 IV 6 2 8 2001 12a +12b 48+38 39+38 163 Total 33 49 7 89 92 310 305 615 NE = not examined, the kits died before weaning afemales were mated during the first oestrus of the season bfemales were mated during the second oestrus of the season

Figure 7a. Freshly flushed Day 9 blastocysts. 7b. Frozen-thawed Day 9 blastocysts. Photos: Sergei Amstislavsky.

Figure 8. Farmed European polecats born after transfer of OPS vitrified-warmed embryos. Photo: Heli Lindeberg.

6 DISCUSSION

6.1 Early embryonic development

In this study, morulae were recovered between 5 and 9 d (120 h and 216 h) after the first mating and blastocysts from Day 6 (144 h) onwards. In earlier studies where embryos were flushed both for revealing early embryonic development or for other purposes, morula stage embryos have been recovered between 117.5 - 192 h after mating in the ferret - ferret/polecat hybrids (Robinson 1918, Hammond and Walton 1934, Hamilton 1934, Chang 1968, Daniel 1970, Kidder et al. 1999b). In these above studies and others (Marston and Kelly 1969, Chang 1969, McRae 1994), blastocysts have been recovered starting from 138 h after mating and oviductal blastocysts have been recovered as late as 192 h after mating in the ferret and ferret/polecat hybrids. These results are in agreement with our results in the farmed European polecat.

More than two thirds of the embryos had entered the uterus between Days 6 and 7 after the first mating, and nearly all of the embryos were recovered from the uterus on Day 8 after the first mating. Chang (1969) recovered 42% and 99% of ferret embryos from the uterus 5 d and 7 d after hCG treatment, respectively. Embryos started entering the uterus and completed their oviductal passage one day earlier in Chang's ferrets than in the farmed European polecats in the present study. This may be due to the fact that Chang (1969) used hCG to induce ovulation. Treatment with eCG has been reported to accelerate tubal transport in the stoat (Mustela erminea) (Amstislavsky et al. 1997a,b). It has also been demonstrated that after hCG treatment, unfertilized oocytes in the ferret were transported at a slower rate than embryos (Mead et al. 1988a).

Less than 10% of all females did not produce embryos after mating at the beginning of the breeding season. Starting on the second week of April, females were mated successfully so that vulval swelling receded indicating that ovulation had been induced. This is 1-2 weeks later than for a population of farmed European polecat females housed in typical fur animal houses with out-door cages in the research station. The low amount of natural light entering through the windows of the barn where our experimental animals were kept may have delayed the onset of fertile

75 oestrus. It has been demonstrated that female ferrets are completely dependent on the light ratio for their sexual cycles (Bissonnette 1932, Harvey and MacFarlane 1958).

Uterine swellings and implantation were observed in 4 females on Days 11, 12, 13 and 14 after the first mating (1 female on each day). Embryos of a fifth female were found to float freely in the uterus 13 d after first mating. Implantation in the ferret has been reported to occur between 12 and 13 d post coitum (Enders and Schlafke 1972). Mead et al. (1988b) demonstrated that 2 out of 4 females flushed 11 d after mating had implanted embryos in their uteri, which supports the findings of this study. On Days 11 and 12 it was possible to flush the uterine horns and find remnants of trophoectoderm and embryonic coats in the flushings, but on Days 13 and 14 it was impossible to pass the flushing medium through the uterine horns.

In conclusion, the timing of the recovery of 1- to 16-cell stage embryos, morulae and blastocysts and the timing of implantation are in agreement with earlier results, which have been achieved using animals exposed to artificial light conditions. The results of this study indicate that the timing of early embryonic development is similar between ferrets exposed to an artificial photoperiod and farmed polecats exposed to a natural photoperiod.

6.2 Evaluation of embryos

The shape of embryos in this study was either spherical or ovoid, as reported by Robinson (1918) in the domestic ferret and Amstislavsky et al. (1993b) in the stoat. Under the stereomicroscope, the same embryos may seem spherical from one angle and ovoid from another angle. The majority of the recovered embryos were of excellent or good quality. It is typical for in vivo produced embryos that the embryos representing the correct stage of development when compared to the days after first mating are usually of good quality. The sizes of Day 7-9 blastocysts measured without embryonic coat have been reported to be 200-850 µm (Chang 1968, Robinson 1918). Thus, the diameters of the polecat embryos are in agreement with earlier results. Blastocysts expand in distinct stages: during the first stage between 400-700 µm and in the second stage to >1 mm (McRae 1992). On Days 7 and 8, expanded blastocysts in mustelids rarely exceed 1 mm in diameter (Chang 1965a, 1968, Daniel 1970 and

76 personal observations), but after Day 9, the diameters of the large expanded blastocysts tend to exceed 1 mm (Marston and Kelly 1969, Daniel 1970).

During the expansion of the blastocysts, their coverings thinned. Retrospective calculations of volume of the coat showed that it increased as the blastocyst expanded and the coat thinned. In the ferret, it has been calculated that there is a 4-fold increase in volume of the coat from the morula to the late preimplantation stage (Enders 1971). However, the cause of this increase is unknown; it could be due to the volume of extra material which becomes attach to the coat or the swelling of the coat, this will represent a subject for further investigations in the polecat.

The morphology of the trophectoderm changed prior to implantation. The Day 13 expanded blastocysts that had not yet implanted had a brownish, opaque trophectoderm and a coat which was almost invisible under the stereomicroscope. Some of these Day 13 embryos had a horizontal belt-like bulge surrounding the conceptus. These structures may have been analogous to outgrowing endoderm and mesoderm. In the ferret, this sheet of endodermal cells is exceedingly thin and continuous although not yet forming a yolk sac by day 12 (Enders 1971).

Dissolution of the embryonic coat occurred between Days 11 and 14. During Days 11 and 12 collapsed empty coverings and remnants of brownish trophectoderms were apparent in the flushings. Empty coverings seemed to have ruptured from one location or occasionally some coverings were torn and had several fractures. This was different from that observed when culturing expanded blastocysts in vitro in this study. Shedding of the coats of expanded blastocysts was not seen in vitro as no empty coverings were observed in the media. Instead the coats seemed to have lysed as they were not detectable under the stereomicroscope. However, it is also possible that the coverings became very tightly attached to the trophectoderm and therefore were invisible at this stage of the development. The mechanism of hatching in vivo remains to be investigated in the polecat but it seems that it may occur through outgrowth from the coat rather than by lysis, possibly via the lateral walls of the antimesometrial regions where the thinning of the coat has been reported to be the greatest (Enders and Schlafke 1972).

6.3 Embryo recovery techniques

The post mortem recovery rate of 73% (I, II) in this study was similar to that achieved earlier in the domestic ferret (68%, Kidder et al. 1999a). In earlier post mortem recovery reports, the average number of flushed embryos per ferret has been 8.2 (Mead et al. 1988a) and 9.0 (Kidder et al. 1999a), compared to 9.8 in the present study.

This study describes the first surgical embryo collections in ferrets/polecats. Non- surgical uterine collection has resulted in the recovery of 8.7 embryos per ferret 6-7 days after hCG treatment and two matings 12 h apart (Kidder et al. 1999b) compared to 9.5 (III) and 7.8 (IV) embryos per polecat in this study with surgical collection from the uterus. The recovery rates of 60% (III, n=12 Day 8-9 donors) and 40% (IV, n=5 Day 6 donors) for surgical methods reported in this study are not the same as in the non-surgical method by Kidder et al. (1999b) who recovered 71% (324 embryos/389 corpora lutea; n = 37 donors). In the present study, CL numbers were not counted from all flushed donors and recovery from Day 6 donors was poor. The higher recovery rate in the study of Kidder et al. (1999b), may be attributable to the use of hCG resulting in a higher proportion of embryos in the uterus on Day 6 and by transcervical flushing excluding the interference resulting from insertion of cannulae during surgical recovery.

Surgical flushing was performed once with 20 ml of flushing medium in the present study. Repeating the flushing would probably not have increased the embryo yield as has been reported by Kidder et al. (1999b) who flushed transcervically 10 times with 1-3 ml. We have noticed that most of the embryos are expelled from the uterus along with the first 5 ml of flushing medium (personal unpublished observations).

The linea alba method (III) caused irreversible damage to the uteri so that this technique cannot be recommended for embryo recovery in mustelids. The uterine horns had adhered along the insertion line of the cannulae or sometimes adhered together. This reaction was assumed to be due to sensitivity of the progesterone dominated uterus, stretching of the uterine horns during the operation, touching them with latex gloves and the flushing medium which was at room temperature, factors

78 which may have contributed to these irreversible changes. It has been reported in the domestic cat that the number of adhesions declined as the degree of tissue handling decreased (Dresser et al. 1987). It is worth mentioning that the same embryo recovery technique during the same breeding season did not cause equivalent trauma into the uterine horns of blue fox females. The uterus of the cat and the polecat under progesterone dominance seems to be more sensitive than the uterus of the blue fox.

Surgical embryo recovery using the flank method (IVu) was used to recover embryos from donors on Day 6 to obtain non-expanded blastocysts, as we considered them better candidates for cryopreservation than the expanded blastocysts in the earlier cryopreservation study (III) using the conventional slow freezing technique. However, Day 6 does not seem to be a good choice as a flushing day because the embryo recovery results were disappointing. Embryos of Day 6 donors may still be in the oviducts or migrating into the uterus, but located in the very tips of the horns so that the surgical flushing does not reach them. Possibly the cannulae inserted in the horns remain in such a position that the flushing medium does not circulate optimally through the entire uterus, especially to the tip of the horns where the embryos are located on Day 6. Post mortem recovery of Day 6 donors does not seem to be an alternative, probably due to the fact that embryos are migrating into the uterus and are located at the junction between the horn and the oviduct. This is the location where the tissue is cut for flushing the oviduct and the horn separately, and the incision may have resulted in loss of embryos. Embryo recovery results improved on Days 7-8 indicating that embryos had migrated from the tips of uterine horns and become more readily recoverable. In addition, the use of a larger and longer i.v. cannula in our unpublished experiments has improved recovery results.

Seven (I, II, IV) or eight (III) transferable embryos were recovered from the uterus per single donor between Days 6 and 9 after the first mating. The embryo yield increased from Day 6 to Day 8, when flushing resulted in 8 (I, II, III) or 9 (IV) transferable uterine embryos per donor. The results of different methods are not fully comparable because the embryo flushings were performed on different years, which may have influenced the embryo yield. The lower embryo yields after post mortem recovery (I and II) may be due to the technique of cutting uterine horns into three pieces, causing loss of embryos. The surgical linea alba method (III) yielded high numbers of

79 embryos but was detrimental to the donors. The surgical flank method (IVu) did not harm the uterus and yielded 9 embryos on Day 8 which was one embryo more than obtained using the other methods suggesting that this might be the method of choice.

Post mortem and surgical techniques may be less time consuming than techniques traversing the cervix. However, post mortem embryo recovery is not acceptable when embryo transfer is used in an endangered species. Surgical invasive techniques of embryo transfer can cause tissue damage and complications, which may prevent the animals from reproducing in the future. This was the outcome of the surgical linea alba method (III) in this study. The surgical flank method (IVu) maintained the reproductive capacity of the donors indicating that this method can successfully be used for collection of embryos in the endangered European mink. This has recently been confirmed by Amstislavsky et al. (in press).

Development of a transcervical embryo recovery method is a possible option to overcome surgical complications and has already been reported in the domestic ferret by Kidder et al. (1999b). They used an expensive videoendoscope which may not be practical under farm conditions. Even the transcervical technique is not without its limitations, as it may result in cervical complications, which may prevent the donors from reproducing subsequently. Development of techniques which are both efficient and practical but which cause minimal inconvenience to the donors is always warranted in conservation programmes where loss of even a single individual can pose a threat to the existence of the entire species.

In conclusion, for a small donor, such as the polecat and particularly the even smaller European mink, the surgical embryo recovery using the flank method, worked perfectly in skillful hands, and the donors were able to reproduce after the operations. The flank method is undoubtedly a better option than post mortem collection for embryo recovery in an endangered species and may have application in experiments and in individual cases with a small captive population. A donor that has been flushed may conceive again during the second oestrus of the season which both ferret/polecat and the European mink females experience after the pseudopregnancy period.

6.4 In vitro culture

The in vivo fertilized 1-cell stage embryos required 120 h (5 d) to develop to blastocysts in vitro, which is in agreement with earlier reports (Whittingham 1975, Li et al. 2001). During the in vitro culture, 68% of the in vivo produced 1- to 16-cell stage embryos developed to blastocysts. Of these, 5 of 10 one to 2-cell stage embryos (50%), developed to blastocysts, which agrees with Li et al. (2001), who reported 64.5% and 47.1% of 1- to 2-cell stage embryos cultured in CZB or TCM-199 + 10% FBS, respectively, to develop to blastocysts. Whittingham (1975) cultured in vivo produced 1- to 8-cell stage ferret embryos and observed 75.6% (155/205) of the embryos to develop to blastocysts compared to 50% (7/14) in this study.

In this study the expansion of the in vitro cultured 1- to 16-cell stage embryos and morulae which developed into blastocysts was compromised when compared to their in vivo counterparts. This is in agreement with earlier results of in vitro culture of 1- to 2-cell stage ferret embryos (Li et al. 2001, Ziyi Li, personal communication ). In the domestic cat, blastocysts derived from in vivo morulae have been reported to expand to a greater extent than blastocysts derived from earlier stages of development (Roth et al. 1994, William Swanson, personal communication). The inability to expand in vitro may be due to the hardening of the zona pellucida (HZP). It has been reported that HZP significantly reduces the developmental competence of bovine IVM/IVF oocytes (Katska et al. 1999), and oviductal factors cause specific changes in the zona pellucida components (Kania et al. 2001). Shorter exposure of 1- to 16-cell stage embryos and morulae to oviductal environment may have caused changes in the zona and disturbed the in vivo like expansion of these embryos in vitro.

The diameter of the in vivo developed expanded blastocysts decreased during the in vitro culture. It is possible that due to suboptimal culture conditions, fluid transport mechanisms may have been disturbed, interfering with the accumulation of water inside the blastocoel (Barcroft et al. 2003). The composition of the culture medium was not defined for embryos close to implantation and these embryos may have different substrate and/or nutrient preferences than younger embryos (Bavister 1995). The embryos, especially those that were close to normal time of hatching and implanting in vivo, may have suffered from the absence of uterine signals (growth

81 factors, uterine proteolysins). It has been reported that hatching in vitro was delayed by more than 1 day in in vivo produced hamster blastocysts which were subjected to in vitro culture close to the normal time of hatching in vivo (Gonzales and Bavister 1995).

Although the in vitro cultured 1- to 16-cell and morula stage polecat embryos in this study developed readily into blastocysts, the culture system did not support the expansion of these blastocysts. It was not possible to count the cell numbers of the cultured blastocysts. In our unpublished experiments 1- to 16-cell stage embryos were flushed post-mortem on Day 2 (Day 0 = day of mating) from oviducts of the donors. These embryos were cultured in vitro in TCM-199 with the same ingredients as in this study. On Day 8 or 9, i.e. the embryos were cultured for 6 or 7 days in vitro, the cultured embryos (all blastocysts) were transferred into recipients. For the remaining embryos, which were not chosen for transfers but were stained, the cell numbers varied from 30 to 90. For Day 6 blastocysts of this study (i.e. developed to blastocysts in vivo, were flushed surgically, vitrified and warmed and then in vitro cultured), which were not chosen for embryo transfer but were stained, the cell numbers varied between 90 and 165. In this respect it may be speculated that regardless of the cell numbers the embryos were programmed to convert into blastocysts in vitro between Days 6 and 9 after the first mating which is also normal timing of polecat blastocyst formation in vivo. Cell numbers of embryos which develop into blastocysts in vitro may have been considerably smaller than of embryos forming blastocoel and expanding in vivo between Days 6 and 9 after the first mating. Cell numbers of in vivo blastocysts recovered between Days 6 and 9 after the first mating have been reported to range from 77 to 1,965 (Kidder et al. 1999a). Inadequate in vitro culture environment may have prevented the embryos from cleaving to adequate cell numbers needed for successful expansion. In addition, changing the microenvironment of the embryos during in vitro culture may have attributed to the loss of metabolic control (Gopichandran and Leese 2003) and increased the culture-induced stress of the embryos.

In this study the in vitro hatching of fresh in vivo blastocysts of polecat was reported for the first time, albeit at a low rate. Hatching in vitro was studied because it is generally considered as a viability test for frozen-thawed bovine morulae and

82 blastocysts if transfer into recipients is not possible. However, it is somewhat illogical to use hatching (i.e. escape from all blastocyst coverings) as a criterion of good development in vitro in species like carnivores, the horse and the rabbit, in which the blastocyst hatches from the coverings after a considerable expansion, and notably, in the ferret, in which the blastocysts have been reported to implant without total dissolution of the coverings (Enders and Schlafke 1972). Therefore hatching may not be considered as a good criterion of viability in vitro for the polecat embryos. A better viability test in vitro for in vivo derived blastocysts in the polecat would be the in vivo like expansion of the blastocysts rather than the hatching.

In conclusion, in order to maintain the developmental capacity of polecat embryos during in vitro culture, the culture period should be limited to 24 or 48 h with the medium used in this study. Further studies will be needed to determine which substrates and nutrients can support complete polecat embryo development in vitro and whether these embryos are capable of development to term after transfer.

6.5 Conventional slow freezing

A slow cooling process with a rate of 0.3 °C/min in a programmable freezer has been used successfully in freezing of embryos from a variety of species (Leibo and Songsasen 2002) and was therefore chosen also in this study. Intracellular freezing has not been observed when cooling mouse ova at a rate of 1.2 °C/min (Leibo et al. 1975) but has been calculated to occur when cooling mouse embryos at a rate faster than 1.5 °C/min (Mazur 1963). If the cooling is too fast, the embryos cannot lose water because of osmosis and therefore still contain intracellular water which might freeze. In the domestic cat (Dresser et al. 1988), blastocysts cooled with glycerol at 0.5 °C/min, thawed rapidly in a water bath of 37 °C and transferred into recipients survived equally well (11%) as the blastocysts in the present study (11%). Optimal cooling rates have not been determined for expanded blastocysts in species like the cat, the dog and the ferret whose embryos expand extensively during early embryonic development. Day 8 and Day 9 blastocysts of polecats are large in diameter (300-400 µm in this study), but individual cells of the trophectoderm and the inner cell mass are small. It is known that the larger the cells, the lower the cooling rate at which

83 intracellular ice formation occurs (Mazur 1963), and thus polecat blastocysts may even tolerate a higher cooling rate than that used in this study.

Rapid thawing of polecat embryos was conducted with a rate of 400-500 °C/min by warming the straws with exhaled air, which increased the thawing rate when compared to thawing at room temperature. If embryos are cooled slowly enough to contain no water, or only innocuous amounts, at the time of plunging into LN2 when their temperature is between -32 and -35 °C, they are able to tolerate wide ranges of warming rates as evidenced by Leibo et al. (1974) with mouse 2-cell stage embryos. This was probably not the case with polecat blastocysts which may have contained ice in the large fluid-filled blastocoel cavity. Rapid thawing might have prevented any ice crystals formed during slow cooling from agglomerating into lethal large crystals (Mazur 1977a). The survival rates of 56-58% following thawing of rabbit and sheep embryos at 650-1200 °C/min (Landa 1982, Heyman et al. 1987) suggest that an increased thawing rate may have rescued more polecat embryos.

Some frozen-thawed expanded blastocysts in this study were fragmented into pieces at thawing, probably due to the presence of intracellular ice. However, it may be possible that the fate of different sized blastocysts varied in the freezing process and some became injured in the slow cooling process by solution effects, i.e. mechanisms which are not connected to location of ice but associated with solutions and movement of water during freezing (Mazur 1977b). This may happen through severe volume shrinkage and long-term exposure to high electrolyte concentrations before all components in the embryos have solidified (Gao and Critser 2000). During thawing, the slowly cooled and shrunken embryos may swell and eventually lyse (Mazur et al. 1972). Kizilova et al. (1998) studied the morphology of steppe polecat (Mustela eversmanni) blastocysts after freezing with glycerol and DMSO, and linked the poor survival of the frozen-thawed blastocysts to the inability of glycerol and DMSO to provide sufficient protection against slow-freezing injury (i.e. "solution effects") during the step-wise addition and dilution of the cryoprotectants.

Sensitivity to chilling injury has been assumed to be connected to the amount of lipid in embryos of species like pigs and carnivores that contain numerous intracellular

84 lipid droplets. These droplets may either disrupt or induce heterogenous intracellular ice nucleation during freezing and thawing and have been considered to be the reason for the low survival after freezing and thawing of porcine and steppe polecat embryos (Toner et al. 1986, Kashiwazaki et al. 1991, Dobrinsky 1996, Kizilova et al. 1998). However, results with domestic cat embryos which also contain high amounts of lipid droplets have shown that even in vitro produced embryos have been successfully frozen using a slow, controlled rate freezing method and propanediol as the cryoprotectant. Furthermore, live kittens have been obtained after transfer of in vitro derived, frozen-thawed embryos (Pope et al. 1993, 1994, Swanson et al. 1999). Therefore, it remains to be studied whether the lipid droplets of polecat embryos increase the sensitivity of these embryos to chilling injury.

Ethylene glycol (EG) was easy to use under field conditions because it was added and diluted from sucrose-treated embryos at room temperature. Working at room temperature has resulted in a high survival rate of 73.0% (8 live lambs/11 transferred embryos) after transfer of frozen-thawed sheep embryos with one-step addition and removal of EG (McGinnis et al. 1993). EG may provide a better choice for a cryoprotectant than glycerol and DMSO in earlier transfer experiments of frozen- thawed European polecat embryos (Sergei Amstislavsky, unpublished results and personal communication) because none of the glycerol or DMSO-treated blastocysts of earlier studies but some of the EG-treated blastocysts of the present study developed to full term kits.

An intact zona pellucida is not necessary for successful cryopreservation because hatched and zona-dissected embryos survive through low temperatures (Vajta et al. 1996a,b). In the 1970s Willadsen et al. (1976) showed that sheep embryos containing zonal cracks but no visible damage to the blastomeres, could survive freezing and thawing. Of the frozen-thawed polecat blastocysts, one third had damaged embryonic coats and these were mainly expanded blastocysts. Of these embryos, only 4 expanded blastocysts had actual cracks in their coats after thawing but the majority had dents which made the shape of the blastocysts irregular. The irregularly shaped blastocysts had only partially expanded trophoectoderms inside their dented coats immediately after thawing. It was noted, however, that the partially expanded blastocysts looked unaltered inside their dented coats. The fully expanded

85 trophoectoderms were limited to those blastocysts with intact round coats. Whether these deformations have had an influence on embryo survival is unknown but, in the domestic cat, embryos with disrupted zonae at thawing did not develop past the 20- cell stage in vitro (Pope et al. 1994). Bovine embryos regained the original shape of the zonae when the ice crystals thawed (Lehn-Jensen and Rall 1983) but in the polecat the dented shape persisted until transfer in groups thawed with or without sucrose. The large diameter of polecat blastocysts may have increased their susceptibility to coat deformations during the freezing process.

Seven of the 18 donors that had their blastocysts frozen-thawed had brownish oviductal or uterine material on the embryonic coat. None of these blastocysts developed to term kits after embryo transfer which raised a question whether this material affects the freezability of polecat embryos. The coverings of later stage polecat embryos may be analogous with the equine capsule. On the other hand, the brownish material may be dead cell mass attached to the coat and therefore not directly analogous to equine capsular material which has been reported to interfere with the freezability of large equine embryos by impeding penetration of cryoprotectants (Legrand et al. 2000). Entsymatic treatment of the capsule allows for successful cryopreservation of large equine blastocysts (Legrand et al. 2002).

The frozen-thawed polecat embryos were transferred either straight after thawing without dilution of EG or after a one-step dilution in 0.5 M sucrose. Viable offspring have been produced by direct transfer of embryos without EG dilution in cattle (Dochi et al. 1995) and in the horse (Ulrich and Nowshari 2002). After sucrose treatment, 2 polecat kits were born compared to 7 kits after direct transfer without EG dilution suggesting that sucrose treatment may not be necessary for frozen-thawed polecat embryos. In addition, direct transfer without dilution is clearly more convenient under farm conditions.

In conclusion, the results of this study suggest that the protocol we used may be more suitable for non-expanded blastocysts than for expanded blastocysts, because retrospective examination of the size of the embryos revealed that more than half of the non-expanded blastocysts (12/21) were transferred into those 3 recipients that gave birth. During the cryopreservation process, some embryos will always lose their

86 viability, but in these studies the survival rate of only 11% after transferring frozen- thawed embryos was almost four times lower than the survival rate of 42% after transferring fresh embryos, indicating either that the cryoprotectant had not been able to provide enough protection, or that the rate of cooling and thawing were suboptimal. Nonetheless, some of the embryos did survive and produced the first mustelids born from frozen-thawed embryos.

6.6 Vitrification

In this study, the in vitro survival rate of vitrified-warmed embryos was 51% whereas in vivo survival after transfer of in vitro survived embryos was 16%. This agrees with the earlier results in which post-warming in vitro survival rates for in vivo embryos after OPS vitrification have been reported to vary between 27-93.5% (pig: 27-67%; Berthelot et al. 2000, mouse: 93.5%; Kong et al. 2000, pig: 33-59%; Beebe et al. 2002, rabbit: 44-88%; López-Béjar and López-Gatius 2002). In the pig, the in vivo survival rate has varied between 10-13% (Berthelot et al. 2000, 2001) whereas in the rabbit it has been higher, 42-52% (López-Béjar and López-Gatius 2002) and even higher in the sheep, 58-73% (Isachenko et al. 2003a) and in the goat 64% (El-Gayar et al. 2001).

In the present study, in vitro survival of Day 6 and Day 7 embryos was better than that of Day 8 embryos. The embryos were pooled after warming for in vitro culture and cultured to blastocysts, so it was not possible to determine which day embryos developed to term after transfer. However, it did seem that expanded blastocysts in particular failed to survive vitrification with the method used. Survival rates of vitrified-warmed in vivo blastocysts of mice have been shown to decrease as the blastocoel enlarges (Shaw et al. 1991, Miyake et al. 1993). Expanded blastocysts may be more sensitive to high concentrations of EG than embryonic cells at other stages of development. In the mouse, toxicity tests revealed that blastocysts were more sensitive than morulae to high concentrations of EG (Kasai et al. 1990, Zhu et al. 1993). On the contrary, perihatching blastocysts in the pig (Berthelot et al. 2000, Beebe et al. 2002) and expanded blastocysts in the sheep (Isachenko et al. 2003a) have developed to term after OPS vitrification. In the bovine, the re-expansion of hatched in vitro produced blastocysts vitrified on Day 8 has been reported to be 81%

87 by the OPS method (Vajta et al. 1998a). In this study, three bull calves were achieved following transfer after OPS vitrification at both the oocyte and blastocyst stage.

It seems that each stage of development has different demands for vitrification. The permeability of an embryo to the cryoprotectant changes with the stage of development (Kasai 1995). In order to obtain viable offspring after transfer of a wide range of developmental stages, the vitrification solution should be optimised, i.e. one has to determine the optimal temperature, equilibration time, composition and concentration, for each stage of development separately.

We incubated Day 8 expanded blastocysts for 3 min in EG + DMSO and then 25 - 45 sec in EG, DMSO and sucrose. The incubation time for early morulae, morulae, and Day 6 and Day 7 non-expanded blastocysts was longer, 4 min and 30 sec. Since individual cells of blastocysts are smaller in size, the cryoprotectant permeates them more rapidly, and therefore blastocysts might not need to be incubated as long as cells at younger stages of development. Both of the cryoprotectants used, EG and DMSO, are highly permeant (Kasai et al. 1990, Kasai 1996). First they enter the cells and then the blastocoel cavity. However, the incubation period of expanded blastocysts may have been too short to allow sufficient amounts of cryoprotectants to permeate into the cells and the blastocoel cavity. In fact, most of the expanded Day 8 blastocysts were totally destroyed during warming, i.e. they shattered into small pieces either inside the embryonic coat or including the coat, indicating massive cryodamage. Longer exposure to cryoprotectants has been reported to improve the survival of in vivo blastocysts at a lower incubation temperature in the mouse (Shaw et al. 1991) and at 39 °C in the pig (Berthelot et al. 2000). An increase in the exposure time in the vitrification solution could have provided better survival for Day 8 expanded blastocysts than was observed in the present study. However, on the other hand, a longer exposure period increases the possibility of toxic actions of EG and DMSO.

In conclusion, OPS vitrification can be considered as a promising technique for cryopreservation of mustelid embryos. Compared to the conventional slow freezing, OPS vitrification does not require a freezing machine but does require craftmanship in processing the embryos through vitrification and warming. After solving technical

88 problems in cryopreserving the expanded blastocyst stage embryos in the polecat, OPS vitrification will become the method of choice for cryopreservation under farm conditions.

6.7 Surgical transfer of fresh, frozen-thawed and vitrified-warmed embryos

A total of 89 kits were born to 28 recipient females. The overall survival rate of 30% including fresh (42% survival, II), frozen-thawed (11% survival, III) and vitrified- warmed (16% survival, IV) embryos was similar to those reported earlier after transfer of fresh embryos in the domestic ferret (Chang 1968: survival rate 33%, surgical transfer; Kidder et al. 1999b: survival rate 26%, nonsurgical transfer) and in the American mink (Zhelezova and Golubitsa 1978: survival rate 26%, surgical transfer; Adams 1982: survival rate 25%, surgical transfer). The overall kit result (an average of 3 kits/recipient) after the transfer of an average of 10 embryos/recipient was higher or similar to those obtained in earlier studies in the ferret, except that Li et al. (2001) obtained an average of 9 kits/recipient after transferring an average of 18 embryos/recipient.

Reasons for not producing kits after embryo transfer may be related to several factors: weight of the recipients, synchronization of donors and recipients, cryopreservation of embryos, number of transferred embryos, embryo processing before transfer or the transfer technique itself. During the first year of this study it was noticed that better kit results (6.4 versus 3 kits/recipient) and higher survival rates (52% versus 32%) were achieved when the recipient weighed >1 kg (12.4 embryos transferred per recipient) compared to <1 kg (9.4 embryos transferred per recipient). Females weighing under 1000 g were excluded from embryo transfer programmes in the subsequent years.

Induction of ovulation was used to synchronize donors and recipients. More kits were born (6.8 versus 3.1 kits/recipient) and higher survival rates achieved (52% versus 33%) after transfer of embryos from multiple donors (13.2 embryos transferred per recipient) than from a single donor (9.3 embryos transferred per recipient). Vicente and García-Ximénez (1993) transferred frozen-thawed rabbit embryos from a single donor versus two donors into recipients (9 embryos versus 16 embryos). The survival

89 rates were higher but not significantly higher for the group of two donors than for the group of single donors. Li et al. (2001) pooled the ferret embryos, and 50% continued embryonic development to full term kits. The use of multiple donors seemed to increase the number of transferred embryos per recipient as did synchrony between donors and recipients, which is required to obtain normal development as demonstrated by Chang (1969) in the domestic ferret. Additionally, altered embryo development conditions after embryo transfer may have a positive effect on survival as has been reported in the pig (Kvasnitski 2001).

In this study, all of the recipients which received ≥12 transferred embryos gave birth. More embryos were required for birth of kits if the embryos had been frozen-thawed or vitrified-warmed (13 embryos on average) rather than if the embryos were fresh (11 embryos on average). Pope et al. (1993) reported more pregnancies in the domestic cat after transfer of ≥12 embryos than <12 embryos. However, live offspring have been born after transfer of 3 and 6 fresh embryos (McRae 1994, Li et al. 2001). In our study, ≥12 embryos were transferred only to 36% (10/28) of the recipients. It seems that increasing the number of transferred embryos per recipient could well have increased the survival rate (= live kits/transferred embryos).

When transferring vitrified-warmed embryos, in vitro culture for a couple of hours did not affect the synchronization but culture periods longer than 24 hours seemed to compromise further development. The 7 embryos of the first OPS recipient were warmed too early by mistake and were cultured for 3 days. This may have accelerated development during these 3 days, and thus, the embryos were actually Day 11 embryos which were being transferred into a Day 8 uterus, or perhaps the culture period had compromised the developmental capacity of these embryos and they were no longer competent at transfer. An asynchrony of 2 days (but not 3 days) between donor and recipient has been reported to be acceptable for production of viable offspring (Chang 1969; own personal observations). Also, culture periods longer than 24-48 hours in TCM-199 medium in this study compromised the developmental capacity of the polecat in vivo embryos when compared to development in vivo. This indicates that extended in vitro culture of embryos after vitrification decreases the chances of survival. Therefore direct transfer without in vitro culture or a short culture

90 period <24 hours may rescue the transferred vitrified-warmed embryos and increase the kit yield.

In this study the technique of surgical embryo transfer was less traumatic to the females than the surgical embryo recovery. The recipients which did not whelp have been subsequently used by mating them during the second oestrus of the season. All that have conceived (75%, 9/12) have reproduced normally. This indicates that the technique of embryo transfer used throughout this study is not detrimental to further reproduction and can be applied in programmes designed to preserve endangered mustelids (Amstislavsky et al., in press).

The recipients gave birth to more male than female offspring after embryo transfer. During the corresponding years 1998-99 and 2001 on the research farm, there were no significant differences between the sexes of the kits of the breeding females and the embryo transfer kits. However, when combining the three years it seems that the treatment of embryos significantly favors male offspring (P=0.040, nonparametric binomial test). Also, several reports have described male-biased sex ratio of cattle and mouse preimplantation embryos among blastocyst stage after in vitro culture (Valdivia et al. 1993, Sato et al. 1995, Gutiérrez-Adán et al. 1996, Carvalho et al. 1996, Pegoraro et al. 1998, Peippo 2001). For conservation purposes, excess production of male offspring by assisted reproductive technology is undesirable, as increased efforts are required to produce sufficient number of breeding females.

7 CONCLUSIONS

Early embryonic development in the farmed European polecat is a process that is typical of all carnivores: oviductal passage is slow, the embryos enter the uterus on Day 6 after the first mating and implantation occurs late, between Days 11 and 14 after the first mating. The embryos enter the uterus mainly as morulae, some as early morulae and some as early blastocysts. On Days 6 and 7, nearly 80% of embryos can be flushed from the uterus and on Day 8 almost all embryos have arrived in the uterus. It can be concluded that for the best recovery results, surgical embryo recovery ought to be performed on Day 8. However, 50% of polecat embryos have already expanded to a large size on Day 8 and become difficult to handle and cryopreserve using the methods described in this study. Embryo recovery on Day 7 results in smaller blastocysts which are more suitable for embryo transfer but 20% of the recovered blastocysts have already expanded too much to stand cryopreservation. Embryo recovery on Day 6 would be the best solution for cryopreservation because blastocysts have not yet expanded, but the recovery rate on Day 6 is only around 30%. In conclusion, in the present situation, the best compromise seems to be the selection of Day 7 after the first mating as the optimal day to harvest embryos for cryopreservation in the farmed European polecat using surgical embryo recovery method and cryopreservation methods described in this study. However, after development of a reliable method for cryopreservation of expanded blastocyst stage embryos in the polecat, embryo recoveries can be performed on Day 8 which results in good embryo recovery rates.

Earliest embryonic stages that developed to term after transfer into the uterus consisted of 8- to 16-cell stages. Kits were produced also from fresh, frozen-thawed and vitrified-warmed morulae and blastocysts and both fresh and frozen-thawed expanded blastocysts. All recipients receiving ≥12 embryos produced kits.

An asynchrony of ±1 day between a donor and a recipient did not affect the birth outcome. A reasonable survival rate (= live kits/transferred embryos) of 52% was achieved when fresh embryos were transferred to recipients weighing more than 1 kg. Polecat recipients weighing more than 1 kg gave birth to an average of 6 kits

92 compared to 3 kits for recipients weighing less than 1 kg suggesting that light recipients (< 1 kg) should not be used in embryo transfer programs.

Cryopreservation seemed particularly harmful for expanded blastocysts as the majority of these embryos did not develop to term after freezing, thawing and transfer and most vitrified-warmed expanded blastocysts were totally destroyed. The compromised results of OPS vitrified-warmed expanded blastocyst stage embryos may have been a consequence of a minor technical problem (incubation time, temperature, concentration of cryoprotectans) that will undoubtedly be solved in future studies.

Surgical flushing is more sensitive to complications than surgical transfer, and further developmental work needs to be done to improve the flushing and transfer methods. The surgical flank method results in high embryo yields from Day 7 and Day 8 donors, does not cause adhesions or jeopardize further reproduction of the donors and is undoubtedly a good option for embryo recovery.

Timing of blastocyst formation in vitro did not differ from that in vivo. Expansion of blastocysts in vitro was comparable to expansion in vivo during the first 24-h period for embryos placed in culture as morulae, early blastocysts or as expanded blastocysts which were considered to expand further. Embryos placed in culture at earlier stages of development failed to expand at all after reaching the blastocyst stage in vitro. These results indicate that polecat in vivo embryos can be maintained in vitro, in culture medium designated for cattle, for a period of 24-h.

If one is to use large-scale embryo transfer programs effectively in a conservation programme, the kit yield should be increased. Our results are encouraging and the techniques may be applicable in experimental populations or in the management of small populations of endangered European mink in individual cases. Reliable methods of embryo collection and transfer already exist for immediate embryo transfer, and methods of semen collection, cryopreservation and transcervical artificial insemination are being developed.

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