Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Cryopreservation and transplantation of gonadal tissue for genetic conservation and biological research… Liu, Jianan 2013

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2013_fall_liu_jianan.pdf [ 2.96MB ]
Metadata
JSON: 24-1.0165518.json
JSON-LD: 24-1.0165518-ld.json
RDF/XML (Pretty): 24-1.0165518-rdf.xml
RDF/JSON: 24-1.0165518-rdf.json
Turtle: 24-1.0165518-turtle.txt
N-Triples: 24-1.0165518-rdf-ntriples.txt
Original Record: 24-1.0165518-source.json
Full Text
24-1.0165518-fulltext.txt
Citation
24-1.0165518.ris

Full Text

  CRYOPRESERVATION AND TRANSPLANTATION OF GONADAL TISSUE FOR GENETIC CONSERVATION AND BIOLOGICAL RESEARCH IN AVIAN SPECIES  by  JIANAN LIU  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENT FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Applied Animal Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2013  ? JIANAN LIU, 2013 ii  ABSTRACT Avian researchers and the poultry industry have experienced a massive loss of genetic resources due to the high cost of maintaining live flocks.  Cryobanking of germplasm is economical and ensures long-term availability of genetic resources.  Cryopreservation and transplantation of avian gonadal tissue allow effective preservation and recovery of female germplasm and provide an alternative to semen cryobanking.  A vitrification protocol including dimethyl sulphoxide, ethylene glycol and sucrose as cryoprotective agents and the use of acupuncture needles to facilitate tissue handling has been successful in preserving Japanese quail ovarian tissue.  Its effectiveness in preserving testicular tissue is unknown and an efficient storage system is needed.   The vitrification protocol was first adapted to a 0.5-ml straw system and used to cryopreserve quail ovarian tissue, which showed no significant impairment of viability or vascularization compared to fresh tissue after in ovo culture.  This system was then replaced by a simpler 2-ml straw system and applied to quail testicular and ovarian tissue.  Normal morphology of testicular tissue was observed after in ovo culture and live offspring were produced by intramagnal insemination of the extrusion retrieved from allotransplants of cryopreserved testicular tissue.  Donor-derived offspring were also efficiently produced from cryopreserved and allotransplanted ovarian tissue.    Gonadal transplantation is critical to functional recovery of cryopreserved tissue but can be limited by tissue rejection.  Donor thymic tissue was implanted into recipient embryos and gonadal tissue from the same donor was transplanted ectopically to the recipient after hatching.  Transplant viability and histology was examined.  Thymic implantation may iii  improve survival of the allogeneic gonadal transplants in chickens but not the xenotransplants from quail to chickens.  Investigations of avian ovarian transplantation have led to intriguing additional observations.  Donor-derived offspring were produced from transplanted adult quail ovarian tissue, although delayed age at first egg and reduced reproductive longevity were observed with the transplants.  As well, offspring with chimeric plumage coloration were produced from cryopreserved and transplanted chicken ovarian tissue, indicating chimeric folliculogenesis. This dissertation provides a model of cryobanking avian gonadal tissue using a simple vitrification method, and suggests future directions in improving transplantation tolerance and using gonadal transplantation in avian research.     iv  PREFACE For this dissertation, I was the lead investigator responsible for major areas of concept formation, data collection and manuscript composition.  Cheng KM was the academic supervisor who was involved in concept formation and manuscript preparation.  Silversides FG was the research supervisor who was involved throughout the project in concept formation and manuscript preparation.   A version of Chapter 1 has been published [Liu, J., K. M. Cheng, and F. G. Silversides. 2013. Fundamental principles of cryobiology and application to conservation of avian species. Avian Biol. Res. 6 (3):187-197].   A version of Chapter 2 has been published [Liu, J., K. M. Cheng, and F. G. Silversides. 2012. Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica). Anim. Reprod. Sci. 134:197-202].  The research protocol was approved by the Animal Care Committee of the University of British Columbia (Certificate Number A11-0045). A version of Chapter 3 has been published [Liu, J., K. M. Cheng, P. H. Purdy, and F. G. Silversides. 2012. A simple vitrification method for cryobanking avian testicular tissue. Poult. Sci. 91:3209-3213].  Purdy PH was involved in the early stages of concept formation and contributed to manuscript edits.  The research protocol was approved by the Animal Care Committee of the University of British Columbia (Certificate Number A11-0045). v  A version of Chapter 4 has been published [Liu, J., K. M. Cheng, and F. G. Silversides. 2013. Production of live offspring from testicular tissue cryopreserved by vitrification procedures in Japanese quail (Coturnix japonica). Biol. Reprod. 88:124, 1-6].  The research protocol was approved by the Animal Care Committee of the University of British Columbia (Certificate Number A11-0045). A version of Chapter 5 has been accepted for publication [Liu, J., K. M. Cheng, and F. G. Silversides. 2013. A model for cryobanking female germplasm in Japanese quail (Coturnix japonica). Poult. Sci. (in press)].  The research protocol was approved by the Animal Care Committee of the University of British Columbia (Certificate Number A11-0045). A version of Chapter 7 has been presented to 2013 Poultry Science Association Annual Meeting [Liu, J., K. M. Cheng, and F. G. Silversides. 2013. Production of donor-derived chicks from transplantation of adult ovarian tissue in Japanese quail (Coturnix japonica). Poult. Sci. 92 (E-Supplement 1): 18.] and submitted to Reproduction, Fertility and Development for publication [Liu, J., K. M. Cheng, and F. G. Silversides. 2013. Recovery of fertility from adult ovarian tissue transplanted into week-old Japanese quail chicks. Reprod. Fertil. Dev. (Manuscript No. RD 13256)].  The research protocol was approved by the Animal Care Committee of the University of British Columbia (Certificate Number A11-0045). A version of Chapter 8 has been published [Liu, J., M. C. Robertson, K. M. Cheng, and F. G. Silversides. 2013. Chimeric plumage coloration produced by ovarian transplantation in chickens. Poult. Sci. 92:1073-1076].  Robertson MC contributed to data vi  collection and manuscript edits.  The research protocol was approved by the Animal Care Committee of the University of British Columbia (Certificate Number A11-0045).   vii  TABLE OF CONTENTS  Abstract ........................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of contents........................................................................................................................... vii List of tables .................................................................................................................................... x List of figures ................................................................................................................................ xii List of abbreviations .................................................................................................................... xiv Acknowledgements........................................................................................................................ xv Dedication ................................................................................................................................... xvii 1 Introduction ................................................................................................................................... 1 1.1 Fundamental cryobiology and its application in preserving avian germplasm ..................... 1 1.1.1 Fundamental principles of cryobiology .......................................................................... 1 1.1.2 Cryopreservation strategies in general............................................................................ 3 1.1.3 Cryobiology of animal and human germplasm .............................................................. 6 1.1.4 Cryopreservation of germplasm in avian species ......................................................... 15 1.2 Transplantation immunology ............................................................................................... 20 1.2.1 Immunity and tolerance ................................................................................................ 21 1.2.2 Mechanisms of transplant rejection .............................................................................. 25 1.2.3 Prevention of transplant rejection ................................................................................. 27 1.2.4 Induction of tolerance in avian gonadal transplantation ............................................... 33 1.3 Functional recovery of avian gonadal transplants ............................................................... 34 1.3.1 Transplantation of avian testicular tissue ..................................................................... 35 1.3.2 Transplantation of avian ovarian tissue ........................................................................ 37 1.4 Objectives ............................................................................................................................ 39 2 Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica)  ................ 48 2.1 Introduction .......................................................................................................................... 48 2.2 Materials and methods ......................................................................................................... 50 2.2.1 Birds, chemicals and tissue preparation........................................................................ 50 2.2.2 Vitrification and warming procedures .......................................................................... 51 2.2.3 Histological examination .............................................................................................. 52 2.2.4 Tissue grafting onto chicken embryo chorioallantoic membrane ................................. 52 viii  2.2.5 Statistical analyses ........................................................................................................ 53 2.3 Results and discussion ......................................................................................................... 54 3 A simple vitrification method for cryobanking avian testicular tissue ....................................... 63 3.1 Introduction .......................................................................................................................... 63 3.2 Materials and methods ......................................................................................................... 65 3.2.1 Birds, chemicals and tissue preparation........................................................................ 65 3.2.2 Vitrification and warming procedures .......................................................................... 66 3.2.3 Tissue grafting onto chicken embryonic chorioallantoic membrane ............................ 67 3.2.4 Histological examination .............................................................................................. 68 3.2.5 Statistical analyses ........................................................................................................ 68 3.3 Results and discussion ......................................................................................................... 68 4 Production of live offspring from testicular tissue cryopreserved by vitrification procedures in Japanese quail (Coturnix japonica)......................................................................... 75 4.1 Introduction .......................................................................................................................... 75 4.2 Materials and methods ......................................................................................................... 77 4.2.1 Birds, chemicals and tissue preparation........................................................................ 77 4.2.2 Vitrification and warming procedures .......................................................................... 77 4.2.3 Testicular allografting ................................................................................................... 78 4.2.4 Histological examination .............................................................................................. 79 4.2.5 Intramagnal insemination ............................................................................................. 80 4.2.6 Statistical analysis ......................................................................................................... 81 4.3 Results .................................................................................................................................. 81 4.3.1 Survival and growth of testicular transplants ............................................................... 81 4.3.2 Production of offspring by intramagnal insemination .................................................. 82 4.4 Discussion ............................................................................................................................ 82 5 A model for cryobanking female germplasm in Japanese quail (Coturnix japonica) ................ 94 5.1 Introduction.......................................................................................................................... 94 5.2 Materials and methods ......................................................................................................... 95 5.3 Results and discussion ......................................................................................................... 97 6 Induction of immunological tolerance for gonadal transplantation in avian species by implanting donor thymic tissue into recipient chorioallantoic membrane .................................. 102 6.1 Introduction........................................................................................................................ 102 6.2 Materials and methods ....................................................................................................... 104 6.2.1 Birds and chemicals .................................................................................................... 104 6.2.2 Preparation of donor gonadal and thymic tissue ......................................................... 104 ix  6.2.3 Implanting donor thymic tissue into recipient embryos ............................................. 104 6.2.4 Gonadal transplantation and histological examination ............................................... 106 6.2.5 Statistical analysis ....................................................................................................... 107 6.3 Results ................................................................................................................................ 107 6.4 Discussion .......................................................................................................................... 108 7 Recovery of fertility from adult ovarian tissue transplanted into week-old Japanese quail chicks ........................................................................................................................................... 118 7.1 Introduction........................................................................................................................ 118 7.2 Materials and methods ....................................................................................................... 119 7.3 Results and discussion ....................................................................................................... 120 8 Chimeric plumage coloration produced by ovarian transplantation in chickens ...................... 127 8.1 Introduction........................................................................................................................ 127 8.2 Materials and methods ....................................................................................................... 128 8.3 Results and discussion ....................................................................................................... 130 9 General discussion .................................................................................................................... 135 9.1 Cryoconservation of avian germplasm .............................................................................. 135 9.2 Use of avian gonadal transplantation for biological research ............................................ 138 9.3 Conclusion ......................................................................................................................... 140 Bibliography ................................................................................................................................ 143     x  LIST OF TABLES Table 1.1.  A general comparison of innate and adaptive immunity ...................................... 41 Table 1.2.  Embryonic transplantation experiments conducted by Le Douarin's group ......... 42 Table 2.1.  Viability and vascularization of ovarian tissues grafted onto chicken chorioallantoic membrane ...................................................................................................... 58 Table 3.1.  Vascularization of the testicular grafts and viability of the host embryos ........... 71 Table 4.1.  Transplantation of cryopreserved and warmed testicular tissue in Japanese quail ................................................................................................................................................ 87 Table 4.2.  Production of live offspring by intramagnal insemination using spermatozoa retrieved from the testicular transplants (mean ? SEM) ......................................................... 88 Table 4.3.  Histological examination of the testicular transplants (mean ? SEM) ................. 89 Table 4.4.  Performance of hens inseminated with spermatozoa retrieved from testicular transplants (mean ? SEM) ...................................................................................................... 90 Table 4.5.  Production of eggs and chicks from hens producing live offspring ..................... 91 Table 5.1.  Body weight and 56-day egg production (mean ? SEM) starting at 17-20 wk old quail transplanted with cryopreserved ovarian tissue ........................................................... 100 Table 5.2.  Four wk of progeny test of quail recipients transplanted with ovarian tissue that were cryopreserved by vitrification ...................................................................................... 101 Table 6.1.  Hatchability of recipient embryos according to the implantation techniques and transplantation types ............................................................................................................. 112 Table 6.2.  Macroscopic appearance and histological examination of gonadal transplants in allotransplantation in chickens ............................................................................................. 113 xi  Table 6.3.  Macroscopic appearance and histological examination of xenotransplants from quail to chickens ................................................................................................................... 114 Table 7.1.  Reproductive performance of recipients that laid eggs and control birds (mean ? SEM) ..................................................................................................................................... 124 Table 7.2.  Reproductive performance of recipients that produced offspring during progeny test ......................................................................................................................................... 125 Table 9.1. Status of germplasm cryopreservation in avian species ...................................... 142   xii  LIST OF FIGURES Figure 1.1.  Schematic phase diagram of a cryoprotective agent (CPA) solution .................. 43 Figure 1.2.  Cycle of germline development .......................................................................... 44 Figure 1.3.  A schematic drawing of major structural features of spermatozoa from different species ..................................................................................................................................... 45 Figure 1.4.  A possible method of inducing allo- and xenogeneic tolerance for avian gonadal transplantation......................................................................................................................... 46 Figure 1.5.  Schematic diagram of the oviduct of a mature hen ............................................. 47 Figure 2.1.  Needle-in-straw (NIS) vitrification device .......................................................... 59 Figure 2.2.  Histology of ovarian tissue .................................................................................. 60 Figure 2.3.  Grafting ovarian tissue onto chicken chorioallantoic membrane (CAM) ........... 61 Figure 2.4.  The ratio of normal follicles to total visible follicles .......................................... 62 Figure 3.1.  A demonstration of the vitrification device used in this study ............................ 72 Figure 3.2.  Japanese quail testes grafted to chicken chorioallantoic membrane (CAM) ...... 73 Figure 3.3.  Appearance of Japanese quail testes at the time of isolation and 9 d after grafting ................................................................................................................................................ 74 Figure 4.1.  Testicular transplants and chicks produced by intramagnal insemination using the extruded fluid .................................................................................................................... 92 Figure 4.2.  Histological examination of testicular tissue ...................................................... 93 Figure 6.1.  Macroscopic appearance and histology of allotransplants ................................ 115 Figure 6.2.  Survival curves based on the histology of the allogeneic gonadal transplants . 116 Figure 6.3.  Macroscopic appearance and histology of xenotransplants .............................. 117 Figure 7.1.  Production of offspring from transplantation of adult ovarian tissue ............... 126 xiii  Figure 8.1.  Chicks produced from progeny test .................................................................. 133 Figure 8.2.  Possible mechanisms for chimeric folliculogenesis .......................................... 134    xiv  LIST OF ABBREVIATIONS B cell  Bursa-derived cell BPR  Barred Plymouth Rock  CAM  Chorioallantoic membrane CPA  Cryoprotective agent  DMSO  Dimethyl sulphoxide  DPBS  Dulbecco?s phosphate buffered saline E  Extended black allele  EG  Ethylene glycol  Fab  Fragment antigen binding Fc  Fragment crystallisable HM  Handling medium  I  Dominant white allele  MHC  Major histocompatibility complex  NIS  Needle-in-straw NIV  Needle immersed vitrification  RIR  Rhode Island Red RT  Room temperature S  Silver allele    T cell  Thymus-derived cell Tg   Glass transition temperature  Th   Homogeneous nucleation temperature Tm   Melting temperature  WB  White Breasted WL  White Leghorn xv  ACKNOWLEDGEMENTS One of the very useful strategies I learned from Dr. Fred Silversides is to regard giving a presentation or writing a paper as an opportunity to share a story.  As what Dr. Wilmut said in his book The Second Creation (2000) about the creation of Dolly, ?The story may seem a bit messy, but that?s because life is messy, and science is a slice of life.? Scientific progress, which can be as huge as the birth of Dolly or as humble as what you are going to read in this dissertation, cannot come into being without involvement and interconnections of many people.  I sincerely thank anyone of them for coming into a very valuable period of my life to make this story happen, including those who are not highlighted here.    I would like to thank my research supervisor Dr. Fred Silversides, who guided and trained me with penetrating questions, constant encouragements, and enormous patience.  He was always the first person to whom I shared with my delightfulness of discovery and my perplexity about our research or my career.  As a hatchling to the academia, I?ve learned so much from him, the most important is to do curiosity-driven research with a practical style and an open mind, which will have profound and positive impacts on my academic life.  I would like to thank my academic supervisor Dr. Kim Cheng.  It is his offer that enables me to start my academic life in a fascinating field with such an adventure.  His candid comments on the manuscripts not only made them better but also helped me to improve my skills in preparing scientific manuscripts.  I thank Drs. Ronaldo Cerri and Anthony Cheung for being on my supervisory committee and providing guidance and advice on my research and the draft of this dissertation.  The Poultry Industry Council, Canadian Poultry Research Council and Egg Farmers of Canada are acknowledged for providing a grant to support this research.   I wish to thank Dr. Yonghong Song, whose pioneering work on avian gonadal transplantation has laid the foundation of this field, which I cite extensively in this dissertation.  Thanks to the poultry staff of Agassiz Research Centre for taking care of the experimental birds.  Thanks to Dr. Dave Gillespie for providing administrative support to me.  Drs. Tom Forge and Sheila Fitzpatrick, and Carol Koch are also acknowledged for providing the equipment for microphotography. Thanks to all the fellow students and researchers in Dr. Kim Cheng?s lab for all the encouragements that helped me to get through the difficulties and for being the witnesses of many important moments in my life in Canada.  xvi  Last, but certainly not least, I am indebted to my parents and my brother who have been feeling a lot of anxiety about my oversea life but have been showing absolute support and incredible understanding.    xvii  DEDICATION  To my grandma, Yuhua Lian 1  1 INTRODUCTION1 1.1 Fundamental cryobiology and its application in preserving avian germplasm The term cryobiology was first coined by Parkes (1964), who gave a definition as ?the study of the biological effects of low temperatures?.  The principles of cryobiology are fundamental to divergent areas including preservation of biological materials, understanding natural cold tolerance, food sciences and cryosurgery (Mazur, 1970).  This section will focus on understanding the effects of physical-chemical events on cellular systems during cooling and warming procedures and the application of this knowledge in preserving animal germplasm, with special reference to avian species.    1.1.1 Fundamental principles of cryobiology Water is the most important component of a cellular system and the phase transition of water and its biological effects is the central topic of cryobiology.  When temperature is lowered below the melting temperature (Tm) or equilibrium freezing point at a given pressure, freezing will not take place unless a nucleator with a critical size is present to trigger crystallization.  Unfrozen water that is below the melting point is supercooled (Mishima and Stanley, 1998), which is defined as the deviation of its temperature from Tm.  The number of water molecules that is required to form a critically sized nucleator decreases with temperature (Mazur, 2004), so when the temperature is sufficiently low, supercooled water freezes spontaneously.  The temperature at this point is called the homogeneous nucleation temperature (Th) (Mishima and Stanley, 1998).  In reality, homogeneous                                                  1 A version of Chapter 1 has been published. Liu, J., K. M. Cheng, and F. G. Silversides. 2013. Fundamental principles of cryobiology and application to conservation of avian species. Avian Biol. Res. 6 (3):187-197. 2  nucleation is a rare event and crystallization is usually initiated by minor perturbations (heterogeneous nucleation) at a point between Tm and Th.  Water can also exist as a noncrystalline solid known as glass or glassy water at temperatures below the glass transition temperature (Tg; Figure 1.1).  The arrangement of water molecules in glassy water is similar to that of liquid water but the molecules in glassy water are immobilized, and glassy water exhibits an amorphous solid rather than liquid state (Mishima and Stanley, 1998). When cells are exposed to subzero temperatures, the cytoplasm will be supercooled to an extent even when ice formation takes place in the external medium.  Mazur (1965, 1970) ascribed this phenomenon to the presence of cell membranes and lack of effective intracellular nucleators.  At a given subzero temperature, the supercooled water has a higher vapour pressure than that of ice or water in a solution in equilibrium with ice, so cells respond to the disequilibrium across the membrane by losing water.  The rate and extent of the resulting cellular dehydration depend on the cooling rate and the inherent characteristics of cells, including the permeability of their membrane to water, the activation energy and the surface-to-volume ratio.  With assumptions for simplification, these relationships were described elegantly by mathematic methods in Mazur?s work (1963).   The probability of nucleation in a solution increases as the amount of supercooling increases (Muldrew et al., 2004).  If the cooling rate is greater than a critical value, cells cannot lose sufficient supercooled water across their membranes and will complete their equilibration by intracellular ice formation, which is associated with cell death (Mazur, 1970).  Therefore, theoretically, intracellular ice formation and subsequent cell death can be circumvented by cooling cells at a sufficiently low rate.  However, plots of survival versus cooling rate of various types of cells commonly take the shape of an inverted ?U? (Mazur, 3  1970), with very slow cooling also causing cell death.  This means that another class of events with respect to very slow cooling rate may be harmful to cells. These are collectively called ?solution effects? and are associated with long exposure of cells to concentrated intracellular and extracellular components as a result of cell dehydration and extracellular ice formation.  Various hypotheses are proposed to elucidate their mechanisms (Muldrew et al., 2004) but none of these has been rigorously proven or refuted. 1.1.2 Cryopreservation strategies in general Effective cryopreservation protocols minimize cryoinjuries associated with cellular response to subzero temperatures.  According to a two-factor hypothesis, intracellular ice formation is responsible for injury when cooling is faster than optimum in terms of survival while solution effects are responsible for injury when cooling is slower than optimum, which is the foundation of the slow equilibrium freezing strategy (Mazur et al., 2008).  Several studies (Acker and McGann, 2003; Mazur, 2004) showed that small intracellular ice crystals might be innocuous, but the growth of these crystals can have lethal effects, which is an important consideration for warming procedures (see below).  Slow (equilibrium) freezing minimizes intracellular ice formation with cooling rates that are slow enough to yield sufficient osmotic dehydration to keep the chemical potential of intracellular water and the partly frozen extracellular medium near to equilibrium.  By the end of cooling processes, the highly concentrated intracellular solution will form glass while the extracellular solution is frozen (Mazur, 1970).  Solution effects are diminished by cryoprotective agents (CPA), which have cryoprotective properties. CPAs are categorized into penetrating (permeating) and nonpenetrating (nonpermeating) groups (Muldrew et al., 2004).  Penetrating CPAs are nonionic molecules 4  with low molecular weight that have a high solubility in water at low temperatures and can enter and equilibrate in the cytoplasm.  The freezing point of the intracellular solution is depressed by the presence of these CPAs as solutes (freezing point depression), which reduces the extent of supercooling and thus the probability of freezing internally (Mazur, 1963).  In addition, according to colligative theory, they reduce the solution effects by lowering the concentration of damaging solutes such as electrolytes (Meryman et al., 1977).  Non-colligative mechanisms of penetrating CPAs such as stabilizing cellular structures have also been proposed (Crowe et al., 1990).  Glycerol (Polge et al., 1949) and dimethyl sulphoxide (DMSO, Lovelock and Bishop, 1959) are examples of widely used penetrating CPAs.  Nonpenetrating CPAs include sugars and macromolecules that are soluble in water and have limited ability to cross cell membranes.  Their large osmotic coefficients indicate that they can facilitate cell dehydration at a low concentration, which reduces the extent of supercooling of the cytoplasm and the chance of freezing (Muldrew et al., 2004).  Mechanisms of their protection against solution effects remain to be elucidated. Slow freezing has been successful in preserving specific cell types but has not been effective for multicellular systems such as cell aggregates, tissue and organs.  Cells can be very different in their permeability to water, activation energy and surface-to-volume ratio, which in turn can lead to a possible 1000-fold difference in the optimal cooling rate (Mazur, 1970).  Therefore, a cooling rate that produces high survival of one type of cell may not guarantee high survival of the others in a multicellular system.  In addition, with slow freezing, the formation of ice in the extracellular or intercellular area is detrimental.  If extracellular freezing is eliminated, the consequent stresses such as the probability of 5  intracellular ice formation and solution effects can be avoided, which can be achieved by vitrification procedures. Fahy et al. (1984) defined vitrification as the process of solidification of a liquid by extreme enhancement of viscosity instead of crystallization with the resultant amorphous solid being called glass.  Theoretically, this process can be achieved by ultra-rapid cooling of biological materials subjected to high concentrations of glass-promoting CPAs.   The outcome is that both intracellular and extracellular components convert to glass in a very short time, which can be explained by the phase diagram in Figure 1.1.  The intersection between the homogeneous nucleation curve (Th) and the glass transition curve (Tg) gives a critical concentration of a solution, above which it is possible to cool the solution directly to the glass transition temperature without freezing (Rasmussen and Luyet, 1970).   In addition, a higher cooling rate lowers the required concentration of solute (Fahy et al., 1987).  Current technologies are not able to verify whether true vitrification of cellular systems is obtained; Seki and Mazur (2009) suggested using the term ?vitrification procedure? to refer to cryopreservation procedures that approach vitrification by using an ultra-high cooling rate and highly concentrated CPAs. Cryopreserved biological materials must survive warming procedures before they can be used.  Warming rate is of critical importance (Seki and Mazur, 2008).  As the temperature rises above Tg, glass will convert to a highly viscous liquid (Mishima and Stanley, 1998), which has a tendency to form crystalline ice through devitrification.  At an appropriate higher temperature, small ice crystals with high surface energies will enlarge when sufficient time is given, in a process known as recrystallization.  The large crystals themselves or their formation process may lead to cell death (Mazur, 2004).  If cells are preserved by slow 6  freezing, the effects of warming rate on cell survival are very complex and depend on cell type, CPAs and cooling rate.  In some cases, the warming rate may have no effect, whereas rapid or slow warming is favoured in other cases (summarized by Mazur, 2004).  For materials preserved by vitrification procedures, rapid warming is essential because the growth of small ice crystals that may have formed during the cooling process or during devitrification and subsequent recrystallization can be prevented by sufficiently high warming rate (Seki and Mazur, 2008).  The other critical factor for warming is osmotic stress associated with the removal of penetrating CPAs, which needs to be minimized.  The concentration of penetrating CPAs in the extracellular solution is lowered progressively using stepwise dilutions, which permits cells to resume equilibrium gradually.  As an alternative, a nonpenetrating CPA such as sucrose can be used alone in the warming solution to facilitate the efflux of penetrating CPA and reduce excessive water influx (Muldrew et al., 2004). 1.1.3 Cryobiology of animal and human germplasm 1.1.3.1 Cryobiology of germ cells and embryos An important application of cryobiology is long-term preservation of animal and human germplasm.  Mazur et al. (2008) defined ?germplasm? as cells that result in the development of offspring, singly or in combination, but principally referred to spermatozoa and preimplantation embryos.  The present dissertation will expand the view of Mazur et al. (2008) to also include female gametes, primordial germ cells, and gonadal tissue because genetic variation can be accessed at various points in the germline cycle (Figure 1.2) and successful preservation of any component of this cycle and subsequent functional recovery will lead to successful ex situ conservation.   7  Investigations into preserving animal germplasm at subzero temperatures started as early as the mid-19th century, when the mechanism of fertilization began to be appreciated (Leibo, 2004).  Progress has been made since then but has largely been based on empirical approaches.  Revolutionary events such as the introduction of glycerol (Polge et al., 1949) and DMSO (Lovelock and Bishop, 1959) as effective penetrating CPAs, and especially, the publication of  Mazur?s physical-chemical models (1963, 1965) led to significant advances in fundamental cryobiology and its application in preserving animal germplasm.  An example is the successful cryopreservation of murine and bovine embryos (Mazur et al., 2008).  However, using mathematical approaches to design protocols to preserve germplasm appears to be challenging, although the physical-chemical models can predict the behaviour of various somatic cell types and a limited number of oocytes and embryos during the cooling procedures with reasonable degrees of accuracy.  This can be explained by the biological characteristics of germ cells.  For many vertebrates, the precursors of germ cells and those of somatic cell types separate at an early stage of development.  Compared to most somatic cells, of which only a specific portion of their genome is expressed at specific stages in an individual?s life, germ cells are designed to pass the entire complement of genetic materials and instructive information needed for differentiation and development to the next generation.  During gametogenesis, germ cells complete or partially complete meiosis and they obtain very specialized cellular and subcellular features that are essential for fertilization and initiation of subsequent events.  This can cause a great deviation of their cytoplasm from an ideal dilute solution and subsequent violations of the assumptions required by physical-chemical modeling (Mazur, 1970).  In addition, high viability and cellular integrity resulting from a 8  low level of lethal injuries may not guarantee functional recovery after cryopreservation.  Sublethal injuries that alter structures and components of germ cells can lead to unpredictable effects on their functions.  An example is high motility but low fertilization rates that are often observed in cryopreserved swine spermatozoa (Mazur et al., 2008).  Another feature of germ cells and embryos is that they can be extensively damaged by exposure to temperatures approaching 0?C.  This type of damage is known as chilling injury (Mazur et al., 2008) and is different from the ?two factors? previously described, although its physiological nature remains ill defined.  1.1.3.2 Cryopreservation of male germplasm Semen cryopreservation is the most commonly used method of preserving male mammalian germplasm and has been integrated as an assisted reproductive technology in human reproductive medicine.  It also contributes to genetic improvement in cattle breeding and maintenance of mouse strains bearing specific genotypes.  Basic structures of spermatozoa are highly conserved among species, including a haploid nucleus, a propulsion system and an acrosome.  The nucleus contains the genetic materials that need to be conveyed to the female gamete for fertilization while the propulsion system and acrosome enable the nucleus to move to and enter the female gamete.  The purpose of cryopreservation is to ensure functional resumption of these components after warming to achieve normal fertilization.  The specialized structures of spermatozoa are obtained during spermatogenesis, in which most of the cytoplasm is eliminated and the nucleus is condensed, leading to a high surface-to-volume ratio and low water content.  According to Mazur?s model (1963), spermatozoa can be cryopreserved at a relatively high cooling rate without intracellular ice formation.  The threshold for mouse spermatozoa was 9  estimated to be greater than 250?C/min by experimental inference (Mazur and Koshimoto, 2002), compared to less than 1?C/min for mouse ova (Leibo et al., 1978).  In addition, chilling injury has been reported in various species.  There is general agreement that unejaculated spermatozoa are more resistant to chilling injury than ejaculated spermatozoa (Leibo, 2004). To date, slow-freezing, vitrification and freeze-drying are strategies that are available for semen cryopreservation, of which slow-freezing is the most broadly practiced.  Briefly, spermatozoa suspended in a diluent containing CPA(s) are cooled at a controlled rate until the spermatozoa can be stored in liquid nitrogen.  The procedures used today are very similar to those developed around 60 years ago and the history of adding egg yolk to diluents (Phillips and Lardy, 1940) is even longer.  The procedures may not be optimal, but optimization is rarely practical because there are so many variables, and high variation exists among species and individuals in response to slow-freezing procedures (Mazur et al., 2008).  Slow freezing has been used to preserve human, murine and bovine spermatozoa with satisfactory efficiency, and good fertility has recently been reported from frozen/thawed boar semen (Didion et al., 2013).  The alternative, vitrification procedures, provides a potential solution because the sensitive temperature zone can be bypassed by using an ultra-rapid cooling rate.  In addition, penetrating CPAs that have toxic and osmotic effects can be excluded because the cytoplasm of spermatozoa is very condensed.  Vitrification procedures using only nonpenetrating CPAs have been successfully used to preserve human spermatozoa and these spermatozoa have produced healthy babies (Isachenko et al., 2012).  A third strategy for long-term preservation of mammalian spermatozoa is to use freeze drying (Wakayama et al., 1998) or evaporative drying (Li et al., 2007), in which the 10  spermatozoa are killed and fertilization is achieved by intracytoplasmic sperm injection subsequent to rehydration of the dead spermatozoa.  This may be a promising strategy for preservation of male germplasm of species for which intracytoplasmic sperm injection is available. The prerequisite for semen preservation is the presence of spermatozoa, which is a limitation for individuals before maturation whose fertility needs to be preserved, such as young patients who suffer sterility as a result of cancer treatments.  The solution is to preserve their testicular tissue before the onset of the treatments, which can be recovered and allowed to resume maturation in vivo or in vitro at a later time.  This may allow the germline of a valuable domestic or wild animal to be preserved regardless of its developmental stage.  Slow freezing is conventionally used, following basic procedures similar to those of semen cryopreservation, and live offspring have been obtained from cryopreserved testicular tissue of lab rodents and chickens (Ehmcke and Schlatt, 2008). From a cryobiological perspective, vitrification procedures might be better for cryopreserving testicular tissue than slow freezing because resumption of spermatogenesis and steroidogenesis of testicular tissue depends on at least three types of cells, including germ cells which bear the potential of fertility and are in different stages of maturation; Sertoli cells which support the germ cells physically and biochemically; and Leydig cells, which are the major source of male hormones.  Variables of slow-freezing procedures, such as the cooling rate, are cell-specific so the optimal protocol for one cell type will not necessarily be optimal for other cell types and their different developmental stages.  More importantly, these cells must be organized properly by their extracellular matrix in and around testicular tubules which are vulnerable to damage by ice crystals induced by slow 11  freezing (Woods et al., 2004).  Investigation of the use of vitrification to preserve testicular tissue has shown promising results in humans (Curaba et al., 2011a) and various animal models (Abrishami et al., 2010; Curaba et al., 2011b). 1.1.3.3 Cryopreservation of female germplasm  For most mammalian and avian species, mitotic proliferation of oogonia ceases by the time around birth (Rothchild, 2003).  A small portion of these oogonia enter the first meiotic division and are called primary oocytes and their meiosis is arrested at Prophase I.  Primary oocytes, the surrounding single layer of epithelial granulosa cells and a layer of basement membrane form primordial follicles.  With the onset of sexual maturity, groups of follicles periodically enter folliculogenesis, which includes maturation of oocytes (oogenesis) and proliferation and maturation of the surrounding granulosa cells.  During oogenesis, meiosis is resumed by oocytes and is arrested again at Metaphase II until fertilization. In most mammalian species, the ovulated ovum is enclosed in a glycoprotein membrane named the zona pellucida, which is surrounded by cumulus cells derived from the innermost granulosa cells of the follicle.  During fertilization, the spermatozoon binds to the zona pellucida and initiates the acrosomal reaction which releases enzymes that facilitate the penetration of a spermatozoon.  Subsequent binding of a spermatozoon to the plasma membrane of the ovum enables the entry of the spermatozoa nucleus.  The ovum is then activated to trigger modification of the zona pellucida (zona hardening) to prevent polyspermy and the pronuclei are formed (Bedford, 2004).  These events make embryos more resistant to the negative effects of cryopreservation than oocytes, although the mechanisms remain to be clarified (Fuller et al., 2004). 12  The first success in cryopreservation of embryos was achieved by Whittingham et al. (1972) using a slow-freezing protocol following the principles addressed by Mazur?s model.  Success was also achieved by Wilmut (1972) independently around the same time.  A modified protocol was used by Willadsen et al. (1978) for ruminants, and this has since been refined and widely used.  Using a similar protocol, Chen (1986) first reported pregnancy from cryopreserved human oocytes.  Generally, embryos and oocytes are equilibrated with CPAs and cooled at a controlled rate to a seeding temperature where extracellular ice nucleation is induced.  The temperature is then lowered slowly until the samples can be stored in liquid nitrogen.  The cooling rate for embryos and oocytes is very low compared to that used for semen (less than 0.5?C/min compared to 10 to 100?C/min commonly used for spermatozoa).  This is because the surface-to-volume ratio and the permeability of oocytes and embryos are very low compared to spermatozoa, leaving them very susceptible to intracellular ice formation.  Leibo et al. (1978) estimated the upper threshold of cooling rate without intracellular ice formation to be 1?C/min.  However, a slow cooling rate can still cause cryoinjury to oocytes and embryos because both are very sensitive to chilling injury (Saragusty and Arav, 2011).  Disruption of the meiotic spindle and zona hardening caused by cooling procedures increase the challenges for preservation of oocytes (Mazur et al., 2008). Attention has been devoted to vitrification procedures since their first successful application to preserving embryos (Rall and Fahy, 1985).  Vitrification solves problems caused by high chilling sensitivity and high intracellular ice nucleating temperature exhibited by oocytes and embryos.  It also ameliorates adverse effects of extracellular ice and long exposure to concentrated intracellular and extracellular solutions.  There is consensus regarding methods of reducing the toxicity of high concentrations of CPAs.  The first is to 13  combine both penetrating and nonpenetrating CPAs, which reduces overall toxicity and promotes both internal and external glass transition.  Ethylene glycol is the most widely used penetrating CPA, and is sometimes used in combination with other penetrating CPAs such as DMSO and 1, 2-propanediol.  Sugars such as sucrose are used in many studies as nonpenetrating CPAs and different types of sera are common additives (Chen and Yang, 2007).  The second aspect is stepwise equilibration of CPAs whereby oocytes or embryos are first equilibrated with a low concentration of penetrating CPAs and then briefly with nonpenetrating CPAs and then more concentrated penetrating CPAs.  The third aspect is to maximize the cooling rate so that the concentrations of CPAs can be lowered accordingly (Fahy et al., 1987).  Many specialized devices have been developed (Saragusty and Arav, 2011), some of which allow direct contact of liquid nitrogen to oocytes or embryos and others (mainly straws) are modified to reduce insulation.  Note that the advantage shown by these devices is not entirely due to the high cooling rate that they allow, but that they also make it possible to achieve a very high warming rate, which may be more important than the cooling rate for recovery of oocytes or embryos preserved in this manner (Seki and Mazur, 2009). An important observation of oocyte cryopreservation is that immature oocytes survive cryopreservation procedures better than mature oocytes (Woods et al., 2004).  However, the conditions for achieving subsequent in vitro maturation of the surviving immature oocytes can be very demanding.  In this regard, cryopreservation of ovarian tissue enables preservation of the abundant primordial follicles enclosed in the tissue and their recovery in their natural micro environment.  For mammals, including humans, ovarian cryopreservation provides an option when normal ovulation cannot be achieved due to 14  physiological or pathological reasons.  For many non-mammalian vertebrates, cryopreservation of ovarian tissue could be the only effective way of preserving female germplasm because their oogenesis involves deposition of large amounts of yolk into the ooplasm, resulting in a large egg with a very low permeability to water and CPAs and thus a very high intracellular nucleating temperature, making it technically impossible to prevent intracellular freezing and chilling injury at the same time. Slow-freezing procedures for ovarian tissue were first developed in the 1990?s and used in various animal models following the principles for preserving embryos.  In humans, the original purpose of preserving fertility of young patients subjected to gonadotoxic treatments has been fulfilled.  Silber (2012) described the birth of 28 healthy babies after transplantation of fresh or frozen-thawed ovarian tissue.  By contrast, live births in humans reported from cryopreserved oocytes are very limited (Silber, 2012).  Vitrification procedures have been adopted by different groups to preserve human ovarian tissue (Silber, 2012), although the efficiency has been questioned by some (Isachenko et al., 2007).  Strategies such as combining penetrating and nonpenetrating CPAs and multi-step equilibration have been adopted by most researchers and special methods have been developed to facilitate rapid cooling and warming (Chen et al., 2006; Wang et al., 2008).  It is foreseeable that vitrification procedures will be widely used because of their feasibility and simplicity.   15  1.1.4 Cryopreservation of germplasm in avian species 1.1.4.1 Cryopreservation of male germplasm in avian species Avian testes are located in the body cavity, attached to the body wall, ventral to the cephalic part of the kidneys (Johnson, 1986a).  There is no pampiniform plexus, which is important for thermoregulation in mammals, indicating that avian testicular tissue can function at a core body temperature of around 40?C.  The duct system includes seminiferous tubules, rete tubules, vasa efferentia, epididymes and the vasa deferentia, where semen is stored in most avian species.  In many passerines, coils of terminal vasa deferentia form a cloacal protuberance during the breeding season (Gee et al., 2004).  Birds have no organs comparable to mammalian accessory reproductive glands such as the prostate gland, the bulbourethral gland and the seminal vesicle; seminal plasma in birds is from seminifierous tubules and vasa efferentia (Johnson, 1986a).  Therefore avian semen shows very different physiological and biochemical properties than mammalian semen (Blesbois, 2011; Long, 2006).   In general, an avian spermatozoon consists of a straight or slightly curved head resembling a long, slender cylinder and a long tail (Romanoff, 1960).  The proximal end of the head is covered by an acrosome, the shape of which varies among species (Gee et al., 2004).  Avian spermatozoa can be broadly categorized into the simple sauropsid form and the complex helical form, with the exception of the American kestrel, in which the spermatozoa are round to slightly flattened (Gee et al., 2004).  All of these are distinguished from mammalian spermatozoa (Figure 1.3).  The simple sauropsid form is common in non-passerine species, in which the middle piece of the tail is relatively long and sometimes spiral (Romanoff, 1960).  The complex helical form is characterized in passerine birds with a 16  predominantly spiral configuration in each portion of the cell, which usually has a ribbon-like structure that covers the acrosome or the entire cell (Gee et al., 2004).   Fowl spermatozoa were the first biological material successfully cryopreserved (Polge, 1949).  Optimization of cryopreservation in terms of CPA type, cooling rate and packaging for storage using slow-freezing procedures was done primarily based on empirical approaches.  Two protocols have been adopted for gene banking of local chicken breeds in Europe; one (Blesbois, 2007) uses glycerol as the CPA and a relatively slow cooling rate (7?C/min) and the other (Woelders et al., 2006) uses dimethylacetamide as the CPA and a relatively high cooling rate (around 200? C /min).  In addition, live chicks have been produced from cryopreserved semen of a number of nondomestic avian species (Gee et al., 2004) and semen cryopreservation has successfully contributed to species recovery, such as cranes and Golden eagles (Blanco et al., 2009). Avian spermatozoa might be better preserved by vitrification procedures than slow freezing.  Fragile structures such as long tails and spiral configurations can be preserved by minimizing extracellular ice formation, which is important for avian species because in vitro fertilization is not practical and structural integrity is crucial for survival of spermatozoa in the female reproductive tract and fertilization.  Vitrification procedures also arrest solution effects by ultra-rapid cooling, eliminating the need for penetrating CPAs that have contraceptive (glycerol) or toxic effects (dimethyl acetamide).  The benefits of eliminating penetrating CPAs have been demonstrated for mouse semen (Koshimoto et al., 2000) and penetrating CPAs have been eliminated successfully in cryopreservation of human semen (Isachenko et al., 2012).  The spermatozoa of some avian species such as the Sandhill crane exhibit relatively high osmotic tolerance (Blanco et al., 2008), making it possible to use 17  nonpenetrating CPAs with a relatively high concentration to facilitate glass transition.  In addition, the nucleus of an avian spermatozoon is highly condensed and the intracellular water content is very low compared to other vertebrate cells (Blesbois, 2011) so that the intracellular components naturally favour glass transition if the cooling rate is rapid enough, as was demonstrated for fowl semen pellets preserved by direct immersion in liquid nitrogen (Tselutin et al., 1999).   An alternative to semen cryopreservation is to preserve testicular tissue, which also provides an option for some wild species in which semen quality is too low to be used for cryopreservation and subsequent artificial insemination because of a short reproductive season and the stress related to collection procedures (Blanco et al., 2009).  Furthermore, considering that spermatogonia are present in the testicular tissue regardless of the male?s age, cryopreservation of testicular tissue should make it possible to recover the germline of a valuable male bird even after an unexpected death.  Successful cryopreservation and recovery of testicular tissue has been demonstrated in chickens (Song and Silversides, 2007a) using slow-freezing procedures, demonstrating the possibility of cryobanking avian testicular tissue.  Efforts are needed in developing a vitrification method because of the aforementioned advantages in tissue cryopreservation.  In Chapter 2, a vitrification protocol successful in preserving Japanese quail ovarian tissue was adapted to a straw system and an in ovo tissue culture method was used to evaluate viability and potential of vascularization of cryopreserved tissue.  Chapter 3 describes how the vitrification protocol was further optimized with a 2-ml macrotube straw system and applied to quail testicular tissue.  The in ovo culture method described in Chapter 2 was used for a preliminary evaluation.  In Chapter 18  4, the functional recovery of quail testicular tissue cryopreserved by vitrification method is demonstrated by testicular transplantation and subsequent progeny test.   1.1.4.2 Cryopreservation of female germplasm in avian species In most avian species, only the left ovary and oviduct are functional (Golden and Arbona, 2012).  The ovary is located at the cephalic end of the kidney and consists of an outer cortex and an inner medulla.  In mature female birds, the functional ovarian cortex possesses numerous follicles arranged in a hierarchical manner.  The maturation of the avian oocyte is characteristic of hormone-controlled yolk deposition or vitellogenesis.  The precursors of yolk contents are synthesized in the liver and are transported to the follicles.  The cell layers surrounding the oocyte and yolk include the oocyte plasma membrane, perivitelline membrane, granulosa cells, basal lamina, theca interna and theca externa.  Each follicle is connected with the ovary through a long stalk upon maturation, which is different from that of mammals.  During ovulation, the ovum containing a large amount of yolk is expelled from the follicle through the less vascularized stigma of the follicle.   The ovulated ovum is engulfed by the infundibulum of the oviduct (Figure 1.5), which is the location of fertilization (Johnson, 1986b).  As the fertilized ovum enters different sections of the oviduct, layers of albumin and eggshell are deposited around it.  Embryonic development starts while the egg is still in the oviduct and by the time of oviposition or egg-laying, the embryo is in a disk shape residing on the surface of the yolk.  The embryo at this stage is called the blastoderm and contains around 20,000 cells in the chicken (Gilbert, 2010).  At a later stage of embryonic development, primordial germ cells originating from an extraembryonic region named the germinal crescent move to and colonize the gonads, where the germ cells mature to start the germline cycle again through 19  fertilization.  The complex structure of ovulated avian ova and embryos prevents the application of cryopreservation procedures and female germplasm can only be preserved in the upstream forms of the germline cycle, namely, primordial germ cells and ovarian tissue (Figure 1.2).  Circulating and newly colonized primordial germ cells can be collected at early stages of donor embryonic development and cryopreserved (see Petitte, 2006; Silversides and Liu, 2012 for reviews).  Germ line chimeras can then be produced by transplantation of cryopreserved donor primordial germ cells into recipient embryos at an appropriate developmental stage.  The recipient embryos (chimeras) then need to hatch and mature and can be mated inter se to produce offspring that represent the donor line.  To date, rigorous verification of cryopreservation efficiency in preserving avian primordial germ cells is scarce, probably because the procedures involved are complex and require significant resources and training to carry out.  All examples of reconstitution including use of both fresh and cryopreserved primordial germ cells demonstrate very low efficiency, and thousands of prepared primordial germ cells and hundreds of manipulated recipient embryos have produced only a handful of donor-derived chicks (Silversides and Liu, 2012).  The FAO (2012) suggests an effective population size of 50 for regeneration of a population, so although primordial germ cells may be valuable for research on genetic manipulation, they are not suitable for cryobanking of avian germplasm.  Cryopreservation of immature oocytes can be achieved by preserving ovarian tissue, which can be recovered by orthothopic transplantation (Song and Silversides, 2006, 2007b).   The efficiency of this strategy was demonstrated by Liu et al. (2010), who cryopreserved ovarian tissue from immature female Japanese quail using slow-freezing and vitrification 20  procedures and recovered it by orthotopic transplantation with successful production of donor-derived offspring.  The vitrification protocol used in this study was more efficient than the slow-freezing protocol in in vitro and in vivo tests.  The simple protocol adapted from mammalian studies (Chen et al., 2006; Wang et al., 2008) has been successfully used in various species including mice, felids, ungulates (Comizzoli et al., 2012), Japanese quail (Liu et al., 2010) and chickens (unpublished data).  Investigation and evaluation of various straw systems to be used along with this protocol to facilitate ovarian tissue cryobanking are detailed in Chapter 2 and Chapter 5.  The protocol described in Chapter 5 is now used for cryobanking of avian gonadal tissue by Canadian and U.S. government animal genetic resources programs. 1.2 Transplantation immunology In mammals, functional recovery of cryopreserved gonadal tissue can be achieved using in vitro techniques such as in vitro maturation and in vitro insemination.  Application of these techniques in avian species is very limited.  Gonadal transplantation can be used as a special form of tissue culture (Paris and Schlatt, 2007) for resumption of gametogenesis in the tissue, and has been successful in various avian species (summarized by Silversides and Liu, 2012). When the recipient and donor are from the same species, but are not genetically identical, the transplantation is referred to as allotransplantation.  Transplantation between different species is called xenotransplantation (Lakkis, 2012).  In both cases, grafts are subjected to the risk of being rejected by the immune system of the recipient.  Many attempts have been made to prevent rejection in allo- and xenotransplantation in mammalian animal models and in human organ or tissue transplantation.  This section will review the functions 21  of the immune system in immunity and tolerance, mechanisms of graft rejection, and possible strategies to prevent rejection that can be used to facilitate avian gonadal transplantation. 1.2.1 Immunity and tolerance 1.2.1.1 Immunity Innate immunity and adaptive immunity are used by vertebrates to protect the host from pathogens (Iwasaki and Medzhitov, 2010).  Table 1.1 provides a general comparison between the two.  Cells and molecules in the two systems need to work in a coordinated manner to provide effective protection.  Avian innate immune cells include macrophages, dendritic cells, natural killer cells and granulocytes such as heterophils, eosinophils and basophils. The mammalian counterparts of heterophils are neutrophils (Schijns et al., 2008).  Cells in the innate immune system carry multiple pattern recognition receptors that recognize pathogen associated molecular patterns, which are characteristic and conserved for most microorganisms of a given class (Iwasaki and Medzhitov, 2010).  Recognition of pathogen associated molecular patterns will trigger these cells to produce cytokines and activate the complement system, resulting in an inflammatory response, in which phagocytic cells including macrophages, neutrophils and immature dendritic cells are induced to remove pathogens (Banchereau and Steinman, 1998).  Another consequence of innate recognition is activation of adaptive immunity through antigen presentation by professional antigen-presenting cells such as mature dendritic cells and macrophages, which process the ingested pathogens and their components and present antigens in the context of major histocompatibility complex (MHC) molecules on their surfaces (Iwasaki and Medzhitov, 2010; Janeway et al., 2005). 22  Adaptive immunity can be broadly categorized into cell-mediated immunity and humoral immunity and depends on the functions and interactions of thymus-derived (T) cells and bursa-derived (B) cells (Davison, 2008).  In mammals, which do not possess a bursa of Fabricius, bone marrow is the major source of postnatal B cells (Halverson et al., 2004).   Cell-mediated immunity is directed by T cells, which express T cell receptors to detect antigen-bound MHC molecules.  Activation and clonal expansion of na?ve T cells are facilitated with antigen-presenting cells such as dendritic cells, macrophages and B cells, which present antigens and provide necessary costimulatory signals (Janeway et al., 2005).  The MHC class I molecules are recognized by CD8+ T cells, which are known as cytotoxic T lymphocytes and induce apoptosis of infected and transformed cells.  The MHC class II molecules are recognized by CD4+ T cells, which are called helper T cells and facilitate immune responses including activation of macrophages and antibody production (Kaufman, 2008).   Humoral immunity is responsible for production of circulating antibodies, which bind to antigens and effector molecules or cells through Fab (Fragment antigen binding) and Fc (Fragment crystallizable) fragments, respectively (Janeway et al., 2005).  Na?ve B cells express receptors on their surface that can recognize unprocessed antigens (Ratcliffe, 2008).  These B cells then process and present antigens through their MHC class II molecules to helper T cells that recognize the same antigen, which in turn induce the differentiation of B cells into plasma cells to produce circulating antibodies (Janeway et al., 2005).  Pathogens or their toxic products can be neutralized by binding to specific antibodies.  Circulating antibodies facilitate effector mechanisms through opsonisation, in which antibody-bond pathogens are recognized by cells that possess Fc receptors, including macrophages and 23  neutrophils that ingest pathogens through phagocytosis or natural killer cells, eosinophils and basophils that release the contents of their granules for exocytosis.  A third mechanism recruited by antigen binding is the complement system, which opsonizes the pathogens or directly destroys the pathogens (Chen et al., 2010; Janeway et al., 2005).   1.2.1.2 Induction and maintenance of self-tolerance Genetic modulation mechanisms enable the adaptive immune system to express receptors with great diversity to recognize almost all antigens that they are exposed to (Janeway et al., 2005).  However, the immune system must maintain self-tolerance to prevent the adaptive immune system from responding to a self- or autologous-antigens, resulting in damaging autoimmunity (Erf, 2008).  Tolerance is the state of immunological non-responsiveness to a given antigen and self-tolerance is achieved by coordinated central and peripheral processes (Griesemer et al., 2010). Central tolerance refers to the process of eliminating autoreactive lymphocytes during maturation in central lymphoid organs (Hogquist et al., 2005).  Thymic epithelial cells especially the medullary thymic epithelial cells conduct promiscuous gene expression, by which self-antigens representing most parenchymal organs are expressed.   Self-antigens are presented to T-cell precursors by thymic epithelial cells and other antigen-presenting cells such as dendritic cells (Kyewski and Derbinski, 2004) in the form of self-peptide-MHC ligands in the thymus.  The fate of T-cell precursors depends on the affinity of their receptor to self-peptide-MHC ligands.  About 95% of the precursors undergo apoptosis due to lack of specificity of their receptors for an MHC ligand, which is known as death by neglect (Griesemer et al., 2010).  Precursors of which the receptors show mild affinity to MHC ligands receive signals that rescue them from apoptosis and permit their further 24  differentiation.  This process is positive selection.  High affinity to MHC ligands has two outcomes.  One is negative selection which is mainly achieved by apoptosis of selected precursors (clonal deletion; Griesemer et al., 2010).  The rest of the precursors that show high affinity to self MHC ligands are the sources of regulatory T-cell populations (Hogquist et al. 2005) which are involved in peripheral tolerance.  Other mechanisms such as anergy and receptor editing that weaken the specificity of T cells are also documented (Hogquist et al. 2005).   Autoreactive T cells that escape negative selection require regulation by peripheral tolerance.  One mechanism is bystander suppression, in which peripheral dendritic cells that present self-antigen-MHC ligands to autoreactive T cells can recruit regulatory T-cell cells by co-presentation of ligands that bind to regulatory T-cell cells, which suppress the autoreactive T cells (Kyewski and Derbinski, 2004).  In addition, tolerogenic dendritic cells that recognize autoreactive T cells can induce functional unresponsiveness (clonal anergy) or peripheral deletion (Mueller, 2010). The antigens detected by B cells do not have to be presented by MHC molecules.  However, if the surface receptor can bind to self MHC molecules, the B cell might be eliminated by clonal deletion (Janeway et al., 2005).  Another outcome of an autoreactive B cell is receptor editing through gene recombination or somatic hypermutation so that the receptor is replaced by one that is not autoreactive (Halverson et al., 2004).  A third mechanism is anergy, in which intrinsic factors dampen the sensitivity of the autoreactive B cell receptor (Janeway et al., 2005).  The survival and activation of autoreactive B cells in peripheral lymphoid organs is suppressed by limiting necessary signals.  Intrinsic anergy is also involved in peripheral B-cell tolerance (Goodnow et al., 2005).   25  1.2.2 Mechanisms of transplant rejection 1.2.2.1 Initiation of rejection by transplants In addition to pathogen-associated antigens, cell receptors of the innate immune system can also recognize markers of tissue damage (Medzhitov and Janeway, 2002).  In transplantation, such markers can be produced by transplants that are subjected to tissue injuries associated with tissue isolation, manipulation and implantation.  Recognition of these markers by the recipient innate immune system will induce inflammatory responses and complement activation and eventually trigger responses from the adaptive immune system (Murphy et al., 2011).   1.2.2.2 Hyperacute rejection Hyperacute rejection is observed in allotransplantation of primarily vascularized organs (Chinen and Buckley, 2010).  Preformed recipient circulating antibodies target antigens of donor vascular endothelial cells and subsequently activate the complement system and blood clotting cascade, resulting in endothelial damage and thrombosis.  Similarly, in xenotransplantations, species-specific carbohydrates and proteins especially those on the endothelium of transplants can induce hyperacute rejection, which is very common in swine-to-primate models but not in transplantation between closely related species (Pierson et al., 2009; Samstein and Platt, 2001). 1.2.2.3 Acute and chronic rejection In allotransplantation, acute rejection by the adaptive immune response to a transplant starts with allorecognition (Wood and Goto, 2012).  In direct allorecognition, intact donor peptide-MHC complexes are presented to recipient T cells by donor antigen-presenting cells 26  residing in the transplants.  Indirect allorecognition is detection of donor peptides derived from donor MHC antigens that are captured, processed and displayed by recipient-derived antigen-presenting cells.  A third pathway is semi-direct allorecognition, in which intact donor peptide-MHC complexes are captured by recipient antigen-presenting cells and presented to recipient T cells.  Evidence suggests that both direct and indirect pathways are used in xenorecognition (Pierson et al., 2009).   Allorecognition enables the T cell receptors complex on the T-cell membrane to convey a signal (Signal 1) into the cytoplasm.  Activation of T cells requires a second type of signal (Signal 2) which is produced by interaction of costimulatory ligands provided by antigen-presenting cells with their receptors on T cells.  Signal 1 and Signal 2 trigger signal transductions to facilitate production of a Signal 3 that is necessary for clonal expansion and differentiation of specific T cells (Wood and Goto, 2012).  Cytotoxic CD8+ T cells are the major effectors in the destruction of allotransplants while CD4+ T cells provide costimulatory signals for B cells to mature to produce donor-specific antibodies (Chinen and Buckley, 2010).  In addition, donor-specific CD4+ T cells recruit innate immune cells causing damage to transplants, known as delayed-type hypersensitivity (Wood and Goto, 2012).  The major mechanism of xenorejection at the acute stage is CD4+ T cell associated damage (Pierson et al., 2009).  The B cells also contribute to antigen presentation and the production of signal molecules necessary for activation and amplification of innate and adaptive responses (Balin et al., 2009). Chronic rejection is a rare occurance in allotransplantation and xenotransplantation mainly because most transplants or patients do not survive the acute rejections until this stage (Samstein and Platt, 2001).  Chronic rejection observed in kidney transplantation is primarily 27  mediated by antibodies (Chinen and Buckley, 2010) produced by B-cell derived plasma cells.  Other studies demonstrate the involvement of T cell mediated mechanisms, especially the delayed-type hypersensitivity in chronic vasculopathy of the transplants (Bedi et al., 2010).   1.2.3 Prevention of transplant rejection Rejection of transplants by immunocompetent recipients must be prevented to achieve successful transplantation (Wood and Goto, 2012).  Current treatments almost entirely rely on immunosuppressive drugs, which broadly suppress immune responses of recipients, especially T-cell mediated mechanisms.  As indicated earlier, T cells play a critical role in recognition and rejection of transplants and in coordination of other effector mechanisms.  However, none of the drugs are specific and all of them have side effects on recipients (Post et al., 2005).  As an alternative, it is possible to induce donor-specific tolerance in recipients so that donor-specific recognition and subsequent rejection can be prevented and the immune system of the recipient retains the potential to respond to invasion of pathogens in a normal pattern.  However, treatments based on this strategy are still experimental, largely because the knowledge of the mechanisms involved in induction and maintenance of immunological self-non-self discrimination remains limited. 1.2.3.1 Immunosuppressive drugs and their functions Calcineurin inhibitors including cyclosporine and tacrolimus are heavily used in human organ transplantation.  Calcineurin is a crucial downstream product of the pathways induced by Signal 1 and Signal 2 in T cell activation mentioned above, which facilitates translocation of a transcription factor named the nuclear factor to the nucleus by dephosphorylation.  Transcription factors, including the nuclear factor, upregulate the T-cell growth factor interleukin-2 and its receptor, which facilitate T-cell proliferation (Wood and 28  Goto, 2012).  Cyclosporine and tacrolimus inhibit calcineurin by forming a complex engaging different immunophilins (Rosen, 2008).  Another drug, rapamycin (sirolimus) shows structural similarity to tacrolimus but functions in blocking the downstream pathway after the production of interleukin-2 (Wood and Sakaguchi, 2003).  In addition, rapamycin may selectively preserve regulatory T cells in vivo and in vitro (Rosen, 2008) and promote tolerance of transplants. Non-calcineurin inhibitors include corticosteroids, anti-metabolite agents and antibodies.  Corticosteroids have long been used as a treatment to supress acute immune responses and as a maintenance therapy.  The main function is to inhibit expression of interleukins produced by T cells and antigen-presenting cells that are crucial in signal transduction (Post et al., 2005).  Anti-metabolite agents include azathioprine, mycophenolate mofetil and mycophenolic acid, which inhibit purine synthesis and therefore block T-cell and B-cell differentiation and proliferation (Rosen, 2008).  Various antibodies targeting lymphocytes and cytokines involved in signal transductions are now available (Chinen and Buckley, 2010).   To date, use of the immunosuppressive drug mycophenolate mofetil is the only demonstrated solution for prevention of tissue rejection in avian gonadal tissue transplantation (Silversides and Liu, 2012).  Long-term survival and normal functioning of transplants subsequent to a short course of immunosuppressive treatment have been observed in chicken ovarian allotransplantation (Song and Silversides, 2008a), which might be explained by the theory of infectious tolerance (see below).  This treatment has also been used in xenotransplantation of ovarian tissue in anseriformes (Song et al., 2012).  The empirical dose that has been effective in previous studies in Japanese quail (Song and 29  Silversides, 2008b; Liu et al., 2010) and in chickens (Song and Silversides, 2007b) is followed and described in Chapters 4, 5 and 7 and Chapter 8 of this dissertation, respectively. 1.2.3.2 Actively acquired tolerance Naturally occurring acquired tolerance was observed in fraternal calf twins (Owen, 1945).  These twins possess chimeric blood cells at birth and in later life and are therefore tolerant to each other?s self-antigens carried by blood cells, which can be explained by the anastomotic circulation between placentae during fetal life.  This explanation was tested by Billingham et al. (1953) who first obtained artificially induced tolerance to allotransplants in mammalian and avian models. Billingham et al. (1953) demonstrated that, in inbred mice, intra-embryonic injection of cell suspensions derived from donor tissues into fetal recipients permitted long-term tolerance of skin transplantation conducted after the birth of the recipients.  Similarly, if whole blood from a donor was injected intravenously into the chorioallantoic membrane (CAM) of a recipient chicken embryo or if donor tissues were transplanted onto the CAM, the post-hatch recipient might not reject a skin graft from the same donor.  Based on these results, Billingham et al. (1953) introduced the hypothesis of actively acquired tolerance, that is, tolerance of an allotransplant can be induced in mammals and birds if the recipient is exposed to donor-derived tissue or cells at a sufficiently early stage of development.  Some important conclusions were summarized in a later paper (Billingham et al., 1956).  First, actively acquired tolerance is specific, which means that the recipient has the potential of rejecting the antigens from a third party.  Second, tolerance can only be induced in a small interval restricted to the embryonic and perinatal period depending on the species.  Third, the effective tolerogenic components of donor tissues are live lymphocytes.  Finally, actively 30  acquired tolerance reflects a failure of a mechanism at the central level rather than the peripheral level.    Billingham et al. (1956) also attempted to induce xenogeneic tolerance between chickens and ducks by CAM injection or embryonic parabiosis.  The later approach was originally developed by Ha?ek and colleagues (Ha?ek et al., 1958), in which anastomosis of chorioallantoic circulation was facilitated by allowing direct contact of CAMs of donor and recipient embryos during incubation.  Neither approach induced complete tolerance in skin transplantation between chickens and ducks.   Ha?ek et al. (1959) demonstrated that a chicken skin transplant survived 80 d with growing feathers on a turkey recipient that had been treated with embryonic parabiosis.  These observations suggest that it might be easier to induce xenogeneic tolerance between species that are closer in their taxonomic relationship, which is supported by serological evidence comparing chicken-turkey and chicken-goose parabiosis (Simonsen 1955). Induction of long-term xenogeneic tolerance was achieved in a chicken-quail model by Le Douarin?s group in the 1980s.  Table 1.2 summarizes the transplantations conducted by Le Douarin and colleagues. Allo- and xeno-transplantation were conducted at early embryonic stages of donors and recipients before the colonization of thymic and bursal rudiments by hematopoietic precursors.  In all these experiments (Table 1.2), transplants were accepted and resumed normal embryonic development but some xenotransplants were rejected shortly after hatching depending on the experimental design (Le Douarin et al., 1996; Sala?n et al., 2005).  Post hatching tolerance of xenotransplants was achieved by recipients that were transplanted with donor thymic rudiments at the same time but not by bursectomized recipients or recipients that were transplanted with donor bursal rudiment.  Le 31  Douarin et al. (1996) concluded that T-cell mediated mechanisms are dominant to B-cell mediated mechanisms in rejection and tolerance of tissue transplants.  In addition, donor thymic epithelium is necessary for induction of xenogeneic tolerance.   The thymectomy of the recipients was not complete in most of these experiments so the reconstructed recipient thymus was chimeric, consisting of donor- and recipient-derived epithelium and recipient-derived T cells and dendritic cells, which were descendants of hematopoietic precursors.  Therefore the possibility of producing donor-specific T cells that might recognize and reject the transplants was not eliminated.  Le Douarin et al. (1996) proposed that donor thymic epithelium mediated the selection of regulatory T cells that were involved in suppression of potential recognition and rejection.  A population of CD4+ T cells was identified that has regulatory properties in later experiments in mice (Modigliani et al., 1996).  The mechanism of thymic epithelium mediated selection remains enigmatic.  Considering that the specificity of T cell receptors depends on that of antigens and the MHC molecules (Zerrahn et al., 1997), which are encoded by a complex of highly polymorphic genes, the presence of donor thymic epithelium might allow recipient T cell precursors to be selected according to the specificity of their T cell receptors to donor MHC molecules. 1.2.3.3 Mixed chimerism The model of actively acquired tolerance has very limited clinical application because it restricts recipients to those that were manipulated in the embryonic and perinatal period.  One model that can be used for immunocompetent recipients is mixed chimerism, in which tolerance is induced by establishing chimerism in hematopoietic stem cells in the recipient subsequent to deletion and suppression of pre-existed lymphocytes that are donor-antigen responsive.  Chimeric hematopoietic stem cells give rise to chimeric precursors of T-cells 32  and cells that mediate negative selection such as dendritic cells, and new cohorts of T cells after selection will not react to either donor or recipient self-antigens (Kurtz et al., 2004). Early studies in mice suggest that life-long chimerism is required for maintenance of tolerance.  However, studies in pigs, non-human primates and humans demonstrate that transient chimerism is sufficient for long-term tolerance, indicating the involvement of peripheral mechanisms (Fehr and Sykes, 2008; Sachs et al., 2011).  Evidence has shown that thymic chimerism rather than peripheral chimerism is associated with maintaining tolerance and thymic chimerism can be macrochimerism at the cellular level or microchimerism at the molecular level (Horner et al., 2006).  Therefore long-term tolerance derived from transient chimerism could be ascribed to long-term microchimerism.  In addition, it is possible to build thymic chimerism directly by intrathymic inoculation of donor antigens in the form of allopeptides or dendritic cells (Griesemer et al., 2010).  Note that hematopoietic stem cell chimerism is likely to share the same mechanism to Le Douarin?s thymic chimerism, the difference is that the reconstructed recipient thymus in the hematopoietic stem cell chimerism model contains recipient-derived thymic epithelium and donor- and recipient-derived T cells and dendritic cells.   1.2.3.4 Infectious tolerance Some immunosuppressive treatments may lead to long-term tolerance in transplantation in mammalian models (Waldmann, 2008; Chappert and Schwartz, 2010).  A similar phenomenon also exists in allotransplantation of chicken ovarian tissue (Song and Silversides, 2008a).    Qin et al. (1993) proposed a model of infectious tolerance that rejuvenated Gershon and Kondo?s observation of T-cell mediated suppression (Gershon and Kondo, 1971).  If recipient peripheral T cells are chronically exposed to donor antigens but 33  are not able to collaborate with antigen-presenting cells sufficiently, they might develop into tolerant T cells including anergic T cells and regulatory T cells, which may interfere with collaboration between new T cells and antigen-presenting cells, preventing them from being activated and maturing into effector T cells, and causing them to form new tolerant T cells.  Long-term tolerance can be established by this infectious effect (Waldmann, 2008).  Therefore, compared to other models in which tolerant T cells are produced by ?educated? thymus cells, this model proposes that tolerant T cells can be produced at the peripheral level. The initial insufficient collaboration of T cells and antigen-presenting cells can be induced by a blockage of surface molecules by monoclonal antibodies, reducing effector T-cell clones by selective suppression, reducing avidity of T cell receptors to antigen by antigen modification or reducing the dose of antigen (Kendal and Waldmann, 2010; Waldmann et al., 2006).  In addition, interaction between regulatory T-cell and cells of the transplant produces chemokines that attract more regulatory T-cell and molecules involved in tissue protection and repair (Waldmann et al., 2006).  Promising results have been achieved in murine models using regulatory T-cell therapy based on this theory combined with mixed chimerism (Pilat and Wekerle, 2010).         1.2.4 Induction of tolerance in avian gonadal transplantation As previously described, Billingham et al. (1953) and Le Douarin?s group (Corbel et al., 1990) have demonstrated that allogeneic tolerance can be induced by implanting donor tissue into recipient embryos.  Donor thymic tissue is preferable for induction of tolerance because the promiscuous gene expression of thymic epithelial cells is independent of gene regulation that is tissue-specific, or development- or sex-dependent and therefore ensures the 34  selection of T cells to be sufficient (Kyewski and Derbinski, 2004).  Induction and maintenance of xenogeneic tolerance require thymic chimerism (Le Douarin et al., 1996; Zhao et al., 1996), which can be achieved by direct implantation of donor thymic tissue into recipient thymic rudiment.  A possible alternative method is to implant donor thymic tissue that contains precursors of T cells and dendritic cells into the extraembryonic structures, after which the cells may migrate into recipient thymus and facilitate selection.  Because the developing avian embryos in shelled eggs are very easy to access compared to the implanted mammalian embryos, it is possible to induce donor-specific tolerance using embryonic treatments to ?educate? the recipient immune system (Figure 1.4).  After hatching of the recipients, the gonadal transplantation can be conducted and the transplants should be accepted by the recipient because those T cells that may recognize the donor tissue should be eliminated at the central level or suppressed by the regulatory T cells at the peripheral level.  This is tested in allotransplantation in chickens and xenotransplantation between chickens and Japanese quail in Chapter 6.  1.3 Functional recovery of avian gonadal transplants Transplantation allows functional recovery of cryopreserved gonadal tissue and is a useful tool to study developmental mechanisms.  Other than immunological tolerance, survival and function of the gonadal transplants are affected by other factors with respect to special anatomical and physiological characteristics of the avian reproductive system, which will be reviewed in this section. 35  1.3.1 Transplantation of avian testicular tissue During embryonic development, the avian embryo possesses a pair of male ducts named the Wolffian ducts and a pair of female ducts named the M?llerian ducts.  In male embryos, M?llerian ducts degenerate and Wolffian ducts give rise to vasa deferentia which convey spermatozoa to the cloaca in mature birds.  A connection is formed between the embryonic gonad and the Wolffian duct by the rete testis, which is derived from the rete cord in the anterior portion of each gonad (Romanoff, 1960).  Because it is difficult to surgically rebuild this connection, transplantation of avian testicular tissue can only be achieved by heterotopic procedures, in which the transplantation site is different from the normal anatomical site of the testes.  Implanting the transplants into the dorsal subcutaneous tissue of recipients is relatively non-invasive and has been widely used in mammalian models (Paris and Schlatt, 2007).  In chickens, fresh testicular tissue that had been transplanted under the dorsal skin or in the abdominal cavity of recipients showed comparable efficiency in producing donor-derived offspring (Song and Silversides, 2007c).  The techniques of subcutaneous transplantation in chickens have been refined in a recent study (Silversides et al., 2013a).  Recovery of cryopreserved testicular tissue by subcutaneous transplantation has not been achieved, nor has it been tested in other avian species.  These are investigated in Japanese quail and are described in Chapter 4. Castration of recipients affects viability of subcutaneous testicular transplants in chickens (Silversides et al., 2013a).  In addition, fertile spermatozoa can only be obtained from the transplant when the recipient has been completely castrated (Song and Silversides, 2007a,c).  This is probably due to increased gonadotropin secretion caused by removal of negative feedback from recipient testicular tissue.  An increased gonadotropin level favours 36  proliferation of Sertoli cells of transplants, which is essential for spermatogenesis in mammals and in birds (Guibert et al., 2011; Johnson et al., 2008).  However, the requirement of complete castration of recipients might not be as strict for xenotransplantation in mammals (Abbasi and Honaramooz, 2010; Paris and Schlatt, 2007; Shinohara et al., 2002).  Observations in Japanese quail are presented in Chapter 4.  Xenotransplantation of testicular tissue has been successful in a rabbit-mouse model with the production of live offspring from rabbit testicular tissue transplanted into mice (Shinohara et al., 2002).  The same study also demonstrated the feasibility of using cryopreserved testicular tissue.  This combination of cryopreservation and xenotransplantation provides an applicable method of preserving and regenerating male germplasm of endangered species.  Testicular transplantation across the xenogeneic barrier has not been attempted in avian species.  In Chapter 6, a possible approach to inducing xenogeneic tolerance to facilitate testicular transplantation between chickens and Japanese quail is described. Fertilization using testicular spermatozoa in mammals requires in vitro insemination such as intracytoplasmic sperm injection (Nakai et al., 2011).  This is not necessary for at least some avian species because intramagnal insemination of extrusion from testicular transplants that are fresh or frozen-thawed can produce live offspring in chickens (Song and Silversides 2007 a,c).  A similar approach is used in Japanese quail, which is described and discussed in Chapter 4.  37  1.3.2 Transplantation of avian ovarian tissue  In female embryos, the connection between embryonic gonads and the ducts does not appear.  The Wolffian ducts and the right M?llerian duct degenerate in most avian species while the left M?llerian duct fuses with the embryonic cloaca and differentiates into the oviduct (Romanoff, 1960).  In an adult female bird, the functional oviduct is divided into five sections: infundibulum, magnum, isthmus, shell gland (uterus) and vagina (Figure 1.5).  Each section shows distinctive histological and morphological features related to their interactions with the passing ovum (Golden and Arbona, 2012).  After ovulation, the ovum enters the infundibulum, where fertilization takes place in the presence of spermatozoa.  The outer layer of the vitelline membrane and the thick albumen are then deposited in sequence.  The egg is coated with the thin albumen and the shell membranes in the magnum and isthmus, respectively.  In the shell gland, where the egg spends around 20 h, the size increases rapidly due to the influx of fluid into the albumen and the calciferous shell is formed.  The time that the egg spends in the vagina is negligible (Johnson, 1986b). The in vivo processes of egg formation in the oviduct cannot be obtained in vitro, so the ovarian tissue of avian species must be transplanted into the normal anatomical location, which is known as orthotopic transplantation.   Orthotopic transplantation of chicken ovarian tissue was attempted several times in the past without success (Davenport, 1911; Guthrie, 1908; Grossman and Siegel, 1966).  Song and Silversides (2006) developed a transplantation technique in which one-day old chicks were ovariectomized through a transverse cut into the abdominal cavity and the donor ovarian tissue was replaced in the original location of recipient ovary.  This technique has been successfully used to produce live offspring from fresh ovarian transplants in chickens 38  (Song and Silversides 2007b), Japanese quail (Song and Silversides 2008b; Liu et al., 2010) and ducks (Song et al., 2012) and from cryopreserved ovarian transplants in Japanese quail (Liu et al., 2010).  Chapter 5 summarizes the improvement of this technique in Japanese quail, which has also been used in chickens as described in Chapters 6 and 8. In mammals, fertility recovery has been achieved from ovarian tissue of sexually mature and immature donors and the recipients used in most studies are sexually mature (Gosden, 2008).  To date, sexually immature donors and recipients have been used in all the ovarian transplantation in avian species. Whether fertility of adult ovarian tissue can be recovered by transplantation using sexually immature recipients is unknown but could be important for conservation and developmental research.  This is tested in Japanese quail in Chapter 7.   Ovariectomized recipients are used in most studies of ovarian transplantation in mammals.  Intact recipients and male recipients are also used (Cleary et al., 2003; Weissman et al., 1999).  The increased levels of follicle-stimulating hormone and luteinizing hormone in ovariectomized recipients or in male recipients that received gonadotropin administration may favour the maturation of dominant follicles in the transplants (Bols et al., 2010).  In avian species, complete ovariectomy is challenging because the left ovary is located close to the aorta and vena cava.  Chapter 8 shows an intriguing observation resulting from incomplete ovariectomy and ovarian transplantation in chickens that may reveal mechanisms in avian folliculogenesis and embryonic development.  This also suggests that in addition to being used for functional recovery of cryopreserved ovarian tissue for genetic conservation, ovarian transplantation could be used to investigate mechanisms involved in avian ovarian 39  development and function.  Another example of this is shown in Chapter 7, in which ovarian tissue from sexually mature donors are recovered in immature recipients in Japanese quail.  1.4 Objectives  The first objective of this dissertation is to develop a vitrification method that allows avian gonadal tissue to be preserved in straw systems in liquid nitrogen and recovered at a later time to produce donor-derived offspring, using the Japanese quail as a model.  Two types of straw systems were investigated and their efficiency in preserving tissue viability, morphology and the potential of revascularization was evaluated using in ovo tissue culture. These were described in Chapters 2 and 3.  The simpler and effective 2-ml straw system was used in the subsequent studies.  In Chapter 4, functional recovery of quail testicular tissue cryopreserved by this vitrification method was tested by allotransplantation of cryopreserved tissue and subsequent intramagnal insemination using the extrusion from the transplants.  Functional recovery of quail ovarian tissue cryopreserved using this vitrification method was evaluated by allotransplantation and subsequent progeny test as described in Chapter 5.  The second objective of this dissertation is to optimize avian gonadal transplantation in terms of preventing immunological rejection of the transplants, using adult birds as ovarian donors and applying ovarian vitrification and transplantation to chickens.  In Chapter 6, the possibility of inducing immunological tolerance for post-hatching gonadal transplantation by implanting donor thymic tissue onto recipient CAM was tested in chickens for allotransplantation and between chickens and quail for xenotransplantation.  Graft viability and morphology were examined.  The possibility of recovering fertility of ovarian tissue from sexually mature donors by transplatation was tested in quail by transplanting ovarian tissue from adult birds into week-old recipients and subsequent progeny test as 40  described in Chapter 7.  Chapter 8 describes the use of the vitrification and transplantation of ovarian tissue in chickens, which provides an example of using these techniques for recovery of genetic resources and also suggests their potential use in avian biology research.     41  Table 1.1.  A general comparison of innate and adaptive immunity1  Innate immunity Adaptive immunity Specificity of recognition Non-specific  Specific  Speed of response Immediate  Delayed  Immunological memory No  Yes Species specificity Universal Specific to jawed vertebrates 1 Adapted from Janeway et al., 2005 and Schijns et al., 2008.   42  Table 1.2.  Embryonic transplantation experiments conducted by Le Douarin's group Embryonic transplants Treatment of recipient chicken embryo  Post-hatching tolerance or rejection References Chicken limb bud  Limbectomy Tolerance  Corbel et al., 1990 Quail limb bud  Limbectomy Rejection  Ohki et al., 1987 Quail limb bud and  thymic rudiment   Limbectomy and thymectomy  Tolerance  Ohki et al., 1987 Quail limb bud   Limbectomy and bursectomy  Rejection Le Douarin et al., 1996 Quail bursal rudiment  Bursectomy  Rejection Corbel et al., 1987 Quail bursal rudiment and thymic rudiment  Bursectomy and thymectomy Tolerance  Belo et al., 1989    43   Figure 1.1.  Schematic phase diagram of a cryoprotective agent (CPA) solution.  Tm: melting temperature curve; Th: homogeneous nucleation temperature curve; Tg: glass transition temperature curve.  The intersection between Th and Tg gives a critical CPA concentration; above this concentration, it is possible to cool the solution directly to the glass transition temperature without freezing.  Adapted from Fahy et al. (1984).   44   Figure 1.2.  Cycle of germline development. Successful preservation of any component of this cycle and subsequent functional recovery will lead to successful ex situ conservation.   45   Figure 1.3.  A schematic drawing of major structural features of spermatozoa from different species.  Spermatozoa are adapted from Garner and Hafez (2000) and Romanoff (1960) and are not drawn to scale.   46                Thymic tissue  Recipient embryo Recipient Gonadal tissue Donor  Cryopreservation Implantation  Transplantation  Hatch  Figure 1.4.  A possible method of inducing allo- and xenogeneic tolerance for avian gonadal transplantation. 47          Infundibulum Magnum Isthmus Shell gland Vagina Figure 1.5.  Schematic diagram of the oviduct of a mature hen. 48  2 NOVEL NEEDLE-IN-STRAW VITRIFICATION CAN EFFECTIVELY PRESERVE THE FOLLICLE MORPHOLOGY, VIABILITY, AND VASCULARIZATION OF OVARIAN TISSUE IN JAPANESE QUAIL (COTURNIX JAPONICA)  2 2.1 Introduction Avian researchers and the poultry industry have faced a drastic reduction of avian genetic resources in the past few decades, largely because of the lack of an inexpensive and reliable preservation-reconstitution regimen.  Until recently, the only effective method of preserving avian genetic resources has been in living flocks but the high cost has resulted in a dramatic and continuous decline in numbers of research flocks (Fulton and Delany, 2003).  Meanwhile, the modern poultry breeding industry uses very few highly selected lines, leaving it vulnerable to threats such as disease or unable to meet market diversification.  Cryopreservation of germplasm has been successfully used in the dairy industry, which provides an example for poultry.  However, it has been extremely challenging in birds, especially for females.  Avian eggs are characterized by the presence of a large amount of yolk (polylecithal), and the polar position of the developing embryo (telolecithal).  These characteristics prevent the application of the cryopreservation techniques that have been used for mammals.  Efforts have been made to cryopreserve                                                  2 A version of Chapter 2 has been published. Liu, J., K. M. Cheng, and F. G. Silversides. 2012. Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica). Anim. Reprod. Sci. 134:197-202. 49  primordial germ cells to generate chimeras, but the feasibility of this strategy for genetic conservation is limited due to the low efficiency and high cost (Petitte, 2006).   Ovarian tissue contains a large pool of primordial follicles with the potential to generate mature follicles (Johnson and Woods, 2009), providing that the tissue can be recovered.  This has been confirmed by recent success in ovarian transplantation in chickens (Song and Silversides, 2006, 2007b) and Japanese quail (Song and Silversides, 2008b).   In addition, we have demonstrated that ovarian tissue of Japanese quail can be successfully cryopreserved and recovered by transplantation (Liu et al., 2010).  Liu et al. (2010) showed that a vitrification method was more efficient than a conventional slow-freezing method.  Vitrification likely provides better preservation of the integrity of the tissue, which is important for its functional recovery, through the process of solidification without ice crystallization (glass formation).  With the conventional slow-freezing method, ice formation is induced in the extracellular space (Nawroth et al., 2007).   The vitrification method that we used previously was based on successful studies in mammals (Chen et al., 2006; Wang et al., 2008).  The use of acupuncture needles as tissue carriers minimized the handling of individual samples and enhanced the efficiency of processing a large quantity of samples.  However, when tissue is stored in cryovials for cryobanking, there are practical limitations such as difficulty in handling and security concerns (Kuwayama, 2007).  The objective of this study was to test the efficiency of vitrification and warming procedures using straws instead of cryovials to preserve the ovarian tissue of Japanese quail.  Histology of the cryopreserved and warmed ovarian tissue and its viability and 50  vascularization after being transplanted onto the chicken chorioallantoic membrane (CAM) were examined and analyzed.  2.2 Materials and methods 2.2.1 Birds, chemicals and tissue preparation Quail ovarian tissue was obtained from one-week-old female chicks of White Breasted (WB) and QO lines of Japanese quail (Liu et al., 2010).  Fertile chicken eggs were from the Minnesota Marker line (Pisenti et al., 2001).  Both the quail and chicken lines were maintained at the Agassiz Research Centre.  The research protocol was approved by the Animal Care Committee of the Agassiz Research Centre following principles described by the Canadian Council on Animal Care (2009).  All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) unless otherwise indicated.  One-week-old quail chicks were used because a previous study (Song and Silversides, 2008b) found that recipients with ovarian transplantation at this age survived better than those at one-day-old age.  Ovarian tissue was obtained immediately after euthanasia of the birds by cervical dislocation and immersed in Dulbecco?s phosphate buffered saline (DPBS) with 20% fetal bovine serum on ice.  The surrounding connective tissue was then gently removed and each ovary was cut to a size of approximately 2.5 mm ? 2.5 mm under a dissecting microscope.  All tissue fragments were kept on ice before further treatment within 2 h. 51  2.2.2 Vitrification and warming procedures For the needle-in-straw (NIS) method, four to six ovarian tissue fragments were  transfixed on an acupuncture needle (Cloud & Dragon Medical Device Co. Lid, Jiangsu Province, China), which was modified slightly to fit the straw.  Tissue fragments carried by the needle (Figure 2.1A) were first submerged in DPBS with 20% fetal bovine serum containing 7.5% (v/v) dimethyl sulphoxide (DMSO) and 7.5 % (v/v) ethylene glycol (EG) for 10 minutes, then a similar solution containing 15% (v/v) DMSO, 15% (v/v) EG and 0.5 M sucrose for 2 minutes. This two-step vitrification protocol had been successfully used for mice (Chen et al., 2006; Wang et al, 2008) and Japanese quail (Liu et al., 2010).  The tissue fragments were blotted briefly with a piece of gauze and rolled in a layer of tin foil.  The tin foil package was then submerged in liquid nitrogen and immediately inserted into a pre-cooled, 0.5-ml straw (CBSTM High Security Straw, Cryo Bio System, Paris, France) with one end pre-sealed by heat.  The other end was sealed with a pre-cooled straw adaptor plug (Bioniche Animal Health, Belleville, ON, Canada) (Figure 2.1B), and the straws were stored in liquid nitrogen.  For the needle immersed vitrification (NIV) method, tissue fragments were treated as described for the NIS method except that the handles of the needles were folded to make the needles shorter  to fit the cryovials (Fisher Scientific, Edmonton, AB, Canada) and tin foil was not involved as was described in previous studies (Wang et al., 2008; Liu et al., 2010).  For warming, the straws and vials were opened while still immersed in the liquid nitrogen.  The needles carrying ovarian fragments were removed from the liquid nitrogen and immediately immersed in 1 M sucrose in DPBS with 20% fetal bovine serum at 52  room temperature (RT) or at 37?C for 5 min.  For the tissue preserved in the straws, the tin foil was removed immediately.  Tissue fragments were subsequently transferred to DPBS with 20% fetal bovine serum containing 0.5 M, 0.25 M and 0 M sucrose for 5 min each at RT or at 37?C.  The tissue was kept on ice before further use.  2.2.3 Histological examination Fresh and cryopreserved and warmed tissue fragments were fixed in Bouin?s solution for 24 h, dehydrated in alcohol and subsequently embedded in paraffin.  The embedded samples were cut into 7 ?m serial sections, mounted on slides and stained with hematoxylin and eosin.  Images were captured and examined using a digital camera (1300R, Qimaging Corp., Burnaby, British Columbia, Canada) mounted on a microscope (Olympus BX51, Olympus Corp., Tokyo, Japan).  In this study, follicles with the following characteristics (Figure 2.2) were defined as normal: (i) intact follicular epithelium; (ii) intact ooplasm with a visible germinal vesicle; (iii) widest diameter greater than 20 ?m.  The ratio of normal follicles to total visible follicles was determined from five widely separated sections for each tissue fragment to prevent any single follicle being counted more than once.  2.2.4 Tissue grafting onto chicken embryo chorioallantoic membrane Fertilized chicken eggs were artificially incubated under normal conditions for 3 d.  The eggs were then positioned horizontally in the incubator for 30 min and the position of the embryo was located by candling and marked on the shell.  After 1 to 1.5 ml albumen were withdrawn through the small end of the egg using an 18-gauge needle, a window of approximately 1cm2 was made in the shell and shell membranes above the 53  embryo with a small drill (Dremel Stylus?, Racine, WI, United States).  The window was sealed with a piece of parafilm, the edges of which were further sealed by surgical tape and the eggs were returned to the incubator.  After an additional 5 d of incubation without turning, the fresh or cryopreserved and warmed quail ovarian tissue fragments were placed onto the developing CAM through the window, with the grafted spot slightly traumatized (Martinez-Madrid et al., 2009) to facilitate tissue attachment and survival.  Each egg was grafted with one piece of tissue.  The window was then re-sealed and the egg was returned to the incubator.  After an additional 5 to 6 d of incubation without turning, the ovarian grafts were retrieved from the CAM of those chicken embryos that survived (Figure 2.3).   The viability and vascularization of the grafts were examined under a dissecting microscope (Figure 2.3B-D).  Live grafts were firmly attached to the CAM with an obvious blood supply from the CAM while the dead grafts were not firmly attached and their surfaces were hardened with a yellowish-brown color.  The vascularization of the live grafts was evaluated in a manner similar to that described by Qureshi et al. (2008).  Three categories of vascularization were observed: (i) well-vascularized, the graft was mostly pink; (ii) partially-vascularized, the graft was pink in isolated spots; (iii) poorly-vascularized, the graft was well attached to the CAM with an obvious blood supply but was mostly pale in appearance.   2.2.5 Statistical analyses All statistical analyses were conducted with SAS Server Interface 2.0.3.  Follicle morphology data were analyzed with ANOVA to compare the five groups using the GLM procedure.  The Likelihood Ratio Chi-square Test in the FREQ procedure was used 54  to analyze the contingency tables of viability and vascularization of the grafts.  Statements of statistical significance are based on P ? 0.05. 2.3 Results and discussion The means of the ratio of normal follicles to total visible follicles of the five treatments were compared (Figure 2.4).  The highest ratio was obtained for the control group and was not different from the NIS device with warming at room temperature.  All other treatment groups had a significantly lower ratio than the control.  The viability and vascularization of the ovarian tissue grafted onto chicken CAM is presented in Table 2.1.  There was no significant difference in the viability and vascularization of the cryopreserved ovarian grafts compared to the fresh grafts.    Vitrification refers to the solidification of a liquid into an amorphous, highly vitreous form known as glass without ice crystallization (Fahy et al., 1984).  Proper vitrification can be achieved under various experimental conditions (Angell, 2002).  There is consensus that when applied to cryopreservation of biological materials, successful vitrification depends on sufficiently high cooling and warming rates and sufficiently high concentrations of cryoprotective agents (CPA) (Mazur et al., 2008). Because of the reciprocal relationship between the cooling rate and the required concentrations of CPA (Fahy et al., 1987), relatively lower concentrations can be used to reduce the toxic and osmotic effects by enhancing the cooling and warming rates.  A simple and effective approach to maximizing the cooling rate is to allow dehydrated tissue to contact liquid nitrogen directly which Chen et al. (2006) termed direct cover vitrification.  The practicality of direct cover vitrification was further improved by Wang et al. (2008) by adapting acupuncture needles as tissue carriers, which they named 55  ?needle immersed vitrification? (NIV).  We have successfully applied NIV to preserve ovarian tissue of Japanese quail (Liu et al., 2010).  In spite of the effectiveness of this method, almost all commercially available cryovials are designed for storage in the vapor phase of liquid nitrogen and putting liquid nitrogen in the cryovials creates a potential for explosion (Kuwayama, 2007), making the long-term storage difficult.   As an alternative, variations of the plastic straw were developed to facilitate ultra-rapid cooling of oocytes and embryos by modifying the straws or using special carriers (Kuwayama, 2007; Vajta et al., 1998; Yavin et al., 2009), some of which are commercially available.  No comparable device is available for preserving tissue by vitrification.  The insulation of the straw and the surrounding solution reduces the cooling rate and increases the chance of ice crystal nucleation and formation (Kuwayama, 2007), which damages the tissue structures.  Abrishami et al. (2010) improved this by placing the tissue fragments in small tin foil boats floating on top of liquid nitrogen.  In this study, using a tissue carrier and tin foil, which has a low insulation value, ensured a rapid cooling rate and enabled storage in straws, which may account for the high efficiency in preserving the morphology of ovarian follicles.  In addition, the NIS system is enclosed and takes little space, making it practical for the establishment and utilization of a cryobank.  During the last decade, attention has been devoted almost exclusively to the development of devices that facilitate a rapid cooling rate, but the importance of the warming rate to the vitrification method has only begun to be addressed.  Based on their work on mouse oocytes, Seki and Mazur (2008, 2009) and Mazur and Seki (2011) postulated that the warming rate is more important than the cooling rate for the survival 56  of the cells.  Fast warming was superior to slow warming because the latter allowed small ice crystals that had formed during the cooling procedure to grow into crystals that passed the lethal threshold for the cells.  The use of needles to hold the tissue made it convenient to submerge a number of tissue fragments in the warming solution immediately after removal from the liquid nitrogen which is essential to achieve rapid warming rates.  In this study, ovarian tissue was warmed in sucrose solution at RT and at 37?C and warming at a higher temperature was expected to improve tissue recovery.  However, the experimental data do not appear to agree with the theoretical prediction, possibly because of the interaction between the device and the warming temperature.  Alternatively, the difference between the two warming temperatures may have been too small to reveal the actual effect.  Reproduction of avian species is characterized by the offspring developing in hard-shelled eggs, with limited embryonic development in the maternal body before oviposition.  Therefore techniques such as in vitro maturation and in vitro fertilization of mammalian follicles retrieved from ovarian tissue that are used in combination with embryo transfer cannot be used for birds.  Ovarian transplantation allows functional recovery of fresh (Song and Silversides, 2007b, 2008b) and cryopreserved (Liu et al., 2010) ovarian tissue in chickens and Japanese quail.    A chicken CAM model was used in this study to evaluate the effects of vitrification methods on the viability and vascularization of quail ovarian tissue after transplantation.  This model provided an effective simulation for ovarian transplantation surgery in birds because the grafts were placed onto a traumatized, highly vascularized 57  surface, which is similar to ovarian transplantation (Song and Silversides, 2006).  More importantly, the number of follicles that resume folliculogenesis is not only dependent on the number of normal follicles that have been preserved but also on whether the tissue can survive and re-establish vascularization (Liu et al., 2002). The effect of the vitrification methods on the viability and vascularization of the ovarian grafts was not significant, indicating that cryopreservation had a negligible effect on the recovery of the grafts.   In conclusion, the results of this study suggest that the novel NIS vitrification device can effectively preserve the follicle morphology of quail ovarian tissue and has no negative effect on the vascularization of the ovarian grafts subsequent to cryopreservation. Therefore, it can be used to replace cryovials to store tissue samples following vitrification procedures in a safe and practical manner.   58  Table 2.1.  Viability and vascularization of ovarian tissues grafted onto chicken chorioallantoic membrane Groups Total Surviving embryos Vascularization of live ovarian grafts* Dead ovarian grafts* Well Partially Poorly Control 17 17 4 5 3 5 NIS/37?C 12 12 1 5 4 2 NIS/RT 15 14 2 4 5 3 NIV/37?C 15 13 1 3 4 5 NIV/RT 12 10 2 3 2 3 * No significant difference was seen between control and any treatment groups (P>0.05).   59   Figure 2.1.  Needle-in-straw (NIS) vitrification device. A: ovarian fragments carried by a needle; B: A demonstration of the device: tin foil including tissue fragments on a needle was inserted into a straw, with the end of the straw sealed by an adaptor plug.   60   Figure 2.2.  Histology of ovarian tissue. In this field, the arrows point to the examples of normal follicles with intact follicular epithelium, intact ooplasm, a visible germinal vesicle, and the widest diameter greater than 20 ?m. Some examples of follicles that were not considered as normal because of non-intact structures listed above and/or invisible germinal vesicle are indicated by crosses. Bar = 20?m.   61   Figure 2.3.  Grafting ovarian tissue onto chicken chorioallantoic membrane (CAM). A: In ovo view of a piece of graft shortly before retrieving; B: an example of dead graft; C: an example of live graft that was categorized as well vascularized; D: an example of live graft that was categorized as poorly vascularized.   62   Figure 2.4.  The ratio of normal follicles to total visible follicles. Five groups were the Control group: fresh tissue without treatment (n = 5); NIS/37?C: tissue was preserved using Needle-in-Straw method and warmed at 37?C (n = 4); NIS/RT: tissue was preserved using Needle-in-Straw method and warmed at room temperature (n = 4); NIV/37?C: tissue was preserved using Needle Immersed Vitrification method and warmed at 37?C (n = 4); NIV/RT:  tissue was preserved using Needle Immersed Vitrification method and warmed at room temperature (n = 5). a-d: means with no common superscript differ significantly, P?0.05. 63  3 A SIMPLE VITRIFICATION METHOD FOR CRYOBANKING AVIAN TESTICULAR TISSUE3 3.1 Introduction Cyropreservation of germplasm is an effective approach to conservation and recovery of genetic diversity of domestic animals and wild species because it is not limited by the reproductive life span or the location of the breeders.  In avian species, investigations have long been focused on semen cryopreservation, especially in the domestic fowl.  To date, cryopreserved semen has produced considerable variation in fertility among chicken breeds and individuals (Blesbois et al., 2007).  Efforts have been made to cryopreserve semen of other avian species (Blesbois, 2011) but this strategy requires fertile birds and proper semen collection and artificial insemination methods, which can be challenging for some species.  Alternative strategies for preserving male fertility in avian species are needed. Cryopreservation of testicular tissue allows the enclosed spermatogonia bearing the potential of spermatogenesis to be preserved and recovered when the appropriate conditions are met.  Live offspring have been obtained from cryopreserved murine testicular tissue and a number of investigations have been conducted to use this strategy to preserve male fertility of domestic animals and humans in situations when semen collection and preservation is not an option (Ehmcke and Schlatt, 2008).  In avian species, chicken testicular tissue has been successfully cryopreserved by slow freezing                                                  3 A version of Chapter 3 has been published. Liu, J., K. M. Cheng, P. H. Purdy, and F. G. Silversides. 2012. A simple vitrification method for cryobanking avian testicular tissue. Poult. Sci. 91:3209-3213. 64  procedures and subsequently recovered by heterotopic transplantation (Song and Silversides, 2007a), providing a promising model for preserving and recovering avian genetic resources.   Minimizing intracellular ice formation is the prerequisite for successful preservation of biological materials at subzero temperatures because of the lethal effects of intracellular ice formation (Mazur, 1963).  There are various cryopreservation procedures to circumvent intracellular ice formation (Sakai, 2004) and two of them have been used for animal tissue and organs: slow-freezing and vitrification.  Most of the published procedures for testicular tissue cryopreservation used slow-freezing strategies.  Alternately, vitrification strategies have been investigated.  Vitrification is the transformation of a liquid into an amorphous, highly vitreous solid (glass) (Fahy et al., 1984) which can be achieved using a very high cooling rate and high concentrations of cryoprotective agents (CPA) (Mazur et al., 2008).  Vitrification methods make it possible to protect both intracellular and extracelluar components from ice formation (Fahy et al., 1984) and offer promise for tissue and organ cryopreservation.  Encouraging results have been achieved using this strategy to preserve testicular tissue of mammals including humans (Abrishami et al., 2010; Curaba et al., 2011a,b).  In addition, our recent study in Japanese quail showed the advantage of a vitrification method in preserving ovarian tissue (Liu et al., 2010).  We envision that avian testicular tissue can also be effectively cryopreserved but cooling and warming procedures may need adjustments. Cryopreserved testicular tissue needs to be cultured for resumption of germline development.  In vitro culture has been successful in mammals (Sato et al. 2011) but is not available for birds.  In vivo culture or transplantation was used to recover frozen-65  thawed chicken testicular tissue (Song and Silversides, 2007a) but requires immunosuppressive treatment and sacrifice of the recipients.  Implantation of cryopreserved tissue onto chicken chorioallantoic membrane (CAM) provides a fast and simple alternative that can be used for screening of  cryopreservation methods (Martinez-Madrid et al., 2009).  In this study, a vitrification method was used to preserve the testicular tissue of one-week old Japanese quail.  The effects of cooling and warming procedures on vascularization of cryopreserved and warmed tissue were evaluated by grafting the tissue onto the chicken CAM.   3.2 Materials and methods 3.2.1 Birds, chemicals and tissue preparation Testicular tissue was isolated from one-week-old male chicks of the QO line of Japanese quail (Liu et al., 2010) and fertile chicken eggs were obtained from the Minnesota Marker line (Pisenti et al., 2001). Both lines are maintained at the Agassiz Research Centre.  The research protocol was approved by the Animal Care Committee of the Agassiz Research Centre following principles described by the Canadian Council on Animal Care (2009).  All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) unless otherwise indicated.  Testes were collected from the birds immediately after euthanasia by cervical dislocation and immersed in a handling medium (HM) consisted of Dulbecco?s phosphate buffered saline (DPBS) supplemented with 20% fetal bovine serum on ice.  The tunica vaginalis and tunica albuginea were torn open under a dissecting microscope 66  to expose the testicular tissue.  The tissue was kept in HM on ice before further treatment within 2 h. 3.2.2 Vitrification and warming procedures Five to six testes were transfixed on an acupuncture needle (Seirin Corporation, Shizuoka, Japan, Figure 3.1A).  Testes carried by the needle were first submerged at room temperature (RT) in an equilibration solution (Chen et al., 2006) containing 7.5% (v/v) dimethyl sulphoxide (DMSO) and 7.5 % (v/v) ethylene glycol (EG) in HM for 10 min, then a vitrification solution composed of 15% (v/v) DMSO, 15% (v/v) EG and 0.5 M sucrose in HM for 2 min.  This two-step protocol has been successfully used for ovarian tissue of mice (Chen et al., 2006; Wang et al., 2008) and Japanese quail (Liu et al., 2010).  The testes were blotted briefly with a piece of gauze, plunged into liquid nitrogen and immediately inserted into a modified pre-cooled 2 ml macrotube straw with one end pre-sealed with a glass sealing ball (Minit?b GmbH, Tiefenbach, Germany, Figure 3.1B).  The other end was sealed with a pre-cooled glass ball and the straws were stored in liquid nitrogen. For warming, the straws were opened while still immersed in the liquid nitrogen.  After removal from the liquid nitrogen, the testes carried by the needles were immediately immersed in the HM with 1 M sucrose at either RT or 40?C for 5 min.  They were subsequently transferred to 0.5 M, 0.25 M and 0 M sucrose solutions for 5 min each at RT.  The testes were then suspended in HM on ice before further use. Transfixed fresh tissue was used as a control treatment. 67  3.2.3 Tissue grafting onto chicken embryonic chorioallantoic membrane Fertilized chicken eggs were artificially incubated under standard conditions for 3 d.  The eggs were then positioned horizontally in the incubator for 30 min and the position of the embryo was located by candling and marked on the shell.  After 1 to 1.5 ml albumen were withdrawn through the small end of the egg using an 18-gauge needle, a window of approximately 1 cm2 was made in the shell and shell membranes above the embryo with a small drill (Dremel Stylus?, Racine, WI, United States).  The window was sealed with a piece of parafilm, the edges of which were further sealed by surgical tape and the eggs were returned to the incubator.  After an additional 5 d of incubation without turning, fresh or cryopreserved quail testes were placed onto a slightly traumatized area of the developing CAM of the windowed eggs (Martinez-Madrid et al., 2009).  The trauma facilitates tissue attachment and survival.  Each egg was grafted with one testis.  The window was then sealed as before and the egg was returned to the incubator for an additional 8 to 9 d of incubation without turning.  The viability of the embryos was checked daily and the dead embryos were culled.  The grafts from the CAM of those chicken embryos that survived were retrieved after the incubation period.  The vascularization of the grafts was examined under a dissecting microscope (Wild Leitz Canada, Ltd., Willowdale, ON, Canada) and the images were recorded by an attached camera (Infinity2, Lumenera Corp., Ottawa, ON, Canada).  All the grafts were attached to the CAM and had an obvious blood supply (Figure 3.2A).  The vascularization of the grafts was evaluated using criteria similar to those described by Qureshi et al. (2008).  Three categories of vascularization were observed in this study: (i) well-vascularized, the graft was mostly pink (Figure 3.2B); (ii) partially-vascularized, the 68  graft was pink with some pale areas; (iii) poorly-vascularized, the graft was well attached to the CAM with an obvious blood supply but was mostly pale in appearance (Figure 3.2C).   3.2.4 Histological examination Grafts retrieved from the CAM were fixed in Bouin?s solution for 24 h, dehydrated in alcohol and embedded in paraffin.  The embedded samples were cut into 5 ?m serial sections, mounted on slides and stained with hematoxylin and eosin.  Fresh tissue was treated in a similar fashion.  Slides were observed with a light microscope (Olympus BX51, Olympus Corp., Tokyo, Japan) and images were recorded using an attached digital camera (1300R, Qimaging Corp., Burnaby, BC, Canada)  3.2.5 Statistical analyses All statistical analyses were performed using SAS Server Interface 2.0.3.  The Fisher?s Exact Test in the FREQ procedure was used for pair-wise comparisons of the vascularization of the grafts and viability of the host embryos in the three experimental groups.  The criterion for statistical significance was P ? 0.05.  3.3 Results and discussion All of the grafted quail testes in the chicken embryos that survived were supported by the CAM and had an obvious blood supply.  The vascularization of the grafts and the viability of the host embryos are shown in Table 3.1.  The tissue warmed at RT, but not the tissue warmed at 40 ?C, had significantly less vascularization when compared to the grafts of fresh tissue (control).  There was no significant difference in mortality of host embryos among treatments.  69   The vascularization of the grafts agreed with the histology in that large areas of abnormal structures were observed in poorly-vascularized grafts but were very rare in well-vascularized grafts (Figure 3.3).  Compared with the untreated testicular tissue, the change in spermatogenic development and the seminiferous tubules of well-vascularized graft is in accordance with normal testicular development described by Mather and Wilson (1964). Most previous studies in cryopreservation of testicular tissue have used slow-freezing procedures, in which ice nucleation is induced in the extracelluar space and the difference in chemical potential between intracellular and extracellular water drives the intracellular water out of the cell (dehydration) to form ice.  The cooling rate is slow enough to maintain the equilibrium between extracellular ice formation and cell dehydration to prevent intracellular ice formation (Mazur, 1963).  However, extracellular ice formation involved in slow-freezing procedures is detrimental to multicellular systems such as testicular tissue (Woods et al., 2004), which may be better preserved by vitrification.  In addition, the cooling processes are simplified, making it a practical approach to tissue cryobanking.  The present study provided evidence that a simple vitrification method could allow testicular tissue from an avian species to survive vitrification and warming procedures and resume vascularization. A number of vitrification protocols such as ?direct cover vitrification? (Chen et al., 2006) and ?needle immersed vitrification? (NIV, Wang et al., 2008) have been developed for ovarian tissue to maximize the cooling rate so that the concentrations of CPA could be decreased concordantly to reduce the toxic and osmotic effects (Fahy et al., 1987).  Quail ovarian tissue has been successfully preserved using NIV (Liu et al., 70  2010) with an acupuncture needle as a tissue carrier to minimize repeated tissue handling and facilitate fast cooling and warming. The use of macrotube straws and glass sealing balls in the current study allowed tissue to be stored in a closed system that can be adapted to any standard cryobanking system.  Fast warming was achieved by warming tissue at 40 ?C, which is the core body temperature of birds.  The importance of fast warming to vitrification methods was investigated by Mazur and Seki (2011), who postulated that slow warming allows time for lethal ice crystallization resulting from recrystallization or devitrification during warming procedures.  Our results suggest a beneficial effect of warming tissue samples at 40 ?C.   The CAM model used in the current study provides a useful tool to study short-term viability and vascularization of cryopreserved testicular tissue after implantation. The chick?s immune system at this stage is immature and is not able to mount a specific or nonspecific response to the implants (Ribatti, 2008) so that the viability and vascularization of the implants can be used to rapidly evaluate the efficiency of cryopreservation protocols.  However, the CAM model is limited by the incubation period of the host embryo (Yuan et al., 2009) and maturation and production of fertile spermatozoa by the cryopreserved testicular tissue can only be tested by transplantation (Song and Silversides, 2007a).   In conclusion, we provided evidence that by using a simple and practical method, testicular tissue from an avian species can be cryopreserved using vitrification and stored without significant loss of the ability to establish vascularization after recovery, which paves the way for further investigations of cryopreservation of male germplasm for avian species when semen preservation is not applicable.  71   Table 3.1.  Vascularization of the testicular grafts and viability of the host embryos Groups Vascularization of live  testicular grafts Live host embryos1 Dead host embryos1 Well Partly Poorly Controla 6 3 0 9 2 Warming at 40 ?Cab 8 3 3 14 3 Warming at RTb 4 1 5 10 4 a,b Groups with no common superscript differ (P < 0.05) in the degree of vascularization of testicular grafts. 1 There was no difference (P > 0.05) between groups in mortality of host embryos.   72     Figure 3.1.  A demonstration of the vitrification device used in this study. A: Testes held by the needles were suspended in the handling media; B: A straw containing a needle was sealed by glass balls at both ends. 73     Figure 3.2.  Japanese quail testes grafted to chicken chorioallantoic membrane (CAM). A: an in ovo view of a graft shortly before retrieval, which is indicated by the arrow; B: a graft that was categorized as well-vascularized; C: a graft that was categorized as poorly-vascularized.   74   Figure 3.3.  Appearance of Japanese quail testes at the time of isolation and 9 d after grafting. A: a section of testis isolated from one-week old quail; B: a section of a graft that was categorized as well-vascularized; C: a section of a graft that was categorized as poorly-vascularized, notice the abnormal structure in the center. D-F: a view of A-C with a higher degree of magnification. Scale bars = 100 ?m. 75  4 PRODUCTION OF LIVE OFFSPRING FROM TESTICULAR TISSUE CRYOPRESERVED BY VITRIFICATION PROCEDURES IN JAPANESE QUAIL (COTURNIX JAPONICA)4 4.1 Introduction Cryopreservation of testicular tissue preserves the enclosed spermatogonial stem cells which can produce functional spermatozoa in their normal cellular environment.  It is a promising strategy for preservation of fertility of human patients in situations when semen is not available (Rodriguez-Sosa et al., 2012), such as before the donor is sexually mature.  Restoration of spermatogenesis in testicular tissue that has been cryopreserved and subsequently recovered by transplantation has been reported in various mammalian species (Ehmche and Schlatt, 2008) with the production of live offspring in laboratory rodents (Shinohara et al., 2002).  Cryopreserved testicular tissue of newly-hatched chickens can survive transplantation and can produce live chicks (Song and Silversides, 2007a).  This provides a practical approach to ex situ germplasm conservation and the management of male germplasm of avian species such as Japanese quail for which semen cryopreservation has not been successful (Fulton and Delany, 2003).     In most previous studies, slow freezing procedures have been used to preserve testicular tissue.  This usually includes induction of extracellular ice nucleation at a particular seeding temperature and controlling the cooling rate to minimize lethal intracellular ice formation.  Cryoprotective agents are used to ameliorate the detrimental                                                  4 A version of Chapter 4 has been published. Liu, J., K. M. Cheng, and F. G. Silversides. 2013. Production of live offspring from testicular tissue cryopreserved by vitrification procedures in Japanese quail (Coturnix japonica). Biol. Reprod. 88:124,1-6. 76  solution effects which are associated with slow cooling (Mazur et al., 2008).  However, this strategy provides limited protection to multicellular structures (Mazur, 2004) such as testicular tissue, and vitrification procedures have been investigated.  Vitrification is the process of solidification of a liquid without crystallization, resulting in an amorphous solid called glass (Fahy et al, 1984).  This process can be approached by using ultra-rapid cooling combined with sufficiently concentrated cryoprotective agents so that intracellular and extracellular water is converted to glass (Mazur et al., 2008).  The application of vitrification for preservation of testicular tissue has been attempted in mammalian species, including humans (Abrishami et al., 2010; Curaba et al., 2011a,b), and fish (Bono-Mestre et al., 2009), with encouraging results although no offspring have been produced.  The effectiveness of vitrification procedures in cryopreserving ovarian tissue of Japanese quail has been demonstrated (Liu et al., 2010).  Testicular tissue of Japanese quail has also been cryopreserved using vitrification procedures and shows normal macro- and microscopic structures after short-term in ovo culture (Liu et al., 2012a).  Whether these findings can be extended to functional recovery requires confirmation.  Functional recovery of mammalian testicular tissue cryopreserved by slow freezing could be achieved by transplantation and subsequent intracytoplasmic spermatozoa injection using spermatozoa extracted from the testicular transplants (Ehmche and Schlatt, 2008).   Although intracytoplasmic spermatozoa injection is available for birds such as Japanese quail (Hrabia et al., 2003), it is unlikely to be practical for conservation programs.  Intramagnal insemination using fluid extrusion retrieved from testicular transplants has been used in chickens (Song and Silversides, 77  2007a,c), but has not been described for Japanese quail.  In this study, testicular tissue of week-old Japanese quail was cryopreserved using a vitrification protocol (Liu et al., 2012a).   Functional recovery of the cryopreserved testicular tissue was evaluated by ectopic transplantation and intramagnal insemination using the fluid extrusion retrieved from the transplants after maturation of the recipients. 4.2 Materials and methods 4.2.1 Birds, chemicals and tissue preparation One-week old male chicks of the QO and White-breasted (WB) lines (Liu et al., 2010) were used as testicular donors and recipients, respectively.  Adult hens from the QO line and the JWT line (the UBC-N line described by Pisenti et al., 2001) were inseminated intramagnally.  All of these lines are maintained at Agassiz Research Centre.  The research protocol was approved by the Animal Care Committee of the Agassiz Research Centre following principles described by the Canadian Council on Animal Care.  All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) unless otherwise indicated. Testes were removed from the donor birds immediately after euthanasia by cervical dislocation and immersed in a handling medium (HM) composed of Dulbecco?s phosphate buffered saline (DPBS) supplemented with 20% fetal bovine serum on ice.  The tunica vaginalis and tunica albuginea were cut to expose the testicular tissue.   4.2.2 Vitrification and warming procedures The cryopreservation procedures used in this study followed a protocol that has recently been adapted for avian testicular tissue (Liu et al., 2012a).  Five testes from 78  different males were transfixed on an acupuncture needle (Seirin Corporation, Shizuoka, Japan).  At room temperature, testes carried by the needle were submerged in an equilibration solution of HM with 7.5% (v/v) dimethyl sulphoxide (DMSO) and 7.5 % (v/v) ethylene glycol (EG) for 10 min, then a vitrification solution of HM with 15% (v/v) DMSO, 15% (v/v) EG, and 0.5 M sucrose (VWR, Mississauga, ON, Canada) for 2 min.  This protocol has been successfully used for ovarian tissue of mice (Chen et al., 2006; Wang et al., 2008) and Japanese quail (Liu et al., 2010).  The testes were blotted on a piece of gauze, plunged into liquid nitrogen, and inserted into a modified pre-cooled 2 ml straw with one end pre-sealed with a sealing ball (Minit?b GmbH, Tiefenbach, Germany).  The other end was sealed with a sealing ball and the straws were stored in liquid nitrogen. For warming, the straws were opened with the end containing tissue still immersed in the liquid nitrogen.  The needles carrying tissue were removed from the straws and immersed in HM with 1 M sucrose for 5 min at either room temperature or at 40?C.  Tissue was transferred to 0.5 M, 0.25 M and 0 M sucrose solutions in sequence for 5 min each at room temperature, and the testes were suspended in HM on ice before further use within 4 h.  4.2.3 Testicular allografting Surgical procedures used for ovariectomy of day-old chickens (Liu et al., 2013a) were adapted for castration of week-old quail.  Recipient WB chicks were anesthetized by administration of isoflurane gas.  The chick was placed on its back on a heated surgical surface.  The feathers were removed from the left abdominal area, the skin was disinfected, and a 1.5-cm incision was made 1 cm left of the medial plane.  The 79  abdominal organs were displaced to the right to expose the testes, which were removed whole by cutting the mesorchium with a pair of fine forceps, and the incision was closed by simple interrupted stitches.  Immediately after castration, two testes from two different males that were cryopreserved and warmed were inserted together under the dorsal skin of each recipient through a small incision, which was closed by a single stitch.  Fresh transfixed testicular tissue was also transplanted into castrated recipients using the same procedures.  After surgery, the recipient chicks were administered an immunosuppressant, mycophenolate mofetil (CellCept, Hoffmann-La Roche Ltd., Mississauga, ON, Canada), orally at 100 mg/kg per day for 2 wk.  Numbers of recipients in each treatment group are shown in Table 4.1. 4.2.4 Histological examination At the age of 22 or 31 wk, 11 of the recipients with visible transplants were euthanized by cervical dislocation and the transplants were isolated.  For each recipient, the transplant appeared to be one piece and whether each transplant was derived from one of the transplanted testes or from both of them is unknown.  Pieces of transplant tissue of approximately 3 mm3 were fixed in Bouin?s solution and the rest of the transplant tissue was used for intramagnal insemination (see below).  Similar samples of testicular tissue from three sexually mature males were also fixed and used as controls.  The tissue was embedded in paraffin, sectioned at 5 ?m and stained with hematoxylin and eosin for histological examination.  Images were captured using a digital camera (QICAM; QImaging Corp., Surrey, BC, Canada) mounted on a microscope (EL-Einsatz; Carl Zeiss Group, Jena, Germany).  Ten seminiferous tubules from 10 different sections for each transplant or control testes were sampled.  The shortest diameters of the sampled tubules 80  and their lumens were measured using an ocular micrometer, and half of the difference between the two was considered to be the depth of the seminiferous epithelium. 4.2.5 Intramagnal insemination The width (lateral) and the height (dorsoventral) of the proctodeal gland were measured for each recipient sacrificed and the product of the width and the height was used as an index of gland size (Siopes and Wilson, 1975).  Each of the 11 transplants was minced with a scalpel in a separate petri dish.  The resultant fluid extrusion was collected in a syringe equipped with a 16-gauge needle.  Intramagnal insemination procedures that have been used in chickens (Engel et al., 1991; Song and Silversides, 2007a,c) were adapted for Japanese quail.  The hen was anesthetized with isoflurane gas and placed on its right side on a heated surgical surface.  The left leg was stretched caudally to expose the lateral abdominal wall, feathers were removed from the area, and the skin was disinfected.  A 2-cm incision was made behind the last vertebral rib, and the incision was spread using a retractor.  A section of the magnum was externalized and 0.15 to 0.30 ml of the fluid extrusion, depending on the amount available which varied among transplants, was injected using a 1-ml syringe and an 18-guage needle.  For some hens, the testicular extrusion was mixed with foam produced by the proctodeal gland from the same recipient immediately before injection. The magnum was then released and the incision was closed by interrupted stitches.  Eggs were collected for 2 wk before and after insemination and incubated.  Numbers of hens inseminated and their performance are shown in Table 4.4.  81  4.2.6 Statistical analysis All statistical analyses were conducted with SAS Server Interface 2.0.3.  The Chi-square analysis in the FREQ procedure was used to test the significance (P ? 0.05) of differences in the numbers of surviving recipients with viable transplants, recipients producing live offspring, and hens producing live offspring.  It was also used to test the correlation between the frequency of recipients that showed viable transplants and the frequency of recipients that showed functional proctodeal foam glands.  The GLM procedure was used to compare the differences among groups in the transplant weight, proctodeal gland index, and fluid extrusion volume, as well as the seminiferous tubules, lumens, and epithelium.  The TTEST procedure was used to compare egg production before and after insemination.   4.3 Results 4.3.1 Survival and growth of testicular transplants Fresh testicular tissue and that which had been cryopreserved and warmed at room temperature or 40?C was transplanted into 21 recipients (Table 4.1), of which 20 survived the surgical transplantation procedures.  At the age of 17 wk, 18 of the surviving recipients showed healthy testicular transplants growing on the back (Figure 4.1A-C).  No significant difference was observed among groups in the number of surviving recipients or recipients showing viable transplants.  A significant correlation was seen between the frequency of recipients showing viable transplants and the frequency of recipients showing functional proctodeal foam glands (Table 4.1).   82  4.3.2 Production of offspring by intramagnal insemination   One of the three transplants derived from fresh tissue and two of the four transplants from each cryopreservation group that were used for intramagnal insemination produced live offspring (Table 4.2).  No significant difference was seen among groups.  The weight and volume of extrusion obtained from the transplants from the cryopreserved tissue were not different from those of the fresh tissue, and the proctodeal gland index was similar among groups.  Seminiferous tubules containing spermatogenesis were seen in the transplants (Figure 4.2B-C), which was confirmed by the presence of motile spermatozoa (Figure 2D).  The average size of the seminiferous tubule, lumen and epithelium of the transplants was similar to that of normal testes (Table 4.3).   Five of the 12 hens that were inseminated with the fluid extrusion without proctodeal foam produced live offspring whereas the 10 hens inseminated with extrusion mixed with foam were not fertile (Table 4.4).  Overall, egg production was significantly lower after insemination than before.  Table 4.5 summarizes the performance of those hens that produced live offspring.  4.4 Discussion This study demonstrates that testicular tissue of an avian species can be cryopreserved using a simple vitrification method and recovered using allogeneic ectopic transplantation.  Live offspring were obtained by intramagnal insemination using the fluid extrusion retrieved from the transplants. Cryopreservation of testicular tissue is an important strategy for preserving male fertility, especially in situations where semen is not available or cannot be cryopreserved.  83  Production of live offspring using this strategy has been limited to rodents and chickens (Ehmcke and Schlatt, 2008; Song and Silversides, 2007a) and these studies used slow-freezing procedures.  Slow-freezing has been used to preserve various types of cells and simple cell aggregations but it is limited for preserving complex multicellular structures such as tissue.  The optimal values of the variables for a specific slow-freezing protocol are dependent on cell-specific properties such as surface-to-volume ratio and membrane permeability (Mazur, 2004).  In addition, the extracellular ice formation induced by slow-freezing procedures is detrimental to the interstitial tissue compartments which are essential for functional recovery of testicular tissue (Rodriguez-Sosa et al., 2012).  These limitations can be circumvented by vitrification procedures, and their advantage in preserving testicular tissue viability and integrity has been demonstrated in pigs (Abrishami et al., 2010), mice (Curaba et al., 2011b), humans (Curaba et al., 2011a), and fish (Bono-Mestre et al., 2009).  The vitrification protocol used in this study has been effective for preserving ovarian tissue in mice (Chen et al., 2006; Wang et al., 2008) and Japanese quail (Liu et al., 2010).  It has recently been optimized for cryobanking avian testicular tissue using a simple and secure storage system (Liu et al., 2012a).  Tissue culture using the chicken chorioallantoic membrane showed that it preserved testicular tissue viability.  The efficiency of this protocol was confirmed in the current study by the high percentage of recipients that grew testicular transplants and the similarity of macroscopic and microscopic properties of the transplants derived from cryopreserved tissue to those of fresh tissue and normal testicular tissue.  The production of healthy offspring further validated the feasibility of using vitrification for cryobanking of avian testicular tissue.  84  The beneficial effect of rapid warming seen in a previous study (Liu et al., 2012a) was not apparent in this study, which could be attributed to the small sample size or the difference in the culture systems (in ovo versus in vivo) used.  More rigorous assessments of warming procedures could be conducted in the future.   Testicular allografting is a means of in vivo maturation which is essential for recovery of the reproductive potential preserved in the tissue.  The high viability of the recipients and the transplanted tissue demonstrated the efficiency of the refined surgical castration and transplantation procedures applied to Japanese quail.  In addition, the presence of testosterone-dependent proctodeal glands (Biswas et al., 2007) in most recipients indicated that steroidogenesis of the transplants was properly established.  In this study, each testis was removed from the body cavity in one piece.  In chickens, this reduced the chance of fatal exsanguination and increased the success rate of castration (Silversides et al., 2013a) compared to previous methods (Song and Silversides, 2007a,c).  Castration appears to be crucial to the survival and functional recovery of testicular transplants in chickens (Song and Silversides, 2007a,c) and some mammals (Shinohara et al., 2002), possibly because it creates a favourable endocrine milieu through the recipient?s hypothalamic-pituitary-gonadal axis (Rodriguez-Sosa and Dobrinski, 2009).  These data showed that complete castration was not essential in Japanese quail, as was suggested in the rabbit-mice xenotransplantation model (Schlatt et al., 2003).  Whether the necessity of complete castration is species-specific needs more investigation. The production of offspring is the ultimate objective of testicular cryopreservation and this was achieved in this study using transplantation and intramagnal insemination.  Egg production of the hens was lower after insemination, which is consistent with a 85  previous study in chickens (Engel et al., 1991).  This disturbance in egg production was ascribed to the use of ketamine and xylazine as anaesthetics (Engel et al., 1991) and the volatile anaesthetic isoflurane used here may have had a similar effect on quail egg production, or the surgery itself could have a negative effect.   The procotodeal gland foam had an adverse effect on the fertility of the spermatozoa from transplants used for intramagnal insemination.  Cheng et al. (1989) found that the deposition of foam into the female proctodeum during copulation had a positive effect on the percentage of females fertilized and the duration of fertility.  However, Che?mo?ska et al. (2006) reported that mixing foam with ejaculated semen diluted with extender and dimethylacetamide was detrimental to spermatozoa morphology and fertility.  In the present study, foam was added to the testicular exudate and the mixture was deposited in the magnum.  The foam may favour the spermatozoa only in certain physiological situations, such as when combined with seminal plasma of ejaculated semen or when it is in the distal part of the oviduct, or it could have differential effects on ejaculated or testicular spermatozoa.     The subcutaneous testicular transplants were not connected to the epididymis and vas deferens and the enclosed spermatozoa likely resembled testicular spermatozoa.  Chicken testicular spermatozoa can fertilize eggs when deposited into the magnum but not into the vagina of the hen (Howarth, 1983), probably because of selective mechanisms in the uterovaginal junction of the oviduct (Birkhead and Brillard, 2007).  Ahammad et al. (2011a,b) proposed that chicken spermatozoa experienced morphological and biochemical maturation as they passed through the male reproductive tract, and that this was critical for their survival in the spermatozoa storage tubules in the 86  oviduct.  The fertility of one quail hen extended to the second week after insemination, indicating that the spermatozoa from the transplants can survive in the sperm storage tubules as long as that of ejaculated spermatozoa (Birkhead and Fletcher, 1994), posing the question of whether maturation is necessary for quail spermatozoa to survive in the oviduct and to fertilize eggs.  On the other hand, the microenvironment of the oviduct could induce maturation and a third possibility is that intrinsic factors could trigger maturation, making it dependent on time alone.  Ectopic subcutaneous transplantation of testicular tissue could provide a useful tool to test these hypotheses.  In conclusion, the production of normal chicks demonstrates that cryopreservation of testicular tissue using vitrification procedures and subsequent transplantation is a feasible option for ex situ conservation of male germplasm in Japanese quail.  The vitrification procedures could be used for cryobanking of male germplasm of other avian species and systematic studies of species-specific questions with respect to fundamental reproductive physiology are needed.     87  Table 4.1.  Transplantation of cryopreserved and warmed testicular tissue in Japanese quail Groups  Recipients Surviving recipients (4 wk after transplantation) Surviving recipients showing viable transplants (17-week old)ab Surviving recipients showing functional proctodeal foam glands (17-week old)b Fresh tissue  3 3 3 3 Warming at 40?C 9 9 7 5 Warming at RTc 9 8 8 8 a No significant difference was seen between any two groups. b Variables are significantly correlated (P ? 0.05). c RT, room temperature.   88  Table 4.2.  Production of live offspring by intramagnal insemination using spermatozoa retrieved from the testicular transplants (mean ? SEM) Groups Recipients used for insemination Recipients producing live offspringa Recipients with complete castration Transplant weight (g)a Proctodeal foam gland index  (mm2)a Fluid extrusion volume (ml)a Fresh tissue 3 1 2 1.50 ? 0.27 177.59 ? 1.69 0.40 ? 0.05 Warming at 40?C 4 2 1 1.79 ? 0.43 141.52 ? 21.71 0.59 ? 0.15 Warming at RTb 4 2 3 1.93 ? 0.21 165.16 ? 22.58 0.63 ? 0.03 a No significant difference was seen between any two groups. b RT, room temperature.   89  Table 4.3.  Histological examination of the testicular transplants (mean ? SEM) Groups Recipients sampled Diameter of seminiferous tubule (?m)a Diameter of lumen (?m)a Depth of seminiferous epithelium (?m)a Control  3 231.1 ? 9.9 59.3 ? 5.4 85.9 ? 4.4 Fresh tissue 3 280.4 ? 9.1 67.0 ? 3.3 106.7 ? 3.3 Warming at 40?C 4 239.1 ? 34.4 87.5 ? 7.2 75.8 ? 13.9 Warming at RTb 4 242.7 ? 17.6 98.4 ? 22.2 72.1 ? 19.1 a No significant difference was seen between any two treatments. b RT, room temperature.   90  Table 4.4.  Performance of hens inseminated with spermatozoa retrieved from testicular transplants (mean ? SEM) Groups Hens inseminated Hens producing live offspring Two-week egg production Before insemination After insemination With foam  10 0a 13.5 ? 0.4 9.1 ? 1.4 Without foam 12 5b 13.2 ? 0.5 9.4 ? 1.2 Total 22 5 13.3 ? 0.3a 9.3 ? 0.9b a, b  Values are significantly different.  91  Table 4.5.  Production of eggs and chicks from hens producing live offspring Groups/Hen ID Fertile eggs produced Live chicks produced Two-week egg production after insemination The first week The second week Fresh tissue     63593 5 2 1 12 Warming at 40?C     63583 2 2 0 13 63594 2 1 0 13 Warming at RT     63580 1 1 0 12 63586 1 1 0 5 92   Figure 4.1.  Testicular transplants and chicks produced by intramagnal insemination using the extruded fluid.  A) A recipient with testicular transplant growing under the skin (feathers removed).  B) A transplant with the covering skin removed.  C) A transplant isolated from the recipient.  D) Chicks hatched from the eggs collected during the first week after insemination.   93   Figure 4.2.  Histological examination of testicular tissue.  A)  Intact male.  B) Testicular transplant.  C) Seminiferous tubule with spermatogenesis in transplanted tissue.  D) Spermatozoa (indicated by arrows) in the fluid extrusion of cryopreserved and transplanted testicular tissue.  Bars are equal to 50 ?m. 94  5 A MODEL FOR CRYOBANKING FEMALE GERMPLASM IN JAPANESE QUAIL (COTURNIX JAPONICA)5 5.1 Introduction Cryobanking of poultry germplasm allows economical and sustainable maintenance of genetic resources for the poultry industry and avian research (Silversides et al., 2012; Liu et al. 2013b).  Cryobanking of female germplasm in birds is important for maintaining genetic information carried by the W chromosome and mitochondrial DNA (Silversides and Liu, 2012).  However, the structure of the avian egg limits the cryobanking of oocytes and embryos that has been used in mammals (Hagedorn, 2006).  As an alternative, avian ovarian tissue can be cryopreserved and functional recovery can be achieved by orthotopic transplantation at a later time (Liu et al., 2013b).   Liu et al. (2010) demonstrated that ovarian tissue from one-week old Japanese quail can survive cooling and warming procedures and produce donor-derived offspring after transplantation.  Compared to a conventional slow-freezing protocol, the vitrification protocol developed by Liu et al. (2010) based on previous studies in mammals (Chen et al., 2006; Wang et al., 2008) was more efficient and simple.  This protocol was adapted to various straw systems (Liu et al., 2012a,b) to facilitate conservation programs (Silversides et al. 2013b).  Further investigations in avian gonadal transplantation (Liu et al., 2013a,c; Silversides et al., 2013a) have contributed to improving the functional recovery of cryopreserved gonadal tissue.                                                     5 A version of Chapter 5 has been accepted for publication. Liu, J., K. M. Cheng, and F. G. Silversides. 2013. A model for cryobanking female germplasm in Japanese quail (Coturnix japonica). Poult. Sci. (in press). 95  The practicality of a cryobanking program is affected by the success of each of the cryobiological and reproductive techniques involved (Comizzoli et al., 2012).  The overall success rate in producing donor-derived offspring is critical to ensure an adequate effective population (FAO, 2012) for genetic restoration.  In this report, the current vitrification and surgical transplantation procedures used by Agriculture and Agri-Food Canada were applied to the ovarian tissue of one-week old Japanese quail and the overall efficiency was evaluated by a progeny test.  5.2 Materials and methods One-week old female Japanese quail chicks from a White-Breasted (WB) line and a QO line were used as donors and recipients, respectively.  Fertile adult male quail from the WB line were used for the progeny test.  The WB quail are homozygous for the recessive white-breasted gene (wb/wb) and the QO quail are wild-type (+/+) at this locus.  Both of these lines are maintained at the Agassiz Research Centre (Liu et al. 2010).  The research protocol was approved by the Animal Care Committee of the Agassiz Research Centre following principles described by the Canadian Council on Animal Care (2009).  All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) unless otherwise indicated. The tissue handling and vitrification and warming procedures were described by Silversides et al. (2013b) and applied to ovaries from one-week old Japanese quail.  Ovaries were removed from the donor birds immediately after euthanasia by cervical dislocation and immersed in a handling medium (HM) containing Dulbecco?s phosphate buffered saline (DPBS) supplemented with 20% fetal bovine serum on ice.  Two whole ovaries were transfixed on an acupuncture needle (Seirin Corporation, Shizuoka, Japan).  96  At room temperature, ovaries carried by the needle were submerged in an equilibration solution of HM with 7.5% (v/v) dimethyl sulphoxide (DMSO) and 7.5 % (v/v) ethylene glycol (EG) for 10 min, then a vitrification solution of HM with 15% (v/v) DMSO, 15% (v/v) EG, and 0.5 M sucrose (VWR, Mississauga, ON, Canada) for 2 min.  The ovaries were blotted on a piece of gauze and plunged into liquid nitrogen directly to facilitate vitrification.  They were then quickly inserted into a pre-cooled, 2 ml straw with one end pre-sealed with a sealing ball (Minit?b GmbH, Tiefenbach, Germany).  The other end was sealed with a sealing ball and the straws were stored in liquid nitrogen.  For warming, the straws were opened with the end containing ovaries still immersed in the liquid nitrogen.  The needles carrying ovaries were removed from the straws and immersed in HM with 1 M sucrose for 5 min at room temperature.  Ovaries were then transferred to 0.5 M, 0.25 M and 0 M sucrose solutions in sequence for 5 min each, and were suspended in HM on ice before further use.  Within 3 h after warming, each ovary was transplanted into one recipient after ovariectomy using the surgical procedures described by Liu et al. (2013c).  The recipient chick was anaesthetized using isoflurane gas and placed on its back on a heated surface during surgery.  Feathers were removed from the left abdominal area, the skin was disinfected, and a 1.5-cm incision was made 1 cm left of the medial plane.  The left ovary was exposed and removed in small pieces with a pair of fine forceps.  Only the left ovary is functional in Japanese quail (Brunstr?m et al., 2009).  Immediately, each donor ovary was placed on the site of the removed ovary. No suture was needed to affix the transplanted ovary.  The incision was then closed.  After transplantation, the recipient chicks were administered an immunosuppressant, mycophenolate mofetil (CellCept, 97  Hoffmann-La Roche Ltd., Mississauga, ON, Canada), orally at 100 mg/kg per day for 2 wk.  Fifteen recipients survived the surgery and five untreated week-old female QO chicks were raised with the recipients as controls.  Chicks were raised in a brooder for 2 wk after surgery and in a floor pen until the age of 17 to 20 wk, when they were caged and paired with males from the WB line to test the maternal origin of the resulting chicks.  From this cross, chicks with white-breasted plumage coloration (wb/wb) are derived from WB donor tissue whereas chicks with wild-type coloration (+/wb) originate from regenerated ovarian tissue of the QO recipients.   All statistical analyses were conducted with SAS Server Interface 2.0.3.  Body weight and egg production were compared between the control group and the experimental group using the TTEST procedure.  The significance (P ? 0.05) of differences in the numbers of recipients producing offspring, and the numbers of donor- and recipient-derived offspring was tested by the Chi-square analysis in the FREQ procedure. 5.3 Results and discussion The body weight and egg production of the experimental birds and those in the control group were comparable (Table 5.1), which indicates that the vitrification and transplantation procedures may not affect the growth of the recipient chicks and their reproductive performance after sexual maturation.  As shown in Table 5.2, seven recipients produced donor-derived offspring and five of these produced 100% donor-derived offspring which was a significant improvement compared to a previous study (Liu et al., 2010).  In addition, the proportion of donor-derived offspring was higher (P < 98  0.01) in the current study (Table 5.2).  No functional ovarian development was observed in the remaining eight recipients.    Liu et al. (2010) cut ovaries from week-old Japanese quail in half for vitrification and transplantation.  Small tissue pieces may facilitate equilibration of cryoprotective agents and revascularization of the tissue after transplantation (Kagawa et al., 2007) and therefore attenuate the potential loss of primordial follicles caused by cryopreservation or transplantation-associated ischemic stress (Demeestere et al., 2009).  In this study ovaries were cryopreserved and transplanted in one piece, but depletion of follicles appeared to be negligible.  The high proportion of donor-derived offspring showed that nearly all of the eggs produced by the recipients were from the surviving transplants, and egg production was not different from that of the controls, demonstrating that the donor ovaries were able to support a normal level of egg production.  Therefore ovaries of one-week old Japanese quail can be cryopreserved and transplanted in one piece without compromising reproduction, and this is recommended in the future practice to simplify tissue handling and to minimize damage.     The model described in this report has been demonstrated for one-week old Japanese quail (Liu et al., 2010) and newly-hatched chickens (unpublished data).  Here we provide evidence of a high and consistent success rate of this model in quail which can be used for cryobanking female germplasm of experimental and commercial lines.  This could also provide an approach to reproducing lines that are subfertile or high in post-hatching mortality such as inbred quail (Kim et al., 2007), as has been described for certain transgenic mouse lines (Shaw and Trounson, 2002).  In addition, recent success in producing offspring from transplanted adult quail ovarian tissue (Liu et al., 2013c) 99  suggests that birds that are to be eliminated in a breeding or experimental program can be used as ovarian donors for genetic conservation.  Application of this model in sexually mature birds and in other poultry species will be important avenues for future investigations.    This model may also contribute to ex situ conservation of endangered avian species.  Cryobanking of germplasm in these species has been limited to the use of semen (Gee et al., 2004), which does not allow preservation of w chromosome or the mitochondrial genome.  The simple vitrification procedures described here can be applied in harsh and remote environments where endangered species are usually found (Comizzoli et al., 2012).  Recovery of  adult ovarian tissue (Liu et al., 2013c) suggests that gonadal tissue from a valuable individual in an endangered species could be collected and preserved  after an unexpected death and be used for research in species-specific reproductive physiology, of which our knowledge may be meager (Comizzoli et al., 2012).  Moreover, successful xenotransplantation (Song et al., 2012) provides the possibility of regenerating the germplasm of endangered species using domestic birds as recipients.        100  Table 5.1.  Body weight and 56-day egg production (mean ? SEM) starting at 17-20 wk old quail transplanted with cryopreserved ovarian tissue Groups Number  Body weight (g)2 Birds producing eggs Egg production2 Day 8 Day 12 Day 16 Control 5 25.9 ? 2.1 45.9 ? 3.3 70.3 ? 3.9 5 48.0 ? 4.5 Experimental1 15 29.4 ? 1.2 47.4 ? 2.2 70.4 ? 2.4 7 36.7 ? 6.3 1 Birds in this group were transplanted with cryopreserved ovarian tissue.  2 No significant difference was seen between control and experimental groups.     101  Table 5.2.  Four wk of progeny test of quail recipients transplanted with ovarian tissue that were cryopreserved by vitrification Groups1 Recipients producing offspring Recipients producing 100% donor-derived offspring Donor-derived offspring Recipient-derived offspring 2009 7 0a 29A 64A 2013 72 5b 92B 3B 1 Data for Group 2009 are from a previous study (Liu et al., 2010). 2 All seven recipients produced donor-derived offspring. a, b Values in a column with different superscripts differ (P < 0.05). A, B Values in a column with different superscripts differ (P < 0.01).  102  6 INDUCTION OF IMMUNOLOGICAL TOLERANCE FOR GONADAL TRANSPLANTATION IN AVIAN SPECIES BY IMPLANTING DONOR THYMIC TISSUE INTO RECIPIENT CHORIOALLANTOIC MEMBRANE   6.1 Introduction Gonadal transplantation can be used for functional recovery of gonadal tissue to produce donor-derived offpring, which is an important step towards successful cryobanking of avian genetic resources and provides a useful tool to investigate avian development and reproductive physiology.   Live offspring have been obtained from allotransplantation of fresh and cryopreserved ovarian tissue (Song and Silversides, 2007b; Song and Silversides, 2008b; Liu et al., 2010) and testicular tissue (Song and Silversides, 2007a; Liu et al., 2013d) in chickens and Japanese quail.  In addition, ovarian transplantation across the xenogeneic barrier has been achieved in ducks (Song et al., 2012).  Immunodeficient rodents are usually used as recipients in mammalian gonadal transplantation to prevent rejection of the transplants.  Immunodeficient birds are not available so an immunosuppressant, mycophenolate mofetil, has been used in previous gonadal transplantation in birds, which broadly inhibits the immune responses of the recipients (Lipsky, 1996).  Although long-term performance of fresh ovarian transplantation in chickens did not appear to be affected (Song and Silversides, 2008b), the potential side effects (Vanhove et al., 2013) of this treatment in birds are not known.  103  As an alternative, specific tolerance of organ transplants can be actively induced through embryonic manipulation.  Billingham et al. (1953) reported that allogeneic tolerance can be actively acquired by the recipient by implantation of donor tissue into recipient embryos, which was demonstrated by acceptance of post-hatching skin transplants from the same donor.  Unfortunately, this was not successful in the xenogeneic models described by Billingham et al. (1953).  Oki et al. (1987) transplanted donor embryonic thymus and wing bud tissue to recipient embryos and observed long-term xenogeneic tolerance of the wing bud transplants after the hatching of the recipients in chickens and Japanese quail.  They postulated that the presence of donor thymic epithelium was required for induction of xenogeneic tolerance.   Embryonic manipulation described above could be used to induce immunological tolerance to facilitate avian gonadal transplantation.  However, in situ thymic transplantation can lead to high mortality of the recipient embryos, which is common in embryonic transplantation (Le Douarin et al., 1996).  A less invasive approach could be to implant donor thymic tissue onto the chorioallantoic membrane (CAM) of the developing recipient embryos, which has been used for tissue culture in our previous studies (Liu et al., 2012a,b) and ensures higher embryonic viability compared to that of embryonic transplantation.  Billingham et al. (1953) used this approach in their allogeneic model but the donor tissue they used didn?t include the thymus.  The objective of this study was therefore to test the possibility of inducing immunological tolerance for post-hatching gonadal transplantation by implanting donor thymic tissue onto recipient CAM.    104  6.2 Materials and methods 6.2.1 Birds and chemicals Chicks from Barred Plymouth Rock (BPR) and White Leghorn (WL) lines (Silversides, 2010) were used as donors and recipients, respectively, for allotransplantation.  For xenotransplantation, quail chicks from a QO line (Liu et al., 2010) and WL chicks were used as donors and recipients, respectively.  All of these lines are maintained at the Agassiz Research Centre.  The research protocol was approved by the Animal Care Committee of the Agassiz Research Centre following principles described by the Canadian Council on Animal Care (2009).  All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) unless otherwise indicated. 6.2.2 Preparation of donor gonadal and thymic tissue For allotransplantation, ovaries and testes from day-old BPR chicks were collected and cryopreserved following the procedures that have been previously described (Silversides et al., 2013b).  Immediately after gonadoectomy, thymic lobes from each donor chick were isolated and kept in Dulbecco?s phosphate buffered saline on ice before implantation (Figure 1.4).  For xenotransplantation, gonadal and thymic tissue of week-old quail chicks was prepared using the above procedures, except that the ovaries were cut in half before cryopreservation.  6.2.3 Implanting donor thymic tissue into recipient embryos For allotransplantation, the procedures of implanting donor thymic tissue into recipient embryos were similar to those used by Liu et al. (2012a,b).  Briefly, fertilized 105  WL eggs were artificially incubated for 3 d, which were then positioned horizontally and the position of the embryo was located by candling.  Approximately 1 ml albumen was withdrawn through the small end of the egg using an 18-gauge needle and a 1 cm2 window was made in the shell and shell membranes above the embryo with a small drill (Dremel Stylus?, Racine, WI, United States).  The window was sealed with parafilm and the egg returned to the incubator.  After an additional 2 d of incubation of the recipient eggs without turning, donor thymic lobes were cut in half and two to three pieces were placed onto a slightly traumatized (Martinez-Madrid et al., 2009) area of the developing CAM of the recipient embryo through the window on the egg.  The window was then sealed and the egg was returned to incubator.  Thymic tissue from each donor was implanted into three to five recipient embryos.  The eggs were turned carefully around the long axes starting from the seventh day of incubation until shortly before hatching.     For xenotransplantation, thymic tissue from some donors was implanted into chicken embryos using the above procedures, which was designated the ?windowed? group.  Another technique described by Qureshi et al. (2008) was also used with modifications.  In general, fertilized WL eggs were artificially incubated for 5 d and the CAM was marked on the shell by candling while keeping the egg positioned vertically.  A hole of 1mm x 2mm was drilled into the shell above the CAM near the blood vessels.  A small slit was made on the exposed shell membrane, through which two to three pieces of donor thymic tissue were wedged in the junction between the CAM and the shell membrane.  The drilled area was sealed with parafilm.  The implanted eggs were then 106  incubated under standard conditions until hatching of the recipients.  This group is referred to as ?wedged?.  6.2.4 Gonadal transplantation and histological examination Hatched recipients were kept in a brooding box for 4 to 7 d before gonadal transplantation (Figure 1.4).  Ovariectomy and castration of the recipients were conducted using the procedures described by Liu et al. (2013c) and Silversides et al. (2013a).  In general, recipient chicks were anaesthetized with isoflourane gas and the down on the left abdomen was removed and the skin was cleaned with 70% EtOH.  A 2-cm vertical incision was made approximately 1 cm left of the median plane.  The residual yolk sac and the viscera were gently displaced to the chick?s right to expose the gonads.  The left ovary of the female recipient was removed piece by piece with a pair of fine forceps.  For the male recipient, each testicle was removed in one piece with a pair of fine forceps.  The removed testicles were placed in Dulbecco?s phosphate buffered saline with 20% fetal bovine serum to be used for autotransplantation or transplantation into another recipient (see below).  The incision was closed using interrupted stitches.    Subcutaneous transplantation used in Japanese quail (Liu et al., 2013d) was adapted to chicken recipients by making a small cut on the skin of the back into which the transplants were placed, and the cut was closed by a single stitch.  For allotransplantation, eight recipients were transplanted with one testis each from the matched thymic donor.  Eight recipients were transplanted with one testis of their own (autotransplantation) and five recipients were transplanted with one testis from another male recipient (unrelated donor).  For xenotransplantation, four recipients from the windowed group and two recipients from the wedged group received testicular 107  transplantation from the matched thymic donors.  Four recipients from the windowed group and one recipient from the wedged group received ovarian transplants from the matched thymic donors.  At 10-13, 20-21 and 30-31 d after transplantation, recipients with transplants under the skin were euthanized by cervical dislocation and the transplants were fixed in Bouin?s solution.  Fixed tissue were embedded in paraffin, sectioned at 5 ?m and stained with hematoxylin and eosin for histological examination.  Microscopic images were taken by a digital camera (QICAM; QImaging Corp., Surrey, BC, Canada) mounted on a light microscope (EL-Einsatz; Carl Zeiss Group, Jena, Germany).   6.2.5 Statistical analysis   Statistical analysis was conducted with SAS Server Interface 2.0.3.  The differences in the numbers of hatched recipients was tested by the Chi-square analysis in the FREQ procedure.  The survival curves based on the histology of the allotransplants were compared with the LIFETEST procedure.  The criterion for statistical significance was P ? 0.05. 6.3 Results When donor thymic tissue was implanted in windowed eggs, those in allotransplantation group showed higher (P < 0.01) hatchability than those in xenotransplantation group.  No difference (P = 0.74) was observed between the two implantation techniques in hatchability of eggs that were used for xenotransplantation (Table 6.1).  108   For allotransplantation of gonadal tissue in chickens (Table 6.2), transplants from the matched thymic donors showed normal macroscopic and histological appearance (Figure 6.1A, B) for at least 31 d after transplantation, which was comparable to those of autotransplants (Figure 6.1C, D).  Although one transplant from an unrelated donor appeared normal macroscopically and contained a small number of seminiferous tubules after 13 d from transplantation (Figure 6.1E, F), none of the transplants from this group appeared to survive to 21 d (Table 6.2).  No difference (P = 0.58) among the three groups was seen in their survival curves (Figure 6.2).   For xenotransplantation, one ovarian transplant demonstrated normal macroscopic appearance and had histologically normal follicular structures (Figure 6.3A, B, Table 6.3) after 10 d.  There was evidence in a testicular transplant that by 10 d after transplantation, the seminiferous tubules were dissociated and infiltrated with non-testicular cells (Figure 6.3D).  Only dead tissue was found in transplants sampled at 20 d (Figure 6.3F) despite normal macroscopic appearances (Figure 6.3C, E).   6.4 Discussion The hatchability of the recipient embryos in this study was higher than that of embryonic transplantation reported in previous literature (Oki et al., 1987; Le Douarin et al., 1996).  Recipient embryos that had been treated with different implantation techniques (windowed and wedged) in xenotransplantation groups exhibited similar hatchability, but the ?wedged? technique is simpler and allows the eggs to be incubated under standard conditions and therefore is preferable in future use.  Its effectiveness in induction of allogeneic tolerance should be tested in future studies.  The higher hatchability observed in the allotransplantation group compared to that of the 109  xenotransplantation group that received the same implantation treatment could be ascribed to the graft-versus-host reactions (Murphy, 1916; Simonsen, 1957) in the latter,  perhaps indicating that the immune system of week-old quail donors is more competent than that of the day-old chicken donors.  If so, after implantation, the immune cells contained in the quail thymus can recognize the recipient embryo as non-self and induce immunological responses that may negatively impact the development of the recipient embryos.   Discrimination of self and non-self tissue by the immune system has been investigated by Billingham et al. (1953), who postulated that mammals and birds are not able to effectively induce an immunological response to non-self, allogeneic tissue if they have been exposed to it sufficiently at an early stage of development.  This was later supported by the central tolerance theory when the central role of thymus and T cells in induction of tolerance of self-antigen began to be appreciated (Hogquist et al., 2005).  According to this, T cell precursors colonizing the thymus are subjected to selection that is dependent on the affinity of their T cell receptors to the self-antigen presented by the major histocompatibility complex (MHC) of the antigen presenting cells.  Affinity higher than a threshold can lead to elimination of these precursors, and is known as clonal deletion.  Therefore the antigens expressed by the tissue that is implanted into the recipient embryos are involved in T cell selection and will not be regarded as non-self later in the recipient?s development.     The above theory of actively acquired tolerance does not seem to be sufficient to induce xenogeneic tolerance.  Le Douarin and colleagues (1996) showed that non-thymic transplants from quail to chicken embryos were rejected after hatching of the chicken 110  recipients.  However, co-transplantation of donor thymic tissue can lead to long-term tolerance of non-thymic xenotransplants, probably because of a role of donor thymic epithelium in selecting a population of donor-specific regulatory T cells that are important in induction and maintenance of immunological tolerance (Sala?n et al., 2005).  Recent studies revealed that the medullary thymic epithelial cells conduct promiscuous expression of tissue-restricted antigens (Klein, 2009), which are presented to the T cell precursors for selection, resulting in clonal deletion or production of regulatory T cells.  How the self-antigen-MHC is presented to the T cell precursors remains controversial (Klein et al., 2011).    In this study, the implanted donor thymic tissue could provide the epithelium for selection of recipient T cells.  This may be true in the allotransplantation model but is unlikely to happen in the xenotransplantation model because the majority of the xenotransplants were rejected during 10 to 20 d after transplantation, which is in accordance with previously described examples of xenogeneic rejection (Billingham et al., 1956; Oki et al., 1987).  One possible reason is that the quail thymic tissue used here could not efficiently induce differentiation of recruited recipient hematopoietic cells that give rise to T cells and antigen presenting cells in the thymus because of an age-related change in their medullary epithelium (Gui et al., 2007).  It could also be because donor thymic tissue implanted to the CAM may not be as efficient as the thymic tissue transplanted in situ, but in situ transplantation of thymic tissue can lead to high mortality of recipient embryos.  Intrathymic injection (Sala?n et al., 2002) of donor thymic epithelial cell suspension to newly-hatched recipients might be considered in the future.  111   Although no significant difference was detected from the surviving curves of the allogeneic gonadal transplants based on a limited number of observations, the curves in Figure 6.2 suggests that tolerance of gonadal transplants from the matched thymic donor is similar to that of the gonadal transplants from self.  In contrast, the gonadal transplants from an unrelated donor tend to be rejected as described in immunological rejection of gonadal transplants in mammals (Gosden, 2008).  This indicates that implanting donor thymic tissue can contribute to a lasting state of tolerance of testicular transplants from the same donor, which was confirmed by histology.  This also provides evidence that testes-restricted tissue antigens are expressed in the thymus as part of the promiscuous gene expression in chickens.   The antigens expressed by donor thymus could be presented to recruited recipient T cell precursors or could be delivered to the developing recipient thymus by antigen presenting cells (Bonasio et al., 2006).  Cellular and molecular profiles of donor and recipient thymus before hatching of recipients could help to reveal the underlying mechanism and therefore contribute to resolving the controversy in antigen presentation in the central tolerance mechanism (Klein et al., 2011).  112  Table 6.1.  Hatchability of recipient embryos according to the implantation techniques and transplantation types Implantation/ transplantation Implanted Hatched Windowed/ Allotransplantation  53 42 (79.2%)A Windowed/ Xenotransplantation 21 10 (47.6%)B Wedged/ Xenotransplantation 12 5 (41.6%)B A, B Values in a column with different superscripts differ (P < 0.01).   113  Table 6.2.  Macroscopic appearance and histological examination of gonadal transplants in allotransplantation in chickens Groups Days after transplantation Number examined Surviving transplants Macroscopically normal   Histologically normal  Transplant from the thymic tissue donor 13 4 3 1  21 2 2 1  30-31 2 2 2 Autotransplantation  12 2 2 1  20-21 4 2 2  30-31 2 2 2 Transplant from unrelated donor 12 2 2 1  21 2 0 0    114  Table 6.3.  Macroscopic appearance and histological examination of xenotransplants from quail to chickens Groups Days after transplantation Number Surviving transplants Macroscopically normal  Histologically normal Windowed 10-11 4 1 0  20-21 2 1 0 Wedged 10-11 2 2 1  20-21 2 2 0  115                 Figure 6.1.  Macroscopic appearance and histology of allotransplants. A: Macroscopic appearance of a transplant from the thymic donor after 31 d of transplantation.  B: Histology of the transplant in Figure A, 100X.  C: Macroscopic appearance of an autotransplant after 31 d of transplantation.  D: Histology of the transplant in Figure C, 100X.  E: Macroscopic appearance of a transplant from an unrelated donor after 13 d of transplantation.  F: Histology of the transplant in Figure E, 100X.  Arrows in A, C, E point to the transplants. The arrow in F indicates tubule-like structures.   A B  A C D E F 116   Figure 6.2.  Survival curves based on the histology of the allogeneic gonadal transplants.  Sampled gonadal transplants that did not show normal histology were considered to fail to survive (censored).  Groups 1, 2, and 3 refer to gonadal transplants that were from self (autotransplantation), from matched thymic donor, and from an unrelated donor, respectively.  The numbers of subjects at risk shown above the horizontal axis refer to the numbers of gonadal transplants in each group that were surviving at the time shown on the axis.  The curves show that the gonadal transplants from self or from matched thymic donor can survive beyond 30 d after transplantation whereas the probability for gonadal transplants from an unrelated donor surviving to 20 d is zero, although the difference of the survival curves among the groups is not different (P = 0.58) statistically.   117              Figure 6.3.  Macroscopic appearance and histology of xenotransplants. A: Macroscopic appearance of an ovarian transplant after 10 d of transplantation.  B: Histology of the transplant in Figure A, showing follicles, 100X.  C: Macroscopic appearance of a testicular transplant after 10 d of transplantation.  D: Histology of the transplant in Figure C, showing abnormal structures infiltrated by non-testicular cells, 100X.  E: Macroscopic appearance of a testicular transplant 20 d of transplantation.  F: Histology of the transplant in Figure E, showing dead tissue, 100X.  Arrows in A, C, E point to the transplants. A B  A C D E F 118  7 RECOVERY OF FERTILITY FROM ADULT OVARIAN TISSUE TRANSPLANTED INTO WEEK-OLD JAPANESE QUAIL CHICKS6 7.1 Introduction Transplantation of ovarian tissue can be used to recover the reproductive potential of primordial follicles that are preserved in their natural microenvironment, and it has been accomplished in various mammalian species including humans (Gosden, 2008).  Along with cryopreservation techniques, ovarian transplantation has been successfully used in maintenance of lines of laboratory mice (Shaw and Trounson, 2002) and restoration of fertility for human patients (Kondapalli, 2012).  Recent progress in ovarian transplantation in birds (Liu et al., 2013b) may contribute to research on cryobanking female germplasm for non-mammalian vertebrates, in which cryobanking of oocytes and embryos is challenging because yolk accumulates in the follicles during vitellogenesis (Rothchild, 2003).   In mammals, the donors of ovarian tissue can be of any age but published research describes only donors and recipients of the same age or recipients that are sexually mature (Gosden, 2008).  In birds, live offspring have been produced from fresh (Song and Silversides, 2007b, 2008b; Song et al., 2012) or cryopreserved (Liu et al., 2010) ovarian tissue of sexually immature donors transplanted into age-matched                                                  6 A version of Chapter 7 has been presented to 2013 Poultry Science Association Annual Meeting [Liu, J., K. M. Cheng, and F. G. Silversides. 2013. Production of donor-derived chicks from transplantation of adult ovarian tissue in Japanese quail (Coturnix japonica). Poult. Sci. 92 (E-Supplement 1):18.] and submitted to Reproduction, Fertility and Development for publication [Liu, J., K. M. Cheng, and F. G. Silversides. 2013. Recovery of fertility from adult ovarian tissue transplanted into week-old Japanese quail chicks. Reprod. Fertil. Dev. (Manuscript No. RD 13256)]. 119  recipients, but fertility of ovarian tissue from adult donors transplanted into immature recipients has not been demonstrated.  In the current study, ovarian tissue from sexually mature Japanese quail was transplanted into one-week old recipient Japanese quail chicks to test the possibility of recovering fertility of mature ovarian tissue by transplantation.   7.2 Materials and methods Twelve- to 13-week old Japanese quail hens from a white-breasted (WB) line and one-week old female quail chicks from a QO line were used as donors and recipients, respectively, and adult male quail from the WB line were used for the progeny test.  The WB quail are homozygous for the recessive white-breasted gene (wb/wb) and the QO quail are wild-type (+/+) at this locus.  Both of these lines are maintained at the Agassiz Research Centre (Liu et al., 2010).  The research protocol was approved by the Animal Care Committee of the Agassiz Research Centre following principles described by the Canadian Council on Animal Care (2009).   Ovaries were collected from three WB Japanese quail hens immediately after euthanasia by cervical dislocation.  The yellow follicles and most of the large white follicles were removed and the remaining tissue was trimmed into 3- to 4-mm2 pieces, which were transplanted into the recipients within 2 h.  Surgical procedures of ovariectomy and transplantation were modified slightly from those described previously (Liu et al., 2010).  The recipient chick was anesthetized by administration of isoflurane gas, and was placed on its back on a heated surface during surgery.  Feathers were removed from the left abdominal area and the skin was disinfected with 70% EtOH.  A 1.5-cm incision was made 1 cm left of the medial plane.  The abdominal organs were displaced to the chick?s right to expose the left ovary, which was removed in small pieces 120  with a pair of fine forceps.  Immediately after ovariectomy, each piece of adult donor tissue was placed on the wound and the incision was closed by simple interrupted stitches.  Only the left ovary is functional in Japanese quail as in most other avian species (Golden and Arbona, 2012).   After transplantation, the recipient chicks were given an immunosuppressant, mycophenolate mofetil (CellCept, Hoffmann-La Roche Ltd., Mississauga, ON, Canada), orally at 100 mg/kg per day for 2 wk.  Untreated week-old female QO chicks were raised with the recipients as controls.  After sexual maturity, the recipients were paired with fertile males from the WB line to determine the maternal origin of the resulting chicks.  White-breasted chicks (wb/wb) from this cross are derived from WB donor tissue whereas dark-colored wild-type chicks (+/wb) originate from regenerated ovarian tissue of the QO recipients.  A t-test was used to compare the age at first egg and the egg production between the two groups, with statistical significance set at P ? 0.05. 7.3 Results and discussion Ten of 12 recipients of adult ovarian transplants survived to maturity and seven of these laid eggs, but all stopped laying by 17 weeks of age.  The age at first egg of those recipients that laid eggs was later than that of control hens and their egg production was less (Table 7.1).  Two hens with tissue from the same donor laid eggs during the test period and together they produced 15 chicks, all of which were derived from the transplanted adult tissue (Table 7.2, Figure 7.1).   The age at first egg, which reflects the onset of puberty, was significantly delayed for recipients with adult ovarian transplants.  A similar tendency was observed in 121  Japanese quail in a previous study (Liu et al., 2010) using sexually immature ovarian donors.  The age of the recipients was the same in both studies, indicating that the endogenous ovarian clock might be affected by the transplantation procedures.  Vascular reanastomosis in transplantation is challenging and has never been conducted in ovarian transplantation in birds.  The perfusion of the transplants is therefore dependent entirely on revascularization, which takes three days to be initiated in mammalian models (Demeestere et al., 2009).  A report that included heterotopic ovarian transplantation in rats demonstrated that this delay in perfusion of the transplants affects the rhythmic expression of a clock gene (Yoshikawa et al., 2009).  The homologues of clock genes have also been identified in the ovaries of chickens and Japanese quail (Sellix and Menaker, 2010).  These regulate the physiological rhythms such as steroidogenesis through a transcription-translation oscillatory loop and the ovarian clock may be involved in the onset of puberty in rodents (Tolson and Chappell, 2012).  Future studies of ovarian transplantation may reveal the potential role of the ovarian clock in puberty initiation in birds by characterizing the post-transplantation hormonal profile of the recipients and the expression of the clock genes in the transplants.   Recipients with adult ovarian transplants had reduced egg production and stopped laying early, which was not the case in previous studies using immature ovarian transplants (Song and Silversides, 2008b; Liu et al., 2010).  Both immature and adult transplants may be subjected to follicular depletion induced by ischemic injury resulting from the delayed vascularization (Demeestere et al., 2009), but the adult transplants in this study represented only a small portion of the entire adult ovary and therefore contained a limited ovarian reserve.  In female mammals, the functional reproductive 122  longevity is determined by the initial finite size (although this is challenged by Johnson et al., 2004) and a species-specific threshold of the ovarian reserve (Finch and Holmes, 2010).  This could also be true in Japanese quail because the preovulatory follicles are arranged in a hierarchy and more follicles are needed than those that are ultimately used to establish the hierarchy (Golden and Arbona, 2012).  Holmes et al. (2003) showed that aged Japanese quail that stopped laying were able to respond to extraneous luteinizing hormone to mount a preovulatory hierarchy in spite of a reduced follicular pool.  The current study suggests that reproductive aging is not initiated by aging of the neuroendocrine system (Ottinger, 2010) that controls the upper stream of the hypothalamic-pituitary-gonadal axis because the recipients were young.  A reduced ovarian reserve and age-related changes in hormone production and gene expression may affect the neuroendocrine axis for ovulation in aged birds.  To test these hypotheses, future studies using partial ovariectomy or ovarian transplantation with controlled donor age and amount of tissue, along with endocrinological and molecular assessments are needed. In this study, production of donor-derived offspring demonstrated the possibility of rescuing fertility from adult ovarian tissue in sexually immature recipients. To our knowledge, this is the first successful recovery of fertility from adult ovarian donors in a non-mammalian vertebrate.  This may aid in developing reproductive techniques for preservation and recovery of endangered species because it allows restoration of the reproductive potential of ovarian tissue collected from a sexually mature individual that suffers unexpected death (Comizzoli et al., 2012).  This may have practical potential for reptiles, amphibians and fish, in which oogonial stem cells are present throughout the 123  female?s life and proliferate during each breeding season (Rothchild, 2003).  For the maintenance of laboratory strains (Silversides et al., 2012; Liu et al., 2013b), individuals that are to be eliminated at the end of a research program can now be considered as ovarian donors for cryobanking.  Moreover, transplantation of adult ovarian tissue into sexually immature recipients can be a useful tool for investigating mechanisms that underlie life history characteristics in birds such as puberty initiation and ovarian aging.   124  Table 7.1.  Reproductive performance of recipients that laid eggs and control birds (mean ? SEM) Groups Number Age  at first egg (days) Egg production Control 5 51.8 ? 1.7a 60.8 ? 3.5b  Recipient 7 75.7 ? 4.2b 21.7 ? 5.7a a, b Values in a column with different superscripts are different (P<0.05).   125  Table 7.2.  Reproductive performance of recipients that produced offspring during progeny test Bird ID Egg production  Fertile eggs Chicks hatched Donor-derived Host-derived 6499 3 2 2 0 6500 14 13 12a 0 a One unhatched chick was donor-derived.     126   Figure 7.1.  Production of offspring from transplantation of adult ovarian tissue. Ovarian tissue of adult female birds from a WB line (recessive plumage color) was transplanted into one-week old female recipients from a QO line (wild-type plumage color). Donor-derived offspring are produced after the sexual maturation of the recipients. 127  8 CHIMERIC PLUMAGE COLORATION PRODUCED BY OVARIAN TRANSPLANTATION IN CHICKENS7 8.1 Introduction Orthotopic transplantation has been used to recover reproductive potential of ovarian tissue in several avian species, using fresh (Song and Silversides, 2007b, 2008b; Song et al., 2012) or cryopreserved (Liu et al., 2010) ovarian tissue from donor birds surgically implanted to the normal location of the left ovary in ovariectomized recipients.  Incomplete ovariectomy has been common in previous studies, which permits the residual ovarian tissue of the recipients to reach maturity and produce recipient-derived offspring.  Plumage coloration has been used as a marker to distinguish donor-derived offspring from recipient-derived offspring (Song and Silversides, 2007b, 2008b; Liu et al., 2010). In chickens, plumage color is determined by the distribution of melanins including eumelanin and pheomelanin as a result of interactions among a series of gene loci (Smyth, 1990).  The distribution of eumelanin is the first genetic decision, which is primarily controlled by the E locus.  At this locus, the E allele that causes extended black is dominant to other alleles including wild type (e+).  The noneumelanic feather may be pigmented by pheomelanin, which is subjected to modifications by other genes such as the sex-linked silver gene at the S locus.  In White Leghorn (WL) chickens, which were used as recipients in a previous study (Song and Silversides, 2007b), the extended black allele (E) at the E locus is suppressed by the presence of a dominant white allele (I) at the                                                  7 A version of Chapter 8 has been published. Liu, J., M. C. Robertson, K. M. Cheng, and F. G. Silversides. 2013. Chimeric plumage coloration produced by ovarian transplantation in chickens. Poult. Sci. 92:1073-1076. 128  I locus and pheomelanin pigmentation is eliminated by a dominant silver allele (S) at the S locus, resulting in white plumage (Smyth, 1990).  The dominance of the I allele to its recessive allele i+ is incomplete and the plumage of extended black individuals that are heterozygous at this locus (I/i+) show occasional black spots on a white background.  When the ovarian tissue from a donor that is colored at the I locus (i+/i+) is transplanted into a WL recipient (I/I), the origin of the progeny can be tested by crossing recipients with males from the donor (colored) line. Resulting chicks with colored down (i+/i+) are derived from the transplanted ovarian tissue, whereas chicks that are white with black spots (I/i+) are derived from the regenerated recipient ovary.   In this study, birds from a line with colored plumage (i+/i+ e+/e+) were used as ovarian donors, and ovarian recipients were from a WL line (I/I E/E). After sexual maturation, a progeny test was conducted by inseminating the recipients with semen collected from colored roosters (i+/i+ e+/e+), using plumage color as a marker.  This research note reports a chick with chimeric plumage coloration that appears to be derived from both donor and recipient ovarian tissue. 8.2 Materials and methods Newly hatched Rhode Island Red (RIR) chicks carrying the rc mutation (Cheng et al., 1980) and WL chicks from a line maintained at the Agassiz Research Centre (Silversides, 2010) were used as donors and recipients, respectively.  The research protocol was approved by the Animal Care Committee of the Agassiz Research Centre following principles described by the Canadian Council on Animal Care (2009). All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) unless otherwise indicated. 129  Ovaries from newly hatched RIR chicks were isolated immediately after euthanasia by cervical dislocation and immersed in a handling medium that consisted of Dulbecco?s phosphate buffered saline with 20% fetal bovine serum on ice.  The isolated ovaries were kept in handling medium on ice before further treatment within 2 h.  The cryopreservation and warming procedures described by Liu et al. (2010) were used in the current study.  The surgical transplantation described previously (Song and Silversides, 2007b) was improved and used in this study.  Newly hatched WL chicks were anesthetized by administration of isoflurane.  The chick was placed on its back on a heated surgical surface.  The feathers were removed from the left abdominal area, where a 2-cm vertical incision was made approximately 1 cm left of the median plane.  The yolk sac was removed after tying the yolk stalk with surgical suture.  The abdominal organs were pushed to the right with a laboratory spatula to expose the ovary, which was illuminated with a fiber light source, and removed in small pieces using a pair of fine forceps.  A donor ovary was then placed in the position of ovariectomy of the recipient, and the incision was closed.  The recipient chicks were administered an immunosuppressant, mycophenolate mofetil (CellCept, Hoffmann-LaRoche Ltd., Mississauga, Ontario, Canada), orally at 100 mg/kg per day for 2 wk, followed by once a week until the age of 2 mo. Recipient chicks were kept in a brooder for 2 wk at a temperature of 33?C and then in pens at a temperature of 25?C and a day length of 12 h. At 15 wk of age, the pullets were caged individually at the same temperature and day length.  At the onset of egg laying, the recipient hens (I/I E/E) with RIR ovarian transplants (i+/i+ e+/e+) were inseminated with pooled semen from RIR rosters (i+/i+ e+/e+) to test the genetic origin of 130  their offspring.  Brown chicks (i+/i+ e+/e+) produced from this cross were derived from the RIR ovarian transplants, whereas white chicks (I/i+ E/e+) with randomly distributed black spots originated from regenerated recipient WL ovarian tissue. 8.3 Results and discussion Donor- and recipient-derived chicks from the transplantations performed are shown in Figure 8.1A.  One hen from this series of transplantations produced 25 white chicks with randomly distributed black spots, indicating that they were derived from the ovary of the recipient.  This hen also produced one chick that had white plumage with black spots over most of her body along with randomly distributed brown patches (Figure 8.1B) over large areas.  The white areas of this chick are clearly derived from the recipient ovary as confirmed by the presence of black spots, indicating that some cells have the E allele at the E locus (E+/e+), which is incompletely suppressed by the heterozygous I locus (I/i+).  However, the brown areas clearly originate from the donor tissue because they do not carry either I or E.  This chick must therefore be a chimera composed of tissue from both donor and recipient ovaries.  Two other hens produced similar chimeric chicks.  One of these hens also produced 48 recipient-derived chicks and 3 light-colored chicks with no black spots, which may have been donor-derived, and the other hen produced 34 recipient-derived chicks with no donor-derived chicks. Chimerism in plumage coloration cannot occur if the oocyte that contributes to the zygote is completely donor- (i+/_ e+/_) or recipient- (I/_ E/_) derived unless the composition of the oocyte is altered during folliculogenesis.  In the present study, the surgeons conducting ovariectomy were inexperienced and the production of the recipient-derived chicks demonstrated that ovariectomy of the recipients was incomplete.  The 131  production of the chimeric chicks showed that donor tissue survived cryopreservation and transplantation, making it possible for donor and residual recipient ovarian tissue to fuse. Before the formation of follicular epithelium, which starts 4 to 5 d after hatch (Greenfield, 1966), closely positioned donor and recipient primary oocytes could exchange their contents through intercellular bridges (Skalko et al., 1972) or interruptions of their plasma membranes (Chin et al., 1979), resulting in two individual follicles containing oocytes with chimeric compositions at a later stage (Figure 8.2A).  Avian oogenesis is characterized by the accumulation of a large amount of maternal RNA (Olszanska and Stepinska, 2008), some of which may be involved in the signaling pathways of melanogenesis (Kerje et al., 2004; Yoshihara et al., 2012) as regulatory elements.  These regulatory elements could be exchanged between donor and recipient oocytes and be unevenly distributed in the early embryonic cells after fertilization and cleavage to modify the melanogenic pathways of the cells in which they reside through epigenetic regulatory mechanisms (Rassoulzadegan et al., 2006).  As an alternative, donor and recipient oocytes could fuse to form a single follicle with chimeric oocytes (Figure 8.2B) through a process similar to the formation of polyovular follicles that are commonly observed in avian folliculogenesis (Amer and Shahin, 1975; Wakuri and Mutoh, 1986).  Polyspermy during fertilization is normal in birds, but spermatozoa that enter the egg through regions other than the germinal vesicle are subjected to high levels of DNase I and II (Olszanska and Stepinska, 2008).  The chimeric polyovular follicle provides two germinal vesicle regions that could allow fertilization of both ova and thereby generate the chimera.  On the other hand, after 132  fertilization, at least one of the ova in the chimeric polyovular follicle could develop parthenogenically with or without sperm activation (Sarvella, 1973; Rougier and Werb, 2001).  These hypotheses can be further tested by transplantation of ovaries into recipients that are incompletely ovariectomised and subsequent histological and molecular assays.  Orthotopic ovarian transplantation in avian species has been attempted previously with limited success (Guthrie, 1908; Davenport, 1911; Grossman and Siegel, 1966), but Song and Silversides (2007b) improved it and Liu et al. (2010) used it for functional recovery of cryopreserved avian ovarian tissue.  The present study suggests that ovarian transplantation can also be used as a useful in vivo approach for investigating folliculogenesis, ovarian development, and ovarian functioning in avian species.      133   Figure 8.1.  Chicks produced from progeny test. A: a donor-derived chick (left) was brown all over and a recipient-derived chick (right) was white with randomly distributed black spots. B: a chick with chimeric plumage coloration was white with randomly distributed black spots and brown patches.   134   Figure 8.2.  Possible mechanisms for chimeric folliculogenesis. A: closely located donor and recipient oocytes may communicate (indicated by the double arrow) and form chimeric follicles separately at a later stage. B: Donor and recipient oocytes may fuse and  form a chimeric polyovular follicle. 135  9 GENERAL DISCUSSION 9.1 Cryoconservation of avian germplasm Cryoconservation is used here to refer to a strategy of ex situ conservation.  A successful cryoconservation program requires the survival of the germplasm through the cryopreservation procedures and recovery at a success rate that produces sufficient offspring to represent the preserved genetic diversity.  Any of the components shown in Figure 1.2 could be considered for preservation, but the practicality of integration into a conservation program for avian species depends on the availability of cryopreservation techniques and the success rate of functional recovery (Table 9.1).  In principle, primordial germ cells can be isolated, cryopreserved and recovered in chimeras but the application of these in genetic conservation is limited by the technical complexity and low efficiency (Silversides and Liu, 2012).  Fertility produced by cryopreserved semen in some avian species is adequate for conservation purposes (Blackburn et al., 2009) but could be improved (see Chapter 1).  Cryopreservation of avian gonadal tissue provides an alternative for preserving male genetic resources and may be the only practical option for preserving female genetic resources.  Recent progress in cryopreservation and transplantation of avian gonadal tissue for genetic conservation is promising.  The advance of vitrification over slow-freezing procedures in preserving tissue was introduced in Chapter 1 and a simple vitrification protocol that has been successful in preserving quail ovarian tissue (Liu et al., 2010) was used in the studies described by subsequent chapters.  The efficiency of this protocol in preserving testicular tissue is demonstrated by tissue histology (Chapter 3), viability after in ovo culture (Chapter 3) 136  and allotransplantion (Chapter 4) and functional recovery (Chapter 4).  These were also confirmed in ovarian tissue (Chapters 2 and 5).  A method based on this protocol using 2-ml macrotube straws for storage was standardized after these studies and is now used as a routine for cryobanking avian genetic resources in Canada (Silversides et al., 2013b).  The simple steps of the vitrification method make it possible for optimization when it is adapted for other species and  a rapid preliminary screening can be achieved using the in ovo culture system described in the Chapters 2 and 3.  In addition to the histological and angiogenic evaluation used in those studies, markers for cell proliferation (Milazzo et al., 2010) or apoptosis (Yoshimura and Nishikori, 2004) should also be included in this culture system in the future.   For domestic species, in which donors and recipients are available and their production can be artificially controlled, procedures described in Chapters 4 and 5 in Japanese quail can be used as a model for preserving and regenerating a single gene (such as the white breasted gene in Japanese quail) that is of interest.  This should be tested in other domestic species such as chickens, turkeys and waterfowl.  Recovery of an entire line with an effective population size suggested by FAO (2012) has not been achieved by using male and female germplasm that are both cryopreserved.  Although the vitrification method can ensure high viability of testicular and ovarian tissue and high efficiency of functional recovery of ovarian tissue in Japanese quail, functional recovery of cryopreserved testicular tissue is overlaid by the limited efficiency of insemination using the spermatozoa retrieved from the testicular extrusion (Chapter 4).  Intramagnal insemination in Japanese quail described in Chapter 4 appeared to be less efficient and predictable compared to that in chickens (Liu et al., unpublished data), largely because 137  optimal conditions such as the most favourable interval between last oviposition and insemination and processing of testicular spermatozoa are unknown, which need more investigations.  Alternatively, this may be resolved by using cryopreserved semen for those species in which semen cryopreservation is available.  The techniques in these chapters could also benefit conservation of endangered avian species, for which investigations of species-specific reproductive physiology is needed to assist their reproduction for conservation but their population is too small to allow extra individuals sacrificed for research ( Comizzoli et al., 2012).  Nevertheless, birds die inevitably and gonadal tissue from these valuable birds after death should be preserved for future research or repopulation, the possibility of which is demonstrated by recovery of adult ovarian tissue in Japanese quail (Chapter 7).  Future research in cryopreservation of adult ovarian tissue is warranted.  In terms of adult testicular tissue, one could preserve the whole testis and retrieve the testicular spermatozoa from sections of preserved tissue without tissue culture (Ohta et al., 2008).  As an alternative, cell suspensions retrieved from the tissue containing spermatogonial stem cells could be preserved and recovered (Roe et al., 2013) to produce spermatozoa.  Although short-term tissue culture could be achieved by in ovo culture because the chicken embryo at the stage of implantation is not immunologically competent to reject the transplants (Ribatti, 2008), functional recovery requires gonadal transplantation.  Xenotransplantation that may recover preserved gonadal tissue of endangered avian species using domestic species as recipients is limited because immunological deficient lines that can be used as universal recipients (Rodriguez-Sosa and Dobrinski, 2009) are not available for birds.  A possible means of inducing immunological tolerance across the species barrier has been 138  attempted (Chapter 6) but the transplants were not able to survival to functional recovery.  Future studies combining this treatment with immunosuppressive treatment (Song et al., 2012) or using alternative immunosuppressive treatments may be considered.    9.2 Use of avian gonadal transplantation for biological research  Surgical gonadoectomy and transplantation were used in the studies described here for functional recovery of gonadal tissue (Chapters 4-5 and 7-8) and evaluation of immunological tolerance (Chapter 6).  The word ?surgery? may seem to be onerous to some, but the procedures are simple and straightforward with low cost and therefore can easily be adapted to an avian anatomy and physiology teaching/training program with regular lab settings.   The original procedures described by Song and Silversides (2007a,b) have been improved through these and other (Silversides et al., 2013a) studies.  The use of isoflurane anaesthetic has significantly increased the ease of bird handling because the side effects from ketamine and xylazine such as violent and sudden reactions of the sedated birds (Liu et al., personal observation; Engel et al., 1991) were prevented and recovery from isoflurane anaesthetic is almost immediate.  Instead of making a transverse cut across the abdomen to facilitate subsequent externalization of the internal organs to expose the gonads (Song and Silversides, 2007b,c, 2008b; Liu et al., 2010), a small vertical cut was made on the abdominal wall of the recipient above the left gonad, which was exposed by gently moving the viscera to the right of the chick using a small spatula or a pair of large forceps with blunt ends as an extractor.  These changes may contribute to the higher rate of complete ovariectomy in Japanese quail compared to a previous study (Liu et al., 2010) described in Chapter 5.   139  Song and Silversides (2007 a-c) conducted allotransplantation in day-old chicks but a recent study (Silversides et al., 2013a) reported lower mortality in castration and autotransplantation of chicken testicular tissue at 6 d of age.  A similar tendency was also seen in ovariectomy of chicks (Karagen? et al., 2011).  Transplantation was conducted in chickens around one week after hatching in Chapter 6, which resulted in negligible mortality (Liu et al., unpublished data).  This also reduces the surgical time spent on each bird and mortality during surgery because there is no need to remove the yolk sac as in day-old chicks (Song and Silversides, 2007a-c), which is delicate and well vascularized so that removal poses a risk of lethal bleeding.  Transplantation has long been used in embryology to study autonomous and conditional fate determination of cells (Gilbert, 2010).  Similarly, gonadal transplantation provides a way of separating the gonadal tissue from its original biological milieu, which may reveal the intrinsic gonadal factors and mechanisms that affect the reproductive physiology and development in birds.  An example is seen in Chapter 7, in which intriguing observations were obtained by transplantation of adult ovarian tissue into sexually immature recipients.  These observations suggested intrinsic ovarian mechanisms involved in the onset of puberty and ovarian aging that can be tested by comparing gene expressions of ovarian transplants at different developmental stages to that of the in situ ovarian tissue.  In addition, spermatogenesis can be re-established in subcutaneous testicular transplants of fresh (Song and Silversides, 2007c) or cryopreserved (Chapter 4) tissue, which may contribute to the study of controversial questions such as maturation of avian spermatozoa (see the discussion of Chapter 4) by 140  comparing genomics and proteomics of the spermatozoa retrieved from the transplants and those from the vas deferens. Incomplete ovariectomy is not desirable for functional recovery of ovarian transplants because it leads to production of unwanted recipient-derived offspring (Chapters 5, 7, and 8) but controlled incomplete ovariectomy might be a useful tool for biological research, such as in a mammalian study of ovarian aging (Brook et al., 1984).  Its potential in avian research was seen in the serendipitous observation reported in Chapter 8.  Controlled incomplete ovariectomy with or without subsequent transplantation should be examined by endocrinological, histological and molecular tools to investigate questions such as the presence of oogonial stem cells (Johnson et al., 2004) in birds. 9.3 Conclusion      The studies in this dissertation demonstrated that a simple vitrification protocol can be adapted to straw storage systems without compromising its efficiency in preserving the viability, structural integrity and potential of revascularization of gonadal tissue in Japanese quail.  In addition, live offspring can be produced from quail testicular tissue that has been cryopreserved through vitrification and recovered by transplantation, demonstrating the feasibility of using vitrification to preserve testicular tissue.  Similarly, donor-derived offspring can be produced from cryopreserved and transplanted ovarian tissue in quail and chickens using the same vitrification method, which provides a model for cryobanking germplasm for these species and perhaps other avian species.   141  Evidence presented here suggests that implantation of donor thymic tissue onto the chorioallantoic membrane of recipient embryos may lead to a lasting state of tolerance of allogeneic gonadal transplantation from the specific donor, but more studies are needed to demonstrate induction of donor-specific tolerance that would allow functional recovery of gonadal tissue in allo-and xenotransplantation.  This dissertation also showed that the fertility of ovarian tissue from adult quail can be recovered by transplantation into sexually immature recipients and can produce donor-derived offspring.  This makes fertility rescue from an endangered avian species a conceivable prospect, and widens the options of ovarian donors for cryobanking female gerplasm in domestic species.  Production of offspring with chimeric plumage coloration from cryopreserved and transplanted ovarian tissue in chickens suggest possible interactions between surviving transplants and the regenerating host tissue, suggesting the potential of using gonadal transplantation in avian biology research.  Recovery of the donor gene also demonstrated the possibility of cryopreserving ovarian tissue in chickens.   This dissertation established methods for cryopreservation and storage of avian gonadal tissue and demonstrated significant improvements in gonadal transplantation techniques.  It also investigated methods of inducing immunological tolerance which will be necessary for gonadal transplantation across species barriers and it laid the foundation for using gonadal transplantation in the study of avian reproduction and development.    142  Table 9.1. Status of germplasm cryopreservation in avian species Germplasm Status References Gonadal tissue Male Available Song and Silversides, 2007a; Liu et al., 2013 Female Available Liu et al., 2010; Liu et al., 2013 Gametes Male Available Blesbois, 2011; Gee et al., 2004 Female NA NA Primordial germ cells  Limited efficiency Petitte, 2006 Silversides and Liu, 2012 Embryos  NA NA  143  BIBLIOGRAPHY Abrishami, M., M. Anzar, Y. Yang, and A. Honaramooz. 2010. Cryopreservation of immature porcine testis tissue to maintain its developmental potential after xenografting into recipient mice. Theriogenology 73:86-96. doi:10.1016/j.theriogenology.2009.08.004. Abbasi, S., and A. Honaramooz. 2010. Effects of recipient mouse strain, sex and gonadal status on the outcome of testis tissue xenografting. Reprod. Fertil. Dev. 22:1279-1286. doi:10.1071/RD10084.  Acker, J. P., and L. E. McGann. 2003. Protective effect of intracellular ice during freezing? Cryobiology 46:197-202. doi:10.1016/S0011-2240(03)00025-7.  Ahammad, M. U., C. Nishino, H. Tatemoto, N. Okura, Y. Kawamoto, S. Okamoto, and T. Nakada. 2011a. Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract. Theriogenology 76:1100-1109. doi:10.1016/j.theriogenology.2011.05.017.  Ahammad, M. U., C. Nishino, H. Tatemoto, N. Okura, Y. Kawamoto, S. Okamoto, and T. Nakada. 2011b. Maturational changes in the survivability and fertility of fowl sperm during their passage through the male reproductive tract. Anim. Reprod. Sci. 128:129-136. doi:10.1016/j.anireprosci.2011.09.010.  Amer, F. I., and M. A. Shahin. 1975. The post-hatching development of the gonads in the fowl Gallus domesticus. Annls. Zool. 11:1-25.  Angell, C. A. 2002. Liquid fragility and the glass transition in water and aqueous solutions. Chem. Rev. 102:2627-2649. doi:10.1021/cr000689q.  Balin, S. J., J. L. Platt, and M. Cascalho. 2009. Noncognate function of B cells in transplantation. Transplant Int. 22:593-598. doi:10.1111/j.1432-2277.2008.00816.x.  Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245-252. doi:10.1038/32588.  Bedford, J. M. 2004. Enigmas of mammalian gamete form and function. Biol. Rev. 79:429-460. doi:10.1017/S146479310300633X.  Bedi, D. S., L. V. Riella, S. G. Tullius, and A. Chandraker. 2010. Animal models of chronic allograft injury: contributions and limitations to understanding the mechanism of long-term graft dysfunction. Transplantation 90:935-944. doi:10.1097/TP.0b013e3181efcfbc.  144  Belo, M., C. Corbel, C. Martin, and N. M. L. Douarin. 1989. Thymic epithelium tolerizes chickens to embryonic grafts of quail bursa of Fabricius. Int. Immunol. 1:105-112. doi:10.1093/intimm/1.2.105.  Billingham, R. E., L. Brent, and P. B. Medawar. 1953. Actively acquired tolerance of foreign cells. Nature 172:603-606. doi:10.1038/172603a0.  Billingham, R. E., L. Brent, and P. B. Medawar. 1956. Quantitative studies on tissue transplantation immunity. III. Actively acquired tolerance. Philosophical Transactions of the Royal Society of London.Series B, Biological Sciences 239:357-414. doi:10.1098/rstb.1956.0006.  Birkhead, T. R., and J. Brillard. 2007. Reproductive isolation in birds: postcopulatory prezygotic barriers. Trends Ecol. Evol. 22:266-272. doi:10.1016/j.tree.2007.02.004.  Birkhead, T. R., and F. Fletcher. 1994. Sperm storage and the release of sperm from the sperm storage tubules in Japanese quail (Coturnix japonica). Ibis 136:101-104.  Biswas, A., O. S. Ranganatha, J. Mohan, and K. V. H. Sastry. 2007. Relationship of cloacal gland with testes, testosterone and fertility in different lines of male Japanese quail. Anim. Reprod. Sci. 97:94-102. doi:10.1016/j.anireprosci.2005.12.012.  Blackburn, H. D., F. Silversides, and P. H. Purdy. 2009. Inseminating fresh or cryopreserved semen for maximum efficiency: implications for gene banks and industry. Poult. Sci. 88:2192-2198.  Blanco, J. M., D. E. Wildt, U. H?fle, W. Voelker, and A. M. Donoghue. 2009. Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species. Theriogenology 71:200-213. doi:10.1016/j.theriogenology.2008.09.019.  Blanco, J. M., J. A. Long, G. Gee, A. M. Donoghue, and D. E. Wildt. 2008. Osmotic tolerance of avian spermatozoa: Influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability. Cryobiology 56:8-14. doi:10.1016/j.cryobiol.2007.09.004.  Blesbois, E. 2007. Current status in avian semen cryopreservation. Worlds Poult. Sci. J. 63:213-222. doi:10.1017/S0043933907001419.  Blesbois, E. 2011. Freezing avian semen. Avian Biol. Res. 4:52-58. doi:10.3184/175815511X13069413108523.  Bols, P. E. J., J. M. J. Aerts, A. Langbeen, I. G. F. Goovaerts, and J. L. M. R. Leroy. 2010. Xenotransplantation in immunodeficient mice to study ovarian follicular development in domestic animals. Theriogenology 73:740-747. doi:10.1016/j.theriogenology.2009.10.002.  145  Bonasio, R., M. Scimone, P. Schaerli, N. Grabie, A. Lichtman, and U. von Andrian. 2006. Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus. Nat. Immunol. 7:1092-1100. doi:10.1038/ni1385.  Bono-Mestre, C., J. Cardona-Costa, and F. Garcia-Ximenez. 2009. Effects on cell viability of three zebrafish testicular cell or tissue cryopreservation methods. Cryoletters 30:148-148.  Brook, J. D., R. G. Gosden, and A. C. Chandley. 1984. Maternal ageing and aneuploid embryos--evidence from the mouse that biological and not chronological age is the important influence. Hum. Genet. 66:41-45. doi:10.1007/BF00275184.  Brunstr?m, B., J. Axelsson, A. Mattsson, and K. Halldin. 2009. Effects of estrogens on sex differentiation in Japanese quail and chicken. Gen. Comp. Endocrinol. 163:97-103. doi:10.1016/j.ygcen.2009.01.006.  Chappert, P., and R. H. Schwartz. 2010. Induction of T cell anergy: integration of environmental cues and infectious tolerance. Curr. Opin. Immunol. 22:552-559. doi:10.1016/j.coi.2010.08.005.  Chelmonska, B., E. Lukaszewicz, A. Kowalczyk, and A. Jerysz. 2006. The effect of proctodeal gland foam, diluent and dimethylacetamide addition on morphology and fertilising ability of Japanese quail (Coturnix japonica) spermatozoa. J. Poult. Sci. 43:54-59. doi:10.2141/jpsa.43.54.  Chen, C. 1986. Pregnancy after human oocyte cryopreservation. Lancet 1:884-886.  Chen, S., C. Chien, M. Wu, T. Chen, S. Lai, C. Lin, and Y. Yang. 2006. Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice. Hum. Reprod. 21:2794-2800. doi:10.1093/humrep/del210.  Chen, M., M. R. Daha, and C. G. M. Kallenberg. 2010. The complement system in systemic autoimmune disease. J. Autoimmun. 34:J276-J286. doi:10.1016/j.jaut.2009.11.014.  Chen, S., and Y. Yang. 2007. Vitrification of oocytes: various procedures. Pages 129-144 in Vitrification in Assisted Reproduction: A User's Manual and Trouble-Shooting Guide. M. J. Tucker, and Liebermann, J., eds. Informa UK Ltd., London.  Cheng, K. M., and A. R. Hickman. 1989. Role of the proctodeal gland foam of male Japanese quail in natural copulations. Auk 106:279-285.  Cheng, K. M., R. N. Shoffner, K. N. Gelatt, G. G. Gum, J. S. Otis, and J. J. Bitgood. 1980. An autosomal recessive blind mutant in the chicken. Poult. Sci. 59:2179-2182.  146  Chin, S. H., E. R. Gaginskaia, and E. I. Kalinina. 1979. Characteristics of oogenesis in the chick. I. The extrafollicular period in the development of the oocytes. Ontogenez 10:340-349.  Chinen, J., and R. H. Buckley. 2010. Transplantation immunology: solid organ and bone marrow. J. Allergy Clin. Immunol. 125:S324-S335. doi:10.1016/j.jaci.2009.11.014.  Cleary, M., M. C. J. Paris, J. Shaw, G. Jenkin, and A. Trounson. 2003. Effect of ovariectomy and graft position on cryopreserved common wombat (Vombatus ursinus) ovarian tissue following xenografting to nude mice. Reprod. Fertil. Dev. 15:333-342. doi:10.1071/RD03063.  Comizzoli, P., N. Songsasen, M. Hagedorn, and D. E. Wildt. 2012. Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species. Theriogenology 78:1666-1681. doi:10.1016/j.theriogenology.2012.04.008.  Corbel, C., M. Belo, C. Martin, and N. Le Douarin. 1987. A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. II. Immune response of bursectomized chicks and chimeras and post-natal rejection of the grafted quail bursas. J. Immunol. 138:2813-2821.  Corbel, C., C. Martin, H. Ohki, M. Coltey, I. Hlozanek, and N. M. Le Douarin. 1990. Evidence for peripheral mechanisms inducing tissue tolerance during ontogeny. Int. Immunol. 2:33-40. doi:10.1093/intimm/2.1.33.  Crowe, L. M., J. H. Crowe, J. F. Carpenter, and T. J. Anchordoguy. 1990. Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 27:219-231. doi:10.1016/0011-2240(90)90023-W.  Curaba, M., J. Poels, A. van Langendonckt, J. Donnez, and C. Wyns. 2011a. Can prepubertal human testicular tissue be cryopreserved by vitrification? Fertil. Steril. 95:2123.e9-2123.e12. doi:10.1016/j.fertnstert.2011.01.014.  Curaba, M., M. Verleysen, C. A. Amorim, M. Dolmans, A. Van Langendonckt, O. Hovatta, C. Wyns, and J. Donnez. 2011b. Cryopreservation of prepubertal mouse testicular tissue by vitrification. Fertil. Steril. 95:1229-1234.e1. doi:10.1016/j.fertnstert.2010.04.062.  Davenport, C. B. 1911. The transplantation of ovaries in chickens. J. Morphol. 22:111-122.  Davison, F. 2008. The importance of the avian immune system and its unique features. Pages 1-11 in Avian Immunology. F. Davison, Kaspers, B., and Schat, K. A., eds. Elsevier Ltd., London.  147  Demeestere, I., P. Simon, S. Emiliani, A. Delbaere, and Y. Englert. 2009. Orthotopic and heterotopic ovarian tissue transplantation. Hum. Reprod. Update 15:649-665. doi:10.1093/humupd/dmp021.  Didion, B. A., G. D. Braun, and M. V. Duggan. 2013. Field fertility of frozen boar semen: a retrospective report comprising over 2600 AI services spanning a four year period. Anim. Reprod. Sci. 137:189-196. doi:10.1016/j.anireprosci.2013.01.001.  Ehmcke, J., and S. Schlatt. 2008. Animal models for fertility preservation in the male. Reproduction 136:717-723. doi:10.1530/REP-08-0093.  Engel, H. N., D. P. Froman, and J. D. Kirby. 1991. An improved procedure for intramagnal insemination of the chicken. Poult. Sci. 70:1965-1969.  Erf, G. F. 2008. Autoimmune diseases of poultry. Pages 339-358 in Avian Immunology. F. Davison, Kaspers, B., and Schat, K. A., eds. Elsevier Ltd., London.  Fahy, G. M., D. I. Levy, and S. E. Ali. 1987. Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions. Cryobiology 24:196-213. doi:10.1016/0011-2240(87)90023-X.  Fahy, G. M., D. R. MacFarlane, C. A. Angell, and H. T. Meryman. 1984. Vitrification as an approach to cryopreservation. Cryobiology 21:407-426. doi:10.1016/0011-2240(84)90079-8.  FAO.  2012.  Cryoconservation of animal genetic resources.  FAO Animal Production and Health Guidelines No. 12. Rome. Fehr, T., and M. Sykes. 2008. Clinical experience with mixed chimerism to induce transplantation tolerance. Transplant Int. 21:1118-1135. doi:10.1111/j.1432-2277.2008.00783.x.  Finch, C. E., and D. J. Holmes. 2010. Ovarian aging in developmental and evolutionary contexts. Ann. N. Y. Acad. Sci. 1204:82-94. doi:10.1111/j.1749-6632.2010.05610.x.  Fuller, B., S. Paynter, and P. Watson. 2004. Cryoperservation of human gametes and embryos. Pages 505-539 in Life in the Frozen State. B. J. Fuller, Lane, N., and Benson, E. E., eds. CRC Press LLC, Boca Raton.  Fulton, J. E., and M. E. Delany. 2003. Genetics. Poultry genetic resources--operation rescue needed. Science (New York, N.Y.) 300:1667-1668. doi:10.1126/science.1085407.  Gee, G., H. Bertschinger, A. Donoghue, J. Blanco, and J. Soley. 2004. Reproduction in nondomestic birds: Physiology, semen collection, artificial insemination and cryopreservation. Avian Biol. Res. 15:47-101.  148  Gershon, R. K., and K. Kondo. 1971. Infectious immunological tolerance. Immunology 21:903-914.  Gilbert, S. F. 2010. Developmental Biology. 9th ed. Sinauer Associates, Inc., Massachusetts.  Golden, J. B., and D. V. Arbona. 2012. A review of the structure and regulation of the laying hen's reproductive system and the interaction between the stress and reproductive axes. CAB Reviews 7:1-8. doi:10.1079/PAVSNNR20127014.  Goodnow, C. C., J. Sprent, B. Fazekas de St Groth, and C. G. Vinuesa. 2005. Cellular and genetic mechanisms of self tolerance and autoimmunity. Nature 435:590-597. doi:10.1038/nature03724.  Greenfield, M. L. 1966. The oocyte of the domestic chicken shortly after hatching, studied by electron microscopy. J. Embryol. Exp. Morphol. 15:297-316.  Griesemer, A. D., E. C. Sorenson, and M. A. Hardy. 2010. The role of the thymus in tolerance. Transplantation 90:465-474. doi:10.1097/TP.0b013e3181e7e54f.  Grossman, M., and P. B. Siegel. 1966. Orthotopic ovarian transplants in chickens. Poult. Sci. 45:1434-1436.  Gui, J., X. Zhu, J. Dohkan, L. Cheng, P. F. Barnes, and D. Su. 2007. The aged thymus shows normal recruitment of lymphohematopoietic progenitors but has defects in thymic epithelial cells. Int. Immunol. 19:1201-1211. doi:10.1093/intimm/dxm095.  Guibert, E., S. Bri?re, R. Pelletier, J. P. Brillard, and P. Froment. 2011. Characterization of chicken Sertoli cells in vitro. Poult. Sci. 90:1276 -1286.  Guthrie, C. C. 1908. Further results of transplantation of ovaries in chickens. J. Exp. Zool. 5:563-576. doi:10.1002/jez.1400050406.  Hagedorn, M. 2006. Avian genetic resource banking: can fish embryos yield any clues for bird embryos? Poult. Sci. 85:251-254.  Halverson, R., R. M. Torres, and R. Pelanda. 2004. Receptor editing is the main mechanism of B cell tolerance toward membrane antigens. Nat. Immunol. 5:645-650. doi:10.1038/ni1076.  Hasek, M., T. Hraba, and J. Hort. 1958. Embryonic parabiosis and related problems. Ann. N. Y. Acad. Sci. 73:570-575. doi:10.1111/j.1749-6632.1959.tb40834.x.  Hasek, M., T. Hraba, and J. Hort. 1959. Acquired immunological tolerance of heterografts. Nature 183:1199-1200. doi:10.1038/1831199a0.  149  Hogquist, K. A., T. A. Baldwin, and S. C. Jameson. 2005. Central tolerance: learning self-control in the thymus. Nat. Rev. Immunol. 5:772-782. doi:10.1038/nri1707.  Holmes, D. J., S. L. Thomson, J. Wu, and M. A. Ottinger. 2003. Reproductive aging in female birds. Exp. Gerontol. 38:751-756. doi:10.1016/S0531-5565(03)00103-7.  Horner, B. M., R. A. Cina, K. J. Wikiel, B. Lima, A. Ghazi, D. P. Lo, K. Yamada, D. H. Sachs, and C. A. Huang. 2006. Predictors of organ allograft tolerance following hematopoietic cell transplantation. Am. J. Transplant. 6:2894-2902. doi:10.1111/j.1600-6143.2006.01563.x.  Howarth, J., B. 1983. Fertilizing ability of cock spermatozoa from the testis epididymis and vas deferens following intramagnal insemination. Biol. Reprod. 28:586-590. doi:10.1095/biolreprod28.3.586.  Hrabia, A., S. Takagi, T. Ono, and K. Shimada. 2003. Fertilization and development of quail oocytes after intracytoplasmic sperm injection. Biol. Reprod. 69:1651-1657. doi:10.1095/biolreprod.103.019315.  Isachenko, V., E. Isachenko, A. M. Petrunkina, and R. Sanchez. 2012. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod. Fertil. Dev. 24:323-326.  Isachenko, V., E. Isachenko, J. Reinsberg, M. Montag, K. van der Ven, H. van der Ven, C. Dorn, and B. Roesing. 2007. Cryopreservation of human ovarian tissue: Comparison of rapid and conventional freezing. Cryobiology 55:261-268. doi:10.1016/j.cryobiol.2007.08.008.  Iwasaki, A., and R. Medzhitov. 2010. Regulation of adaptive immunity by the innate immune system. Science 327:291-295. doi:10.1126/science.1183021.  Janeway, C. 2005. Immunobiology: the immune system in health and disease. Garland Science, New York.  Johnson, A. L. 1986a. Reproduction in the male. Pages 432-451 in Avian Physiology. 4th ed. P. D. Sturkie, ed. Springer-Verlag Inc., New York.  Johnson, A. L. 1986b. Reproduction in the female. Pages 403-431 in Avian Physiology. 4th ed. P. D. Sturkie, ed. Springer-Verlag Inc., New York.  Johnson, A. L., and D. C. Woods. 2009. Dynamics of avian ovarian follicle development: Cellular mechanisms of granulosa cell differentiation. Gen. Comp. Endocrinol. 163:12-17. doi:10.1016/j.ygcen.2008.11.012.  150  Johnson, J., J. Canning, T. Kaneko, and J. K. Pru. 2004. Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428:145-150. doi:10.1038/nature02316.  Johnson, L., J. Thompson Donald L., and D. D. Varner. 2008. Role of Sertoli cell number and function on regulation of spermatogenesis. Anim. Reprod. Sci. 105:23-51. doi:10.1016/j.anireprosci.2007.11.029.  Kagawa, N., M. Kuwayama, K. Nakata, G. Vajta, S. Silber, N. Manabe, and O. Kato. 2007. Production of the first offspring from oocytes derived from fresh and cryopreserved pre-antral follicles of adult mice. Reprod. Biomed. Online 14:693-699. doi:10.1016/S1472-6483(10)60670-0.  Karagenc, L., M. K. Turkyilmaz, and M. Sankikci. 2011. Timing of the ovariectomy operation affects the survival of newly-hatched chicks. Kafkas. Univ. Vet. Fak. Derg. 17:923-926.  Kaufman, J. 2008. The avian MHC. Pages 159-181 in Avian Immunology. F. Davison, Kaspers, B., and Schat, K. A., eds. Elsevier Ltd., London.  Kendal, A. R., and H. Waldmann. 2010. Infectious tolerance: therapeutic potential. Curr. Opin. Immunol. 22:560-565. doi:10.1016/j.coi.2010.08.002.  Kerje, S., P. Sharma, U. Gunnarsson, H. Kim, S. Bagchi, R. Fredriksson, K. Sch?tz, P. Jensen, G. von Heijne, R. Okimoto, and L. Andersson.. 2004. The Dominant white, Dun and Smoky color variants in chicken are associated with insertion/deletion polymorphisms in the PMEL17 gene. Genetics 168:1507-1518. doi:10.1534/genetics.104.027995.   Kim, S. H., K. M. Cheng, C. Ritland, K. Ritland, and F. G. Silversides. 2007. Inbreeding in Japanese quail estimated by pedigree and microsatellite analyses. J. Hered. 98:378-381. doi:10.1093/jhered/esm034.  Klein, L. 2009. Dead man walking: how thymocytes scan the medulla. Nat. Immunol. 10:809-811. doi:10.1038/ni0809-809.  Klein, L., M. Hinterberger, J. von Rohrscheidt, and M. Aichinger. 2011. Autonomous versus dendritic cell-dependent contributions of medullary thymic epithelial cells to central tolerance. Trends Immunol. 32:188-193. doi:10.1016/j.it.2011.03.002.  Kondapalli, L. A. 2012. Ovarian tissue cryopreservation and transplantation. Pages 63-75 in Oncofertility Medical Practice: Clinical Issues and Implementation. C. Gracia, and Woodruff, T. K., eds. Springer Science+Business Media, New York, NY.  151  Koshimoto, C., E. Gamliel, and P. Mazur. 2000. Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose. Cryobiology 41:204-231. doi:10.1006/cryo.2000.2281.  Kurtz, J., T. Wekerle, and M. Sykes. 2004. Tolerance in mixed chimerism - a role for regulatory cells? Trends Immunol. 25:518-523. doi:10.1016/j.it.2004.08.007.  Kuwayama, M. 2007. Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology 67:73-80. doi:10.1016/j.theriogenology.2006.09.014.  Kyewski, B., and J. Derbinski. 2004. Self-representation in the thymus: an extended view. Nat. Rev. Immunol. 4:688-698. doi:10.1038/nri1436.  Lakkis, F. G. 2012. The immune response to a transplanted organ: an overview. Pages 3-9 in Immunotherapy in Transplantation: Principles and Practice. B. Kaplan, Burkhart, G. J., and Lakkis, F. G., eds. John Wiley & Sons, West Sussex.  Le Douarin, N., C. Corbel, A. Bandeira, V. Thomas-Vaslin, Y. Modigliani, A. Coutinho, and J. Sala?n. 1996. Evidence for a thymus-dependent form of tolerance that is not based on elimination or anergy of reactive T cells. Immunol. Rev. 149:35-53. doi:10.1111/j.1600-065X.1996.tb00898.x.  Leibo, S. P. 2004. The early history of gamete cryobiology. Pages 347-370 in Life in the Frozen State. B. J. Fuller, Lane, N., and Benson, E. E., eds. CRC Press LLC, Boca Raton.  Leibo, S. P., J. J. McGrath, and E. G. Cravalho. 1978. Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate. Cryobiology 15:257-271. doi:10.1016/0011-2240(78)90036-6.  Li, M. W., J. D. Biggers, H. Y. Elmoazzen, M. Toner, L. McGinnis, and K. C. Kent Lloyd. 2007. Long-term storage of mouse spermatozoa after evaporative drying. Reproduction 133:919-929. doi:10.1530/REP-06-0096.  Lipsky, J. J. 1996. Mycophenolate mofetil. Lancet 348:1357-1359. doi:10.1016/S0140-6736(96)10310-X.  Liu, J., K. M. Cheng, P. H. Purdy, and F. G. Silversides. 2012a. A simple vitrification method for cryobanking avian testicular tissue. Poult. Sci. 91:3209-3213. doi:10.3382/ps.2012-02454.  Liu, J., K. M. Cheng, and F. G. Silversides. 2012b. Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica). Anim. Reprod. Sci. 134:197-202. doi:10.1016/j.anireprosci.2012.08.002.  152  Liu, J., K. M. Cheng, and F. G. Silversides. 2013b. Fundamental principles of cryobiology and application to conservation of avian species. Avian Biol. Res. 6 (3):187-197 Liu, J., K. M. Cheng, and F. G. Silversides. 2013c. Recovery of fertility from adult ovarian tissue transplanted into week-old Japanese quail chicks. Poult. Sci. 92 (E-Supplement 1):18. Liu, J., K. M. Cheng, and F. G. Silversides. 2013d. Production of live offspring from testicular tissue cryopreserved by vitrification procedures in Japanese quail (Coturnix japonica). Biol. Reprod. 88:124,1-6. doi:10.1095/biolreprod.113.108951.  Liu, J., M. C. Robertson, K. M. Cheng, and F. G. Silversides. 2013a. Chimeric plumage coloration produced by ovarian transplantation in chickens. Poult. Sci. 92:1073-1076.  Liu, J., Y. Song, K. M. Cheng, and F. G. Silversides. 2010. Production of donor-derived offspring from cryopreserved ovarian tissue in Japanese quail (Coturnix japonica). Biol. Reprod. 83:15-19. doi:10.1095/biolreprod.110.083733.  Long, J. A. 2006. Avian semen cryopreservation: what are the biological challenges? Poult. Sci. 85:232-236.  Lovelock, J. E., and M. W. Bishop. 1959. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183:1394-1395. doi:10.1038/1831394a0.  Martinez-Madrid, B., J. Donnez, A. Van Eyck, A. Veiga-Lopez, M. Dolmans, and A. Van Langendonckt. 2009. Chick embryo chorioallantoic membrane (CAM) model: a useful tool to study short-term transplantation of cryopreserved human ovarian tissue. Fertil. Steril. 91:285-292. doi:10.1016/j.fertnstert.2007.11.026.  Mather, F. B., and W. O. Wilson. 1964. Post-natal testicular development in Japanese quail (Coturnix coturnix japonica). Poult. Sci. 43:860-864.  Mazur, P. 1963. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. Gen. Physiol. 47:347-369. doi:10.1085/jgp.47.2.347.  Mazur, P. 1965. The role of cell membranes in the freezing of yeast and other single cells. Ann. N. Y. Acad. Sci. 125:658-676.  Mazur, P. 1970. Cryobiology: The freezing of biological systems. Science 168:939-949. doi:10.1126/science.168.3934.939.  Mazur, P. 2004. Principles of cryobiology. Pages 3-65 in Life in the Frozen State. B. J. Fuller, Lane, N., and Benson, E. E., eds. CRC Press LLC, Boca Raton.  153  Mazur, P., and C. Koshimoto. 2002. Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates? Biol. Reprod. 66:1485-1490.  Mazur, P., S. P. Leibo, and Seidel, George, E.,Jr. 2008. Cryopreservation of the germplasm of animals used in biological and medical research: importance, impact, status, and future directions. Biol. Reprod. 78:2-12. doi:10.1095/biolreprod.107.064113.  Mazur, P., and S. Seki. 2011. Survival of mouse oocytes after being cooled in a vitrification solution to-196 degrees C at 95 degrees to 70,000 degrees C/min and warmed at 610 degrees to 118,000 degrees C/min: A new paradigm for cryopreservation by vitrification. Cryobiology 62:1-7. doi:10.1016/j.cryobiol.2010.10.159.  Medzhitov, R., and J. Janeway Charles A. 2002. Decoding the patterns of self and nonself by the innate immune system. Science (New York, N.Y.) 296:298-300.  Meryman, H. T., R. J. Williams, and M. S. Douglas. 1977. Freezing injury from "solution effects" and its prevention by natural or artificial cryoprotection. Cryobiology 14:287-302. doi:10.1016/0011-2240(77)90177-8.  Milazzo, J. P., A. Travers, A. Bironneau, A. Safsaf, E. Gruel, C. Arnoult, B. Mac?, O. Boyer, and N. Rives. 2010. Rapid screening of cryopreservation protocols for murine prepubertal testicular tissue by histology and PCNA immunostaining. J. Androl. 31:617-630.  Mishima, O., and H. E. Stanley. 1998. The relationship between liquid, supercooled and glassy water. Nature 396:329-335. doi:10.1038/24540.  Modigliani, Y., A. Coutinho, P. Pereira, N. LeDouarin, V. ThomasVaslin, O. BurlenDefranoux, J. Salaun, and A. Bandeira. 1996. Establishment of tissue-specific tolerance is driven by regulatory T cells selected by thymic epithelium. Eur. J. Immunol. 26:1807-1815.  Mueller, D. L. 2010. Mechanisms maintaining peripheral tolerance. Nat. Immunol. 11:21-27. doi:10.1038/ni.1817.  Muldrew, K., J. P. Acker, J. A. W. Elliott, and L. E. McGann. 2004. The water to ice transition: implications for living cells. Pages 67-108 in Life in the Frozen State. B. J. Fuller, Lane, N., and Benson, E. E., eds. CRC Press LLC, Boca Raton.  Murphy, J. B. 1916. The effect of adult chicken organ grafts on the chick embryo. J. Exp. Med. 24:1-5. doi:10.1084/jem.24.1.1.  Murphy, S. P., P. M. Porrett, and L. A. Turka. 2011. Innate immunity in transplant tolerance and rejection. Immunol. Rev. 241:39-48. doi:10.1111/j.1600-065X.2011.01009.x.  154  Nakai, M., N. Kashiwazaki, J. Ito, N. Maedomari, M. Ozawa, M. Shino, J. Noguchi, H. Kaneko, and K. Kikuchi. 2011. Factors affecting fertilization and embryonic development during intracytoplasmic sperm injection in pigs. J. Reprod. Dev. 57:183-187. doi:10.1262/jrd.10-200E.  Nawroth, F., V. Isachenko, E. Isachenko, and G. Rahimi. 2007. Vitrification of ovarian tissue. Pages 261-272 in  Vitrification in Assisted Reproduction: A User?s Manual and Trouble-Shooting Guide   . M. J. Tucker, and Liebermann, J., eds. Informa UK Ltd., London.  Ohki, H., C. Martin, C. Corbel, M. Coltey, and N. M. Le Douarin. 1987. Tolerance induced by thymic epithelial grafts in birds. Science 237:1032-1035. doi:10.1126/science.3616623.  Ohta, H., Y. Sakaide, and T. Wakayama. 2008. The birth of mice from testicular spermatozoa retrieved from frozen testicular sections. Biol. Reprod. 78:807-811. doi:10.1095/biolreprod.107.065557.  Olszanska, B., and U. Stepinska. 2008. Molecular aspects of avian oogenesis and fertilisation. Int. J. Dev. Biol. 52:187-194. doi:10.1387/ijdb.072329ob.  Ottinger, M. A. 2010. Mechanisms of reproductive aging: conserved mechanisms and environmental factors. Ann. N. Y. Acad. Sci. 1204:73-81.  Owen, R. D. 1945. Immunogenetic consequences of vascular anastomoses between bovine twins. Science (New York, N.Y.) 102:400-401. doi:10.1126/science.102.2651.400.  Paris, M. C. J., and S. Schlatt. 2007. Ovarian and testicular tissue xenografting: its potential for germline preservation of companion animals, non-domestic and endangered species. Reprod. Fertil. Dev. 19:771-782. doi:10.1071/RD07038.  Parkes, A. S. 1964. Cryobiology. Cryobiology 1:3-3. doi:10.1016/0011-2240(64)90014-8.  Petitte, J. N. 2006. Avian germplasm preservation: embryonic stem cells or primordial germ cells? Poult. Sci. 85:237-242.  Phillips, P. H., and H. A. Lardy. 1940. A yolk-buffer pabulum for the preservation of bull semen. J. Dairy Sci. 23:399-404. doi:10.3168/jds.S0022-0302(40)95541-2.  Pierson, R., A. Dorling, D. Ayares, M. Rees, J. Seebach, J. Fishman, B. Hering, and D. Cooper. 2009. Current status of xenotransplantation and prospects for clinical application. Xenotransplantation 16:263-280. doi:10.1111/j.1399-3089.2009.00534.x.  155  Pierson, R., A. Dorling, D. Ayares, M. Rees, J. Seebach, J. Fishman, B. Hering, and D. Cooper. 2009. Current status of xenotransplantation and prospects for clinical application. Xenotransplantation 16:263-280. doi:10.1111/j.1399-3089.2009.00534.x.  Pilat, N., and T. Wekerle. 2010. Combining Treg therapy with mixed chimerism: getting the best of both worlds. Chimerism 1:26-29. doi:10.4161/chim.1.1.12964.  Pisenti, J. M., F. A. Bradley, K. M. Cheng, R. R. Dietert, J. B. Dodgson, A. M. Donoghue, A. B. Emsley, R. J. Etches, R. R. Frahm, R. J. Gerrits, P. F. Goetinck, M. E. Delany, A. A. Grunder, D. E. .Harry, S. J. Lamont, G. R. Martin, P. E. McGuire, G. P. Moberg, L. J. Pierro, C. O. Qualset, M. A. Qureshi, F. T. Shultz, R. L. Taylor, B. W. Wilson, U. K. Abbott, H. Abplanalp, J. A. Arthur, M. R. Bakst, C. Baxter-Jones, J. J. Bitgood, and Avian Genet Resources Task Force. 2001. Avian genetic resources at risk: An assessment and proposal for conservation of genetic stocks in the USA and Canada. Avian Biol. Res. 12:1-2.  Polge, C., A. U. Smith, and A. S. Parkes. 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666-666. doi:10.1038/164666a0.  Post, D. J., D. D. Douglas, and D. C. Mulligan. 2005. Immunosuppression in liver transplantation. Liver Transpl. 11:1307-1314. doi:10.1002/lt.20614.  Qin, S., S. P. Cobbold, H. Pope, J. Elliott, D. Kioussis, J. Davies, and H. Waldmann. 1993. "Infectious" transplantation tolerance. Science 259:974-977. doi:10.1126/science.8094901.  Qureshi, A. I., S. S. Nussey, G. Bano, P. Musonda, S. A. Whitehead, and H. D. Mason. 2008. Testosterone selectively increases primary follicles in ovarian cortex grafted onto embryonic chick membranes: relevance to polycystic ovaries. Reproduction 136:187-194. doi:10.1530/REP-07-0172.  Rall, W. F., and G. M. Fahy. 1985. Ice-free cryopreservation of mouse embryos at -196 ?C by vitrification. Nature 313:573-575. doi:10.1038/313573a0.  Rasmussen, D., and B. Luyet. 1970. Contribution to the establishment of the temperature-concentration curves of homogeneous nucleation in solutions of some cryoprotective agents. Biodynamica 11:33-44.  Rassoulzadegan, M., V. Grandjean, P. Gounon, S. Vincent, I. Gillot, and F. Cuzin. 2006. RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse. Nature 441:469-474. doi:10.1038/nature04674.  Ratcliffe, M. J. H. 2008. B cells, the bursa of Fabricius and the generation of antibody repertoires. Pages 67-89 in Avian Immunology. F. Davison, Kaspers, B., and Schat, K. A., eds. Elsevier Ltd., London.  156  Ribatti, D. 2008. Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis. Int. Rev. Cell Mol. Biol. 270:181-224.  Rodriguez-Sosa, J. R., and I. Dobrinski. 2009. Recent developments in testis tissue xenografting. Reproduction 138:187-194. doi:10.1530/REP-09-0012.  Rodriguez-Sosa, J. R., S. Schlatt, and I. Dobrinski. 2012. Testicular tissue transplantation for fertility preservation. Pages 331-343 in Fertility Preservation: Emerging Technologies and Clinical Applications. E. Seli, and Agarwal, A., eds. Springer Science+Business Media, LLC, New York.  Roe, M., N. McDonald, B. Durrant, and T. Jensen. 2013. Xenogeneic Transfer of Adult Quail (Coturnix coturnix) Spermatogonial Stem Cells to Embryonic Chicken (Gallus gallus) Hosts: A Model for Avian Conservation. Biol. Reprod. 88:129,1-7. doi:10.1095/biolreprod.112.105189.  Roger G Gosden. 2008. Ovary and uterus transplantation. Reproduction 136:671-680. doi:10.1530/REP-08-0099.  Romanoff, A. L. 1960. The avian embryo: structural and functional development. Macmillan, New York.  Rosen, H. R. 2008. Transplantation immunology: what the clinician needs to know for immunotherapy. Gastroenterology 134:1789-1801. doi:10.1053/j.gastro.2008.02.062.  Rothchild, I. 2003. The yolkless egg and the evolution of eutherian viviparity. Biol. Reprod. 68:337-357.  Rougier, N., and Z. Werb. 2001. Minireview: Parthenogenesis in mammals. Mol. Reprod. Dev. 59:468-474. doi:10.1002/mrd.1054.  Sachs, D. H., M. Sykes, T. Kawai, and A. B. Cosimi. 2011. Immuno-intervention for the induction of transplantation tolerance through mixed chimerism. Semin. Immunol. 23:165-173. doi:10.1016/j.smim.2011.07.001.  Sakai, A. 2004. Plant cryopreservation. Pages 329-345 in Life in the Frozen State. B. J. Fuller, Lane, N., and Benson, E. E., eds. CRC Press LLC, Boca Raton, Florida.  Sala?n, J., C. Corbel, and N. M. Le-Douarin. 2005. Regulatory T cells in the establishment and maintenance of self-tolerance: role of the thymic epithelium. Int. J. Dev. Biol. 49:137-142. doi:10.1387/ijdb.041959js.  Sala?n, J., N. Simmenauer, P. Belo, A. Coutinho, and N. M. Le Douarin. 2002. Grafts of supplementary thymuses injected with allogeneic pancreatic islets protect nonobese diabetic mice against diabetes. Proc. Natl. Acad. Sci. U. S. A. 99:874-877. doi:10.1073/pnas.012597499.  157  Samstein, B., and J. L. Piatt. 2001. Xenotransplantation and tolerance. Philosophical Transactions of the Royal Society of London.Series B: Biological Sciences 356:749-758. doi:10.1098/rstb.2001.0850.  Saragusty, J., and A. Arav. 2011. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 141:1-19. doi:10.1530/REP-10-0236.  Sarvella, P. 1973. Adult parthenogenetic chickens. Nature 243:171-171. doi:10.1038/243171a0.  Sato, T., K. Katagiri, A. Gohbara, K. Inoue, N. Ogonuki, A. Ogura, Y. Kubota, and T. Ogawa. 2011. In vitro production of functional sperm in cultured neonatal mouse testes. Nature 471:504-504. doi:10.1038/nature09850. Schijns, V. E. J. C., J. Sharma, and I. Tarpey. 2008. Practical aspects of poultry vaccination. Pages 373-394 in Avian Immunology. F. Davison, Kaspers, B., and Schat, K. A., eds. Elsevier Ltd., London.  Schlatt, S., A. Honaramooz, M. Boiani, H. R. Sch?ler, and I. Dobrinski. 2003. Progeny from sperm obtained after ectopic grafting of neonatal mouse testes. Biol. Reprod. 68:2331-2335. doi:10.1095/biolreprod.102.014894.  Seki, S., and P. Mazur. 2008. Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling. Cryobiology 56:171-180. doi:10.1016/j.cryobiol.2008.02.001.  Seki, S., and P. Mazur. 2009. The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure. Cryobiology 59:75-82. doi:10.1016/j.cryobiol.2009.04.012.  Sellix, M. T., and M. Menaker. 2010. Circadian clocks in the ovary. Trends Endocrinol. Metab. 21:628-636. doi:10.1016/j.tem.2010.06.002.  Shaw, J. M., and A. O. Trounson. 2002. Ovarian tissue transplantation and cryopreservation. Application to maintenance and recovery of transgenic and inbred mouse lines. Pages 229-251 in Methods in Molecular Biology. 2nd ed. A. R. Clarke, ed. Humana Press Inc., Totowa, NJ.  Shinohara, T., K. Kogishi, T. Honjo, A. Ogura, K. Inoue, N. Ogonuki, M. Kanatsu-Shinohara, H. Miki, K. Nakata, M. Kurome, H. Nagashima, and S. Toyokuni. 2002. Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination. Hum. Reprod. 17:3039-3045. doi:10.1093/humrep/17.12.3039.  Silber, S. J. 2012. Ovary cryopreservation and transplantation for fertility preservation. Mol. Hum. Reprod. 18:59-67. doi:10.1093/molehr/gar082.  158  Silversides, F. 2010. Laying performance of six pure lines of chickens and four commercial hybrids at the Agassiz Research Centre. Can. J. Anim. Sci. 90:341-347.  Silversides, F., and J. Liu. 2012. Novel techniques for preserving genetic diversity in poultry germplasm. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 7. doi:10.1079/PAVSNNR20127068.  Silversides, F. G., P. H. Purdy, and H. D. Blackburn. 2012. Comparative costs of programmes to conserve chicken genetic variation based on maintaining living populations or storing cryopreserved material. Br. Poult. Sci. 53:599-607.  Silversides, F. G., M. C. Robertson, and J. Liu. 2013a. Growth of subcutaneous testicular transplants. Poult. Sci. 92:1916-1920.  Silversides, F. G., M. C. Robertson, and J. Liu. 2013b. Cryoconservation of avian gonads in Canada. Poult. Sci. (in press).  Simonsen, M. 1955. Artificial production of immunological tolerance; induced tolerance to heterologous cells and induced susceptibility to virus. Nature 175:763-764. doi:10.1038/175763a0.  Simonsen, M. 2007. The impact on the developing embryo and newborn animal of adult homologous cells. APMIS 115:510-510. doi:10.1111/j.1600-0463.2007.apm_698a.x.  Siopes, T. D., and W. O. Wilson. 1975. The cloacal gland--an external indicator of testicular development in coturnix. Poult. Sci. 54:1225-1229.  Skalko, R. G., J. M. Kerrigan, J. R. Ruby, and R. F. Dyer. 1972. Intercellular bridges between oocytes in the chicken ovary. Zeitschrift f?r Zellforschung und Mikroskopische Anatomie 128:31-41. doi:10.1007/BF00306886.  Smyth, J. R. J. 1990. Genetics of plumage, skin and eye pigmentation in chickens. Pages 109-167 in Poultry Breeding and Genetics. R. D. Crawford, ed. Elsevier, New York.  Song, Y., K. M. Cheng, M. C. Robertson, and F. G. Silversides. 2012. Production of donor-derived offspring after ovarian transplantation between Muscovy (Cairina moschata) and Pekin (Anas platyrhynchos) ducks. Poult. Sci. 91:197-200. doi:10.3382/ps.2011-01672.  Song, Y., and F. G. Silversides. 2006. The technique of orthotopic ovarian transplantation in the chicken. Poult. Sci. 85:1104-1106.  Song, Y., and F. G. Silversides. 2007a. Production of offspring from cryopreserved chicken testicular tissue. Poult. Sci. 86:1390-1396.  159  Song, Y., and F. G. Silversides. 2007b. Offspring produced from orthotopic transplantation of chicken ovaries. Poult. Sci. 86:107-111.  Song, Y., and F. Silversides. 2007c. Heterotopic transplantation of testes in newly hatched chickens and subsequent production of offspring via intramagnal insemination. Biol. Reprod. 76:598-603. doi:10.1095/biolreprod.106.058032.  Song, Y., and F. G. Silversides. 2008a. Long-term production of donor-derived offspring from chicken ovarian transplants. Poult. Sci. 87:1818-1822. doi:10.3382/ps.2008-00103.  Song, Y., and F. G. Silversides. 2008b. Transplantation of ovaries in Japanese quail (Coturnix japonica). Anim. Reprod. Sci. 105:430-437. doi:10.1016/j.anireprosci.2007.12.024.  Tolson, K. P., and P. E. Chappell. 2012. The changes they are a-timed: metabolism, endogenous clocks, and the timing of puberty. Front. Endocrinol. 3:45,1-17.  Tselutin, K., F. Seigneurin, and E. Blesbois. 1999. Comparison of cryoprotectants and methods of cryopreservation of fowl spermatozoa. Poult. Sci. 78:586-590.  Vajta, G., P. Holm, M. Kuwayama, P. J. Booth, H. Jacobsen, T. Greve, and H. Callesen. 1998. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev. 51:53-58. doi:10.1002/(SICI)1098-2795(199809)51:1<53::AID-MRD6>3.0.CO;2-V.  Vanhove, T., D. Kuypers, K. J. Claes, P. Evenepoel, B. Meijers, M. Naesens, Y. Vanrenterghem, T. Cornelis, and B. Bammens. 2013 (in press).  Reasons for dose reduction of mycophenolate mofetil during the first year after renal transplantation and its impact on graft outcome. Transplant Int. doi:10.1111/tri.12133.  Wakayama, T., and R. Yanagimachi. 1998. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat. Biotechnol. 16:639-641. doi:10.1038/nbt0798-639.  Wakuri, H., and K. Mutoh. 1986. Histological observations on polyovular follicles in the domestic chicken, Gallus domesticus. Okajimas Folia Anat. Jpn. 62:323-329.  Waldmann, H. 2008. Tolerance can be infectious. Nat. Immunol. 9:1001-1003. doi:10.1038/ni0908-1001.  Waldmann, H., E. Adams, P. Fairchild, and S. Cobbold. 2006. Infectious tolerance and the long-term acceptance of transplanted tissue. Immunol. Rev. 212:301-313.  Wang, Y., Z. Xiao, L. Li, W. Fan, and S. Li. 2008. Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation. Hum. Reprod. 23:2256-2265. doi:10.1093/humrep/den255.  160  Weissman, A., L. Gotlieb, T. Colgan, A. Jurisicova, E. M. Greenblatt, and R. F. Casper. 1999. Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse. Biol. Reprod. 60:1462-1467. doi:10.1095/biolreprod60.6.1462.  Whittingham, D. G., S. P. Leibo, and P. Mazur. 1972. Survival of mouse embryos frozen to -196 ? and -269 ? C. Science 178:411-414. doi:10.1126/science.178.4059.411.  Willadsen, S., C. Polge, and L. E. Rowson. 1978. The viability of deep-frozen cow embryos. J. Reprod. Fertil. 52:391-393. doi:10.1530/jrf.0.0520391.  Wilmut, I. 1972. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci. 11:1071-1079.  Woelders, H., C. A. Zuidberg, and S. J. Hiemstra. 2006. Animal genetic resources conservation in the Netherlands and Europe: poultry perspective. Poult. Sci. 85:216-222.  Wood, K. J., and R. Goto. 2012. Mechanisms of rejection: current perspectives. Transplantation 93:1-10. doi:10.1097/TP.0b013e31823cab44.  Wood, K. J., and S. Sakaguchi. 2003. Regulatory T cells in transplantation tolerance. Nat. Rev. Immunol. 3:199-210. doi:10.1038/nri1027.  Woods, E. J., J. D. Benson, Y. Agca, and J. K. Critser. 2004. Fundamental cryobiology of reproductive cells and tissues. Cryobiology 48:146-156. doi:10.1016/j.cryobiol.2004.03.002.  Yavin, S., A. Aroyo, Z. Roth, and A. Arav. 2009. Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush. Hum. Reprod. 24:797-804. doi:10.1093/humrep/den397.  Yoshihara, C., A. Fukao, K. Ando, Y. Tashiro, S. Taniuchi, S. Takahashi, and S. Takeuchi. 2012. Elaborate color patterns of individual chicken feathers may be formed by the agouti signaling protein. Gen. Comp. Endocrinol. 175:495-499. doi:10.1016/j.ygcen.2011.12.009.  Yoshikawa, T., M. Sellix, P. Pezuk, and M. Menaker. 2009. Timing of the ovarian circadian clock is regulated by gonadotropins. Endocrinology 150:4338-4347. doi:10.1210/en.2008-1280.  Yoshimura, Y., and M. Nishikori. 2004. Identification of apoptotic oocytes in the developing ovary of embryonic and post-hatched chicks in Japanese quail (Coturnix japonica). J. Poult. Sci. 41:64-68. doi:10.2141/jpsa.41.64.  161  Yuan, J. H., J. S. Gao, Z. F. Zhan, H. W. Liu, W. J. Jin, and Z. D. Li. 2009. Development-promoting effect of chicken embryo membrane on chicken ovarian cortical pieces of different age. Poult. Sci. 88:2415-2421. doi:10.3382/ps.2008-00555.  Zerrahn, J., W. Held, and D. H. Raulet. 1997. The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell 88:627-636. doi:10.1016/S0092-8674(00)81905-4.  Zhao, Y., K. Swenson, J. J. Sergio, J. S. Arn, D. H. Sachs, and M. Sykes. 1996. Skin graft tolerance across a discordant xenogeneic barrier. Nat. Med. 2:1211-1216. doi:10.1038/nm1196-1211.   

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0165518/manifest

Comment

Related Items