Fundamental principles of cryobiology and application to ex situ conservation of avian species
Jianan Liua,b, Kimberly M. Chenga and Frederick G. Silversides b*
a
Avian Research Centre, Faculty of Land and Food Systems, University of British Columbia,
Vancouver, British Columbia, Canada, V6T 1Z4 b
Agassiz Research Center, Agriculture and Agri-Food Canada, Agassiz, British Columbia,
Canada, V0M 1A0 Email:
[email protected] Agassiz Research Center Contribution Number 812
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ABSTRAST
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Cryopreservation of animal germplasm is an important ex situ conservation strategy. Germplasm
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can be preserved at subzero temperatures and recovered in a way that allows germline
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development to be resumed at a later time. This strategy is routinely used for genetic
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improvement of dairy cattle, maintenance of lab strains of rodents and in human reproductive
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medication, and it contributes to genetic conservation of a growing number of wild mammalian
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species. Although there is an urgent need, cryoconservation of germplasm for birds is limited,
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partly because of skepticism with respect to the effectiveness of cryopreservation techniques. An
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understanding of fundamental principles of cryobiology and comparative cryobiological and
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physiological properties of germplasm is essential to the development and optimization of
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cryopreservation protocols. In mammals, slow freezing has been used to cryopreserve gametes
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and embryos but the effectiveness of slow freezing is specific to the cell type and it does not
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protect extracellular structures. Vitrification, which is a process of solidification without
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crystallization, can be used to effectively preserve multicellular structures such as gonadal tissue.
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In some domestic and wild avian species, the fertility produced by cryopreserved semen is
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sufficient for conservation but the w chromosome is lost. Avian eggs and embryos cannot be
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cryopreserved, but gonadal tissue can be cryopreserved and recovered by transplantation.
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Cryopreservation of germplasm allows ex situ conservation of avian species because it allows
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repopulation without the limitations of geographic isolation or reproductive longevity.
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Keywords: avian species, genetic diversity, conservation, cryobiology, cryopreservation,
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germplasm, germline cycle, sperm, gonadal tissue, transplantation
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1. INTRODUCTION
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There is a pressing need to develop effective ex situ conservation programs for avian species that
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are threatened by declining genetic diversity. Approximately 500 species out of the known 9672
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existing avian species are categorized as “vulnerable”, “endangered” or “critically endangered”
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(Gee et al., 2004; Blanco et al., 2009). Priority should be given to in situ conservation but when
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rare individual birds inevitably die, the genetic information that they carry is lost. At the same
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time, avian biology research and the poultry industry are experiencing a massive decline of
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genetic diversity carried by specialized experimental populations and breeding stocks that are
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invaluable to biological research and agriculture (Fulton and Delany, 2003; Silversides and Liu,
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2012). The decline is largely because the development and application of germplasm
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cryopreservation techniques for avian species have been very limited compared to mammalian
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species. The genetic variation represented by living populations of birds is lost with the
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termination of the research or breeding programs.
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Mazur et al. (2008) defined “germplasm” as cells that result in the development of
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offspring, singly or in combination, but principally referred to sperm and preimplantation
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embryos. The present review will expand the view of Mazur et al. (2008) to also include female
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gametes, primordial germ cells (PGCs), and gonadal tissue because genetic variation can be
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accessed at various points in the germline cycle (Figure 1) and successful preservation of any
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component of this cycle and subsequent functional recovery will lead to successful ex situ
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conservation. Although there is species-specificity, commonalities in germplasm cryobiology
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exist among taxa and an understanding of fundamental principles of cryobiology allows potential
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adaptations. The knowledge obtained from research in mammals can be used for conservation of
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wild avian species and sustainable maintenance and use of experimental and breeding domestic
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stocks.
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2. FUNDAMENTAL PRINCIPLES OF CRYOBIOLOGY
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Water is the most important component of a cellular system and the phase transition of water and
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its biological effects is the central topic of cryobiology. When temperature is lowered below the
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melting temperature (Tm) or equilibrium freezing point at a given pressure, freezing will not take
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place unless a nucleator with a critical size is present to trigger crystallization. Unfrozen water
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that is below the melting point is supercooled (Mishima and Stanley 1998), which is defined as
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the deviation of its temperature from Tm. The number of water molecules that is required to
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form a critically sized nucleator decreases with temperature (Mazur 2004), so when the
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temperature is sufficiently low, supercooled water freezes spontaneously. The temperature at
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this point is called the homogeneous nucleation temperature (Th ) (Mishima and Stanley 1998).
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In reality, homogeneous nucleation is a rare event and crystallization is usually initiated by
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minor perturbations (heterogeneous nucleation) at a point between Tm and Th . Water can also
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exist as a noncrystalline solid known as glass or glassy water at temperatures below the glass
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transition temperature (Tg; Figure 2). The arrangement of water molecules in glassy water is
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similar to that of liquid water but the molecules in glassy water are immobilized, and glassy
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water exhibits an amorphous solid rather than liquid state (Mishima and Stanley 1998).
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When cells are exposed to subzero temperatures, the cytoplasm will be supercooled to an
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extent even when ice formation takes place in the external medium. Mazur (1965, 1970)
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ascribed this phenomenon to the presence of cell membranes and lack of effective intracellular 4
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nucleators. At a given subzero temperature, the supercooled water has a higher vapour pressure
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than that of ice or water in a solution in equilibrium with ice, so cells respond to the
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disequilibrium across the membrane by losing water. The rate and extent of the resulting cellular
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dehydration depend on the cooling rate and the inherent characteristics of cells, including the
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permeability of their membrane to water, the activation energy and the surface-to-volume ratio.
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With assumptions for simplification, these relationships were described elegantly by mathematic
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methods in Mazur’s work (1963).
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The probability of nucleation in a solution increases as the amount of supercooling
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increases (Muldrew et al., 2004). If the cooling rate is greater than a critical value, cells cannot
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lose sufficient supercooled water across their membranes and will complete their equilibration by
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intracellular ice formation (IIF), which is associated with cell death (Mazur 1970). Therefore,
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theoretically, IIF and subsequent cell death can be circumvented by cooling cells at a sufficiently
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low rate. However, plots of survival versus cooling rate of various types of cells commonly take
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the shape of an inverted “U” (Mazur 1970), with very slow cooling also causing cell death. This
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means that another class of events with respect to very slow cooling rate may be harmful to cells.
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These are collectively called “solution effects” and are associated with long exposure of cells to
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concentrated intracellular and extracellular components as a result of cell dehydration and
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extracellular ice formation. Various hypotheses are proposed to elucidate their mechanisms
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(Muldrew et al., 2004) but none of these has been rigorously proven or refuted.
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3. CRYOPRESERVATION STRATEGIES IN GENERAL
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Effective cryopreservation protocols minimize cryoinjuries associated with cellular response to
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subzero temperatures. According to a two-factor hypothesis, IIF is responsible for injury when
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cooling is faster than optimum in terms of survival while solution effects are responsible for
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injury when cooling is slower than optimum, which is the foundation of the slow equilibrium
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freezing strategy (Mazur et al., 2008). Several studies (Acker and McGann 2003, Mazur 2004)
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showed that small intracellular ice crystals might be innocuous, but the growth of these crystals
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can have lethal effects, which is an important consideration for warming procedures (see below).
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Slow (equilibrium) freezing minimizes IIF with cooling rates that are slow enough to yield
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sufficient osmotic dehydration to keep the chemical potential of intracellular water and the partly
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frozen extracellular medium near to equilibrium. By the end of cooling processes, the highly
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concentrated intracellular solution will form glass while the extracellular solution is frozen
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(Mazur 1970). Solution effects are diminished by cryoprotective agents (CPA), which have
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cryoprotective properties.
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CPAs are categorized into penetrating (permeating) and nonpenetrating (nonpermeating)
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groups (Muldrew et al., 2004). Penetrating CPAs are nonionic molecules with low molecular
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weight that have a high solubility in water at low temperatures and can enter and equilibrate in
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the cytoplasm. The freezing point of the intracellular solution is depressed by the presence of
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these CPAs as solutes (freezing point depression), which reduces the extent of supercooling and
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thus the probability of freezing internally (Mazur 1963). In addition, according to colligative
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theory, they reduce the solution effects by lowering the concentration of damaging solutes such
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as electrolytes (Meryman et al., 1977). Non-colligative mechanisms of penetrating CPAs such
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as stabilizing cellular structures have also been proposed (Crowe et al., 1990). Glycerol (Polge
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et al., 1949) and dimethyl sulphoxide (DMSO, Lovelock and Bishop 1959) are examples of 6
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widely used penetrating CPAs. Nonpenetrating CPAs include sugars and macromolecules that
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are soluble in water and have limited ability to cross cell membranes. Their large osmotic
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coefficients indicate that they can facilitate cell dehydration at a low concentration, which
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reduces the extent of supercooling of the cytoplasm and the chance of freezing (Muldrew et al.,
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2004). Mechanisms of their protection against solution effects remain to be elucidated.
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Slow freezing has been successful in preserving specific cell types but has not been
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effective for multicellular systems such as cell aggregates, tissue and organs. Cells can be very
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different in their permeability to water, activation energy and surface-to-volume ratio, which in
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turn can lead to a possible 1000-fold difference in the optimal cooling rate (Mazur 1970).
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Therefore, a cooling rate that produces high survival of one type of cell may not guarantee high
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survival of the others in a multicellular system. In addition, with slow freezing, the formation of
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ice in the extracellular or intercellular area is detrimental. If extracellular freezing is eliminated,
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the consequent stresses such as the probability of IIF and solution effects can be avoided, which
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can be achieved by vitrification procedures.
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Fahy et al. (1984) defined vitrification as the process of solidification of a liquid by
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extreme enhancement of viscosity instead of crystallization with the resultant amorphous solid
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being called glass. Theoretically, this process can be achieved by ultra-rapid cooling of
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biological materials subjected to high concentrations of glass-promoting CPAs. The outcome is
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that both intracellular and extracellular components convert to glass in a very short time, which
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can be explained by the phase diagram in Figure 2. The intersection between the homogeneous
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nucleation curve (Th ) and the glass transition curve (Tg) gives a critical concentration of a
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solution, above which it is possible to cool the solution directly to the glass transition
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temperature without freezing (Rasmussen and Luyet 1970). In addition, a higher cooling rate 7
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lowers the required concentration of solute (Fahy et al., 1987). Current technologies are not able
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to verify whether true vitrification of cellular systems is obtained; Seki and Mazur (2009)
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suggested using the term “vitrification procedure” to refer to cryopreservation procedures that
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approach vitrification by using an ultra-high cooling rate and highly concentrated CPAs.
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Cryopreserved biological materials must survive warming procedures before they can be
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used. Warming rate is of critical importance (Seki and Mazur 2008). As the temperature rises
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above Tg, glass will convert to a highly viscous liquid (Mishima and Stanley 1998), which has a
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tendency to form crystalline ice through devitrification. At an appropriate higher temperature,
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small ice crystals with high surface energies will enlarge when sufficient time is given, in a
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process known as recrystallization. The large crystals themselves or their formation process may
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lead to cell death (Mazur 2004). If cells are preserved by slow freezing, the effects of warming
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rate on cell survival are very complex and depend on cell type, CPAs and cooling rate. In some
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cases, the warming rate may have no effect, whereas rapid or slow warming is favoured in other
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cases (summarized by Mazur 2004). For materials preserved by vitrification procedures, rapid
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warming is essential because the growth of small ice crystals that may have formed during the
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cooling process or during devitrification and subsequent recrystallization can be prevented by
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sufficiently high warming rate (Seki and Mazur 2008). The other critical factor for warming is
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osmotic stress associated with the removal of penetrating CPAs, which needs to be minimized.
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The concentration of penetrating CPAs in the extracellular solution is lowered progressively
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using stepwise dilutions, which permits cells to resume equilibrium gradually. As an alternative,
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a nonpenetrating CPA such as sucrose can be used alone in the warming solution to facilitate the
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efflux of penetrating CPA and reduce excessive water influx (Muldrew et al., 2004).
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4. CRYOBIOLOGY OF ANIMAL GERMPLASM
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An important application of cryobiology is long-term preservation of animal and human
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germplasm. Investigations into preserving animal germplasm at subzero temperatures started as
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early as the mid-19th century, when the mechanism of fertilization began to be appreciated
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(Leibo 2004). Progress has been made since then but has largely been based on empirical
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approaches. Revolutionary events such as the introduction of glycerol (Polge et al., 1949) and
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DMSO (Lovelock and Bishop 1959) as effective penetrating CPAs, and especially, the
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publication of Mazur’s physical-chemical models (1963, 1965) led to significant advances in
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fundamental cryobiology and its application in preserving animal germplasm. An example is the
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successful cryopreservation of murine and bovine embryos (Mazur et al., 2008). However, using
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mathematical approaches to design protocols to preserve germplasm appears to be challenging,
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although the physical-chemical models can predict the behaviour of various somatic cell types
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and a limited number of oocytes and embryos during the cooling procedures with reasonable
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degrees of accuracy. This can be explained by the biological characteristics of germ cells.
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For many vertebrates, the precursors of germ cells and those of somatic cell types
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separate at an early stage of development. Compared to most somatic cells, of which only a
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specific portion of their genome is expressed at specific stages in an individual’s life, germ cells
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are designed to pass the entire complement of materials and instructive information needed for
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development and differentiation to the next generation. During gametogenesis, germ cells
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complete or partially complete meiosis and they obtain very specialized cellular and subcellular
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features that are essential for fertilization and initiation of subsequent events. This can cause a
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great deviation of their cytoplasm from an ideal dilute solution and subsequent violations of the
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assumptions required by physical-chemical modeling (Mazur 1970). In addition, high viability 9
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and cellular integrity resulting from a low level of lethal injuries may not guarantee functional
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recovery after cryopreservation. Sublethal injuries that alter structures and components of germ
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cells can lead to unpredictable effects on their functions. An example is high motility but low
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fertilization rates that are often observed in cryopreserved swine spermatozoa (Mazur et al.,
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2008). Another feature of germ cells and embryos is that they can be extensively damaged by
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exposure to temperatures approaching 0˚C. This type of damage is known as chilling injury
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(Mazur et al., 2008) and is different from the “two factors” previously described, although its
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physiological nature remains ill defined.
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4. 1. CRYOPRESERVATION OF MALE GERMPLASM
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Semen cryopreservation is the most commonly used method of preserving male mammalian
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germplasm and has been integrated as an Assisted Reproductive Technology (ART) in human
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reproductive medicine. It also contributes to genetic improvement in cattle breeding and
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maintenance of mouse strains bearing specific genotypes.
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Basic structures of spermatozoa are highly conserved among species, including a haploid
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nucleus, a propulsion system and an acrosome. The nucleus contains the genetic materials that
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need to be conveyed to the female gamete for fertilization while the propulsion system and
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acrosome enable the nucleus to move to and enter the female gamete. The purpose of
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cryopreservation is to ensure functional resumption of these components after warming to
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achieve normal fertilization. The specialized structures of spermatozoa are obtained during
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spermatogenesis, in which most of the cytoplasm is eliminated and the nucleus is condensed,
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leading to a high surface-to-volume ratio and low water content. According to Mazur’s model 10
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(1963), spermatozoa can be cryopreserved at a relatively high cooling rate without IIF. The
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threshold for mouse spermatozoa was estimated to be greater than 250˚C/min by experimental
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inference (Mazur and Koshimoto 2002), compared to less than 1˚C/min for mouse ova (Leibo et
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al., 1978). In addition, chilling injury has been reported in various species. There is general
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agreement that unejaculated spermatozoa are more resistant to chilling injury than ejaculated
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spermatozoa (Leibo 2004).
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To date, slow-freezing, vitrification and freeze-drying are strategies that are available for
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semen cryopreservation, of which slow-freezing is the most broadly practiced. Briefly,
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spermatozoa suspended in a diluent containing CPA(s) are cooled at a controlled rate until the
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spermatozoa can be stored in liquid nitrogen. The procedures used today are very similar to
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those developed around 60 years ago and the history of adding egg yolk to diluents (Phillips and
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Lardy 1940) is even longer. The procedures may not be optimal, but optimization is rarely
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practical because there are so many variables, and high variation exists among species and
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individuals in response to slow-freezing procedures (Mazur et al., 2008). Slow freezing has been
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used to preserve human, murine and bovine spermatozoa with satisfactory efficiency, and good
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fertility has recently been reported from frozen/thawed boar semen (Didion et al., 2013). The
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alternative, vitrification procedures, provide a potential solution because the sensitive
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temperature zone can be bypassed by using an ultra-rapid cooling rate, but success is species
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specific. In addition, penetrating CPAs that have toxic and osmotic effects can be excluded
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because the cytoplasm of spermatozoa is very condensed. Vitrification procedures using only
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nonpenetrating CPAs have been successfully used to preserve human spermatozoa and these
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spermatozoa have produced healthy babies (Isachenko et al., 2012). A third strategy for long-
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term preservation of mammalian spermatozoa is to use freeze drying (Wakayama et al., 1998) or 11
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evaporative drying (Li et al., 2007), in which the spermatozoa are killed and fertilization is
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achieved by intracytoplasmic sperm injection (ICSI) subsequent to rehydration of the dead
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sperm. This may be a promising strategy for preservation of male germplasm of species for
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which ICSI is available.
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The prerequisite for semen preservation is the presence of spermatozoa, which is a
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limitation for individuals before maturation whose fertility needs to be preserved, such as young
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patients who suffer sterility as a result of cancer treatments. The solution is to preserve their
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testicular tissue before the onset of the treatments, which can be recovered and allowed to
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resume maturation in vivo or in vitro at a later time. This also allows the germline of a valuable
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domestic or wild animal be preserved regardless of its developmental stage. Slow freezing is
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conventionally used, following basic procedures similar to those of semen cryopreservation, and
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live offspring have been obtained from cryopreserved testicular tissue of various mammalian
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species including chickens (Ehmcke and Schlatt 2008).
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From a cryobiological perspective, vitrification procedures might be better for
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cryopreserving testicular tissue than slow freezing because resumption of spermatogenesis and
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steroidogenesis of testicular tissue depends on at least three types of cells, including germ cells
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which bear the potential of fertility and are in different stages of maturation; Sertoli cells which
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support the germ cells physically and biochemically; and Leydig cells, which are the major
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source of male hormones. Variables of slow-freezing procedures, such as the cooling rate, are
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cell-specific so the optimal protocol for one cell type will not be optimal for other cell types and
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their different developmental stages. More importantly, these cells must be organized properly
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by their extracellular matrix in and around testicular tubules which are vulnerable to damage by
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ice crystals induced by slow freezing (Woods et al., 2004). Investigation of the use of 12
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vitrification to preserve testicular tissue has shown promising results in humans (Curaba et al.,
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2011a) and various animal models (Abrishami et al., 2010, Curaba et al., 2011b).
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4. 2. CRYOPRESERVATION OF FEMALE GERMPLASM
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For most mammalian and avian species, mitotic proliferation of oogonia ceases by the time
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around birth (Rothchild 2003). A small portion of these oogonia enter the first meiotic division
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and are called primary oocytes and their meiosis is arrested at Prophase I. Primary oocytes, the
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surrounding single layer of epithelial granulosa cells and a layer of basement membrane form
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primordial follicles. With the onset of sexual maturity, groups of follicles periodically enter
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folliculogenesis, which includes maturation of oocytes (oogenesis) and proliferation and
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maturation of the surrounding granulosa cells. During oogenesis, meiosis is resumed by oocytes
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and is arrested again at Metaphase II until fertilization.
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In most mammalian species, the ovulated ovum is enclosed in a glycoprotein membrane
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named the zona pellucida, which is surrounded by cumulus cells derived from the innermost
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granulosa cells of the follicle. During fertilization, the spermatozoon binds to the zona pellucida
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and initiates the acrosomal reaction which releases enzymes that facilitate the penetration of a
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spermatozoon. Subsequent binding of a spermatozoon to the plasma membrane of the ovum
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enables the entry of the sperm nucleus. The ovum is then activated to trigger modification of the
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zona pellucida (zona hardening) to prevent polyspermy and the pronuclei are formed (Bedford
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2004). These events make embryos more resistant to the negative effects of cryopreservation
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than oocytes, although the mechanisms remain to be clarified (Fuller et al., 2004).
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The first success in cryopreservation of embryos was achieved by Whittingham et al.
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(1972) using a slow-freezing protocol following the principles addressed by Mazur’s model.
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Success was also achieved by Wilmut (1972) independently around the same time. A modified
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protocol was used by Willadsen et al. (1978) for ruminants, and this has since been refined and
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widely used. Using a similar protocol, Chen (1986) first reported pregnancy from cryopreserved
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human oocytes. Generally, embryos and oocytes are equilibrated with CPAs and cooled at a
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controlled rate to a seeding temperature where extracellular ice nucleation is induced. The
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temperature is then lowered slowly until the samples can be stored in liquid nitrogen. The
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cooling rate for embryos and oocytes is very low compared to that used for semen (less than
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0.5˚C/min compared to 10 to 100˚C/min commonly used for spermatozoa). This is because the
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surface-to-volume ratio and the permeability of oocytes and embryos are very low compared to
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spermatozoa, leaving them very susceptible to IIF. Leibo et al. (1978) estimated the upper
280
threshold of cooling rate without IIF to be 1˚C/min. However, a slow cooling rate can still cause
281
cryoinjury to oocytes and embryos because both are very sensitive to chilling injury (Saragusty
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and Arav 2011). Disruption of the meiotic spindle and zona hardening caused by cooling
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procedures increase the challenges for preservation of oocytes (Mazur et al., 2008).
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Attention has been devoted to vitrification procedures since their first successful
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application to preserving embryos (Rall and Fahy 1985). Vitrification solves problems caused
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by high chilling sensitivity and high intracellular ice nucleating temperature exhibited by oocytes
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and embryos. It also ameliorates adverse effects of extracellular ice and long exposure to
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concentrated intracellular and extracellular solutions. There is consensus regarding methods of
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reducing the toxicity of high concentrations of CPAs. The first is to combine both penetrating
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and nonpenetrating CPAs, which reduces overall toxicity and promotes both internal and 14
291
external glass transition. Ethylene glycol (EG) is the most widely used penetrating CPA, and is
292
sometimes used in combination with other penetrating CPAs such as DMSO and 1, 2-
293
propanediol (PROH). Sugars such as sucrose are used in many studies as nonpenetrating CPAs
294
and different types of sera are common additives (Chen and Yang 2007). The second aspect is
295
stepwise equilibration of CPAs whereby oocytes or embryos are first equilibrated with a low
296
concentration of penetrating CPAs and then briefly with nonpenetrating CPAs and more
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concentrated penetrating CPAs. The third aspect is to maximize the cooling rate so that the
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concentrations of CPAs can be lowered accordingly (Fahy et al., 1987). Many specialized
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devices have been developed (Saragusty and Arav 2011), some of which allow direct contact of
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liquid nitrogen to oocytes or embryos and others (mainly straws) are modified to reduce
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insulation. Note that the advantage shown by these devices is not entirely due to the high
302
cooling rate that they allow, but that they also make it possible to achieve a very high warming
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rate, which may be more important than the cooling rate for recovery of oocytes or embryos
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preserved in this manner (Seki and Mazur 2009).
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An important observation of oocyte cryopreservation is that immature oocytes survive
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cryopreservation procedures better than mature oocytes (Woods et al., 2004). However, the
307
conditions for achieving subsequent in vitro maturation of the surviving immature oocytes can be
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very demanding. In this regard, cryopreservation of ovarian tissue enables preservation of the
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abundant primordial follicles enclosed in the tissue and their recovery in their natural micro
310
environment. For mammals including humans, ovarian cryopreservation provides an option
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when normal ovulation cannot be achieved due to physiological or pathological reasons. For
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many non-mammalian vertebrates, cryopreservation of ovarian tissue could be the only effective
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way of preserving female germplasm because their oogenesis involves deposition of large 15
314
amounts of yolk into the ooplasm, resulting in a large egg with a very low permeability to water
315
and CPAs and thus a very high intracellular nucleating temperature, making it technically
316
impossible to prevent intracellular freezing and chilling injury at the same time.
317
Slow-freezing procedures for ovarian tissue were first developed in the 1990’s and used
318
in various animal models following the principles for preserving embryos. In humans, the
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original purpose of preserving fertility of young patients subjected to gonadotoxic treatments has
320
been fulfilled. Silber (2012) described the birth of 28 healthy babies after transplantation of
321
fresh or frozen-thawed ovarian tissue. By contrast, live births reported from cryopreserved
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oocytes are very limited. Vitrification procedures have been adopted by different groups to
323
preserve human ovarian tissue (Silber 2012), although the efficiency has been questioned by
324
some (Isachenko et al., 2007). Strategies such as combining penetrating and nonpenetrating
325
CPAs and multi-step equilibration have been adopted by most researchers and special methods
326
have been developed to facilitate rapid cooling and warming (Chen et al., 2006, Wang et al.,
327
2008). It is foreseeable that vitrification procedures will be widely used because of their
328
feasibility and simplicity.
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5. CRYOPRESERVATION OF AVIAN GERMPLASM
331 332
5. 1. CRYOPRESERVATION OF MALE GERMPLASM IN AVIAN SPECIES
333
Avian testes are located in the body cavity, attached to the body wall, ventral to the cephalic part
334
of the kidneys (Johnson 1986a). There is no pampiniform plexus, which is important for
16
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thermoregulation in mammals, indicating that avian testicular tissue can function at a core body
336
temperature of around 40˚C. The duct system includes seminiferous tubules, rete tubules, vasa
337
efferentia, epididymes and the vasa deferentia, where semen is stored in most avian species. In
338
many passerines, coils of terminal vasa defernetia form cloacal protuberance during the breeding
339
season (Gee et al., 2004). Birds have no organs comparable to mammalian accessory
340
reproductive glands such as the prostate gland, the bulbourethral gland and the seminal vesicle;
341
seminal plasma in birds is from seminifierous tubules and vasa efferentia (Johnson 1986a).
342
Therefore avian semen shows very different physiological and biochemical properties than
343
mammalian semen (Blesbois 2011, Long 2006).
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In general, an avian spermatozoon consists of a straight or slightly curved head
345
resembling a long, slender cylinder and a long tail (Romanoff 1960). The proximal end of the
346
head is covered by an acrosome, the shape of which varies among species (Gee et al., 2004).
347
Avian spermatozoa can be broadly categorized into the simple sauropsid form and the complex
348
helical form, with the exception of American kestrel, in which the spermatozoa are round to
349
slightly flattened (Gee et al., 2004). All of these are distinguished from mammalian spermatozoa
350
(Figure 3). The simple sauropsid form is common in non-passerine species, in which the middle
351
piece of the tail is relatively long and sometimes spiral (Romanoff 1960). The complex helical
352
form is characterized in passerine birds with a predominantly spiral configuration in each portion
353
of the cell, which usually has a ribbon-like structure that covers the acrosome or the entire cell
354
(Gee et al., 2004).
355
Fowl spermatozoa are the first biological material that was successfully cryopreserved
356
(Polge 1951). Optimization of cryopreservation in terms of CPA type, cooling rate and
357
packaging for storage using slow-freezing procedures was made primarily based on empirical 17
358
approaches. Two protocols have been adopted for gene banking of local chicken breeds in
359
Europe; one (Blesbois 2007) uses glycerol as the CPA and a relatively slow cooling rate
360
(7˚C/min) and the other (Woelders et al., 2006) uses dimethyl acetamide (DMA) as the CPA and
361
a relatively high cooling rate (around 200˚ C /min). In addition, live chicks have been produced
362
from cryopreserved semen of a number of nondomestic avian species (Gee et al., 2004) and
363
cryopreserved avian semen have successfully contributed to species recovery, such as cranes and
364
Golden eagles (Blanco et al., 2009).
365
Avian spermatozoa might be better preserved by vitrification procedures than slow
366
freezing. Fragile structures such as long tails and spiral configurations can be preserved by
367
minimizing extracellular ice formation, which is important for avian species because in vitro
368
fertilization is not practical and structural integrity is crucial for survival of spermatozoa in the
369
female reproductive tract and fertilization. Vitrification procedures also arrest solution effects by
370
ultra-rapid cooling, eliminating the need for penetrating CPAs that have contraceptive (glycerol)
371
or toxic effects (DMA). The benefits of eliminating penetrating CPAs have been demonstrated
372
for mouse semen (Koshimoto et al., 2000) and penetrating CPAs have been eliminated
373
successfully in cryopreservation of human semen (Isachenko et al., 2012). The spermatozoa of
374
some avian species exhibit relatively high osmotic tolerance (Comizzoli et al., 2012), making it
375
possible to use nonpenetrating CPAs with a relatively high concentration to facilitate glass
376
transition. In addition, the nucleus of an avian spermatozoon is highly condensed and the
377
intracellular water content is very low compared to other vertebrate cells (Blesbois 2011) so that
378
the intracellular components naturally favour glass transition if the cooling rate is rapid enough,
379
as was demonstrated for fowl semen pellets preserved by direct immersion in liquid nitrogen
380
(Tselutin et al., 1999). 18
381
An alternative to semen cryopreservation is to preserve testicular tissue, which also
382
provides an option for some wild species in which semen quality is too low to be used for
383
cryopreservation and subsequent artificial insemination because of a short reproductive season
384
and the stress related to collection procedures (Blanco et al., 2009). Furthermore, considering
385
that spermatogonia are present in the testicular tissue regardless of the male’s age,
386
cryopreservation of testicular tissue should make it possible to recover the germline of a valuable
387
male bird even after an unexpected death. Successful cryopreservation and recovery of testicular
388
tissue has been demonstrated in chickens (Song and Silversides 2007a) using slow-freezing
389
procedures. Liu et al. (2012) have recently developed a practical vitrification protocol for
390
Japanese quail.
391 392
5. 2. CRYOPRESERVATION OF FEMALE GERMPLASM IN AVIAN SPECIES
393
In most avian species, only the left ovary and oviduct are functional (Golden and Arbona 2012,
394
Johnson 1986b). The ovary is located at the cephalic end of the kidney and consists of an outer
395
cortex and an inner medulla. In mature female birds, the functional ovarian cortex possesses
396
numerous follicles arranged in a hierarchical manner. The maturation of the avian oocyte is
397
characteristic of hormone-controlled yolk deposition or vitellogenesis. Precursors of yolk
398
contents are synthesized in the liver and are transported to the follicles. The cell layers
399
surrounding the oocyte and yolk include the oocyte plasma membrane, perivitelline membrane,
400
granulosa cells, basal lamina, theca interna and theca externa. Each follicle is connected with the
401
ovary through a long stalk upon maturation, which is different from that of mammals. During
19
402
ovulation, the ovum containing a large amount of yolk is expelled from the follicle through the
403
less vasularized stigma of the follicle.
404
The ovulated ovum is engulfed by the infundibulum of the oviduct, which is the location
405
of fertilization. As the fertilized ovum enters different sections of the oviduct, layers of albumin
406
and eggshell are deposited around it. Embryonic development starts while the egg is still in the
407
oviduct and by the time of oviposition or egg-laying, the embryo is in a disk shape residing on
408
the surface of the yolk. The embryo at this stage is called the blastoderm and contains around
409
50,000 to 60,000 cells in chicken (Petitte 2006). At a later stage of embryonic development,
410
PGCs originating from an extraembryonic region named the germinal crescent move to and
411
colonize the precursor of the gonads, where the germ cells mature and start the germline cycle
412
again through fertilization. The complex structure of ovulated avian ova and embryos prevents
413
the application of cryopreservation procedures and female germplasm can only be preserved in
414
the upstream forms of the germline cycle, namely, PGCs and ovarian tissue (Figure 1).
415
Circulating and newly colonized PGCs can be collected at early stages of donor
416
embryonic development and cryopreserved (see Petitte 2006, Silversides and Liu 2012 for
417
reviews). Germ line chimeras can then be produced by transplantation of cryopreserved donor
418
PGCs into recipient embryos at an appropriate developmental stage. The recipient embryos
419
(chimeras) then need to hatch and mature and can be mated inter se to produce offspring that
420
represent the donor line. To date, rigorous verification of cryopreservation efficiency in
421
preserving avian PGCs is scarce, probably because the procedures involved are complex and
422
require significant resources and training to carry out. All examples of reconstitution including
423
use of both fresh and cryopreserved PGCs demonstrate very low efficiency, and thousands of
424
prepared PGCs and hundreds of manipulated recipient embryos have produced only a handful of 20
425
donor-derived chicks (Silversides and Liu 2012). The FAO (2011) suggests an effective
426
population size of 50 for regeneration of a population, so although PGCs may be valuable for
427
research on genetic manipulation, they are not suitable for cryobanking of avian germplasm.
428
Cryopreservation of immature oocytes can be achieved by preserving ovarian tissue,
429
which can be recovered by orthothopic transplantation (Song and Silversides 2006, 2007b). The
430
efficiency of this strategy was demonstrated by Liu et al. (2010), who cryopreserved ovarian
431
tissue from immature female Japanese quail using slow-freezing and vitrification procedures and
432
recovered it by orthotopic transplantation with successful production of donor-derived offspring.
433
The vitrification protocol used in this study was more efficient than the slow-freezing protocol in
434
in vitro and in vivo tests. The simple protocol adapted from mammalian studies (Chen et al.,
435
2006, Wang et al., 2008) has been successfully used in various species including mice, felids,
436
ungulates (Comizzoli et al., 2012), Japanese quail and chickens (unpublished data). With
437
refinement (Liu et al., 2012), it is now used for cryobanking of avian gonadal tissue by Canadian
438
and U.S. government animal genetic resources programs.
439 440
6. TRANSPLANTATION OF AVIAN GONADAL TISSUE
441
In mammals, functional recovery of cryopreserved gonadal tissue can be achieved using in vitro
442
techniques such as in vitro maturation and in vitro insemination. Application of these techniques
443
in avian species is very limited. Paris and Schlatt (2007) suggested that gonadal transplantation
444
in mammals can be used as a special form of tissue culture and it is currently the only approach
445
for resumption of gametogenesis for cryopreserved avian reproductive tissue.
21
446
The characteristic, hormone-controlled vitellogenesis in avian folliculogenesis and the in
447
vivo process of producing hard-shelled eggs in the avian oviduct cannot be reproduced in vitro,
448
and the ovarian tissue of avian species must be transplanted into the normal anatomical location,
449
which is known as orthotropic transplantation. Orthotopic transplantation of chicken ovarian
450
tissue was attempted several times in the past without success (Davenport 1911, Guthrie 1908,
451
Grossman and Siegel 1966). Song and Silversides (2006) developed a transplantation technique
452
in which in one-day old chicks were ovariectomized through a transverse cut on the abdominal
453
cavity and the donor ovarian tissue was replaced in the original location of recipient ovary.
454
Mycophenolate mofetil was given to post-surgical birds to prevent tissue rejection. This
455
technique has been successfully used to produce live offspring from fresh ovarian transplants in
456
chickens (Song and Silversides 2007b), Japanese quail (Song and Silversides 2008a) and ducks
457
(Song et al., 2012) and from cryopreserved ovarian transplants in Japanese quail (Liu et al.,
458
2010). We have recently improved this technique by making a smaller cut parallel to the median
459
plane on the left side of the abdominal cavity of the recipients to expose the ovary without
460
externalizing the gizzard and intestines (Liu et al., 2013). In addition, Karagenç et al. (2011)
461
demonstrated that survival of two-day old recipients is better than one-day old recipients and we
462
now conduct the transplantation in older chicks which significantly reduces mortality during
463
surgery and simplifies the technique by eliminating the need to remove the yolk sac
464
(unpublished data).
465
In mammals, immunodeficient rodents can be used as recipients for allogeneic and
466
xenogeneic gonadal transplantation to prevent tissue rejection (Bols et al., 2010, Ehmcke and
467
Schlatt 2008). Immunodeficient birds are not available so an immunosuppressive drug,
468
mycophenolate mofetil, is used in allogeneic and xenogeneic ovarian transplantation (Song and 22
469
Silversides 2007b, 2008a; Liu et al., 2010; Song et al., 2012) and allogeneic testicular
470
transplantation (Song and Silversides 2007a, c). In addition, a short-term immunosuppressive
471
regimen allows long-term tolerance of allogeneic ovarian transplants in chickens (Song and
472
Silversides, 2008b).
473
The connection between testes and vasa deferentia cannot be surgically reconstructed so
474
transplantation of avian testicular tissue is achieved by heterotopic procedures, in which the
475
transplantation site is different from the normal anatomical site of the testes. In chickens, fresh
476
testicular tissue that has been transplanted under the dorsal skin or in the abdominal cavity of
477
recipients showed comparable efficiency in producing donor-derived offspring (Song and
478
Silversides 2007c). Recent experimentation in chickens and Japanese quail (unpublished data)
479
suggests that survival of testicular tissue implanted subcutaneously is very high. Castration of
480
recipients is essential for successful spermatogenesis of chicken testicular transplants (Song and
481
Silversides 2007a, c). A similar tendency is observed in some mammalian models and is
482
probably due to increased gonadotropin secretion caused by removal of negative feedback from
483
recipient testicular tissue (Schlatt et al., 2003). However, the requirement of complete castration
484
of recipients might not be as strict if donors and recipients are of different species (Paris and
485
Schlatt 2007, Shinohara et al., 2002) because the signal transduction may be species-specific at
486
certain levels. Fertilization using testicular spermatozoa in mammals requires in vitro
487
insemination such as intracytoplasmic sperm injection (Nakai et al., 2010). However, this is not
488
necessary for at least some avian species because intramagnal insemination of suspensions from
489
testicular transplants that are fresh or frozen-thawed can produce live offspring in chickens
490
(Song and Silversides 2007 a, c).
491 23
492 493
7. CRYOCONSERVATION OF AVIAN GERMPLASM: FUTURE DIRECTIONS A successful cryoconservation program requires the survival of the germplasm through
494
the cryopreservation procedures and recovery at a success rate that produces sufficient offspring
495
to represent the preserved genetic diversity. Any of the components shown in Figure 1 could be
496
considered for preservation, but the practicality of integration into a conservation program for
497
avian species depends on the availability of cryopreservation techniques and the success rate of
498
functional recovery (Table 1). In principle, PGCs can be isolated, cryopreserved and recovered
499
in chimeras but the application of these in genetic conservation is limited by the complexity and
500
low efficiency. Fertility produced by cryopreserved semen in some avian species is adequate for
501
conservation purposes but could be improved. Cryopreservation of avian gonadal tissue
502
provides an alternative for preserving male genetic resources and may be the only practical
503
option for preserving female genetic resources. Recent progress in cryopreservation and
504
transplantation of avian gonadal tissue for genetic conservation is promising.
505
In theory, cryopreservation and transplantation of avian gonadal tissue should allow
506
preservation and regeneration of germplasm of any endangered species, using recipients from the
507
same or a different species. The vitrification protocol used by Liu et al. (2010) has recently been
508
improved and used in preserving avian testicular tissue. Promising results have been obtained in
509
Japanese quail (Liu et al., 2012) and chickens (unpublished data), which suggests that this
510
method could be used for other species including wild birds with minimal adjustments. In
511
addition, the simple procedures and low cost make this method field-friendly for research in wild
512
avian species, which are usually conducted in harsh, remote environments (Comizzoli et al.,
513
2012). Efforts should be made to build cryobanks of different forms of avian germplasm from as
514
many species as possible. Functional recovery of cryopreserved avian germplasm can be more 24
515
challenging. Reproduction in avian species is regulated by delicate orchestration of many
516
environmental, behavioral and physiological mechanisms, of which our knowledge is meagre.
517
An important venue for future research is comparative and systematic investigations of species-
518
specific, fundamental reproductive mechanisms in avian species, which will allow the genetic
519
resources of these species to be conserved and recovered following the fundamental principles of
520
cryobiology and transplantation described here.
521 522
ACKNOWLEDGEMENTS
523
The authors would like to thank Canadian Poultry Industry Council, Canadian Poultry Research
524
Council and Egg Farmers of Canada for providing funds for avian genetic cryoconservation
525
research. Thanks to Drs. Kiran Soma and Richard Buchholz for valuable and helpful comments
526
on the manuscript.
25
527
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720
Table 1 Status of germplasm cryopreservation in avian species Germplasm
Status
Reference Liu et al., 2012
Male
Available
Gonadal tissue
Song and Silversides 2007a Female
Available
Liu et al., 2010
Male
Available
Blesbois 2011, Gee et al., 2004
Female
NA
NA
NA
NA
Gametes Embryos
Petitte 2006 Primordial germ cells
Limited in efficiency Silversides and Liu et al., 2012
721 722
36
723 724 725
FIGURE LEGENDS Figure 1 Cycle of germline development. Successful preservation of any component of
726
this cycle and subsequent successful functional recovery will lead to successful ex situ
727
conservation.
728
Figure 2 Schematic phase diagram of a CPA solution. Tm: melting temperature curve;
729
Th : homogeneous nucleation temperature curve; Tg: glass transition temperature curve. The
730
intersection between Th and Tg gives a critical CPA concentration; above this concentration, it is
731
possible to cool the solution directly to the glass transition temperature without freezing.
732
Adapted from Fahy et al. (1984)
733
Figure 3 A schematic drawing of major structural features of spermatozoa from different
734
species. Spermatozoa are adapted from Garner and Hafez (2000) and Romanoff (1960) and are
735
not drawn to scale.
37
736
FIGURES
737
Figure 1
738 739
38
740
Figure 2
741
39
742
Figure 3
743
40
