**2. The male**

The male's gametes are produced in very large numbers and are relatively easy to obtain. A wide variety of collection methods have been devised, including: 1) post-coital vaginal collection, either directly (e.g. O'Brien & Roth, 2000) or with the aid of intra-vaginal condom or vaginal sponge (e.g. Bravo et al., 2000), 2) artificial vagina (e.g. Gastal et al., 1996; Asher et al., 2000), 3) manual stimulation of either the rectum (e.g. Schmitt & Hildebrandt, 1998; Schmitt & Hildebrandt, 2000), the abdomen (Burrows & Quinn, 1937), or through stimulation of the penis (e.g. Schneiders et al., 2004; Melville et al., 2008), 4) electroejaculation (e.g. Hermes et al., 2009b), 5) pharmacologically-induced ejaculation by oral imipramine and intravenous xylazine (McDonnell, 2001) or through urethral catheterization after medetomidine administration (Zambelli et al., 2008), 6) aspiration from the cauda epididymis (e.g. Moghadam et al., 2005), and 7) semen retrieval from the cauda epididymis and proximal portion of the vas deference following castration or post mortem (e.g. Jewgenow et al., 1997; Saragusty et al., 2006; Keeley et al., 2011). Whereas techniques one to three above are relatively close to natural ejaculation, they require easy access to the

Genome Banking for Vertebrates Wildlife Conservation 297

sperm is also fairly viscous but it can be enzymatically liquefied (Bravo et al., 2000). Similar enzymatic liquefaction was also helpful when attempting to separate rhinoceros sperm from the seminal plasma, something that cannot be done efficiently with centrifugation alone in some of the ejaculates (Behr et al., 2009b). The volume and concentration also vary by several orders of magnitude among species. In the naked mole rat (*Heterocephalus glaber*) only 5 to 10 µL of sperm can be collected with cells in the hundreds to thousands at the most, many of which are morphologically abnormal (unpublished data). In the European brown hare (*Lepus europaeus*) or the Asiatic black bear (*Ursus thibetanys*), volume of semen collected by electroejaculation is often in the range of 1 mL or less with concentrations that at times can exceed 109 cells/mL (personal observations; Chen et al., 2007). Low volume of up to a few mL and low concentration of few millions per mL is often the case in felids both in captivity and in the wild (Barone et al., 1994; Morato et al., 2001). In the pygmy hippopotamus (*Choeropsis liberiensis*) the sperm-rich fraction can be extremely concentrated. In one case we found as much as 9.85 × 109 spermatozoa per mL (Saragusty et al., 2010a). In some other animals volumes can be very large. In boar, donkey or elephant semen can exceed 100 mL with concentrations of several hundred million cells per mL (unpublished data and e.g. Saragusty et al., 2009e; Contri et al., 2010). Initial motility is expected to be low in sperm collected from the epididymis, as epididymal sperm is immotile in most mammals. This is likely to change after a short incubation time in a suitable media. As many of the cells in the epididymis did not complete their maturation process at the time of extraction, cytoplasmic droplets can be highly prevalent (Saragusty et al., 2010b). Some specific characteristics were also noted in certain species. For example the seminal plasma pH of the black flying fox (*Pteropus alecto*) or the snow leopard (*Panthera uncial*) is high (8.2 and 8.4, respectively) (Roth et al., 1996; Melville et al., 2008) or in the Asian elephant (*Elephas maximus*) osmolarity of the seminal plasma is low, at around 270 mOsm/kg (Saragusty et al., 2009e). Such characteristics demonstrate the need to verify multiple aspects of the semen so that suitable diluents can be made. One should always keep in mind that when dealing with endangered species, many were pushed into a bottleneck situation, resulting in highly inbred populations. Inbreeding comes with a very high price with respect to the soundness of the reproductive system. This can be manifested in sperm quality (Roldan et al., 1998; Gomendio et al., 2000; Ruiz-Lopez et al., 2010) and in the outcome in term of litter size and survival (Rabon & Waddell, 2010). Once proper sample of sufficiently good quality is in

Probably the most popular preservation technique is slow freezing of semen or the cells therein. Spermatozoa are generally small in size and thus have low surface to volume ratio, an important factor in cryopreservation, which influences the movement of cryoprotectants and water in and out of the cells. They also have highly condensed and thus stable nucleus and little cytoplasm, making them relatively easy to freeze. Although problems are still numerous and even after more than 60 years of extensive research, propelled primarily by that related to human infertility and livestock and laboratory animal production, our knowledge about the exact mechanisms that eventually lead to success, failure or anywhere in-between is still very limited (Saragusty et al., 2009a). Thus, much of the progress in this field has been primarily empirical in nature (e.g. Saragusty et al., 2009e). Evolution made

hand, there are several options for its preservation.

**2.1 Semen freezing** 

animal and excessive training (e.g. Robeck & O'Brien, 2004) and are thus limited to only a handful of species and individuals. The pharmacological techniques (5) and aspiration from the epididymis (6) are too invasive to be frequently used, and extraction from the epididymis (7) is a one-time technique, which is often used as a gamete rescue procedure. Epididymal sperm extraction and preservation is a well-documented collection technique. Probably the main advantage of this method is that it enables us to collect sperm post mortem and, if stored, it can be used to extend the reproductive "life span" of that individual. When dealing with endangered species, this may enable us to preserve the spermatozoa of wild and genetically valuable captive males who die in an accident or otherwise. The spermatozoa accumulated in the cauda epididymis is already mature and fertile (Foote, 2000) making it a useful source. Several methods were described as to how to extract the sperm out of the cauda epididymis. These include squeezing the cauda epididymis (Krzywinski, 1981), making cuts in the cauda epididymis (Krzywinski, 1981; Hishinuma et al., 2003; Martinez-Pastor et al., 2006; Saragusty et al., 2006), cutting and squeezing (Quinn & White, 1967), extrusion by air pressure (Kikuchi et al., 1998; Ikeda et al., 2002) and flushing the vas deferens (Martinez-Pastor et al., 2006). Flushing the vas deference, when compared with the cutting method (Martinez-Pastor et al., 2006) , was showed to be superior, yet it seems to be less suitable for field work. For epididymal sperm extraction, spermatozoa stored chilled within the epididymis seem to survive better and for longer periods than those stored in an extender (Ringleb et al., 2011). Still, for *in vivo* sperm collection, electroejaculation became by far the most frequently used method in wildlife species. To be successful, one would need a suitable probe, which often needs to be specifically designed for the animal to be collected based on preliminary knowledge of its anatomy (Hildebrandt et al., 2000; Roth et al., 2005). Even so, ejaculates often come with urine contamination (e.g. Anel et al., 2008) or they may come with or without the relevant secretions from all accessory glands. In elephants this is manifested by occasional ejaculates with very sticky consistency indicating that high level of secretions from the bulbourethral gland are present (personal observation) whereas in rhinoceros it is manifested by high viscosity of the ejaculate (Behr et al., 2009b). In rhinoceros, measuring alkaline phosphatase in the ejaculate was suggested as a mean to identify true ejaculates (Roth et al., 2010). Despite its wide use and success in many species, there is one major drawback to electroejaculation that limits its use on a frequent basis. To conduct electroejaculation, the animal needs to be anesthetized, something many zoos would rather avoid when possible. The need to anesthetize the animal makes it impossible to collect from the same individual on a regular, frequent basis. Anesthesia may also affect the collection procedure (Santiago-Moreno et al., 2010) and the quality of the collected sample (Campion et al., 2011). One should also keep in mind that the collection technique itself may effect the composition of the ejaculate and therefore its quality (Christensen et al., 2011). Thus, the development of preservation protocols for wildlife species progress slowly and often rely on relatively small number of individuals, repeats, and/or ejaculates.

Sperm evaluation also requires understanding of the species under study as sperm competition, for instance, is a major driver behind the wide variety of sperm traits, morphologies and behaviors found in nature (Tourmente et al., 2011). In primates, semen can be as thick as paste, which requires liquefaction and extraction of the cells into a diluent (e.g. Oliveira et al., 2010). In camelids, possibly due to the absence of vesicular glands,

animal and excessive training (e.g. Robeck & O'Brien, 2004) and are thus limited to only a handful of species and individuals. The pharmacological techniques (5) and aspiration from the epididymis (6) are too invasive to be frequently used, and extraction from the epididymis (7) is a one-time technique, which is often used as a gamete rescue procedure. Epididymal sperm extraction and preservation is a well-documented collection technique. Probably the main advantage of this method is that it enables us to collect sperm post mortem and, if stored, it can be used to extend the reproductive "life span" of that individual. When dealing with endangered species, this may enable us to preserve the spermatozoa of wild and genetically valuable captive males who die in an accident or otherwise. The spermatozoa accumulated in the cauda epididymis is already mature and fertile (Foote, 2000) making it a useful source. Several methods were described as to how to extract the sperm out of the cauda epididymis. These include squeezing the cauda epididymis (Krzywinski, 1981), making cuts in the cauda epididymis (Krzywinski, 1981; Hishinuma et al., 2003; Martinez-Pastor et al., 2006; Saragusty et al., 2006), cutting and squeezing (Quinn & White, 1967), extrusion by air pressure (Kikuchi et al., 1998; Ikeda et al., 2002) and flushing the vas deferens (Martinez-Pastor et al., 2006). Flushing the vas deference, when compared with the cutting method (Martinez-Pastor et al., 2006) , was showed to be superior, yet it seems to be less suitable for field work. For epididymal sperm extraction, spermatozoa stored chilled within the epididymis seem to survive better and for longer periods than those stored in an extender (Ringleb et al., 2011). Still, for *in vivo* sperm collection, electroejaculation became by far the most frequently used method in wildlife species. To be successful, one would need a suitable probe, which often needs to be specifically designed for the animal to be collected based on preliminary knowledge of its anatomy (Hildebrandt et al., 2000; Roth et al., 2005). Even so, ejaculates often come with urine contamination (e.g. Anel et al., 2008) or they may come with or without the relevant secretions from all accessory glands. In elephants this is manifested by occasional ejaculates with very sticky consistency indicating that high level of secretions from the bulbourethral gland are present (personal observation) whereas in rhinoceros it is manifested by high viscosity of the ejaculate (Behr et al., 2009b). In rhinoceros, measuring alkaline phosphatase in the ejaculate was suggested as a mean to identify true ejaculates (Roth et al., 2010). Despite its wide use and success in many species, there is one major drawback to electroejaculation that limits its use on a frequent basis. To conduct electroejaculation, the animal needs to be anesthetized, something many zoos would rather avoid when possible. The need to anesthetize the animal makes it impossible to collect from the same individual on a regular, frequent basis. Anesthesia may also affect the collection procedure (Santiago-Moreno et al., 2010) and the quality of the collected sample (Campion et al., 2011). One should also keep in mind that the collection technique itself may effect the composition of the ejaculate and therefore its quality (Christensen et al., 2011). Thus, the development of preservation protocols for wildlife species progress slowly and often rely on relatively small

number of individuals, repeats, and/or ejaculates.

Sperm evaluation also requires understanding of the species under study as sperm competition, for instance, is a major driver behind the wide variety of sperm traits, morphologies and behaviors found in nature (Tourmente et al., 2011). In primates, semen can be as thick as paste, which requires liquefaction and extraction of the cells into a diluent (e.g. Oliveira et al., 2010). In camelids, possibly due to the absence of vesicular glands, sperm is also fairly viscous but it can be enzymatically liquefied (Bravo et al., 2000). Similar enzymatic liquefaction was also helpful when attempting to separate rhinoceros sperm from the seminal plasma, something that cannot be done efficiently with centrifugation alone in some of the ejaculates (Behr et al., 2009b). The volume and concentration also vary by several orders of magnitude among species. In the naked mole rat (*Heterocephalus glaber*) only 5 to 10 µL of sperm can be collected with cells in the hundreds to thousands at the most, many of which are morphologically abnormal (unpublished data). In the European brown hare (*Lepus europaeus*) or the Asiatic black bear (*Ursus thibetanys*), volume of semen collected by electroejaculation is often in the range of 1 mL or less with concentrations that at times can exceed 109 cells/mL (personal observations; Chen et al., 2007). Low volume of up to a few mL and low concentration of few millions per mL is often the case in felids both in captivity and in the wild (Barone et al., 1994; Morato et al., 2001). In the pygmy hippopotamus (*Choeropsis liberiensis*) the sperm-rich fraction can be extremely concentrated. In one case we found as much as 9.85 × 109 spermatozoa per mL (Saragusty et al., 2010a). In some other animals volumes can be very large. In boar, donkey or elephant semen can exceed 100 mL with concentrations of several hundred million cells per mL (unpublished data and e.g. Saragusty et al., 2009e; Contri et al., 2010). Initial motility is expected to be low in sperm collected from the epididymis, as epididymal sperm is immotile in most mammals. This is likely to change after a short incubation time in a suitable media. As many of the cells in the epididymis did not complete their maturation process at the time of extraction, cytoplasmic droplets can be highly prevalent (Saragusty et al., 2010b). Some specific characteristics were also noted in certain species. For example the seminal plasma pH of the black flying fox (*Pteropus alecto*) or the snow leopard (*Panthera uncial*) is high (8.2 and 8.4, respectively) (Roth et al., 1996; Melville et al., 2008) or in the Asian elephant (*Elephas maximus*) osmolarity of the seminal plasma is low, at around 270 mOsm/kg (Saragusty et al., 2009e). Such characteristics demonstrate the need to verify multiple aspects of the semen so that suitable diluents can be made. One should always keep in mind that when dealing with endangered species, many were pushed into a bottleneck situation, resulting in highly inbred populations. Inbreeding comes with a very high price with respect to the soundness of the reproductive system. This can be manifested in sperm quality (Roldan et al., 1998; Gomendio et al., 2000; Ruiz-Lopez et al., 2010) and in the outcome in term of litter size and survival (Rabon & Waddell, 2010). Once proper sample of sufficiently good quality is in hand, there are several options for its preservation.

#### **2.1 Semen freezing**

Probably the most popular preservation technique is slow freezing of semen or the cells therein. Spermatozoa are generally small in size and thus have low surface to volume ratio, an important factor in cryopreservation, which influences the movement of cryoprotectants and water in and out of the cells. They also have highly condensed and thus stable nucleus and little cytoplasm, making them relatively easy to freeze. Although problems are still numerous and even after more than 60 years of extensive research, propelled primarily by that related to human infertility and livestock and laboratory animal production, our knowledge about the exact mechanisms that eventually lead to success, failure or anywhere in-between is still very limited (Saragusty et al., 2009a). Thus, much of the progress in this field has been primarily empirical in nature (e.g. Saragusty et al., 2009e). Evolution made

Genome Banking for Vertebrates Wildlife Conservation 299

being a bit less field-friendly as they require bringing the cold ethanol or liquid nitrogen to the site of work. Still, freezing in liquid nitrogen vapor is currently the most popular one amongst the low-tech, equipment-free freezing techniques. The more sophisticated and more laboratory-bound techniques include the controlled-rate freezing machines (Landa & Almquist, 1979) and the directional freezing machine (Arav, 1999; O'Brien & Robeck, 2006; Si et al., 2006; Saragusty et al., 2007; Reid et al., 2009). When the initial sample is of very poor quality or with very small number of cells, small cell-number or single cell cryopreservation techniques may become useful. Starting in the late 1990's (Cohen & Garrisi, 1997; Cohen et al., 1997), reports on several single sperm cryopreservation techniques showed up in the scientific literature (Walmsley et al., 1998; Gil-Salom et al., 2000; Gvakharia & Adamson, 2001; Just et al., 2004; Herrler et al., 2006; Isaev et al., 2007; Koscinski et al., 2007; Woods et al., 2010). Using these various techniques, researchers reported a wide range of outcomes and recovery efficiency. Time will tell which of these technologies, or others that are currently under development, will emerge as the leading technique that will gain a foothold in sperm banks. Naturally, when sperm banking is considered, single sperm cryopreservation is an option to be considered only if all other possibilities were exhausted. When banking sperm from wildlife, the aim is to bank large number of cells from large

About three decades ago, the thus far only technique that has reached commercial level made it possible to sort sperm according to the sex chromosome they carry (Johnson et al., 1987a; Johnson et al., 1987b). This technique has been tested in various wildlife species such as elephants, rhinoceros, dolphins and non-human primates (O'Brien et al., 2004; O'Brien & Robeck, 2006; Behr et al., 2009a; Behr et al., 2009b; Hermes et al., 2009a). Sperm sorting machines, however, are very expensive, scarce and usually situated far away from where the sperm donor and recipient are located. For this, the double freezing technique has been developed (Arav et al., 2002; Hollinshead et al., 2004; Maxwell et al., 2007; Saragusty et al., 2009c; Montano et al., 2010). This technique allows collection and cryopreservation of sperm sample near the donor, transportation of the frozen sample to the sorting center, thawing it for sorting and then freezing the sorted sample for transportation to the recipient. In this

Although advances were made over the six decades of sperm cryopreservation history, the basic model that will predict behavior of spermatozoa during cryopreservation is still to be devised. Current knowledge is lacking in many respects and thus, when approaching a new species, much empirical work, often based on trial and error, should be conducted. Thanks to the large number of cells in each ejaculate, these can be split into several treatment modalities, thus speeding up the freezing protocol development process. Once better understanding is attained, and predictions can be made for sperm behavior under various freezing-associated conditions, probably the right course to be taken will be tailor-made, individual-based cryopreservation. This will help overcoming considerable differences between males in response to cryopreservation (Thurston et al., 2002; Saragusty et al., 2007; Loomis & Graham, 2008). However, despite all hurdles, and certainly since ICSI made sperm motility and membrane integrity obsolete, sperm banking under liquid nitrogen is probably the most widely used technique in gametes and tissue banking for reproduction preservation. Success in post-thaw survival, and often also in offspring production, has been

respect the advantage of large volume freezing at the sperm donor site is clear.

number of individuals to ensure availability and variability.

each species unique in many respects, one of which is the sperm that comes in different shapes, sizes, membrane composition, and sensitivity to chilling, osmotic pressure, pH and more. This means that any new species is an enigma and the specific characteristics of its spermatozoa and seminal plasma and their interaction with various components of freezing extenders and stages of the freezing and thawing process should all be verified. While this can be done relatively easy in domestic and laboratory animals where samples are ample and easy to get, conducting such studies in endangered species is very difficult. Opportunities to obtain samples are rare and often far apart in terms of time and space. Such samples are thus very valuable and using them for experiments rather than for banking would be a waste of important genetic material. Still, to date, semen from probably upward of 200 species from all five major classes of the Vertebrata subphylum (mammals, birds, fish, reptiles and amphibians) have been cryopreserved. When approaching a new species, several hurdles must be overcome before a successful cryopreservation protocol can be developed. The first step is to determine the specific characteristics of its spermatozoa and seminal plasma mentioned above. The next step would be to determine the composition of the freezing extender. Sensitivity to chilling-, freezing- and thawing-associated damages and cryoprotectant-associated toxic or osmotic damages is species-specific and often even individual-specific (Thurston et al., 2002). Similarly, sensitivity to various aspects of sperm handling in preparation for cryopreservation should also be taken into account. In some species it is better to remove the seminal plasma by centrifugation before freezing [e.g. goat, boar, elephant (Saragusty et al., 2009e; unpublished data)] while in others this is not required [e.g. hare or cattle (Hildebrandt et al., 2009; Saragusty et al., 2009c)]. When the seminal plasma is removed, at times adding at least some back after thawing is needed to facilitate fertilization [e.g. camels (Pan et al., 2001)]. Centrifugation is also used for selection of live, morphologically normal cells. When doing so, one needs to understand the basic species-specific sperm characteristics. For example, in opossum (*Monodelphis domestica*) sperm tend to team into pairs to enhance swimming speed (Moore & Taggart, 1995) or in deer mice (genus Peromyscus) sperm form large aggregates (Fisher & Hoekstra, 2010). One should also keep in mind that the fast forward moving population is not necessarily the right one to choose because in some species the slow and steady ones are the cells to eventually win the race (Dziminski et al., 2009). Some species are highly sensitive to glycerol (e.g. mice, boar) while others require concentrations as high as 28% for freezing to be successful, with even higher concentrations to maintain high DNA integrity (e.g. in marsupials: Johnston et al., 1993; Czarny et al., 2009a). In some cases insemination can be done with the thawed sample [e.g. cattle, rhinoceros (Hermes et al., 2009b)] while in others the glycerol should be removed or else fertilization does not occur (e.g. Poitou donkey: Trimeche et al., 1998). So, on the way to developing a successful cryopreservation protocol, species-specific characteristics should be identified and techniques to protect the cells from all these damaging mechanisms should be devised (Zeron et al., 2002; Saragusty et al., 2005; Pribenszky et al., 2006; Saragusty et al., 2009b; Pribenszky & Vajta, 2010). Several cryopreservation techniques were described. These can be divided into field-friendly and unfriendly ones. The field-friendly techniques include the pellet method [placing a sample drop of ~200 µL directly on carbon dioxide ice ("dry ice")] (Gibson & Graham, 1969), the dry-shipping container technique (Roth et al., 1999), freezing in cold ethanol (Saroff & Mixner, 1955) or in liquid nitrogen vapor (Sherman, 1963; Roussel et al., 1964). The last two

each species unique in many respects, one of which is the sperm that comes in different shapes, sizes, membrane composition, and sensitivity to chilling, osmotic pressure, pH and more. This means that any new species is an enigma and the specific characteristics of its spermatozoa and seminal plasma and their interaction with various components of freezing extenders and stages of the freezing and thawing process should all be verified. While this can be done relatively easy in domestic and laboratory animals where samples are ample and easy to get, conducting such studies in endangered species is very difficult. Opportunities to obtain samples are rare and often far apart in terms of time and space. Such samples are thus very valuable and using them for experiments rather than for banking would be a waste of important genetic material. Still, to date, semen from probably upward of 200 species from all five major classes of the Vertebrata subphylum (mammals, birds, fish, reptiles and amphibians) have been cryopreserved. When approaching a new species, several hurdles must be overcome before a successful cryopreservation protocol can be developed. The first step is to determine the specific characteristics of its spermatozoa and seminal plasma mentioned above. The next step would be to determine the composition of the freezing extender. Sensitivity to chilling-, freezing- and thawing-associated damages and cryoprotectant-associated toxic or osmotic damages is species-specific and often even individual-specific (Thurston et al., 2002). Similarly, sensitivity to various aspects of sperm handling in preparation for cryopreservation should also be taken into account. In some species it is better to remove the seminal plasma by centrifugation before freezing [e.g. goat, boar, elephant (Saragusty et al., 2009e; unpublished data)] while in others this is not required [e.g. hare or cattle (Hildebrandt et al., 2009; Saragusty et al., 2009c)]. When the seminal plasma is removed, at times adding at least some back after thawing is needed to facilitate fertilization [e.g. camels (Pan et al., 2001)]. Centrifugation is also used for selection of live, morphologically normal cells. When doing so, one needs to understand the basic species-specific sperm characteristics. For example, in opossum (*Monodelphis domestica*) sperm tend to team into pairs to enhance swimming speed (Moore & Taggart, 1995) or in deer mice (genus Peromyscus) sperm form large aggregates (Fisher & Hoekstra, 2010). One should also keep in mind that the fast forward moving population is not necessarily the right one to choose because in some species the slow and steady ones are the cells to eventually win the race (Dziminski et al., 2009). Some species are highly sensitive to glycerol (e.g. mice, boar) while others require concentrations as high as 28% for freezing to be successful, with even higher concentrations to maintain high DNA integrity (e.g. in marsupials: Johnston et al., 1993; Czarny et al., 2009a). In some cases insemination can be done with the thawed sample [e.g. cattle, rhinoceros (Hermes et al., 2009b)] while in others the glycerol should be removed or else fertilization does not occur (e.g. Poitou donkey: Trimeche et al., 1998). So, on the way to developing a successful cryopreservation protocol, species-specific characteristics should be identified and techniques to protect the cells from all these damaging mechanisms should be devised (Zeron et al., 2002; Saragusty et al., 2005; Pribenszky et al., 2006; Saragusty et al., 2009b; Pribenszky & Vajta, 2010). Several cryopreservation techniques were described. These can be divided into field-friendly and unfriendly ones. The field-friendly techniques include the pellet method [placing a sample drop of ~200 µL directly on carbon dioxide ice ("dry ice")] (Gibson & Graham, 1969), the dry-shipping container technique (Roth et al., 1999), freezing in cold ethanol (Saroff & Mixner, 1955) or in liquid nitrogen vapor (Sherman, 1963; Roussel et al., 1964). The last two being a bit less field-friendly as they require bringing the cold ethanol or liquid nitrogen to the site of work. Still, freezing in liquid nitrogen vapor is currently the most popular one amongst the low-tech, equipment-free freezing techniques. The more sophisticated and more laboratory-bound techniques include the controlled-rate freezing machines (Landa & Almquist, 1979) and the directional freezing machine (Arav, 1999; O'Brien & Robeck, 2006; Si et al., 2006; Saragusty et al., 2007; Reid et al., 2009). When the initial sample is of very poor quality or with very small number of cells, small cell-number or single cell cryopreservation techniques may become useful. Starting in the late 1990's (Cohen & Garrisi, 1997; Cohen et al., 1997), reports on several single sperm cryopreservation techniques showed up in the scientific literature (Walmsley et al., 1998; Gil-Salom et al., 2000; Gvakharia & Adamson, 2001; Just et al., 2004; Herrler et al., 2006; Isaev et al., 2007; Koscinski et al., 2007; Woods et al., 2010). Using these various techniques, researchers reported a wide range of outcomes and recovery efficiency. Time will tell which of these technologies, or others that are currently under development, will emerge as the leading technique that will gain a foothold in sperm banks. Naturally, when sperm banking is considered, single sperm cryopreservation is an option to be considered only if all other possibilities were exhausted. When banking sperm from wildlife, the aim is to bank large number of cells from large number of individuals to ensure availability and variability.

About three decades ago, the thus far only technique that has reached commercial level made it possible to sort sperm according to the sex chromosome they carry (Johnson et al., 1987a; Johnson et al., 1987b). This technique has been tested in various wildlife species such as elephants, rhinoceros, dolphins and non-human primates (O'Brien et al., 2004; O'Brien & Robeck, 2006; Behr et al., 2009a; Behr et al., 2009b; Hermes et al., 2009a). Sperm sorting machines, however, are very expensive, scarce and usually situated far away from where the sperm donor and recipient are located. For this, the double freezing technique has been developed (Arav et al., 2002; Hollinshead et al., 2004; Maxwell et al., 2007; Saragusty et al., 2009c; Montano et al., 2010). This technique allows collection and cryopreservation of sperm sample near the donor, transportation of the frozen sample to the sorting center, thawing it for sorting and then freezing the sorted sample for transportation to the recipient. In this respect the advantage of large volume freezing at the sperm donor site is clear.

Although advances were made over the six decades of sperm cryopreservation history, the basic model that will predict behavior of spermatozoa during cryopreservation is still to be devised. Current knowledge is lacking in many respects and thus, when approaching a new species, much empirical work, often based on trial and error, should be conducted. Thanks to the large number of cells in each ejaculate, these can be split into several treatment modalities, thus speeding up the freezing protocol development process. Once better understanding is attained, and predictions can be made for sperm behavior under various freezing-associated conditions, probably the right course to be taken will be tailor-made, individual-based cryopreservation. This will help overcoming considerable differences between males in response to cryopreservation (Thurston et al., 2002; Saragusty et al., 2007; Loomis & Graham, 2008). However, despite all hurdles, and certainly since ICSI made sperm motility and membrane integrity obsolete, sperm banking under liquid nitrogen is probably the most widely used technique in gametes and tissue banking for reproduction preservation. Success in post-thaw survival, and often also in offspring production, has been

Genome Banking for Vertebrates Wildlife Conservation 301

Storage of cryopreserved samples under liquid nitrogen is very demanding in terms of maintenance, storage space, storage equipment, specially trained personnel and associated costs. Resulting from the need for constant liquid nitrogen supply in large quantities, such storage facilities have very high carbon footprint. The possibility of discontinuation of the liquid nitrogen supply due to human (e.g. conflict, strike) or natural (e.g. earthquake, hurricane) put these facilities at a constant risk. An alternative that would minimize all these is the dry storage. Drying of cells can be done by either freeze-drying or convective-drying. Freeze-drying is achieved by sublimation of the ice after freezing the sample to subzero temperatures. Convective drying, on the other hand, is achieved by placing the sample in a vacuum oven at ambient temperatures. Sperm drying is, however, damaging to cellular membrane and rehydrated cells are often devoid of biological activity, motility and viability. Some degree of chromosomal damage may also take place due to endogenous nucleases. Attempts to freeze-dry spermatozoa were first reported about six decades ago on animal (Polge et al., 1949; Sherman, 1957; Yushchenko, 1957; Meryman & Kafig, 1959) and human (Sherman, 1954) sperm. Most researchers, however, consider all these early reports, dubious. The definitive proof that freeze-dried spermatozoa retain genetic integrity was established only when microsurgical procedures for bypassing the lack of motility of freeze-dried spermatozoa were developed, and normal mice were produced by intracytoplasmic sperm injection (ICSI) of freeze-dried sperm (Wakayama & Yanagimachi, 1998). To date, embryonic development after ICSI with freeze-dried sperm heads has been reported in humans (Katayose et al., 1992; Kusakabe et al., 2008), hamster (Katayose et al., 1992), cattle (Keskintepe et al., 2002; Martins et al., 2007), pigs (Kwon et al., 2004), rhesus macaque (Sanchez-Partida et al., 2008), cats (Moisan et al., 2005; Ringleb et al., 2011) and fish (Poleo et al., 2005), and live offspring were reported in mice (Wakayama & Yanagimachi, 1998; Kaneko et al., 2003; Ward et al., 2003), rabbits (Yushchenko, 1957; Liu et al., 2004), rat (Hirabayashi et al., 2005; Hochi et al., 2008), fish (Poleo et al., 2005) and horses (Choi et al., 2011). Storage at room temperature would be ideal, and at least for mid-range duration it appear to be fine (3 years storage of somatic cells; Loi et al., 2008a). High-temperature storage, however, might be damaging to DNA integrity according to some (Kaneko & Nakagata, 2005; Hochi et al., 2008) but not all (Li et al., 2007; Klooster et al., 2011) researchers. These differences may be due to differences in the drying technique or related to differences between species (Li et al., 2007; Klooster et al., 2011;

While there are many reports on freeze-drying of sperm and other relevant cells, those on convective drying are scarce. Some researchers, however, consider convective drying to be the better option for the fact that it does not involve the freezing step, thus avoiding freezing-associated damages. This technique has been used to dry fibroblasts, and

Regardless of the drying process used, for now sperm drying will usually be placed way behind sperm freezing or vitrification because of the loss of motility and viability, and the

In many cases, sperm can be collected in the field, away from any fully equipped andrology and cryobiology laboratory or a source for liquid nitrogen, and transferring the samples to a

spermatogonial and hematopoietic stem cells (Katkov et al., 2006; Meyers, 2006).

need for ICSI. Thus, sperm drying is still to be demonstrated in true wildlife species.

**2.4 Liquid phase semen short- to mid-term storage** 

**2.3 Sperm drying** 

Kusakabe & Tateno, 2011).

demonstrated in many vertebrate species. And yet, there are other options to preserve male fertility.

#### **2.2 Semen vitrification**

Ice crystals, both outside and even more so – inside, can be very damaging to any frozen cell or tissue. To avoid ice formation and to minimize the pre-freezing chilling damages, vitrification can be used. Vitrification, also known as ice-free cryopreservation, is a process in which liquid is transformed into an amorphous, glass-like solid, free of any crystalline structures (Luyet, 1937). A major advantage of vitrification over slow freezing is its low-tech, low cost, simple to use, suitable for the field character. For vitrification to be successful, however, much experience in sample handling before cooling and after warming and in loading the sample into or onto the carrier system, are needed. Probability of vitrification depends on the interaction between three factors – cooling rate, sample volume and its viscosity, according to the following general relationships (Saragusty & Arav, 2011):

$$\text{Probability of Virfification} = \frac{\text{Couling rate} \times \text{Viscosity}}{\text{Volume}} \tag{1}$$

Thus, to achieve the state of vitrification, very high viscosity (usually attained through high concentrations of cryoprotectants or low water content), and/or very high cooling rates and/or very small volumes are needed. Since the high cryoprotectant concentrations needed are beyond what spermatozoa from most species can tolerate, the vitrified volume is usually being considerably reduced and techniques to achieve high cooling rate with adequate heat transfer throughout the sample are devised. For example, using the cryoloop vitrification technique, it was calculated that cooling rate as high as 720,000ºC/min has been achieved (Isachenko et al., 2004) or with quartz capillaries, cooling rates of around 250,000ºC/min were reported (Risco et al., 2007; Lee et al., 2010). The technique, though, have several major drawbacks when sperm banking is considered: 1) The small volume that can be vitrified (at best, presently only a few microliters of semen suspended in vitrification solution) is way too small for banking for species conservation purposes and vitrifying large number of samples from each individual is not practical. 2) The small volume, and thus the small sperm number, make vitrified samples impractical for use in artificial insemination or even in standard IVF. Its optimal utilization is through ICSI, a technique that requires specialized equipment and expertise not available in most laboratories dealing with wildlife, and ICSI has not yet been developed for most species. 3) The risk of contamination through the liquid nitrogen prevails in many of the currently available vitrification carrier devices, which are open systems. 4) High permeable cryoprotectant concentrations (up to 50% compared to 3-7% in slow freezing in most species) are still needed in many of the vitrification protocols despite the reduction in volume. Such concentrations are both toxic and cause osmotic damages to the cells. To overcome this, permeating cryoprotectant-free vitrification techniques were developed through a sizable increase in cooling rate (Nawroth et al., 2002; Isachenko et al., 2003; Merino et al., 2011). Sperm vitrified this way can maintain motility (Isachenko et al., 2004) and resulted recently in human live birth (Sanchez et al., 2011).

#### **2.3 Sperm drying**

300 Current Frontiers in Cryobiology

demonstrated in many vertebrate species. And yet, there are other options to preserve male

Ice crystals, both outside and even more so – inside, can be very damaging to any frozen cell or tissue. To avoid ice formation and to minimize the pre-freezing chilling damages, vitrification can be used. Vitrification, also known as ice-free cryopreservation, is a process in which liquid is transformed into an amorphous, glass-like solid, free of any crystalline structures (Luyet, 1937). A major advantage of vitrification over slow freezing is its low-tech, low cost, simple to use, suitable for the field character. For vitrification to be successful, however, much experience in sample handling before cooling and after warming and in loading the sample into or onto the carrier system, are needed. Probability of vitrification depends on the interaction between three factors – cooling rate, sample volume and its viscosity, according to the following general relationships

Cooling rate Viscosity Probability of Vitrification= Volume

Thus, to achieve the state of vitrification, very high viscosity (usually attained through high concentrations of cryoprotectants or low water content), and/or very high cooling rates and/or very small volumes are needed. Since the high cryoprotectant concentrations needed are beyond what spermatozoa from most species can tolerate, the vitrified volume is usually being considerably reduced and techniques to achieve high cooling rate with adequate heat transfer throughout the sample are devised. For example, using the cryoloop vitrification technique, it was calculated that cooling rate as high as 720,000ºC/min has been achieved (Isachenko et al., 2004) or with quartz capillaries, cooling rates of around 250,000ºC/min were reported (Risco et al., 2007; Lee et al., 2010). The technique, though, have several major drawbacks when sperm banking is considered: 1) The small volume that can be vitrified (at best, presently only a few microliters of semen suspended in vitrification solution) is way too small for banking for species conservation purposes and vitrifying large number of samples from each individual is not practical. 2) The small volume, and thus the small sperm number, make vitrified samples impractical for use in artificial insemination or even in standard IVF. Its optimal utilization is through ICSI, a technique that requires specialized equipment and expertise not available in most laboratories dealing with wildlife, and ICSI has not yet been developed for most species. 3) The risk of contamination through the liquid nitrogen prevails in many of the currently available vitrification carrier devices, which are open systems. 4) High permeable cryoprotectant concentrations (up to 50% compared to 3-7% in slow freezing in most species) are still needed in many of the vitrification protocols despite the reduction in volume. Such concentrations are both toxic and cause osmotic damages to the cells. To overcome this, permeating cryoprotectant-free vitrification techniques were developed through a sizable increase in cooling rate (Nawroth et al., 2002; Isachenko et al., 2003; Merino et al., 2011). Sperm vitrified this way can maintain motility (Isachenko et al., 2004)

and resulted recently in human live birth (Sanchez et al., 2011).

(1)

fertility.

**2.2 Semen vitrification** 

(Saragusty & Arav, 2011):

Storage of cryopreserved samples under liquid nitrogen is very demanding in terms of maintenance, storage space, storage equipment, specially trained personnel and associated costs. Resulting from the need for constant liquid nitrogen supply in large quantities, such storage facilities have very high carbon footprint. The possibility of discontinuation of the liquid nitrogen supply due to human (e.g. conflict, strike) or natural (e.g. earthquake, hurricane) put these facilities at a constant risk. An alternative that would minimize all these is the dry storage. Drying of cells can be done by either freeze-drying or convective-drying. Freeze-drying is achieved by sublimation of the ice after freezing the sample to subzero temperatures. Convective drying, on the other hand, is achieved by placing the sample in a vacuum oven at ambient temperatures. Sperm drying is, however, damaging to cellular membrane and rehydrated cells are often devoid of biological activity, motility and viability. Some degree of chromosomal damage may also take place due to endogenous nucleases. Attempts to freeze-dry spermatozoa were first reported about six decades ago on animal (Polge et al., 1949; Sherman, 1957; Yushchenko, 1957; Meryman & Kafig, 1959) and human (Sherman, 1954) sperm. Most researchers, however, consider all these early reports, dubious. The definitive proof that freeze-dried spermatozoa retain genetic integrity was established only when microsurgical procedures for bypassing the lack of motility of freeze-dried spermatozoa were developed, and normal mice were produced by intracytoplasmic sperm injection (ICSI) of freeze-dried sperm (Wakayama & Yanagimachi, 1998). To date, embryonic development after ICSI with freeze-dried sperm heads has been reported in humans (Katayose et al., 1992; Kusakabe et al., 2008), hamster (Katayose et al., 1992), cattle (Keskintepe et al., 2002; Martins et al., 2007), pigs (Kwon et al., 2004), rhesus macaque (Sanchez-Partida et al., 2008), cats (Moisan et al., 2005; Ringleb et al., 2011) and fish (Poleo et al., 2005), and live offspring were reported in mice (Wakayama & Yanagimachi, 1998; Kaneko et al., 2003; Ward et al., 2003), rabbits (Yushchenko, 1957; Liu et al., 2004), rat (Hirabayashi et al., 2005; Hochi et al., 2008), fish (Poleo et al., 2005) and horses (Choi et al., 2011). Storage at room temperature would be ideal, and at least for mid-range duration it appear to be fine (3 years storage of somatic cells; Loi et al., 2008a). High-temperature storage, however, might be damaging to DNA integrity according to some (Kaneko & Nakagata, 2005; Hochi et al., 2008) but not all (Li et al., 2007; Klooster et al., 2011) researchers. These differences may be due to differences in the drying technique or related to differences between species (Li et al., 2007; Klooster et al., 2011; Kusakabe & Tateno, 2011).

While there are many reports on freeze-drying of sperm and other relevant cells, those on convective drying are scarce. Some researchers, however, consider convective drying to be the better option for the fact that it does not involve the freezing step, thus avoiding freezing-associated damages. This technique has been used to dry fibroblasts, and spermatogonial and hematopoietic stem cells (Katkov et al., 2006; Meyers, 2006).

Regardless of the drying process used, for now sperm drying will usually be placed way behind sperm freezing or vitrification because of the loss of motility and viability, and the need for ICSI. Thus, sperm drying is still to be demonstrated in true wildlife species.

#### **2.4 Liquid phase semen short- to mid-term storage**

In many cases, sperm can be collected in the field, away from any fully equipped andrology and cryobiology laboratory or a source for liquid nitrogen, and transferring the samples to a

Genome Banking for Vertebrates Wildlife Conservation 303

ICSI, even early developmental stages such as elongating or elongated spermatids can be utilized for fertilization. Such cells as testicular spermatozoa and earlier developmental stages can be extracted using testicular sperm extraction (TESE) techniques, and then used through ICSI to fertilize oocytes (Schoysman et al., 1993; Devroey et al., 1995; Kimura & Yanagimachi, 1995; Hewitson et al., 2002). These early-stage cells can be used fresh but they can also be cryopreserved and used at a later stage when needed (Hirabayashi et al., 2008). Cells of even an earlier developmental stage than the spermatocytes and spermatids are the spermatogonium or spermatogonial stem cells, which can be collected from any male, including infants and juveniles. Infant mortality rate is known to be relatively high in many populations (e.g. Howell-Stephens et al., 2009; Saragusty et al., 2009d) so methods to preserve germ cells from valuable individuals in certainly called for. Spermatogonial stem cells transplantation was first reported in mice (Brinster & Zimmermann, 1994) when it was demonstrated that such transplantation can lead to spermatogenesis. The transplantation technique was later extended to other species such as pigs (Honaramooz et al., 2002), bovine (Izadyar et al., 2003), goats (Honaramooz et al., 2003a; Honaramooz et al., 2003b), cynomolgus monkeys (Schlatt et al., 2002a), and recently to felids as well (Silva et al., 2011). Xenogeneic transplantation, usually from other mammals to nude, immune-deficient mice, has also been reported. However, the further apart (phylogenitically) the donor and recipient species are, the more difficult it becomes. Using this technique, isolated donor testis cells are infused into the seminiferous tubules of the recipient whose testes have been depleted of all germ cells (by irradiation or chemotherapy). The spermatogonial stem cells establish themselves in the testis and through spermatogenesis, produce spermatozoa carrying the donor genetic material. In 2006 the proof that such xenotransplanted cells can actually produce normal, functioning spermatozoa was reported (Shinohara et al., 2006). In their study, spermatogonial stem cells collected from immature rats were transplanted into chemically sterilized mice and the spermatozoa or spermatids collected from the recipient mice produced normal, fertile rat offspring, both when freshly used and following cryopreservation. The donor stem cells can also be grown in culture to generate more cells for transplantation (Nagano et al., 1998) and they can be cryopreserved for future use (Avarbock et al., 1996). Under very complex *in vitro* culture conditions, and with very low efficiency, morphologically normal and even motile spermatozoa were generated from spermatogonial stem cells (Feng et al., 2002; Hong et al., 2004; Stukenborg et al., 2009).

Tissue cryopreservation is more complex than cellular preservation because tissue is composed of more than one cell type and thus of different water and cryoprotectant permeability coefficient values and different sensitivities to chilling and osmotic challenges. Tissue is also larger in volume and thus cryoprotectant penetration is difficult and heat transfer is not uniform, putting the center of the sample at greater risk of intracellular ice formation and death. This is true for testicular tissue, ovarian tissues and many other types of tissues and whole organs. Testicular tissue preservation can be done in one of three basic forms. The tissue can be cryopreserved for future use, it can be cultured *in vitro* for short to mid-term preservation or it can be transplanted. When preserved in the cryopreserved form, one can freeze the whole organ or even the entire animal. Recently it was demonstrated that

**2.6 Testicular tissue cryopreservation** 

facility for processing may take time. For such cases, or when the sample is destined to be used but not immediately, short- and mid-term supra-zero preservation techniques may help. Nature regularly preserves sperm for months to years in a wide variety of species including members of all vertebrate classes (Holt & Lloyd, 2010; Holt, 2011). The location nature has elected is within the female's reproductive tract. This ability has been described in many species and has been investigated in a few. To date the mechanism has not been discovered although a possible direction has recently emerged. In the greater Asiatic yellow bat (*Scotophilus heathii*), with a regular gap of several months between mating and fertilization, it was shown recently that sperm storage is regulated by androgens (Roy & Krishna, 2011). Administration of flutamine, an androgen antagonist, resulted in loss of sperm storage ability in treated females. It was also suggested that sperm storage duration and survival is the outcome of interplay between expression of B-cell lymphoma 2 (Bcl-2) – an anti-apoptotic factor, and caspase-3 – a promoter of apoptosis. In the absence, as yet, of clear knowledge on how nature does it, *in vitro* techniques were devised in an attempt to achieve this long-term fresh storage goal. In some species, such as the pig, chilled storage is the most widespread method of preservation as thus far sperm cryopreservation has provided only mediocre postthaw results. When planning on extended chilled storage, several sperm energy-metabolism aspects should be taken into consideration. Both glycolysis and the Krebs cycle play an important role in sperm energy metabolism. Sperm from various species stored in a range of solutions, osmolarities and storage temperatures, were shown to be functional when inject into oocytes after storage of weeks to several months (Kanno et al., 1998; Van Thuan et al., 2005; Riel et al., 2007; Riel et al., 2011). An alternative is to simply leave the spermatozoa inside the epididymides and keep these at 4ºC. This epididymal preservation option was demonstrated to produce good results in dogs (Yu & Leibo, 2002), bovine (Martins et al., 2009), gazelles (Saragusty et al., 2006), ram (Tamayo-Canul et al., 2011) and many other species. Short-term epididymal preservation has many advantages when dealing with wildlife. Animals usually have the "tendency" to die at inconvenient time or location. The ability to preserve spermatozoa within the epididymis, till it is transported to a laboratory for processing, helps us buy time for rescue procedures. This can easily be done by non-experts (zoo or park employees for example) by simply cutting off the testicles, putting them in 0.9% saline and keeping them in the refrigerator. Motility preservation for several days can also be done with ejaculated sperm in egg yolk based extenders. For instance, we have recently showed that pygmy hippopotamus (*Choeropsis liberiensis*) spermatozoa preserved some motility for 3 weeks when suspended in the Berliner Cryomedium basic solution (a TEST-egg yolk based extender) (Saragusty et al., 2010a) or, in humans, sperm suspended in PBS supplemented with salts, BSA, antibiotics and glucose had about 15% motility and over 40% viability after 10 days at room temperature (Amaral et al., 2011). During such storage, the reduced metabolism and biological activity, the disintegration of dead cells or the presence of leukocytes in the suspension, all result in the release of reactive oxygen species (ROS) and other damaging components into the solution (Whittington & Ford, 1999). Removal of the leukocytes and periodic exchange of solution should thus be beneficial to the stored cells and extend their life.

#### **2.5 Preservation of other male reproductive-related cells**

Spermatozoa, however, can only be potentially retrieved from adult, relatively healthy, individuals but not from sick, azoospermic, or prepubertal ones, and often these carry valuable genetic material that, if not preserved, will be lost for the population. Thanks to

facility for processing may take time. For such cases, or when the sample is destined to be used but not immediately, short- and mid-term supra-zero preservation techniques may help. Nature regularly preserves sperm for months to years in a wide variety of species including members of all vertebrate classes (Holt & Lloyd, 2010; Holt, 2011). The location nature has elected is within the female's reproductive tract. This ability has been described in many species and has been investigated in a few. To date the mechanism has not been discovered although a possible direction has recently emerged. In the greater Asiatic yellow bat (*Scotophilus heathii*), with a regular gap of several months between mating and fertilization, it was shown recently that sperm storage is regulated by androgens (Roy & Krishna, 2011). Administration of flutamine, an androgen antagonist, resulted in loss of sperm storage ability in treated females. It was also suggested that sperm storage duration and survival is the outcome of interplay between expression of B-cell lymphoma 2 (Bcl-2) – an anti-apoptotic factor, and caspase-3 – a promoter of apoptosis. In the absence, as yet, of clear knowledge on how nature does it, *in vitro* techniques were devised in an attempt to achieve this long-term fresh storage goal. In some species, such as the pig, chilled storage is the most widespread method of preservation as thus far sperm cryopreservation has provided only mediocre postthaw results. When planning on extended chilled storage, several sperm energy-metabolism aspects should be taken into consideration. Both glycolysis and the Krebs cycle play an important role in sperm energy metabolism. Sperm from various species stored in a range of solutions, osmolarities and storage temperatures, were shown to be functional when inject into oocytes after storage of weeks to several months (Kanno et al., 1998; Van Thuan et al., 2005; Riel et al., 2007; Riel et al., 2011). An alternative is to simply leave the spermatozoa inside the epididymides and keep these at 4ºC. This epididymal preservation option was demonstrated to produce good results in dogs (Yu & Leibo, 2002), bovine (Martins et al., 2009), gazelles (Saragusty et al., 2006), ram (Tamayo-Canul et al., 2011) and many other species. Short-term epididymal preservation has many advantages when dealing with wildlife. Animals usually have the "tendency" to die at inconvenient time or location. The ability to preserve spermatozoa within the epididymis, till it is transported to a laboratory for processing, helps us buy time for rescue procedures. This can easily be done by non-experts (zoo or park employees for example) by simply cutting off the testicles, putting them in 0.9% saline and keeping them in the refrigerator. Motility preservation for several days can also be done with ejaculated sperm in egg yolk based extenders. For instance, we have recently showed that pygmy hippopotamus (*Choeropsis liberiensis*) spermatozoa preserved some motility for 3 weeks when suspended in the Berliner Cryomedium basic solution (a TEST-egg yolk based extender) (Saragusty et al., 2010a) or, in humans, sperm suspended in PBS supplemented with salts, BSA, antibiotics and glucose had about 15% motility and over 40% viability after 10 days at room temperature (Amaral et al., 2011). During such storage, the reduced metabolism and biological activity, the disintegration of dead cells or the presence of leukocytes in the suspension, all result in the release of reactive oxygen species (ROS) and other damaging components into the solution (Whittington & Ford, 1999). Removal of the leukocytes and periodic exchange of solution should thus be beneficial to the stored cells and extend their life.

**2.5 Preservation of other male reproductive-related cells** 

Spermatozoa, however, can only be potentially retrieved from adult, relatively healthy, individuals but not from sick, azoospermic, or prepubertal ones, and often these carry valuable genetic material that, if not preserved, will be lost for the population. Thanks to ICSI, even early developmental stages such as elongating or elongated spermatids can be utilized for fertilization. Such cells as testicular spermatozoa and earlier developmental stages can be extracted using testicular sperm extraction (TESE) techniques, and then used through ICSI to fertilize oocytes (Schoysman et al., 1993; Devroey et al., 1995; Kimura & Yanagimachi, 1995; Hewitson et al., 2002). These early-stage cells can be used fresh but they can also be cryopreserved and used at a later stage when needed (Hirabayashi et al., 2008).

Cells of even an earlier developmental stage than the spermatocytes and spermatids are the spermatogonium or spermatogonial stem cells, which can be collected from any male, including infants and juveniles. Infant mortality rate is known to be relatively high in many populations (e.g. Howell-Stephens et al., 2009; Saragusty et al., 2009d) so methods to preserve germ cells from valuable individuals in certainly called for. Spermatogonial stem cells transplantation was first reported in mice (Brinster & Zimmermann, 1994) when it was demonstrated that such transplantation can lead to spermatogenesis. The transplantation technique was later extended to other species such as pigs (Honaramooz et al., 2002), bovine (Izadyar et al., 2003), goats (Honaramooz et al., 2003a; Honaramooz et al., 2003b), cynomolgus monkeys (Schlatt et al., 2002a), and recently to felids as well (Silva et al., 2011). Xenogeneic transplantation, usually from other mammals to nude, immune-deficient mice, has also been reported. However, the further apart (phylogenitically) the donor and recipient species are, the more difficult it becomes. Using this technique, isolated donor testis cells are infused into the seminiferous tubules of the recipient whose testes have been depleted of all germ cells (by irradiation or chemotherapy). The spermatogonial stem cells establish themselves in the testis and through spermatogenesis, produce spermatozoa carrying the donor genetic material. In 2006 the proof that such xenotransplanted cells can actually produce normal, functioning spermatozoa was reported (Shinohara et al., 2006). In their study, spermatogonial stem cells collected from immature rats were transplanted into chemically sterilized mice and the spermatozoa or spermatids collected from the recipient mice produced normal, fertile rat offspring, both when freshly used and following cryopreservation. The donor stem cells can also be grown in culture to generate more cells for transplantation (Nagano et al., 1998) and they can be cryopreserved for future use (Avarbock et al., 1996). Under very complex *in vitro* culture conditions, and with very low efficiency, morphologically normal and even motile spermatozoa were generated from spermatogonial stem cells (Feng et al., 2002; Hong et al., 2004; Stukenborg et al., 2009).
