**1. Introduction**

About 140 years ago, Charles Darwin wrote in his book *The Descent of Man, and Selection in Relation to Sex* the following prediction: "The slowest breeder of all known animals, namely the elephant, would in a few thousand years stock the whole world." (Darwin, 1871). Unfortunately, primarily due to human activities, this prediction will probably not come true. Sadly, not only elephants face the risk of extinction. The number of species listed as endangered is on the rise. The Species Survival Commission (SSC) of the International Union for Conservation of Nature and Natural Resources (IUCN) continuously monitors the planet's fauna and flora and launches the IUCN Red List of Threatened Species (http://www.iucnredlist.org). As of the end of 2010, there were 5491 species of mammals described (IUCN, 2010). Of these, 1,131 species (21%) are now listed as endangered to some degree. In addition, there are 324 species listed as near threatened and another 836 species for which data is deficient and thus could be at risk. Adding all these numbers together, about 42% of the planet's mammalian species are at some level of threat for extinction. The list also reports on 76 species (1.4%) of mammals that became extinct in recent years and two more species that are extinct in the wild and whose survival completely depend on *ex situ* breeding programs. The situation is not distinctively different in other classes of the vertebrata subphylum or in the other subphylums of the animal kingdom. If anything, it is even worse for some such as the reptiles (21% endangered), amphibians (30%), fish (21%) or among the invertebrates: insects (22%), mollusks (41%), crustaceans (28%), anthozoa (corals and sea anemones; 27%) or arachnids (58%). With each extinct species, the stability of the entire ecological system surrounding it and the food chain of which it is an integral part is shaken. Such shaking may lead to the co-extinction of dependent species (Koh et al., 2004).

In 1992 the Convention on Biological Diversity (CBD) was ratified at the United Nations Conference on Environment and Development in Rio de Janeiro. Ten years later, during the 6th meeting of the Conference of the Parties to the CBD in 2002, it was agreed "to achieve by 2010 a significant reduction of the current rate of biodiversity loss at global, regional and national level as a contribution to poverty alleviation and to the benefit of all life on Earth" (Convention on Biological Diversity, 2002). However, 2010, which was named by the United Nations as the "Year of Biodiversity", has arrived and gone and this target not only has not

Genome Banking for Vertebrates Wildlife Conservation 295

Frozen Ark Consortium (http://www.frozenark.org/; Clarke, 2009), the Amphibian Ark (http://www.amphibianark.org/) and the Biological Resource Bank of Southern Africa's Wildlife (Bartels & Kotze, 2006). Long-term preservation of such biological material is almost entirely a matter of how water therein is dealt with. Plant seeds, whose water content is very low, can easily be preserved at relatively high subzero temperatures of -20ºC to -30ºC (Ruttimann, 2006) whereas water content in animal tissues and cells is generally very high,

About 70 years ago, the late Ernst W. Mayr coined one of the currently leading definitions for a species: "Species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups" (de Queiroz, 2005). The uniqueness of reproduction is thus central to the definition of species. It is therefore only natural that we find a wide variety of unique reproductive traits across species. These variations can come in a range of different forms, be it the anatomy of the genital system, morphology of gametes, presence or absence of accessory glands, mechanisms of ovulation, variations in the active hormones, duration of reproductive cycle and gestation and many other aspects of reproductive biology. It is also not surprising that great differences between species are found when it comes to the reaction of their gametes, embryos and tissues to the process of cryopreservation. Thus, when a successful cryopreservation protocol has been devised for a certain species, it will not necessarily be successful in other members of the same family, not to mention species that are phylogenetically further apart. In addition, to date the process of cryopreservation and the mechanisms that cause chilling- and cryodamages are not fully understood. Each new species we approach is thus a *terra incognita* and should be thoroughly studied before a successful protocol can be developed, if at all. Model species are often used for the development of basic techniques but in the end vast experimentation should be conducted in the target species. While this is relatively simple in domestic and laboratory animals, when a rare and endangered species is the target, opportunities to obtain gametes and other relevant cells and tissues are rare and far apart in terms of time and space, making progress extremely slow or practically impossible.

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

in the range of 80%, thus requiring special handling.

**2. The male** 

been met, even some of the indicators needed to measure progress (or regress) have not yet been developed or fully implemented (Walpole et al., 2009).

Based on paleontological data, of the total biota of about 10 million species, the natural or background extinction rate is approximately 1 to 10 species per year (Reid & Miller, 1989). This may be divided into species with restricted ranges for which extinction rate might be higher and those with widespread ranges for which it is considerably lower (Pimm et al., 1995). The expected extinction rate amongst all bird and mammal species is about one species every 100 to 1,000 years, yet the current extinction rate for these and other groups is about one species per year, which is 100 to 1,000 times the natural rate by some estimates (Reid & Miller, 1989; Pimm et al., 1995; Ceballos & Ehrlich, 2002; IUCN, 2004; Living Planet Report, 2008) and even as high as 10,000 times by others (Mace et al., 2005). One of the major problems behind these predictions is that we do not really know how many species are there and this is primarily true for many understudied taxonomic groups (e.g. bacteria, marine invertebrates, insects) and endemic species in many parts of the world, which by them being endemic to limited habitats face much higher risk of extinction (Pimm et al., 1995; Hunter, 2011). Earth history has witnessed 5 major events of mass extinctions in which a significant fraction of the diversity in a wide range of taxa went extinct within relatively short period (Erwin, 2001). The last, and probably the most well known episode, took place during the late Cretaceous era, approximately 65 million years ago, when the dinosaurs became extinct. The current dramatically accelerated rate of species extinction has been likened to these evens and was termed 'the sixth mass extinction event in the history of life on Earth' (Chapin et al., 2000; Wake & Vredenburg, 2008). Various studies have demonstrated the severity of this accelerated extinction process on both the population level (Ceballos & Ehrlich, 2002) and the global biodiversity level (Living Planet Report, 2008; Rockstrom et al., 2009). Not all researchers agree with the definition of mass extinction (Barnosky et al., 2011) but all agree that the current extinction rate is far too fast. Whether we call it mass extinction or not, the cause for the current accelerated extinction rate is anthropogenic in essence, resulting from six major human interference categories: (i) habitat loss or fragmentation, (ii) over exploitation, (iii) species introduction (exotic species and diseases), (iv) pollution of water, soil and air, (v) global warming, and (vi) increasing atmospheric carbon dioxide level and the consequential acidification of the oceans. Based on different projections such as climate change, human population growth or deforestation rate, predictions suggest that large chunks of the world's biodiversity is destined to disappear (Reid & Miller, 1989; Ehrlich & Wilson, 1991; Thomas et al., 2004). For the sake of the entire ecosystem stability and for our and future generations' well being, and because we are the leading driver behind this accelerated decline in biodiversity, it is our obligation to try and slow down the current extinction rate. This, however, is not going to happen overnight, probably not even over a single generation time. So as to "buy time", the establishment of genome resource banks (GRB), which will store and manage collections of gametes (sperm and oocytes), embryos, tissues and organs of endangered species, has been proposed (Veprintsev & Rott, 1979; Benirschke, 1984; Wildt, 1992). By gathering such collections, at the moment primarily through cryopreservation, these institutions, among other services, fulfill their function as a mean to extend the reproductive lifespan of individuals beyond their biological life and prevent the loss of valuable individuals to the gene pool. Several such GRBs are already in existence. These include for example the

been met, even some of the indicators needed to measure progress (or regress) have not yet

Based on paleontological data, of the total biota of about 10 million species, the natural or background extinction rate is approximately 1 to 10 species per year (Reid & Miller, 1989). This may be divided into species with restricted ranges for which extinction rate might be higher and those with widespread ranges for which it is considerably lower (Pimm et al., 1995). The expected extinction rate amongst all bird and mammal species is about one species every 100 to 1,000 years, yet the current extinction rate for these and other groups is about one species per year, which is 100 to 1,000 times the natural rate by some estimates (Reid & Miller, 1989; Pimm et al., 1995; Ceballos & Ehrlich, 2002; IUCN, 2004; Living Planet Report, 2008) and even as high as 10,000 times by others (Mace et al., 2005). One of the major problems behind these predictions is that we do not really know how many species are there and this is primarily true for many understudied taxonomic groups (e.g. bacteria, marine invertebrates, insects) and endemic species in many parts of the world, which by them being endemic to limited habitats face much higher risk of extinction (Pimm et al., 1995; Hunter, 2011). Earth history has witnessed 5 major events of mass extinctions in which a significant fraction of the diversity in a wide range of taxa went extinct within relatively short period (Erwin, 2001). The last, and probably the most well known episode, took place during the late Cretaceous era, approximately 65 million years ago, when the dinosaurs became extinct. The current dramatically accelerated rate of species extinction has been likened to these evens and was termed 'the sixth mass extinction event in the history of life on Earth' (Chapin et al., 2000; Wake & Vredenburg, 2008). Various studies have demonstrated the severity of this accelerated extinction process on both the population level (Ceballos & Ehrlich, 2002) and the global biodiversity level (Living Planet Report, 2008; Rockstrom et al., 2009). Not all researchers agree with the definition of mass extinction (Barnosky et al., 2011) but all agree that the current extinction rate is far too fast. Whether we call it mass extinction or not, the cause for the current accelerated extinction rate is anthropogenic in essence, resulting from six major human interference categories: (i) habitat loss or fragmentation, (ii) over exploitation, (iii) species introduction (exotic species and diseases), (iv) pollution of water, soil and air, (v) global warming, and (vi) increasing atmospheric carbon dioxide level and the consequential acidification of the oceans. Based on different projections such as climate change, human population growth or deforestation rate, predictions suggest that large chunks of the world's biodiversity is destined to disappear (Reid & Miller, 1989; Ehrlich & Wilson, 1991; Thomas et al., 2004). For the sake of the entire ecosystem stability and for our and future generations' well being, and because we are the leading driver behind this accelerated decline in biodiversity, it is our obligation to try and slow down the current extinction rate. This, however, is not going to happen overnight, probably not even over a single generation time. So as to "buy time", the establishment of genome resource banks (GRB), which will store and manage collections of gametes (sperm and oocytes), embryos, tissues and organs of endangered species, has been proposed (Veprintsev & Rott, 1979; Benirschke, 1984; Wildt, 1992). By gathering such collections, at the moment primarily through cryopreservation, these institutions, among other services, fulfill their function as a mean to extend the reproductive lifespan of individuals beyond their biological life and prevent the loss of valuable individuals to the gene pool. Several such GRBs are already in existence. These include for example the

been developed or fully implemented (Walpole et al., 2009).

Frozen Ark Consortium (http://www.frozenark.org/; Clarke, 2009), the Amphibian Ark (http://www.amphibianark.org/) and the Biological Resource Bank of Southern Africa's Wildlife (Bartels & Kotze, 2006). Long-term preservation of such biological material is almost entirely a matter of how water therein is dealt with. Plant seeds, whose water content is very low, can easily be preserved at relatively high subzero temperatures of -20ºC to -30ºC (Ruttimann, 2006) whereas water content in animal tissues and cells is generally very high, in the range of 80%, thus requiring special handling.

About 70 years ago, the late Ernst W. Mayr coined one of the currently leading definitions for a species: "Species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups" (de Queiroz, 2005). The uniqueness of reproduction is thus central to the definition of species. It is therefore only natural that we find a wide variety of unique reproductive traits across species. These variations can come in a range of different forms, be it the anatomy of the genital system, morphology of gametes, presence or absence of accessory glands, mechanisms of ovulation, variations in the active hormones, duration of reproductive cycle and gestation and many other aspects of reproductive biology. It is also not surprising that great differences between species are found when it comes to the reaction of their gametes, embryos and tissues to the process of cryopreservation. Thus, when a successful cryopreservation protocol has been devised for a certain species, it will not necessarily be successful in other members of the same family, not to mention species that are phylogenetically further apart. In addition, to date the process of cryopreservation and the mechanisms that cause chilling- and cryodamages are not fully understood. Each new species we approach is thus a *terra incognita* and should be thoroughly studied before a successful protocol can be developed, if at all. Model species are often used for the development of basic techniques but in the end vast experimentation should be conducted in the target species. While this is relatively simple in domestic and laboratory animals, when a rare and endangered species is the target, opportunities to obtain gametes and other relevant cells and tissues are rare and far apart in terms of time and space, making progress extremely slow or practically impossible.
