**4. Options equally good for both males and females**

Some options, as will be discussed in the following sections, are available for both sexes. These options are still largely experimental in nature, their efficiency is often low and they require well equipped laboratories with highly experienced staff so their widespread implementation in wildlife conservation is probably still years down the road. They are, however, worthy of mentioning because of the great potential they hold. These, and many of the options described in the previous sections, are not and may never become widely used techniques. They are also nowhere near the decades old slow freezing and vitrification and so, to be on the safe side one should probably opt for cryopreservation of gametes and embryos using one of the available techniques. However, by definition endangered species are species whose global population is small and declining. This means that with time the genetic diversity of such populations is dwindling. If we do not set up collections of samples

access. Of course ovary transplanted to another location can produce oocytes that should be harvested for use *in vitro*. First whole ovary cryopreservation reported was in sheep (Revel et al., 2001; Revel et al., 2004). This report used directional freezing technique, which is claimed to provide a solution to many of the issues involved in large volume cryopreservation mentioned above (Arav & Natan, 2009). Most other cryopreservation experiments used controlled-rate freezing equipment to achieve the desired very slow (~0.1ºC/min) cooling rate needed. This first report was followed by reports on cryopreserving ovaries of various other species such as rats (Wang et al., 2002; Qi et al., 2008), mice (Liu et al., 2008), bovine (Arav, 2003), pigs (Imhof et al., 2004), human (Bedaiwy et al., 2006) and another study on sheep (Onions et al., 2009) . In some of these studies, pregnancies were achieved and live young were produced. Interestingly, to date transplantation of cryopreserved whole human ovary has not been reported (Bromer & Patrizio, 2009) despite the fact that ovarian transplantation has been in practice for several years now and whole human ovary cryopreservation was attempted by several researchers. Although vitrification is an attractive procedure for cryopreservation of whole ovaries, the current knowledge in cryobiology is insufficient to overcome the multiple problems involved in large volume vitrification (Fahy et al., 1990), primarily when tissue, rather than suspension, is involved. Keeping in mind the relationship between the three factors determining the probability of vitrification mentioned earlier (see section on semen vitrification and also Saragusty & Arav, 2011), to avoid cryoprotectant toxicity, very high cooling rates and very small sample volume are needed. Attempts at whole ovary vitrification did take place and in some cases, when the ovaries were sufficiently small, were even successful. An attempt to vitrify whole sheep ovary resulted in complete loss of all follicles (Courbiere et al., 2009). On the other hand, in studies on mice and rats, vitrification of whole ovary was successful (Migishima et al., 2003; Hoshina et al., 2009). One study showed acceptable post warming viability by *in vitro* evaluations of mice ovaries (Migishima et al., 2003). In another study follicular growth was demonstrated after autotransplantation under the kidney capsule of vitrified warmed rat ovaries (Sugimoto et al., 2000). In yet another study, live offspring were produced when the donor mice were transgenic so that their ovaries expressed anti-freeze

protein type III as an additional mean of cryoprotection (Bagis et al., 2008).

**4. Options equally good for both males and females** 

With the big potential whole ovary cryopreservation holds for wildlife conservation, this procedure is yet to be reported in any animal other than laboratory or domestic species.

Some options, as will be discussed in the following sections, are available for both sexes. These options are still largely experimental in nature, their efficiency is often low and they require well equipped laboratories with highly experienced staff so their widespread implementation in wildlife conservation is probably still years down the road. They are, however, worthy of mentioning because of the great potential they hold. These, and many of the options described in the previous sections, are not and may never become widely used techniques. They are also nowhere near the decades old slow freezing and vitrification and so, to be on the safe side one should probably opt for cryopreservation of gametes and embryos using one of the available techniques. However, by definition endangered species are species whose global population is small and declining. This means that with time the genetic diversity of such populations is dwindling. If we do not set up collections of samples (gametes, embryos, somatic cells, or anything else we can put your hands on) of the genetic diversity, and just sit and wait for some new technology to come by or for breakthrough in one of the still experimental technologies at hand, genetic diversity within species and possibly entire species will be lost for ever. We should therefore aim to create banks that will hold samples from each endangered species on earth and of as wide a diversity of genetic make up as possible in each. Cryopreservation is a more mature technology for this purpose but many other options are advancing and may one day play an important role in long-term banking for wildlife conservation. New and much better technologies may emerge with time but we cannot sit and watch species going extinct and take no action. Collections should be created with any and all possible technologies in mind.

#### **4.1 Somatic cells cryopreservation for SCNT**

To produce embryos *in vivo* or *in vitro*, conspecific spermatozoa and good quality oocytes are required, both or either of which often prove very difficult to obtain. An alternative that can circumvent this, at least in part, is preservation of somatic cells, to be later used for somatic cell nuclear transfer (SCNT, Wilmut et al., 1997). In SCNT, also known as cloning, nucleus of a somatic cell is microinjected into enucleated oocyte, which is then grown *in vitro* and can be later transferred to recipient females for development to term, with or without a cryopreservation step in between. Somatic cells from a wide variety of sources can be used for this purpose. Such diverse sources include cells from tissues preserved without cryoprotectant at -80ºC for more than a decade, or cells from tissues kept at -20ºC for as long as 16 years (Hoshino et al., 2009), cells isolated from mummified animals (Kato et al., 2009), freeze-dried somatic cells (Loi et al., 2008a; Ono et al., 2008; see next section), semen-derived somatic cells (Nel-Themaat et al., 2008a; Nel-Themaat et al., 2008b; Liu et al., 2010), cells collected postmortem (Oh et al., 2008), cell line (Campbell et al., 1996), and of course both fetal and adult cells are suitable for this purpose (Wilmut et al., 1997). SCNT has indeed an obvious potential for the multiplication of rare genotypes (Corley-Smith & Brandhorst, 1999; Loi et al., 2008a; Loi et al., 2008b), but its wide application is prevented by the currently low efficiency in terms of offspring outcome. To date, successful cloning was reported in sheep (Campbell et al., 1996; Wilmut et al., 1997; Loi et al., 2008a; Loi et al., 2008b), cow (Cibelli et al., 1998), mice (Wakayama & Yanagimachi, 1998), goat (Baguisi et al., 1999), pigs (Polejaeva et al., 2000), cats (Shin et al., 2002), dogs (Jang et al., 2007), rabbits (Chesne et al., 2002), ferrets (Li et al., 2006), mule (Woods et al., 2003), horse (Galli et al., 2003), gaur (*Bos gaurus*) (Lanza et al., 2000), buffalo (*Bubalus bubalis*) (Lu et al., 2005; Shi et al., 2007), mouflon (*Ovis orientalis musimon*) (Loi et al., 2001), African wild cat (*Felis silvestris libica*) (Gómez et al., 2003), wolves (*Canis lupus*) (Kim et al., 2007), mountain bongo antelope (*Tragelaphus euryceros isaaci*) (Lee et al., 2003) and eland (*Taurotragus oryx*) (Nel-Themaat et al., 2008b). When dealing with already extinct species, we can anticipate survival of nucleus DNA but not for viable oocytes. The only hope is then to use oocytes from closely related species. Interspecies SCNT (ISCNT), performed by injecting the nucleus from one species into the oocyte of another has also been carried out in a variety of species (for a recent review see Loi et al., 2011a). These include ISCNT from the endangered mouflon to a domestic sheep (*Ovis aries*) (Loi et al., 2001), from red panda (*Ailurus fulgens*) to rabbit (Tao et al., 2009), from sand cat (*Felis margarita*) to domestic cat (Gómez et al., 2008), from Canada lynx (*Lynx canadensis*) to both domestic cat and caracal (*Caracal caracal*) (Gómez et al., 2009), from water buffalo (*Bubalus bubalis*) to cow (*Bos taurus*) (Srirattana et al., 2011), and most strikingly – from a

Genome Banking for Vertebrates Wildlife Conservation 333

granulosa cells, kept at room temperature for 3 years, were used to direct embryonic development following nuclear transfer into *in vitro* matured enucleated oocytes. The reconstructed oocytes initiated cleavage at similar rates to control embryos generated using fresh granulosa cells. Microsatellite DNA analysis of the cloned blastocysts matched perfectly with the lyophilized donor cells. Later, these results were confirmed by other researchers studying mouse granulosa cells (Ono et al., 2008), human hematopoietic stem and progenitor cells (Buchanan et al., 2010) or porcine fetal fibroblasts (Das et al., 2010). These studies demonstrate for the first time that dry cells maintain the development potential when injected into enucleated oocytes. Naturally, we still have a long way to go before live

Embryos can be a source for primordial germ cells (PGC) which, as was shown in the zebrafish, can be vitrified, warmed and then transplanted into sterilized recipient blastulae to differentiate into males and females that produced gametes carrying the genetic material of the transplanted PGC donor (Higaki et al., 2010). Such PGC can be transplanted, along with gonadal somatic cells, and develop into normal male or female gonadal tissue with normal spermatogenesis or oogenesis. Both mouse round spermatids and GV oocytes derived from such tissues were able to direct embryonic development to term following ICSI (Matoba & Ogura, 2010). In a recent study on felids (Silva et al., 2011) it was shown that such germ line stem cells can be transplanted to the gonads of a different species and still develop normal early stage gametes. In that study, ocelot (*Leopardus pardalis*) spermatogonial stem cells were transplanted into domestic cat testis and thirteen weeks

Going even earlier in the development timeline, embryos can be a source for stem cells. Embryonic stem cells, being pluripotent, can differentiate *in vivo* or *in vitro* into germ cells. They can also be used for nuclear transfer. So, they, too, can be considered an optional venue. In a study on mice, transplanted embryonic stem cells were able to form testicular tissue structures and direct spermatogenesis (Toyooka et al., 2003). These cells, which can be isolated from embryos, can also be cryopreserved (Thomson et al., 1998; Toyooka et al., 2003) or vitrified (Reubinoff et al., 2001; He et al., 2008). Such stem cells can also be derived from embryos generated by nuclear transfer of freeze-dried cells (Ono et al., 2008). Embryonic stem cells can also be derived from isolated blastomeres, and blastomers can also be cryopreserved individually by inserting them into emptied zona pellucida and then vitrifying them (Escriba et al., 2010). If embryonic stem cells are not available, somatic cells can be induced to become embryonic stem cells-like (Takahashi & Yamanaka, 2006), also known as induced pluripotent stem cells or iPS cells (for recent review see: Cox & Rizzino, 2010). Being pluripotent in nature, they are also germ line competent (Okita et al., 2007) and

The fantastic options mentioned above are theoretical and speculative in nature when it comes to wildlife preservation as currently these techniques are in their infancy and were adapted thus far only to laboratory animals, and even in these the unknown is still vast.

With the dramatically accelerated species extinction rate we see in recent decades, it is our obligation to seek any possible venue to bring this biodiversity loss to a halt and, while

offspring will be generated using this technology but the potential is there.

later ocelot spermatozoa were retrieved from the cat's epididymis.

as such can give rise to germ cells of both male and female.

**4.3 Stem cell preservation** 

**5. Conclusion** 

15,000 year-old wooly mammoth (*Mammuthus primigenius*) to a mouse (Kato et al., 2009). While this technique holds much promise for the resurrection of extinct species and saving those on their way there (Loi et al., 2011b), with the exception of a few sporadic instances, all these attempts at ISCNT did not result in live offspring. Cryopreservation of reproductive tissue or any other viable body tissue or, alternatively, of *in vitro* grown cell cultures is routinely done in many places around the world and enough cells survive the process to be used in SCNT. Furthermore, obtaining tissue samples is usually much simpler than collecting gametes or embryos, so a larger and more diverse collection can be accumulated.

While SCNT has the advantage that no genetic drift takes place because recombination does not occur, when considering SCNT for wildlife species preservation, several important issues should be taken into consideration. First, as mentioned above, suitable enucleated oocytes are required. The availability of such oocytes and the ability to access them should thus be part of the program (Loi et al., 2011b). If conspecific oocytes are not available, the issues of mitochondrial inheritance and nucleus-cytoplasmic incompatibility become a problem and ways to overcome these should be sought for. When the donor and recipient are close enough, some of the donor mitochondria get transferred as well (Gómez et al., 2009; Srirattana et al., 2011). As was demonstrated for the famous sheep, Dolly, the telomere is shorter following SCNT (Shiels et al., 1999). Interestingly, it was recently shown that cloned cows with short telomeres produce normal and healthy offspring with normal telomere length following artificial insemination with sperm from normal bulls (Miyashita et al., 2011). This study suggests that cloning does not interfere with the eventual function of the germ line. Cloned offspring, however, are known to show elevated prevalence of developmental abnormalities and high mortality rate, issues that should be kept in mind when initiating a cloning program (e.g. Lanza et al., 2000). One should also keep in mind that the spermatozoa carry more than just genetic material. They come with a whole load of epigenetic factors important for proper embryonic development (Yamauchi et al., 2011). These are missing when SCNT is performed and might be one of the causes behind the relatively low efficiency of the process. As with cryopreservation of other cells and tissues, storage space and costs and environmental impact are major issue pertaining to liquid nitrogen storage so a cheaper alternative would be very attractive for long-term conservation purposes.
