**3. The female**

304 Current Frontiers in Cryobiology

spermatozoa or spermatids retrieved from reproductive tissues (whole testes or epididymides) frozen for up to one year at -80ºC or from whole mice frozen at -20ºC for up to 15 years, can produce normal offspring when used, through ICSI, to fertilize mature oocytes (Ogonuki et al., 2006). This success followed a previous, failed, attempt to cryopreserve the entire testis (Yin et al., 2003). The other option is to cryopreserve testicular tissue slices. This technique is widely used today in both adult and pediatric human medicine as a mean to preserve fertility of patients undergoing cancer treatments. To cryopreserve the tissue, it is cut into tiny pieces, usually in the range of 1-2 mm3 to ensure cryoprotectant penetration, efficient heat transfer and eventual successful grafting. Other alternatives that have been proposed are to mince the tissue and then suspend it in freezing extender to achieve better cryoprotection (Crabbe et al., 1999) or to cut the testicular tissue into thin stripes (e.g. ~9×5×1 mm in sheep) to increase the total number of seminiferous tubules in each graft (Rodriguez-Sosa et al., 2010). Although such tissue samples can be obtained from every individual, infant, juvenile or adult, almost all successful studies to date used immature tissue (Ehmcke & Schlatt, 2008). Like in semen cryopreservation, there are differences between species in the reaction of their testicular tissue to cryoprotectants, chilling and cryopreservation (Schlatt et al., 2002b). The preserved testicular tissue can be handled in several ways. From these tissues, spermatozoa, spermatocytes and round and elongated spermatids can all be retrieved and used to fertilize oocytes through ICSI (Hovatta et al., 1996; Gianaroli et al., 1999). Testicular tissue can also be transplanted back to the donating individual (autografting), to another individual of the same species (allografting) or to individual of a different species, usually to nude or immunodeficient mice (xenografting). After transplantation, the graft may be lost due to tissue rejection or ischemia. If it manages to survive the critical first few days, blood supply will reach the graft, it will be supported by the recipient system and, after some time, will start producing spermatozoa, which can be harvested by surgical excision of all or part of the graft (Schlatt et al., 2002b). Although dependent on the recipient system for support, the spermatogenesis cycle length is assumed to be inherent to the spermatogonial stem cells, which are expected to preserve the donating species spermatogenesis length (Zeng et al., 2006). However other studies showed that in some species, the process is accelerated when their testicular tissue was xenografted into mice (rhesus monkeys; Honaramooz et al., 2004) while in others it is not (domestic cat; Snedaker et al., 2004). Acceleration, when identified, bears special interest for species preservation as it can shorten generation time and thus speed up population growth. This acceleration, however, may also mean abnormal spermatogenesis process that produces abnormal gametes. The sperm produced this way does not go through epididymal maturation process so the only way it can be utilized is by ICSI (Shinohara et al., 2002). One should also keep in mind that it is very costly to keep immunodeficient mice and handle them under germ-free conditions and, of course, repeated transplantations from one mouse to another are required to maintain viable tissue for many years. Still, testicular tissue cryopreservation was done in several species and pregnancies were achieved in mice (Schlatt et al., 2002b; Shinohara et al., 2002), rabbit (Shinohara et al., 2002), human (Hovatta et al., 1996), Djungarian hamsters (Schlatt et al., 2002b) and marmoset monkeys (Schlatt et al., 2002b), to name a few. Testicular tissue can also be cultured *in vitro* to give rise to mature and competent cells. Culture conditions, however, are very complex and, until recently, attempts were encouraging but still unsuccessful (Gohbara et al., 2010). Earlier this year,

In comparison to the male, females' gametes pose several difficulties when it comes to preservation (Table 1). Very small number of gametes is progressing to the more advanced developmental stages during each cycle, and at best only a handful mature and ovulate. When dealing with rare and endangered species in which the number of available individuals for research is extremely limited and often spatially and temporally far apart, progress is very slow and limited by the small numbers. Oocytes and embryos are orders of magnitude larger than spermatozoa, thus bringing down the ratio between surface area and volume. The outcome is slower movement of water and cryoprotectants across the cellular membrane and elevated risk for intracellular ice formation. Unlike in males, *in vivo* collection of oocytes, and to a lesser extent - embryos, is an invasive procedure requiring anesthesia or sedation. Although production of new oocytes exists even in adulthood (Niikura et al., 2009; Tilly et al., 2009), it is very minimal. So, in general terms, the female can be considered as if it is born with a limited life-long supply. Males on the other hand produce sperm continuously, throughout their adult life, sperm that can be collected relatively easy almost any time. All these differences contribute to the fact that while the number of species in which sperm was cryopreserved is in the hundreds, the number of species in which embryo cryopreservation was reported (not all successful) is currently less than 50 and the number of species in which oocyte cryopreservation was attempted is far less than that.


Table 1. comparison between male and female gametes in relation to gamete cryopreservation.

Female gametes can be collected at different time points in their maturation process: 1) as mature oocytes, following ovulation (natural or chemically induced), 2) as mature and immature oocytes, by ovum pick up, either transabdominally, transvaginally or

Genome Banking for Vertebrates Wildlife Conservation 307

(Ptak et al., 2002) or between some small cat species and the domestic cat (Herrick et al., 2010) or even between a cat and a mouse (Xu et al., 2011). To enhance the number of oocytes collected at any ovum pick-up procedure, hormonal stimulation can be used. This, however, will result in both mature and immature oocytes and the quality of both may be compromised (Blondin et al., 1996; Moor et al., 1998; Takagi et al., 2001). Although to date no morphological or other method is able to accurately predict which oocytes have optimal developmental potential (Coticchio et al., 2004), it is clear that oocyte quality is a major determining factor in the success of IVF (Coticchio et al., 2004; Krisher, 2004; Combelles & Racowsky, 2005), early embryonic survival, the establishment and maintenance of pregnancy, fetal development, and even adult disease (reviewed in Krisher, 2004). Once all these hurdles have been overcome and while keeping in mind the importance of oocyte

Oocytes are very different from sperm or embryos with respect to cryopreservation. Oocytes (and embryos) are in the range of three to four orders of magnitude larger than spermatozoa, thus substantially increasing their surface-to-volume ratio and making them sensitive to chilling and susceptible to intracellular ice formation (Arav et al., 1996; Zeron et al., 1999; Chen & Yang, 2009). Oocytes at the MII stage also have a formed fuse that is chilling-sensitive (Chen & Yang, 2009) and their plasma membrane has low (temperature dependent) permeability coefficient, thus making the movement of cryoprotectants and water slower (Jackowski et al., 1980; Ruffing et al., 1993). This, however, may vary between species. Membrane permeability increases after fertilization (Jackowski et al., 1980) and seem to be higher in morula/blastocyst stages as compared to earlier embryonic stages (Jin et al., 2011), thus contributing to the fact that embryos are easier to cryopreserve. The oocyte cytoskeleton is highly sensitive to chilling and gets disorganized at suboptimal temperatures (Trounson & Kirby, 1989). Oocytes also have high cytoplasmic lipid content which increases chilling sensitivity (Ruffing et al., 1993). They have less submembranous actin microtubules (Gook et al., 1993) making their membrane less robust. The meiotic spindle, which has formed by the MII stage, is very sensitive to chilling and may be compromised as well (Ciotti et al., 2009) resulting in uneuploidy (Sathananthan et al., 1988) and oocytes are more susceptible to the damaging effects of reactive oxygen species (Gupta et al., 2010). Many of these parameters change after fertilization, making embryos less chilling sensitive and easier to cryopreserve (Jackowski et al., 1980; Gook et al., 1993; Fabbri et al., 2000). Despite many advances in the field of cryopreservation, oocyte (ovulated, mature or immature) cryopreservation still has a long way to go before it can be routinely utilized in many species. Even in human medicine, fewer than 200 births resulting from cryopreserved oocytes were reported as of 2007 (Edgar & Gook), a number that went up to around 500 by 2009 (Nagy et al.). Yet, despite all these difficulties, some success in oocyte cryopreservation

Two main cryopreservation techniques are used for oocyte cryopreservation – slow (equilibrium) freezing and vitrification. In slow freezing, oocytes are exposed to permeating cryoprotectants in the range of 1.0-1.5 M and are frozen, following equilibration and seeding, at a rate of 0.3ºC to 0.5ºC per minute down to -30ºC or lower. Once at the desired temperature they are plunged into liquid nitrogen to vitrify the intra- and extracellular still unfrozen compartments and for storage. Attempts to improve outcome by altering the components of the freezing extender (e.g. replacing sodium chloride with choline chloride; Stachecki et al., 1998a; Stachecki et al., 1998b; Quintans et al., 2002) suggest that there is still some room for improvements in the standard techniques widely in use. Vitrification usually exposes the oocytes to substantially higher concentration of cryoprotectants, in the range of

quality, the next major hurdle to overcome is oocyte cryopreservation.

has been reported.

transrectally. This can be done during natural estrus cycle or following chemical stimulation, 3) at all developmental stages, mostly immature, following ovariectomy, either when neutering the animal or post mortem, a possibility with time constraint because deterioration is fast *in vitro* and even faster *in vivo* – reasonable quality oocytes can be harvested only up to ~24h after the removal of the ovaries if they were held at 4ºC (Wood et al., 1997; Cleary et al., 2001; Personal experience), 4) after fertilization (natural or by artificial insemination), as embryos. This can be done at any stage prior to implantation. The collected oocytes can be at any level of maturation including oocytes found in primordial, preantral or antral follicles, each presenting its own special requirements and sensitivities. Harvesting and preserving oocytes is almost pointless if all other associated assisted reproductive technologies – *in vitro* maturation, *in vitro* fertilization, *in vitro* culture and embryo transfer, are not mastered (at present or in the future) to support it. Female fertility preservation can be done through preservation of oocytes and/or embryos at various developmental stages, as well as by preservation of ovarian tissue or entire ovaries, all of which will be discussed in details in the following sections.

#### **3.1 Oocyte cryopreservation**

For decades it was believed that females are born with their life supply of oocytes in their ovaries, all dormant at a very early maturation stage (Zuckerman, 1951). This dogma, however, was recently challenged by a number of studies suggesting that the female gonads retain the ability to regenerate oocytes throughout adulthood, albeit at a very limited number (e.g. Niikura et al., 2009; and reviewed in Tilly et al., 2009). The vast majority of oocytes, however, is already in the ovaries at birth and remains dormant at a very early stage of maturation to adulthood and beyond. Once the female reaches puberty, one or more cohorts of oocytes are selected at each estrus cycle to progress in the maturation process and, depending on the species, one or several oocytes are ovulated. The remaining oocytes in these selected cohorts degenerate or luteinize to form accessory corpora lutea. To be fertilized, an oocyte needs to overcome the meiotic block and progress to the metaphase II (MII) stage of maturation or else only very few oocytes will fertilize (Luvoni & Pellizzari, 2000). Thus, an *in vitro* maturation procedure should be in hand to handle immature oocytes. This process is currently developed for only a handful of species and even for these success is often fairly limited (Krisher, 2004). Furthermore, collection of immature oocytes disrupts the natural maturation process and thus compromises the quality of the oocytes even if they are later matured *in vitro*. During oocyte maturation and follicular growth, the oocyte accumulates large quantities of mRNA and proteins needed for the continuation of meiosis, fertilization and embryonic development. In the absence of the entire supporting system in the *in vitro* culture, production of some of these needed components is hampered. The resulting mature oocytes are therefore of inferior quality when compared to *in vivo* matured oocytes. In seasonal animals, oocytes collected out of the season may show resistance to IVM and IVF (Spindler et al., 2000; Berg & Asher, 2003; Comizzoli et al., 2003). In red deer for example, while about 15% of cleaved oocytes collected during the season (April-July) developed *in vitro* to blastocysts, none have developed if collected after July (Berg & Asher, 2003). Comizzoli et al. (2003) showed that anti-oxidants and FSH in the culture media can overcome this problem in the domestic cat model they have studied. Naturally, *in vitro* fertilization and culture should also be developed so that embryos can be generated for transfer. During the development of such techniques, as well as in those cases when conspecific oocytes are not available, interspecific IVF can be considered. This was done, for example between the mouflon (*Ovis orientalis musimon*) and the domestic sheep

transrectally. This can be done during natural estrus cycle or following chemical stimulation, 3) at all developmental stages, mostly immature, following ovariectomy, either when neutering the animal or post mortem, a possibility with time constraint because deterioration is fast *in vitro* and even faster *in vivo* – reasonable quality oocytes can be harvested only up to ~24h after the removal of the ovaries if they were held at 4ºC (Wood et al., 1997; Cleary et al., 2001; Personal experience), 4) after fertilization (natural or by artificial insemination), as embryos. This can be done at any stage prior to implantation. The collected oocytes can be at any level of maturation including oocytes found in primordial, preantral or antral follicles, each presenting its own special requirements and sensitivities. Harvesting and preserving oocytes is almost pointless if all other associated assisted reproductive technologies – *in vitro* maturation, *in vitro* fertilization, *in vitro* culture and embryo transfer, are not mastered (at present or in the future) to support it. Female fertility preservation can be done through preservation of oocytes and/or embryos at various developmental stages, as well as by preservation of ovarian tissue or entire ovaries, all of

For decades it was believed that females are born with their life supply of oocytes in their ovaries, all dormant at a very early maturation stage (Zuckerman, 1951). This dogma, however, was recently challenged by a number of studies suggesting that the female gonads retain the ability to regenerate oocytes throughout adulthood, albeit at a very limited number (e.g. Niikura et al., 2009; and reviewed in Tilly et al., 2009). The vast majority of oocytes, however, is already in the ovaries at birth and remains dormant at a very early stage of maturation to adulthood and beyond. Once the female reaches puberty, one or more cohorts of oocytes are selected at each estrus cycle to progress in the maturation process and, depending on the species, one or several oocytes are ovulated. The remaining oocytes in these selected cohorts degenerate or luteinize to form accessory corpora lutea. To be fertilized, an oocyte needs to overcome the meiotic block and progress to the metaphase II (MII) stage of maturation or else only very few oocytes will fertilize (Luvoni & Pellizzari, 2000). Thus, an *in vitro* maturation procedure should be in hand to handle immature oocytes. This process is currently developed for only a handful of species and even for these success is often fairly limited (Krisher, 2004). Furthermore, collection of immature oocytes disrupts the natural maturation process and thus compromises the quality of the oocytes even if they are later matured *in vitro*. During oocyte maturation and follicular growth, the oocyte accumulates large quantities of mRNA and proteins needed for the continuation of meiosis, fertilization and embryonic development. In the absence of the entire supporting system in the *in vitro* culture, production of some of these needed components is hampered. The resulting mature oocytes are therefore of inferior quality when compared to *in vivo* matured oocytes. In seasonal animals, oocytes collected out of the season may show resistance to IVM and IVF (Spindler et al., 2000; Berg & Asher, 2003; Comizzoli et al., 2003). In red deer for example, while about 15% of cleaved oocytes collected during the season (April-July) developed *in vitro* to blastocysts, none have developed if collected after July (Berg & Asher, 2003). Comizzoli et al. (2003) showed that anti-oxidants and FSH in the culture media can overcome this problem in the domestic cat model they have studied. Naturally, *in vitro* fertilization and culture should also be developed so that embryos can be generated for transfer. During the development of such techniques, as well as in those cases when conspecific oocytes are not available, interspecific IVF can be considered. This was done, for example between the mouflon (*Ovis orientalis musimon*) and the domestic sheep

which will be discussed in details in the following sections.

**3.1 Oocyte cryopreservation** 

(Ptak et al., 2002) or between some small cat species and the domestic cat (Herrick et al., 2010) or even between a cat and a mouse (Xu et al., 2011). To enhance the number of oocytes collected at any ovum pick-up procedure, hormonal stimulation can be used. This, however, will result in both mature and immature oocytes and the quality of both may be compromised (Blondin et al., 1996; Moor et al., 1998; Takagi et al., 2001). Although to date no morphological or other method is able to accurately predict which oocytes have optimal developmental potential (Coticchio et al., 2004), it is clear that oocyte quality is a major determining factor in the success of IVF (Coticchio et al., 2004; Krisher, 2004; Combelles & Racowsky, 2005), early embryonic survival, the establishment and maintenance of pregnancy, fetal development, and even adult disease (reviewed in Krisher, 2004). Once all these hurdles have been overcome and while keeping in mind the importance of oocyte quality, the next major hurdle to overcome is oocyte cryopreservation.

Oocytes are very different from sperm or embryos with respect to cryopreservation. Oocytes (and embryos) are in the range of three to four orders of magnitude larger than spermatozoa, thus substantially increasing their surface-to-volume ratio and making them sensitive to chilling and susceptible to intracellular ice formation (Arav et al., 1996; Zeron et al., 1999; Chen & Yang, 2009). Oocytes at the MII stage also have a formed fuse that is chilling-sensitive (Chen & Yang, 2009) and their plasma membrane has low (temperature dependent) permeability coefficient, thus making the movement of cryoprotectants and water slower (Jackowski et al., 1980; Ruffing et al., 1993). This, however, may vary between species. Membrane permeability increases after fertilization (Jackowski et al., 1980) and seem to be higher in morula/blastocyst stages as compared to earlier embryonic stages (Jin et al., 2011), thus contributing to the fact that embryos are easier to cryopreserve. The oocyte cytoskeleton is highly sensitive to chilling and gets disorganized at suboptimal temperatures (Trounson & Kirby, 1989). Oocytes also have high cytoplasmic lipid content which increases chilling sensitivity (Ruffing et al., 1993). They have less submembranous actin microtubules (Gook et al., 1993) making their membrane less robust. The meiotic spindle, which has formed by the MII stage, is very sensitive to chilling and may be compromised as well (Ciotti et al., 2009) resulting in uneuploidy (Sathananthan et al., 1988) and oocytes are more susceptible to the damaging effects of reactive oxygen species (Gupta et al., 2010). Many of these parameters change after fertilization, making embryos less chilling sensitive and easier to cryopreserve (Jackowski et al., 1980; Gook et al., 1993; Fabbri et al., 2000). Despite many advances in the field of cryopreservation, oocyte (ovulated, mature or immature) cryopreservation still has a long way to go before it can be routinely utilized in many species. Even in human medicine, fewer than 200 births resulting from cryopreserved oocytes were reported as of 2007 (Edgar & Gook), a number that went up to around 500 by 2009 (Nagy et al.). Yet, despite all these difficulties, some success in oocyte cryopreservation has been reported.

Two main cryopreservation techniques are used for oocyte cryopreservation – slow (equilibrium) freezing and vitrification. In slow freezing, oocytes are exposed to permeating cryoprotectants in the range of 1.0-1.5 M and are frozen, following equilibration and seeding, at a rate of 0.3ºC to 0.5ºC per minute down to -30ºC or lower. Once at the desired temperature they are plunged into liquid nitrogen to vitrify the intra- and extracellular still unfrozen compartments and for storage. Attempts to improve outcome by altering the components of the freezing extender (e.g. replacing sodium chloride with choline chloride; Stachecki et al., 1998a; Stachecki et al., 1998b; Quintans et al., 2002) suggest that there is still some room for improvements in the standard techniques widely in use. Vitrification usually exposes the oocytes to substantially higher concentration of cryoprotectants, in the range of

Genome Banking for Vertebrates Wildlife Conservation 309

(*Balaenoptera bonaerensis*) with the Cryotop producing better results in post-warming morphology and rate of maturation (Iwayama et al., 2005) and both carrier systems produced better results compared to an earlier attempt to cryopreserve minke whale oocytes by slow freezing (Asada et al., 2000). Oocytes of the Mexican gray wolf (*Canis lupus baileyi*) and the domestic dog were also vitrified recently using the Cryotop carrier system (Boutelle et al., 2011). Post warming viability was 61% of intact dog oocytes and 57% of intact wolf cells. Open systems were also used to vitrify granulosa-oocyte complexes (GOC) from primary follicles of marsupials. In two different studies the fat-tailed dunnart (*Sminthopsis crassicaudata*) (Czarny et al., 2009b) and the Tasmanian devil (*Sarcophilus harrisii*) (Czarny & Rodger, 2010) GOC were vitrified in self-made OPS. Post-warming viability was about 70% in both studies. Immature oocytes of the lowland gorilla (*Gorilla gorilla gorilla*) were also cryopreserved, using slow freezing. Of the thawed oocytes, 4/6 were morphologically degenerated, one arrested at the GV stage and the other progressed to the MI stage and then arrested (Lanzendorf, 1992). Immature oocytes of chousingha (*Tetracerus quadricorni*) were also vitrified using the OPS as a carrier system but post warming maturation rate (29.4%) was considerably lower than that of fresh oocytes (69.3%) (Rao et al., 2011). What unifies all these studies is the fact that only small number of oocytes were cryopreserved and only *in* 

Species Model Liquid nitrogen Nitrogen slush Sig. Refernce

Bovine MII oocytes 28% cleavage 48% cleavage P<0.05 (Arav &

Ovine GV-oocytes 25% survival 5% survival P<0.05 (Isachenko

Rabbit MII oocytes 83% survive 82% survive NS (Cai et al.,

Bovine MII oocytes 39% survive 48% survive P<0.05 (Santos et

Mouse Blastocysts 10% survive 54% survive P<0.05 (Yavin et al.,

Rabbit Morulae 83% develop 92% develop P<0.05 (Papis et al.,

Mouse MII oocytes 45% survive 90% survive P<0.001 (Lee et al.,

Human MII oocyte 56% survive 92% survive P<0.05 (Criado et

Table 2. When oocytes or embryos from various species were vitrified in liquid nitrogen slush in comparison to regular vitrification, results either showed no difference or, more

Porcine Blastocysts 62% survive 83% survive P<0.05

blastocysts 77% survive 95% survive NS (Cuello et

with biopsy 50% survive 87% survive P<0.05 (Lee et al.,

Bovine MII oocytes 40% cleavage 25% cleavage NS (Martino et

al., 1996)

Zeron, 1997)

et al., 2001)

al., 2004)

al., 2006)

2007)

2009)

2009)

2010)

al., 2010)

2005)

*vitro* post thaw / warming evaluations were conducted.

Porcine Early

Mouse 4-cell embryo

NS = not significant.

frequently, that slush was superior.

5.0 to 7.0 M, and cryopreservation is done at cooling rates of 2,500ºC per minute or more, depending on the technique used. Vitrification can, however, be achieved even at cryoprotectant concentrations similar to those used for slow freezing if sample volume is small enough and/or cooling rate is high enough to achieve vitrification. One advantage of vitrification over slow freezing, when oocytes are concerned, is the higher survival rate that the fast cooling facilitates. To achieve very high cooling rates, a wide variety of carrier systems were developed (reviewed by Saragusty & Arav, 2011). The small-volume sample, with the carrier, is plunged directly into liquid nitrogen or nitrogen slush. For vitrification to be successful, one should be highly experienced in handling the oocytes throughout the dilution process and in loading them onto or into the carrier system. By cooling liquid nitrogen from its boiling temperature (-196ºC) to close to its freezing temperature (-210ºC), nitrogen slush is formed. Vitrification in slush gives at least two major advantages. When a sample is inserted into liquid nitrogen, the nitrogen boils and forms an insulation vapor layer around the sample (the Leidenfrost effect). Boiling is considerably reduced when slush is used. Slush also significantly increases the cooling rate. Several studies have demonstrated the superiority of slush over liquid nitrogen (Arav & Zeron, 1997; Isachenko et al., 2001; Beebe et al., 2005; Santos et al., 2006; Lee et al., 2007; Criado et al., 2010) but some found little or no difference (Martino et al., 1996; Cuello et al., 2004; Cai et al., 2005) (Table 2). When cooled at such high cooling rates, oocytes spend very short interval at their lipid phase transition temperature, thus avoiding, or at least minimizing, chilling injury (Arav et al., 1996). Vitrification also reduces the loss of mRNA from the cryopreserved oocytes (Chamayou et al., 2011), mRNA that is crucial for embryonic development and beyond.

The first human pregnancy from cryopreserved (by slow freezing), *in vitro* fertilized oocyte was reported in 1986 (Chen, 1986) following success in other (laboratory) species that came a few years earlier, such as mice (Whittingham, 1977) and rat (Kasai et al., 1979) oocytes cryopreserved to -196ºC or mice oocytes frozen to -75ºC (Parkening et al., 1976). Still, despite several decades of research and many advances in the field, success is very limited and oocyte cryopreservation is still labeled as experimental even in human medicine (Noyes et al., 2010). Cryopreservation can cause cytoskeleton disorganization (Trounson & Kirby, 1989), chromosome and DNA abnormalities (Van Blerkom, 1989), spindle disintegration (Pickering & Johnson, 1987), plasma membrane disruption (Van Blerkom, 1989) and premature cortical granule exocytosis with its related zona pellucida hardening, making it impermeable to spermatozoa (Johnson et al., 1988). It also hamper, at least to some extant, the ability of oocytes to mature *in vitro* after thawing/warming (Rao et al., 2011). When comparing these parameters, in addition to survival rate, oocyte cryopreservation by vitrification seem to be superior to slow freezing, which explains why oocyte vitrification is gradually replacing slow freezing as the leading technique of preservation. Either open or closed carrier systems are used for vitrification. The closed systems are more secure while the open systems can provide higher cooling rates by direct exposure of the sample to liquid nitrogen. The large number of carrier systems (see Saragusty & Arav, 2011 for a most current list) suggests that the field is still developing and even decision if the open or the closed system is better is still under debate. While in most carrier systems, the volume that enables vitrification limits the number of oocytes that can be contained in it to just a few, some carrier systems such as the electron microscope grid (Steponkus et al., 1990) or nylon mesh (Matsumoto et al., 2001) allow simultaneous vitrification of a large number (as many as 65 in one study) of oocytes. Most reports on oocyte vitrification are, however, sporadic in nature and usually on small number of oocytes. The open system [Cryotop and Open Pulled Straw (OPS)] was used to vitrify germinal vesicle-stage oocytes of the minke whale

5.0 to 7.0 M, and cryopreservation is done at cooling rates of 2,500ºC per minute or more, depending on the technique used. Vitrification can, however, be achieved even at cryoprotectant concentrations similar to those used for slow freezing if sample volume is small enough and/or cooling rate is high enough to achieve vitrification. One advantage of vitrification over slow freezing, when oocytes are concerned, is the higher survival rate that the fast cooling facilitates. To achieve very high cooling rates, a wide variety of carrier systems were developed (reviewed by Saragusty & Arav, 2011). The small-volume sample, with the carrier, is plunged directly into liquid nitrogen or nitrogen slush. For vitrification to be successful, one should be highly experienced in handling the oocytes throughout the dilution process and in loading them onto or into the carrier system. By cooling liquid nitrogen from its boiling temperature (-196ºC) to close to its freezing temperature (-210ºC), nitrogen slush is formed. Vitrification in slush gives at least two major advantages. When a sample is inserted into liquid nitrogen, the nitrogen boils and forms an insulation vapor layer around the sample (the Leidenfrost effect). Boiling is considerably reduced when slush is used. Slush also significantly increases the cooling rate. Several studies have demonstrated the superiority of slush over liquid nitrogen (Arav & Zeron, 1997; Isachenko et al., 2001; Beebe et al., 2005; Santos et al., 2006; Lee et al., 2007; Criado et al., 2010) but some found little or no difference (Martino et al., 1996; Cuello et al., 2004; Cai et al., 2005) (Table 2). When cooled at such high cooling rates, oocytes spend very short interval at their lipid phase transition temperature, thus avoiding, or at least minimizing, chilling injury (Arav et al., 1996). Vitrification also reduces the loss of mRNA from the cryopreserved oocytes (Chamayou et al., 2011), mRNA that is crucial for embryonic development and beyond.

The first human pregnancy from cryopreserved (by slow freezing), *in vitro* fertilized oocyte was reported in 1986 (Chen, 1986) following success in other (laboratory) species that came a few years earlier, such as mice (Whittingham, 1977) and rat (Kasai et al., 1979) oocytes cryopreserved to -196ºC or mice oocytes frozen to -75ºC (Parkening et al., 1976). Still, despite several decades of research and many advances in the field, success is very limited and oocyte cryopreservation is still labeled as experimental even in human medicine (Noyes et al., 2010). Cryopreservation can cause cytoskeleton disorganization (Trounson & Kirby, 1989), chromosome and DNA abnormalities (Van Blerkom, 1989), spindle disintegration (Pickering & Johnson, 1987), plasma membrane disruption (Van Blerkom, 1989) and premature cortical granule exocytosis with its related zona pellucida hardening, making it impermeable to spermatozoa (Johnson et al., 1988). It also hamper, at least to some extant, the ability of oocytes to mature *in vitro* after thawing/warming (Rao et al., 2011). When comparing these parameters, in addition to survival rate, oocyte cryopreservation by vitrification seem to be superior to slow freezing, which explains why oocyte vitrification is gradually replacing slow freezing as the leading technique of preservation. Either open or closed carrier systems are used for vitrification. The closed systems are more secure while the open systems can provide higher cooling rates by direct exposure of the sample to liquid nitrogen. The large number of carrier systems (see Saragusty & Arav, 2011 for a most current list) suggests that the field is still developing and even decision if the open or the closed system is better is still under debate. While in most carrier systems, the volume that enables vitrification limits the number of oocytes that can be contained in it to just a few, some carrier systems such as the electron microscope grid (Steponkus et al., 1990) or nylon mesh (Matsumoto et al., 2001) allow simultaneous vitrification of a large number (as many as 65 in one study) of oocytes. Most reports on oocyte vitrification are, however, sporadic in nature and usually on small number of oocytes. The open system [Cryotop and Open Pulled Straw (OPS)] was used to vitrify germinal vesicle-stage oocytes of the minke whale (*Balaenoptera bonaerensis*) with the Cryotop producing better results in post-warming morphology and rate of maturation (Iwayama et al., 2005) and both carrier systems produced better results compared to an earlier attempt to cryopreserve minke whale oocytes by slow freezing (Asada et al., 2000). Oocytes of the Mexican gray wolf (*Canis lupus baileyi*) and the domestic dog were also vitrified recently using the Cryotop carrier system (Boutelle et al., 2011). Post warming viability was 61% of intact dog oocytes and 57% of intact wolf cells. Open systems were also used to vitrify granulosa-oocyte complexes (GOC) from primary follicles of marsupials. In two different studies the fat-tailed dunnart (*Sminthopsis crassicaudata*) (Czarny et al., 2009b) and the Tasmanian devil (*Sarcophilus harrisii*) (Czarny & Rodger, 2010) GOC were vitrified in self-made OPS. Post-warming viability was about 70% in both studies. Immature oocytes of the lowland gorilla (*Gorilla gorilla gorilla*) were also cryopreserved, using slow freezing. Of the thawed oocytes, 4/6 were morphologically degenerated, one arrested at the GV stage and the other progressed to the MI stage and then arrested (Lanzendorf, 1992). Immature oocytes of chousingha (*Tetracerus quadricorni*) were also vitrified using the OPS as a carrier system but post warming maturation rate (29.4%) was considerably lower than that of fresh oocytes (69.3%) (Rao et al., 2011). What unifies all these studies is the fact that only small number of oocytes were cryopreserved and only *in vitro* post thaw / warming evaluations were conducted.


NS = not significant.

Table 2. When oocytes or embryos from various species were vitrified in liquid nitrogen slush in comparison to regular vitrification, results either showed no difference or, more frequently, that slush was superior.

Genome Banking for Vertebrates Wildlife Conservation 311

thawing and transfer. This abstract, however, seem not to have been followed by a full peerreviewed manuscript so it is not clear if those zygotes really froze and resulted in pregnancies. In 1971 another report on successful mouse embryo cryopreservation to -79ºC, using 7.5% polyvinylpyrolidine (PVP) as cryoprotectant, was published, reporting postthaw *in vitro* development to blastocysts and *in vivo* development to day-18 fetuses (Whittingham, 1971). Several researchers tried to repeat these results but none was successful (Whittingham et al., 1972; Wilmut, 1972; Ashwood-Smith, 1986; Leibo & Oda, 1993). The real start of the embryo cryopreservation era can therefore be considered as the year 1972. During that year two groups reported successful cryopreservation of mouse embryos to -196ºC (Whittingham et al., 1972; Wilmut, 1972). These reports came more than two decades after Polge et al. (1949) reported their chance observation that led to successful freezing of spermatozoa and opened a new era in cryobiology and assisted reproduction. Despite the decades that went by and numerous studies attempting a plethora of protocols and combinations of cryoprotectants, it is amazing to note that besides modification to cooling rate that came a few years later (Willadsen et al., 1976; Willadsen et al., 1978), the same basic protocol is still in vast use today. From conservation standpoint, embryo cryopreservation has the advantage of preserving the entire genetic complement of both parents. Naturally, a number of both male and female embryos should be stored to ensure representation of both sexes and a wide genetic diversity. Since sexing each embryo before cryopreservation is not practical, a large number of embryos should be preserved to increase the probability for sufficient representation of embryos from both sexes. Cryobanking of embryos can thus help establishing founder population with the aim of eventual reintroduction into the wild (Ptak et al., 2002) or revive isolated small population. However, while millions of offspring were born following the transfer of cryopreserved embryos in humans, cattle, sheep and mice, success is very limited in many other, even closely related species. To date the number of species in which embryo cryopreservation has been reported stands at less than 50 mammals (human, domestic and laboratory animals included), with live birth achieved in only about half of them (Table 3). There are also a few reports on nonmammalian embryo cryopreservation, all of them in fish (Table 3). Looking through the table, one can see that the majority of species in which embryo cryopreservation led eventually to pregnancy and live birth are domestic, companion, and laboratory species and species of commercial value. Only very few are truly wildlife species. Much of the knowledge gained came from studies on model animals since endangered species are too rare and studying them directly is often too difficult or practically impossible. By definition, however, each species has a unique reproductive specialization so, no matter how close we get with the aid of model animals, we must in the end gain access to the target species and verify that what worked in the model also works in the target. For example, studies on the domestic cat helped develop various technologies, which were later used in non-domestic cats (Dresser et al., 1988; Pope et al., 1994; Pope, 2000), or cattle served as a model for other ungulates (Dixon et al., 1991; Loskutoff et al., 1995). Too often direct adaptation is not possible and either adjustments to protocols or complete revision are required, forcing researchers to settle for small animal study population, at times comprised of a single animal (e.g. Robeck et al., 2011), and samples that are hard to come by. As in the case of oocytes, slow freezing and vitrification are currently used for embryo cryopreservation. Unlike oocytes, however, slow freezing has been producing good results so vitrification does not occupy as important a role in embryo cryopreservation as it does with oocytes. Two main sources of embryos can be considered – *in vivo* produced embryos and those

Immature oocytes seem to be less prone to damages caused by the chilling, freezing and thawing or warming procedures (Arav et al., 1996) and they, too, can be cryopreserved by slow freezing (Luvoni et al., 1997) or vitrification (Arav et al., 1993; Czarny et al., 2009b). Preantral oocytes can be preserved inside the follicle and about 10% seem to be physiologically active after thawing and one week of culture. Of over 16,000 small preantral oocytes recovered from the ovaries of 25 cats, 66.3% were intact after thawing (Jewgenow et al., 1998). Before freezing 33.9% of the follicles contained viable oocytes while after thawing there were 19.3% if frozen in Me2SO and 18.5% if frozen in 1,2 propanediol. However, culture conditions that will allow these oocytes to grow and reach full maturation are still largely unknown despite attempts in several species (Jewgenow et al., 1998; Nayudu et al., 2003). For example, in the marmoset monkey, oocytes collected from secondary pre-antral follicles of either mature or pre-pubertal females were able to develop *in vitro* to the polar body stage but could not complete the maturation process (Nayudu et al., 2003). The exception is the mouse, in which this was done and embryos were produced following IVF of frozen-thawed primary follicles matured *in vitro* and live young were born after embryo transfer (Carroll et al., 1990). Some, very limited, success was also reported in cats, where following vitrification in 40% ethylene glycol, 3.7% of the *in vitro* matured oocytes were able to develop to the blastocyst stage following IVF (Murakami et al., 2004). The problems associated with maturation of early-stage oocytes *in vitro* are the need to develop the complex endocrine system that support the development at different stages, other culture conditions that will ensure survival (oxygen pressure for example) and, in many species, the duration of time required to keep the follicles in culture – 6 months or more. An alternative to isolated oocyte cryopreservation is cryopreservation of individual primordial follicles and later transplanting them to the ovarian bursa, where they can mature and eventually produce young offspring following natural mating as was shown in mice (Carroll & Gosden, 1993).

Liquid-phase sperm preservation is relatively simple. Doing the same with oocytes was, until recently, much more challenging. A recent report on pig oocytes, however, has demonstrated ambient-temperature (27.5ºC) preservation for 3 days with as many as 65% of the GV oocytes maintaining viability and developmental competence (Yang et al., 2010). This study demonstrated that oocyte preservation without freezing for several days is possible and relatively simple. This is of great importance for wildlife as cryopreservation or IVF of oocytes collected from dead animals in the field often cannot be done on the spot. The ability to keep oocytes alive while transporting them to the laboratory will considerably increase the number of possibilities.

#### **3.2 Embryo cryopreservation**

As discussed earlier with regards to oocytes, the vast difference in size, components and associated structures between spermatozoa on the one hand and oocytes and embryos on the other make cryopreservation of the latter much more complex. The issue of intracellular ice formation becomes a major concern, even at relatively slow cooling rates. To avoid this from happening, small volume cryopreservation and either high cryoprotectant concentration coupled with very fast cooling rate to achieve a state of vitrification or lower cryoprotectant concentration and slow cooling rate (slow freezing) are utilized. The first report on fertilized eggs cryopreservation was on rabbit fertilized ova frozen to -79ºC (Ferdows et al., 1958). Some of these cryopreserved ova resulted in pregnancies after

Immature oocytes seem to be less prone to damages caused by the chilling, freezing and thawing or warming procedures (Arav et al., 1996) and they, too, can be cryopreserved by slow freezing (Luvoni et al., 1997) or vitrification (Arav et al., 1993; Czarny et al., 2009b). Preantral oocytes can be preserved inside the follicle and about 10% seem to be physiologically active after thawing and one week of culture. Of over 16,000 small preantral oocytes recovered from the ovaries of 25 cats, 66.3% were intact after thawing (Jewgenow et al., 1998). Before freezing 33.9% of the follicles contained viable oocytes while after thawing there were 19.3% if frozen in Me2SO and 18.5% if frozen in 1,2 propanediol. However, culture conditions that will allow these oocytes to grow and reach full maturation are still largely unknown despite attempts in several species (Jewgenow et al., 1998; Nayudu et al., 2003). For example, in the marmoset monkey, oocytes collected from secondary pre-antral follicles of either mature or pre-pubertal females were able to develop *in vitro* to the polar body stage but could not complete the maturation process (Nayudu et al., 2003). The exception is the mouse, in which this was done and embryos were produced following IVF of frozen-thawed primary follicles matured *in vitro* and live young were born after embryo transfer (Carroll et al., 1990). Some, very limited, success was also reported in cats, where following vitrification in 40% ethylene glycol, 3.7% of the *in vitro* matured oocytes were able to develop to the blastocyst stage following IVF (Murakami et al., 2004). The problems associated with maturation of early-stage oocytes *in vitro* are the need to develop the complex endocrine system that support the development at different stages, other culture conditions that will ensure survival (oxygen pressure for example) and, in many species, the duration of time required to keep the follicles in culture – 6 months or more. An alternative to isolated oocyte cryopreservation is cryopreservation of individual primordial follicles and later transplanting them to the ovarian bursa, where they can mature and eventually produce young offspring following

natural mating as was shown in mice (Carroll & Gosden, 1993).

increase the number of possibilities.

**3.2 Embryo cryopreservation** 

Liquid-phase sperm preservation is relatively simple. Doing the same with oocytes was, until recently, much more challenging. A recent report on pig oocytes, however, has demonstrated ambient-temperature (27.5ºC) preservation for 3 days with as many as 65% of the GV oocytes maintaining viability and developmental competence (Yang et al., 2010). This study demonstrated that oocyte preservation without freezing for several days is possible and relatively simple. This is of great importance for wildlife as cryopreservation or IVF of oocytes collected from dead animals in the field often cannot be done on the spot. The ability to keep oocytes alive while transporting them to the laboratory will considerably

As discussed earlier with regards to oocytes, the vast difference in size, components and associated structures between spermatozoa on the one hand and oocytes and embryos on the other make cryopreservation of the latter much more complex. The issue of intracellular ice formation becomes a major concern, even at relatively slow cooling rates. To avoid this from happening, small volume cryopreservation and either high cryoprotectant concentration coupled with very fast cooling rate to achieve a state of vitrification or lower cryoprotectant concentration and slow cooling rate (slow freezing) are utilized. The first report on fertilized eggs cryopreservation was on rabbit fertilized ova frozen to -79ºC (Ferdows et al., 1958). Some of these cryopreserved ova resulted in pregnancies after thawing and transfer. This abstract, however, seem not to have been followed by a full peerreviewed manuscript so it is not clear if those zygotes really froze and resulted in pregnancies. In 1971 another report on successful mouse embryo cryopreservation to -79ºC, using 7.5% polyvinylpyrolidine (PVP) as cryoprotectant, was published, reporting postthaw *in vitro* development to blastocysts and *in vivo* development to day-18 fetuses (Whittingham, 1971). Several researchers tried to repeat these results but none was successful (Whittingham et al., 1972; Wilmut, 1972; Ashwood-Smith, 1986; Leibo & Oda, 1993). The real start of the embryo cryopreservation era can therefore be considered as the year 1972. During that year two groups reported successful cryopreservation of mouse embryos to -196ºC (Whittingham et al., 1972; Wilmut, 1972). These reports came more than two decades after Polge et al. (1949) reported their chance observation that led to successful freezing of spermatozoa and opened a new era in cryobiology and assisted reproduction. Despite the decades that went by and numerous studies attempting a plethora of protocols and combinations of cryoprotectants, it is amazing to note that besides modification to cooling rate that came a few years later (Willadsen et al., 1976; Willadsen et al., 1978), the same basic protocol is still in vast use today. From conservation standpoint, embryo cryopreservation has the advantage of preserving the entire genetic complement of both parents. Naturally, a number of both male and female embryos should be stored to ensure representation of both sexes and a wide genetic diversity. Since sexing each embryo before cryopreservation is not practical, a large number of embryos should be preserved to increase the probability for sufficient representation of embryos from both sexes. Cryobanking of embryos can thus help establishing founder population with the aim of eventual reintroduction into the wild (Ptak et al., 2002) or revive isolated small population. However, while millions of offspring were born following the transfer of cryopreserved embryos in humans, cattle, sheep and mice, success is very limited in many other, even closely related species. To date the number of species in which embryo cryopreservation has been reported stands at less than 50 mammals (human, domestic and laboratory animals included), with live birth achieved in only about half of them (Table 3). There are also a few reports on nonmammalian embryo cryopreservation, all of them in fish (Table 3). Looking through the table, one can see that the majority of species in which embryo cryopreservation led eventually to pregnancy and live birth are domestic, companion, and laboratory species and species of commercial value. Only very few are truly wildlife species. Much of the knowledge gained came from studies on model animals since endangered species are too rare and studying them directly is often too difficult or practically impossible. By definition, however, each species has a unique reproductive specialization so, no matter how close we get with the aid of model animals, we must in the end gain access to the target species and verify that what worked in the model also works in the target. For example, studies on the domestic cat helped develop various technologies, which were later used in non-domestic cats (Dresser et al., 1988; Pope et al., 1994; Pope, 2000), or cattle served as a model for other ungulates (Dixon et al., 1991; Loskutoff et al., 1995). Too often direct adaptation is not possible and either adjustments to protocols or complete revision are required, forcing researchers to settle for small animal study population, at times comprised of a single animal (e.g. Robeck et al., 2011), and samples that are hard to come by. As in the case of oocytes, slow freezing and vitrification are currently used for embryo cryopreservation. Unlike oocytes, however, slow freezing has been producing good results so vitrification does not occupy as important a role in embryo cryopreservation as it does with oocytes. Two main sources of embryos can be considered – *in vivo* produced embryos and those

Genome Banking for Vertebrates Wildlife Conservation 313

Pregnancy to

Details not provided Stillbirth (Kramer et al., 1983;

Transferred but no pregnancy

Transferred to both cow and gaur. Pregnancy at day 135 in

Transferred but outcome not reported

Pregnancy to term from -35ºC, no pregnancy from -196ºC

Transferred but no pregnancy

Pregnancy by ultrasound

no pregnancy

Pregnancy to

Pregnancy by ultrasound at day 45.

Outcome not reported

Freezing – pregnancy by ultrasound, vitrification – pregnancy to

term

term

8-cell freezing Transferred but

(Yamamoto et al., 1982; Slade et al., 1985)

Dresser et al., 1984; both cited in Schiewe, 1991)

(Stover & Evans, 1984; Armstrong et al., 1995)

(Dresser et al., 1985)

(Hayashi et al., 1989)

(Schiewe et al., 1991a)

(Dixon et al., 1991)

(Kasiraj et al., 1993)

(Morrow et al., 1994)

(Vendramini et al., 1997)

(Skidmore & Loskutoff, 1999; Nowshari et al.,

2005)

(Schiewe, 1991)

(Durrant, 1983)

term

cow

Species Procedure Outcome References

blastocysts freezing

*In vivo*-produced morula freezing

blastocysts freezing

*In vivo*-produced blastocysts freezing

*In vivo*-produced morula and blastocysts

*In vivo*-produced blastocysts freezing

*In vivo*-produced morula and blastocysts

*In vivo*-produced morula and blastocysts

*In vivo*-produced blastocysts freezing

*In vivo*-produced blastocysts freezing and vitrification

freezing

freezing

freezing

blastocysts freezing to - 35ºC and -196ºC

Horse (*Equus caballus*) *In vivo*-produced

Gaur (*Bos gaurus*) *In vivo*-produced

Swine (*Sus domestica*) *In vivo*-produced

African eland

*oryx*)

*leucoryx*)

*euryceros*)

antelope (*Taurotragus* 

Arabian Oryx (*Oryx* 

Bongo (*Tragelphus* 

Scimitar-horned Oryx (*Oryx dammah*)

Water buffalo (*Bubalis* 

Fallow deer (*Dama* 

Domestic donkey (*Equus acinus*)

Dromedary camel (*Camelus dromedarius*)

Red deer (*Cervus* 

Suni Antelope (*Neotragus moschatus* 

*elaphus*)

*zuluensis*)

*bubalis*)

*dama*)

produced *in vitro*. These two embryo groups can develop *in vivo* to produce live offspring but the *in vivo* produced embryos seem to be superior to the *in vitro* ones in many respects, including their sturdiness and ability to survive cryopreservation (Rizos et al., 2002). Obtaining *in vivo*-produced embryos from an endangered species for cryopreservation is a difficult ethical question. If pregnancy has already occurred, shouldn't we let it proceed? Still, because of their superiority, *in vivo*-produced embryos were used in many of the studies on embryo cryopreservation in wildlife.


produced *in vitro*. These two embryo groups can develop *in vivo* to produce live offspring but the *in vivo* produced embryos seem to be superior to the *in vitro* ones in many respects, including their sturdiness and ability to survive cryopreservation (Rizos et al., 2002). Obtaining *in vivo*-produced embryos from an endangered species for cryopreservation is a difficult ethical question. If pregnancy has already occurred, shouldn't we let it proceed? Still, because of their superiority, *in vivo*-produced embryos were used in many of the

> Pregnancy, Pregnancy to

> Pregnancy to

Pregnancy to

Pregnancy *In vitro* survival

Pregnancy to

Pregnancy to

IVF, 2-cell freezing Not reported (Pope et al., 1997a)

Pregnancy to

Pregnancy to

Pregnancy to

term

term

term

(Trounson & Mohr, 1983; Zeilmaker et al., 1984)

(Hearn & Summers, 1986; Summers et al., 1987)

(Balmaceda et al., 1986; Curnow et al., 2002)

(Cranfield et al., 1992)

(Wilmut & Rowson, 1973; Willadsen et al., 1978)

(Willadsen et al., 1974,

(Bilton & Moore, 1976)

1976)

et al., 2001)

(Wolf et al., 1989; Yeoman

(Pope et al., 1984)

term

term

term

term

term

term

IVF, 2-cell freezing Pregnancy to

Species Procedure Outcome References

4 to 8-cell, freezing, 4 to 16-cell freezing

to blastocyst, freezing

*In vivo*-produced 4 to 10-cells and morulae

IVF, 4 to 8-cell freezing 2 to 8-cell vitrification

IVF, early-stage freezing

ICSI blastocysts vitrification

blastocysts freezing

morula and blastocyst

morula and blastocyst

freezing

freezing

studies on embryo cryopreservation in wildlife.

Baboon (*Papio* sp.) *In vivo*-produced 6-cell

freezing

**Primates** 

Human (*Homo sapiens*)

Marmoset monkey (*Callithrix jacchus*)

Cynomolgus monkey (*Macaca fascicularis*)

Hybrid macaque [pig-

tailed (*Macaca nemestrina*) & liontailed (*M. silenus*)]

Western lowland gorilla (*Gorilla gorilla* 

Bovine (*Bos taurus*) *In vivo*-produced

Sheep (*Ovis aries*) *In vivo*-produced

Goat (*Capra aegagrus*) *In vivo*-produced

*gorilla*)

**Ungulates** 

Rhesus macaque (*Macaca mulatta*)


Genome Banking for Vertebrates Wildlife Conservation 315

Transferred but no pregnancy

Transferred but no pregnancy

Confirmed pregnancy and later pregnancy to term by freezing and *in vitro* survival for vitrification

Live fetuses to

Confirmed pregnancy on day 18 for freezing, pregnancy to term for vitrification

Confirmed pregnancy on day 14 for freezing and pregnancy to term for vitrification

Pregnancy to

*In vitro* survival in both systems

term

term

Pregnancy to term from 8 to 16-cell embryos (Pope et al., 2005)

(Suzuki et al., 2009)

(Pope et al., 2009)

(Ferdows et al., 1958; Bank & Maurer, 1974; Whittingham & Adams, 1974, 1976; Popelkova et

(Whittingham et al., 1972;

(Ridha & Dukelow, 1985;

Lane et al., 1999)

(Mochida et al., 2005)

(Breed et al., 1994)

al., 2009)

Wilmut, 1972)

(Whittingham, 1975; Kono et al., 1988)

Species Procedure Outcome References

IVF morula and blastocysts freezing

to blastocyst vitrification

freezing

freezing

Rat (*Rattus norvegicus*) *In vivo*-produced 2 to 8-

*In vivo*-produced 1-cell

IVF and ICSI day-five

Fertilized ova frozen to -79ºC, later 4 to 16-cell and morula freezing to -196ºC and vitrification.

*In vivo*-produced 8-cell

*In vivo*-produced 1-cell to morula freezing and 1 to 2-cell vitrification

*In vivo*-produced 2-cell, morula and

*In vivo*-produced day 2 to 4 freezing and vitrification

blastocyst vitrification

cell freezing and blastocysts vitrification

Serval (*Leptailurus* 

Dog (*Canis lupus familiaris*)

Clouded leopard (*Neofelis nebulosa*)

European rabbit (*Oryctolagus cuniculus*)

Mouse (*Mus musculus*)

Syrian hamster (*Mesocricetus auratus*)

Mongolian gerbil

Fat-tailed dunnart (*Sminthopsis crassicaudata*)

(*Moriones unguieulatus*)

**Marsupials** 

*serval*)

**Glires** 


term

Outcome not reported

Pregnancy by ultrasound after vitrification

term in red deer surrogate hind

Pregnancy to

Pregnancy to term in domestic

development of vitrified only

Both transferred and implanted but not carried to

Pregnancy to

IVF 2 to 8-cell freezing Not evaluated (Swanson et al., 2002)

Transferred but no pregnancy

Pregnancy to term in both cryopreservation techniques

Outcome not mentioned

term

cat

*In vitro*

term

term

term

(cited in Rall, 2001)

(Ptak et al., 2002)

et al., 2002)

Not evaluated (Thundathil et al., 2007)

(Aller et al., 2002; Lattanzi

(Locatelli et al., 2008)

(Dresser et al., 1988)

(Crichton et al., 2000; Crichton et al., 2003)

(cited in Farstad, 2000a)

(Swanson, 2001, 2003)

(Miller et al., 2002)

(Lindeberg et al., 2003; Piltti et al., 2004)

(cited in Swanson, 2003; Pope et al., 2006)

(Swanson & Brown, 2004)

(Pope et al., 2000)

Species Procedure Outcome References

IVF blastocysts vitrification

blastocysts freezing and vitrification

IVF morula and blastocysts vitrification

*In vivo*-produced blastocysts freezing

IVF morula and blastocysts freezing

IVF 2 to 4-cell freezing and vitrification

Frozen and vitrified embryos, stage and source not mentioned

IVF (stage not reported) freezing

blastocyst freezing

Source and technique not mentioned

IVF day 5 to 6 freezing Pregnancy to

*In vivo*-produced morula and blastocysts

freezing and vitrification.

Details not mentioned Pregnancy to

IVF blastocysts freezing Pregnancy to

Wapiti (*Cervus canadensis*)

European mouflon (*Ovis orientalis musimon*)

Wood bison (*Bison bison athabascae*)

Sika deer (*Cervus nippon nippon*)

Domestic cat (*Felis* 

African wildcat (*Felis* 

**Carnivores** 

*catus*)

*silvestris*)

*lagopus*)

*pardalis*)

*tigrinus*)

Siberian Tiger (*Panthera tigris altaica*)

Blue fox (*Alopex* 

Ocelot (*Leopardus* 

Tigrina (*Leopardus* 

European Polecat (*Mustela putorius*)

Caracal *(Felis caracal* or *Caracal caracal*)

Geoffroy's cat (*Felis* 

*geoffroyi*)

Bobcat (*Lynx rufus*) *In vivo*-produced

Llama (*Lama glama*) *In vivo*-produced


Genome Banking for Vertebrates Wildlife Conservation 317

causing severe osmotic damage. Relying on success in IVF followed by embryo transfer (Balmaceda et al., 1984), pregnancies resulting from frozen-thawed IVF-produced embryos in cynomolgus monkeys (*Macaca fascicularis*) were reported (Balmaceda et al., 1986). Fifty-six cynomolgus macaque embryos were cryopreserved at the four- to eight-cell stage using 1.5 M Me2SO as cryoprotectant and the slow-freezing technique. After thawing, 39 embryos (70%) were still viable. Of these, 25 were transferred to nine synchronized recipients 24 to 48 h after ovulation, resulting in three pregnancies. Report on pregnancy carried to term from frozen-thawed transferred embryo in the rhesus macaque (*Macaca mulatta*) came not too long after that (Wolf et al., 1989). Using hormonal stimulation to achieve superovulation, oocytes (68% mature) were retrieved and inseminated *in vitro*. Embryos were then cryopreserved at the three- to six-cell stage following a propanediol-based freezing protocol, originally developed for humans. Embryo post-thaw survival was high (100%; 11/11). After transferring two embryos to each of three recipients during the early luteal phase of spontaneous menstrual cycles, one pregnancy was achieved and was carried to term. The same group also attempted *in vitro* maturation (IVM) of oocytes prior to IVF, freezing and transfer (Lanzendorf et al., 1990). Oocytes collected at the germinal vesicle (GV) stage did not fertilize *in vitro* and fertilization rate of those collected at the metaphase I (MI) stage was low (32%), even if these were matured *in vitro* to the metaphase II (MII) stage. Fertilization rate of oocytes collected at the MII stage was high (93%) and eight embryos frozen and transferred at the two- to six-cell stage to four recipients (two embryos to each) resulted in three pregnancies culminating in the delivery of three twins. Cross-species IVF was also attempted using *in vitro*-matured oocytes from the non-endangered pig-tailed macaque (*Macaca nemestrina*) and sperm from the endangered lion-tailed macaque (*M. silenus*) (Cranfield et al., 1992). Of the 65 oocytes collected, 25 (38%) were fertilized and 15 (24%) have developed to good quality embryos. These embryos were cryopreserved in propandiol-based extender and the slow freezing technique. Nine embryos were transferred to naturally cycling *M. nemestrina* foster mothers, one of which delivered a healthy hybrind male infant. In Western lowland gorilla (*Gorilla gorilla gorilla*), associated *in vitro* techniques (IVM, IVF, IVC) were adopted successfully from humans (Pope et al., 1997a). Of eight embryos at the two-cell stage produced *in vitro*, three were transferred to a single female, leading to a pregnancy and birth of a female infant. The other five embryos were cryopreserved in 1.5 M 1,2-propanediol containing cryoprotectant. Regrettably,

Vitrification is a good alternative to the slow freezing. Following the lead of human and laboratory and farm animals' embryo cryopreservation, the use of vitrification was attempted and compared to slow freezing in non-human primates as well (Yeoman et al., 2001; Curnow et al., 2002). Early-stage (two- to eight-cells) cynomolgus macaque embryos were used to compare vitrification using open pulled straw (OPS) as a carrier system to slow freezing (Curnow et al., 2002). Vitrification proved to be inferior to slow freezing in cell survival rate (18 to 29% vs. 82%), embryo survival (26 to 32% vs. 90%) and cleavage rate (29 to 38% vs. 83%). In another study, on rhesus monkey blastocysts cryopreservation, vitrification using the cryoloop as a carrier system was compared to slow freezing (Yeoman et al., 2001). Embryos were produced *in vitro* by ICSI into mature oocytes and then *in vitro*  cultured to the blastocyst stage. Cryopreservation was carried out by either the slow freezing technique or vitrification using two different cryoprotectant combinations – 2.8M

cryopreservation outcome was not reported.


Table 3. Embryo cryopreservation in vertebrates. The table make it clear that attempts were made almost only in mammals and success in terms of pregnancy carried to term was achieved almost only in domestic, laboratory or companion species and species of commercial value.
