**3.2.1.1 Non-human primates**

The number of cryopreserved human embryos successfully transferred since the first report on birth resulting from a transfer of a frozen-thawed embryo (Trounson & Mohr, 1983) is probably over half a million. Yet, despite the fact that non-human primates are used as laboratory models for humans in many studies, progress in primate embryo cryopreservation has been very limited (Mazur et al., 2008) and reports are scarce but with promising results. Observing the progress in non-human primate embryo cryopreservation, it seems that in this field humans act as models for other primates rather than the other way around. The first report on the birth of a non-human primate (baboon; *Papio* sp.) following transfer of frozen-thawed embryo came in 1984, about a year after similar report in humans (Pope et al., 1984). Six *in vivo* produced embryos were retrieved and frozen using glycerol as cryoprotectant. All six embryos survived the freeze-thaw procedure and resulted in two pregnancies (33.3%) after being transferred to six recipients. A similar report on cryopreservation of *in vivo* produced embryos in marmoset monkey (*Callithrix jacchus*) showed higher pregnancy rates (Hearn & Summers, 1986; Summers et al., 1987). In one of these studies, for example, 70% (7/10) of cryopreserved four- to 10-cell embryos and 56% (5/9) of cryopreserved morulae resulted in pregnancies (Summers et al., 1987). Five pregnancies of the first and four of the latter were carried to term resulting in six babies in each group. These authors noted that 1.5M Me2SO was superior to 1.0M glycerol; the latter

No viability (Zhang & Rawson, 1996)

(Chen & Tian, 2005)

(Fornari et al., 2011)

No viability (Robles et al., 2003)

No viability (Cabrita et al., 2006)

No viability (Ding et al., 2007)

Post-warming hatching

Post-thaw hatching

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

The number of cryopreserved human embryos successfully transferred since the first report on birth resulting from a transfer of a frozen-thawed embryo (Trounson & Mohr, 1983) is probably over half a million. Yet, despite the fact that non-human primates are used as laboratory models for humans in many studies, progress in primate embryo cryopreservation has been very limited (Mazur et al., 2008) and reports are scarce but with promising results. Observing the progress in non-human primate embryo cryopreservation, it seems that in this field humans act as models for other primates rather than the other way around. The first report on the birth of a non-human primate (baboon; *Papio* sp.) following transfer of frozen-thawed embryo came in 1984, about a year after similar report in humans (Pope et al., 1984). Six *in vivo* produced embryos were retrieved and frozen using glycerol as cryoprotectant. All six embryos survived the freeze-thaw procedure and resulted in two pregnancies (33.3%) after being transferred to six recipients. A similar report on cryopreservation of *in vivo* produced embryos in marmoset monkey (*Callithrix jacchus*) showed higher pregnancy rates (Hearn & Summers, 1986; Summers et al., 1987). In one of these studies, for example, 70% (7/10) of cryopreserved four- to 10-cell embryos and 56% (5/9) of cryopreserved morulae resulted in pregnancies (Summers et al., 1987). Five pregnancies of the first and four of the latter were carried to term resulting in six babies in each group. These authors noted that 1.5M Me2SO was superior to 1.0M glycerol; the latter

Species Procedure Outcome References

stage vitrification

14-somite to prehatching stage vitrification

Heart beat stage vitrification

for 6h

Tail bud and tail bud free stages vitrification

Tail bud and tail bud free stages vitrification

Blastoporous closing stage freezing to -8ºC

**Others (fish)** 

Turbot (*Psetta maxima*)

*olivaceus*)

*major*)

Flounder (*Paralichthys* 

Gilthead seabream (*Sparus aurata*)

Cascudo preto (*Rhinelepis aspera*)

commercial value.

**3.2.1 Mammals** 

**3.2.1.1 Non-human primates** 

Red seabream (*Pagrus* 

Zebrafish (*Danio rerio*) 6-somite and heartbeat

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, cryopreservation outcome was not reported.

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

Genome Banking for Vertebrates Wildlife Conservation 319

technology worthwhile pursuing, other associated technologies should also reach a level of

The European mouflon (*Ovis orientalis musimon*) is a wild sheep threatened by extinction. During the efforts to develop the necessary assisted reproductive technologies, the domestic sheep was used as a model. Using 25% glycerol and 25% ethylene glycol as cryoprotectants, *in vitro* produced embryos at the expanded blastocyst stage were vitrified (Ptak et al., 2002). Twenty blastocysts were transferred to domestic sheep foster mothers (two embryos each). At 40 days, seven of the sheep were pregnant and three carried the pregnancy to term, delivering four normal mouflon offspring. In another study, *in vivo* produced embryos were vitrified following embryo vitrification protocol developed for sheep (Naitana et al., 1997; Naitana et al., 2000). Of the five vitrified blastocysts, four survived and were transferred to four synchronized domestic sheep ewes, two of which became pregnant and one pregnancy was carried to term. The domestic sheep, and in part the cow as well, acted as a model for the scimitar-horned Oryx (*Oryx dmmah*) as well. After developing the needed methods, including embryos collection, cryopreservation and transfer, in the sheep, the gained knowledge was used in the scimitar-horned Oryx. *In vivo* produced embryos were frozen in propylene glycol or glycerol but no specific results were reported (Wildt et al., 1986). In another, later study performed on scimitar horned Oryx embryos, thirty late morula- to blastocyst-stage embryos were frozen in cryoprotectant containing Me2SO, glycerol, or propylene glycol, 10 embryos in each (Schiewe et al., 1991a). Survival was higher in the Me2SO and glycerol groups. Although the majority (67%) of *in vitro*-cultured embryos developed into hatched blastocysts after 48 h, no pregnancies were established following nonsurgical (n = 8) or laparoscopic (n = 1) transfer of the remaining transferable embryos. Another Oryx species in which an attempt to cryopreserve embryos was made is the Arabian Oryx (*Oryx leucoryx*). Morula-stage *in vivo*-produced embryos were collected and one was frozen in 1.5M Me2SO. After thawing, the embryo was rated as having a good quality grade. It was transferred to a scimitar-horned Oryx foster female but failed to produce a pregnancy following surgical transfer (Durrant, 1983). Another failed attempt concerns cryopreservation of suni antelope (*Neotragus moschatus zuluensis*) eight-cell stage embryos (N. Loskutoff, personal communication cited in Schiewe, 1991). Of the 18 embryos frozen, nine completely degenerated after thawing. The other nine embryos were transferred by laparoscopy despite the fact that all of them exhibited partial blastomere degradation. No pregnancies were achieved. Attempts were also carried out to freeze *in vivo* produced embryos of African eland antelope (*Taurotragus oryx*) and bongo (*Tragelaphus euryceros*) using glycerol as cryoprotectant. Post-thaw evaluations indicated that six of seven eland (Dresser et al., 1984) and bongo (Dresser et al., 1985) embryos were considered viable and of good enough quality for transfer. Damage to the zona pellucida was noted in one of the eland embryos. Only one pregnancy was carried to term but resulted in a stillborn eland offspring due to dystocia. This attempt was followed by subsequent transfer attempts that resulted in a live eland offspring (B.L. Dressen, personal communication cited in Schiewe,

The red deer (*Cervus elaphus*), an animal of commercial value in various parts of the world, can also act as a model animal for other closely related species. Slow freezing of red deer *in vivo*-produced embryos in 1.4M glycerol followed by embryo transfer in another country resulted in pregnancy rate ranging between 50 and 72% in different farms, with an average pregnancy rate of 61.2% (153/247) (Dixon et al., 1991). In another study, slow freezing was

maturation to support it.

1991).

Me2SO with 3.6M EG (combination A) or 3.4M glycerol with 4.5M EG (combination B). Similar results were achieved when blastocysts were cryopreserved by slow freezing [8/22 (36.4%) embryos survived and 1/22 (4.5%) hatched following co-culture] or combination A of cryoprotectants [6/16 (37.5%) embryos survived and 1/16 (6.3%) hatched]. In comparison, using vitrification with cryoprotectant combination B 28/33 (84.8%) of the blastocysts survived and 23/33 (69.7%) hatched. This last study not only achieved high embryonic survival using vitrification, it has also demonstrated the suitability of this technique to overcome the problem of advanced-stage embryo preservation. Six embryos vitrified with cryoprotectant combination B and transferred to three recipients (two to each) resulted in a twin pregnancy carried to term.

#### **3.2.1.2 Ungulates**

Embryo cryopreservation has reached a commercial level in the cattle industry and to a lesser extant in sheep and goats. According to a report of the International Embryo Transfer Society (IETS), 297,677 *in vivo*-derived frozen-thawed bovine embryos were transferred in 2008 worldwide, representing 55.2% of all transferred *in vivo*-derived bovine embryos in that year (Thibier, 2009). There were also 26,914 frozen-thawed IVF embryos, comprising 10.6% of all transferred bovine IVF embryos in 2008. The actual numbers are most probably much larger since not all transfers are reported to IETS. Major Asian countries such as China, India, Korea and Thailand as well as some of the South American countries did not report their activities to IETS and the reports from Oceania are only partial. At least four important factors are responsible for this success: availability of almost unlimited number of oocytes for research, the possibility to collect *in vivo* produced embryos non-surgically and without the need for anesthesia or sedation, the availability of financial resources to finance overwhelming body of studies and the needs of the cattle industry. Because none of these factors is helping to push studies on endangered ungulates, situation is dramatically less developed in other species of this group. Statements in reviews on assisted reproductive technologies in non-domestic ungulates from only a decade ago were to the effect that by that time only one successful embryo cryopreservation has been achieved (Holt, 2001). Nondomestic ungulates usually do not show discernable signs of estrous and their receptive period is fairly short. This requires a thorough understanding of the estrus cycle endocrine activity, methods for it's monitoring in each species under study and the development of species-specific hormonal administration for ovarian stimulation. As in all other wildlife species, one should always keep in mind that what works for one species not necessarily will also work for another, even closely related species. For example, the bovine IVC protocol works well for the water buffalo (*Bubalis bubalis*) but when this protocol was used for the African buffalo (*Syncerus caffer*), embryos did not develop beyond the morula stage (Loskutoff et al., 1995). Hormonal monitoring can be achieved non-invasively through fecal or urine analysis but even developing such techniques is not always eventless and not always successful (Paris et al., 2008). Hormonal administration requires stress-afflicting activities such as repeated darting, general anesthesia or movement restriction by chute. Thus, progress in this field has been slow and efficiency in *in vitro* technologies (IVM, IVF, IVC) has been low. For example, in the Kudu (*Tragelaphus* sp.), of 397 oocytes collected, 79 zygotes cleaved yet only two blastocysts were achieved (0.5%) (Loskutoff et al., 1995). Another example is the Mohor gazelle (*Gazella dama mhorr*) in which embryos produced by IVF with frozen-thawed semen did not develop beyond the six- to eight-cell stage (Berlinguer et al., 2008). These studies suggest that while embryo cryopreservation is a

Me2SO with 3.6M EG (combination A) or 3.4M glycerol with 4.5M EG (combination B). Similar results were achieved when blastocysts were cryopreserved by slow freezing [8/22 (36.4%) embryos survived and 1/22 (4.5%) hatched following co-culture] or combination A of cryoprotectants [6/16 (37.5%) embryos survived and 1/16 (6.3%) hatched]. In comparison, using vitrification with cryoprotectant combination B 28/33 (84.8%) of the blastocysts survived and 23/33 (69.7%) hatched. This last study not only achieved high embryonic survival using vitrification, it has also demonstrated the suitability of this technique to overcome the problem of advanced-stage embryo preservation. Six embryos vitrified with cryoprotectant combination B and transferred to three recipients (two to each)

Embryo cryopreservation has reached a commercial level in the cattle industry and to a lesser extant in sheep and goats. According to a report of the International Embryo Transfer Society (IETS), 297,677 *in vivo*-derived frozen-thawed bovine embryos were transferred in 2008 worldwide, representing 55.2% of all transferred *in vivo*-derived bovine embryos in that year (Thibier, 2009). There were also 26,914 frozen-thawed IVF embryos, comprising 10.6% of all transferred bovine IVF embryos in 2008. The actual numbers are most probably much larger since not all transfers are reported to IETS. Major Asian countries such as China, India, Korea and Thailand as well as some of the South American countries did not report their activities to IETS and the reports from Oceania are only partial. At least four important factors are responsible for this success: availability of almost unlimited number of oocytes for research, the possibility to collect *in vivo* produced embryos non-surgically and without the need for anesthesia or sedation, the availability of financial resources to finance overwhelming body of studies and the needs of the cattle industry. Because none of these factors is helping to push studies on endangered ungulates, situation is dramatically less developed in other species of this group. Statements in reviews on assisted reproductive technologies in non-domestic ungulates from only a decade ago were to the effect that by that time only one successful embryo cryopreservation has been achieved (Holt, 2001). Nondomestic ungulates usually do not show discernable signs of estrous and their receptive period is fairly short. This requires a thorough understanding of the estrus cycle endocrine activity, methods for it's monitoring in each species under study and the development of species-specific hormonal administration for ovarian stimulation. As in all other wildlife species, one should always keep in mind that what works for one species not necessarily will also work for another, even closely related species. For example, the bovine IVC protocol works well for the water buffalo (*Bubalis bubalis*) but when this protocol was used for the African buffalo (*Syncerus caffer*), embryos did not develop beyond the morula stage (Loskutoff et al., 1995). Hormonal monitoring can be achieved non-invasively through fecal or urine analysis but even developing such techniques is not always eventless and not always successful (Paris et al., 2008). Hormonal administration requires stress-afflicting activities such as repeated darting, general anesthesia or movement restriction by chute. Thus, progress in this field has been slow and efficiency in *in vitro* technologies (IVM, IVF, IVC) has been low. For example, in the Kudu (*Tragelaphus* sp.), of 397 oocytes collected, 79 zygotes cleaved yet only two blastocysts were achieved (0.5%) (Loskutoff et al., 1995). Another example is the Mohor gazelle (*Gazella dama mhorr*) in which embryos produced by IVF with frozen-thawed semen did not develop beyond the six- to eight-cell stage (Berlinguer et al., 2008). These studies suggest that while embryo cryopreservation is a

resulted in a twin pregnancy carried to term.

**3.2.1.2 Ungulates** 

technology worthwhile pursuing, other associated technologies should also reach a level of maturation to support it.

The European mouflon (*Ovis orientalis musimon*) is a wild sheep threatened by extinction. During the efforts to develop the necessary assisted reproductive technologies, the domestic sheep was used as a model. Using 25% glycerol and 25% ethylene glycol as cryoprotectants, *in vitro* produced embryos at the expanded blastocyst stage were vitrified (Ptak et al., 2002). Twenty blastocysts were transferred to domestic sheep foster mothers (two embryos each). At 40 days, seven of the sheep were pregnant and three carried the pregnancy to term, delivering four normal mouflon offspring. In another study, *in vivo* produced embryos were vitrified following embryo vitrification protocol developed for sheep (Naitana et al., 1997; Naitana et al., 2000). Of the five vitrified blastocysts, four survived and were transferred to four synchronized domestic sheep ewes, two of which became pregnant and one pregnancy was carried to term. The domestic sheep, and in part the cow as well, acted as a model for the scimitar-horned Oryx (*Oryx dmmah*) as well. After developing the needed methods, including embryos collection, cryopreservation and transfer, in the sheep, the gained knowledge was used in the scimitar-horned Oryx. *In vivo* produced embryos were frozen in propylene glycol or glycerol but no specific results were reported (Wildt et al., 1986). In another, later study performed on scimitar horned Oryx embryos, thirty late morula- to blastocyst-stage embryos were frozen in cryoprotectant containing Me2SO, glycerol, or propylene glycol, 10 embryos in each (Schiewe et al., 1991a). Survival was higher in the Me2SO and glycerol groups. Although the majority (67%) of *in vitro*-cultured embryos developed into hatched blastocysts after 48 h, no pregnancies were established following nonsurgical (n = 8) or laparoscopic (n = 1) transfer of the remaining transferable embryos. Another Oryx species in which an attempt to cryopreserve embryos was made is the Arabian Oryx (*Oryx leucoryx*). Morula-stage *in vivo*-produced embryos were collected and one was frozen in 1.5M Me2SO. After thawing, the embryo was rated as having a good quality grade. It was transferred to a scimitar-horned Oryx foster female but failed to produce a pregnancy following surgical transfer (Durrant, 1983). Another failed attempt concerns cryopreservation of suni antelope (*Neotragus moschatus zuluensis*) eight-cell stage embryos (N. Loskutoff, personal communication cited in Schiewe, 1991). Of the 18 embryos frozen, nine completely degenerated after thawing. The other nine embryos were transferred by laparoscopy despite the fact that all of them exhibited partial blastomere degradation. No pregnancies were achieved. Attempts were also carried out to freeze *in vivo* produced embryos of African eland antelope (*Taurotragus oryx*) and bongo (*Tragelaphus euryceros*) using glycerol as cryoprotectant. Post-thaw evaluations indicated that six of seven eland (Dresser et al., 1984) and bongo (Dresser et al., 1985) embryos were considered viable and of good enough quality for transfer. Damage to the zona pellucida was noted in one of the eland embryos. Only one pregnancy was carried to term but resulted in a stillborn eland offspring due to dystocia. This attempt was followed by subsequent transfer attempts that resulted in a live eland offspring (B.L. Dressen, personal communication cited in Schiewe, 1991).

The red deer (*Cervus elaphus*), an animal of commercial value in various parts of the world, can also act as a model animal for other closely related species. Slow freezing of red deer *in vivo*-produced embryos in 1.4M glycerol followed by embryo transfer in another country resulted in pregnancy rate ranging between 50 and 72% in different farms, with an average pregnancy rate of 61.2% (153/247) (Dixon et al., 1991). In another study, slow freezing was

Genome Banking for Vertebrates Wildlife Conservation 321

As for other members of this group, some but very modest success have been reported on cryopreservation of domestic species like the horse (Yamamoto et al., 1982; Slade et al., 1985; Barfield et al., 2009; Choi et al., 2009) and swine (Nagashima et al., 1995; Dobrinsky et al.,

The order Carnivora includes two suborders – Caniformia (dog-like species) and Feliformia (cat-like species). Similar to cows among the ungulates, the domestic dog (*Canis lupus familiaris*) and cat (*Felis catus*) are representatives of these two suborders and are highly accessible in terms of their frequent use as laboratory animals and the availability of large number of ovaries from neutered or euthanized animals. Still, despite these similarities and their being members of the same order, embryo cryopreservation and all associated technologies are highly developed for cats but lagging far behind in dogs. The domestic cat was found to be a very suitable model for other felid species, which may partially explain why things are more advanced among felids. Felines are induced ovulators (the release of LH that leads to ovulation is induced by mating) and mostly seasonal breeders. The first report on successful IVF and IVC to the blastocyst stage in a cat came in 1977 (Bowen, 1977). Eleven years later the first in-depth study on cat IVF and the first report on birth of live kittens after embryo transfer of cryopreserved, *in vivo*-derived embryos at the morula stage were published (Dresser et al., 1988; Goodrowe et al., 1988). Cryopreservation was carried out using the slow freezing technique and glycerol as cryoprotectant. However, success rate of embryo transfer was relatively low (14.4%, 17/118), most probably because all thawed embryos were transferred, regardless of their grade. Subsequently, production of offspring after transfer of *in vitro*-derived embryos from *in vivo* and *in vitro* matured oocytes and with or without post thaw culture were described (Pope et al., 1994; Wolfe & Wildt, 1996; Pope et al., 1997b; Wood & Wildt, 1997; Pope et al., 2002). Recently it was suggested that removing some of the lipids from the embryo before cryopreservation, a process known as delipidation, result in higher survival rate and higher rates of post-thaw development to

Differences between the domestic cat and other feline species still exist and transfer of knowledge is not entirely straightforward. Still, following the success in the domestic cat, maturation and *in vitro* fertilization of oocytes from a large number of feline species was demonstrated (Johnston et al., 1991). This included tiger (*Panthera tigris*), lion (*Panthera leo*), leopard (*Panthera pardus*), jaguar (*Panthera onca*), snow leopard (*Panthera uncia*), puma (*Felis concolor*), cheetah (*Acinonyx jubatus*), clouded leopard (*Neofelis nebulosa*), bobcat (*Lynx rufus*), serval (*Felis serval*), Geoffroy's cat (*Felis geoffroyi*), Temminck's golden cat (*Felis temmincki*), and leopard cat (*Felis bengalensis*). A total of 846 oocytes were recovered from ovaries of 35 individuals from these 13 species, 508 of them were of fair to excellent quality, yet only 4 (0.8%) cleaved – one of leopard using homologous sperm and three of puma using domestic cat sperm. Matured oocytes were achieved in all but fertilization was not achieved in jaguar, cheetah, clouded leopard, bobcat and Temminck's golden cat. In another study, on puma, 6/25 recovered oocytes fertilized and five of them cleaved (Jewgenow et al., 1994). Success in IVF came at about the same time in other species, e.g. in the tiger (Donoghue et al., 1990), the Indian desert cat (*Felis silvestris ornata*) (cited in Pope, 2000) or leopard cat (*Felis bengalensis*) (Goodrowe et al., 1989). Pope (2000) also mentions IVF/ET in African wild cat

2000) but very little success have been reported in other species.

morula and blastocyst stages (Tharasanit & Techakumphu, 2010).

(*Felis sylvestris lybica*) but pregnancy here ended with stillbirths.

**3.2.1.3 Carnivores** 

compared to vitrification by the OPS technique and fresh embryos as control (Soler et al., 2007). Pregnancy rates were 64.3% (18/28), 53.3% (8/15) and 70.0% (7/10) for fresh, vitrified and frozen embryos, respectively. The knowledge accumulated through experiments on red deer was used to freeze embryos from fallow deer (*Dama dama*) (Morrow et al., 1994). *In vivo*-produced embryos resulting from AI were collected surgically from fallow deer and transferred either fresh or following cryopreservation to recipients. Pregnancy rate of frozen-thawed embryos was half that of fresh (26% vs. 53%) and the overall efficiency of the program was low (0.9 to 1.0 surrogate pregnancy per donor). Another deer species in which embryos cryopreservation was attempted is sika deer (*Cervus nippon nippon*). Here, too, the protocol developed for the red deer (Dixon et al., 1991) was used. Of 142 oocytes collected following chemical synchronization, 57 (40.1%) cleaved after IVF and 14 of them reached the blastocyst stage. These embryos were cryopreserved by slow freezing and were later transferred (two per recipient) to synchronized red deer hinds. One of the seven recipients delivered a healthy young sika deer fawn after 224 days of pregnancy (Locatelli et al., 2008).

The domestic cow has acted as a model for other members of the Bovinae subfamily. The gaur (*Bos gaurus*), a member of this subfamily living in the forested areas of South and South East Asia is classified in the IUCN red list as vulnerable. Following protocols developed for the cow, nine *in vitro* produced blastocysts were cryopreserved. One embryo was transferred to a domestic cow which was confirmed pregnant on day 135 (Armstrong et al., 1995). Cryopreservation of gaur embryos was reported more than a decade earlier (Stover & Evans, 1984) however that report did not elaborate on the freezing protocol nor was any information provided as to the outcome of the procedure. Another member of this subfamily is the wood bison (*Bison bison athabascae*), a sub species of the North American bison. Using IVM, IVF, IVC and vitrification protocols developed for bovine, *in vitro*-produced embryos were vitrified (Thundathil et al., 2007). Regettably, protocols that works very well for cattle, gave fairly poor results in wood bison. Only 6.9% (11/160) of the embryos reached the blastocyst stage. Morula-stage (n=27) and blastocyst-stage (n=6) embryos were vitrified. Disappointingly, the researchers failed to report on the evaluation of the embryos after warming.

Camelids are seasonal breeders and induced ovulators. *In vivo*-produced embryos of dromedary camel (*Camelus dromedarius*), collected at the blastocyst stage, were vitrified. Post-warming survival and intact morphology were high (92%) and following transfer of 45 embryos (20 during the breeding season and 25 off-season), three pregnancy were obtained, one of which was carried to term (Nowshari et al., 2005). This report follows a previous one in which cryopreserved embryos did not lead to a pregnancy after transfer (Skidmore & Loskutoff, 1999). Among the South American camelids, attempts have reached some level of success in the Llama (*Lama glama*) whose embryos were found to be three- to five-fold larger than bovine embryos of the same stage (Lattanzi et al., 2002). In one attempt, *in vivo* produced hatched blastocysts were either vitrified or frozen slowly (Lattanzi et al., 2002). After 24 h of *in vitro* culture, 64% (21/33) of the vitrified embryos and in 63% (12/19) of the slow freezing embryos re-expanded. In another attempt to vitrify llama embryos, 10/40 embryos re-expanded after warming (von Baer et al., 2002). Three fresh-chilled and two vitrified-warmed embryos were transferred to synchronized recipients but only one of the fresh embryos resulted in a pregnancy. In yet another report from about the same time, by a different group, success was achieved. *In vivo* produced embryos were collected nonsurgically and vitrified at the expanded blastocyst stage. Eight embryos were transferred after warming to four recipients (two embryos, each) and two of them became pregnant, delivering two offspring (Aller et al., 2002).

As for other members of this group, some but very modest success have been reported on cryopreservation of domestic species like the horse (Yamamoto et al., 1982; Slade et al., 1985; Barfield et al., 2009; Choi et al., 2009) and swine (Nagashima et al., 1995; Dobrinsky et al., 2000) but very little success have been reported in other species.

#### **3.2.1.3 Carnivores**

320 Current Frontiers in Cryobiology

compared to vitrification by the OPS technique and fresh embryos as control (Soler et al., 2007). Pregnancy rates were 64.3% (18/28), 53.3% (8/15) and 70.0% (7/10) for fresh, vitrified and frozen embryos, respectively. The knowledge accumulated through experiments on red deer was used to freeze embryos from fallow deer (*Dama dama*) (Morrow et al., 1994). *In vivo*-produced embryos resulting from AI were collected surgically from fallow deer and transferred either fresh or following cryopreservation to recipients. Pregnancy rate of frozen-thawed embryos was half that of fresh (26% vs. 53%) and the overall efficiency of the program was low (0.9 to 1.0 surrogate pregnancy per donor). Another deer species in which embryos cryopreservation was attempted is sika deer (*Cervus nippon nippon*). Here, too, the protocol developed for the red deer (Dixon et al., 1991) was used. Of 142 oocytes collected following chemical synchronization, 57 (40.1%) cleaved after IVF and 14 of them reached the blastocyst stage. These embryos were cryopreserved by slow freezing and were later transferred (two per recipient) to synchronized red deer hinds. One of the seven recipients delivered a healthy young sika deer fawn after 224 days of pregnancy (Locatelli et al., 2008). The domestic cow has acted as a model for other members of the Bovinae subfamily. The gaur (*Bos gaurus*), a member of this subfamily living in the forested areas of South and South East Asia is classified in the IUCN red list as vulnerable. Following protocols developed for the cow, nine *in vitro* produced blastocysts were cryopreserved. One embryo was transferred to a domestic cow which was confirmed pregnant on day 135 (Armstrong et al., 1995). Cryopreservation of gaur embryos was reported more than a decade earlier (Stover & Evans, 1984) however that report did not elaborate on the freezing protocol nor was any information provided as to the outcome of the procedure. Another member of this subfamily is the wood bison (*Bison bison athabascae*), a sub species of the North American bison. Using IVM, IVF, IVC and vitrification protocols developed for bovine, *in vitro*-produced embryos were vitrified (Thundathil et al., 2007). Regettably, protocols that works very well for cattle, gave fairly poor results in wood bison. Only 6.9% (11/160) of the embryos reached the blastocyst stage. Morula-stage (n=27) and blastocyst-stage (n=6) embryos were vitrified. Disappointingly, the

researchers failed to report on the evaluation of the embryos after warming.

delivering two offspring (Aller et al., 2002).

Camelids are seasonal breeders and induced ovulators. *In vivo*-produced embryos of dromedary camel (*Camelus dromedarius*), collected at the blastocyst stage, were vitrified. Post-warming survival and intact morphology were high (92%) and following transfer of 45 embryos (20 during the breeding season and 25 off-season), three pregnancy were obtained, one of which was carried to term (Nowshari et al., 2005). This report follows a previous one in which cryopreserved embryos did not lead to a pregnancy after transfer (Skidmore & Loskutoff, 1999). Among the South American camelids, attempts have reached some level of success in the Llama (*Lama glama*) whose embryos were found to be three- to five-fold larger than bovine embryos of the same stage (Lattanzi et al., 2002). In one attempt, *in vivo* produced hatched blastocysts were either vitrified or frozen slowly (Lattanzi et al., 2002). After 24 h of *in vitro* culture, 64% (21/33) of the vitrified embryos and in 63% (12/19) of the slow freezing embryos re-expanded. In another attempt to vitrify llama embryos, 10/40 embryos re-expanded after warming (von Baer et al., 2002). Three fresh-chilled and two vitrified-warmed embryos were transferred to synchronized recipients but only one of the fresh embryos resulted in a pregnancy. In yet another report from about the same time, by a different group, success was achieved. *In vivo* produced embryos were collected nonsurgically and vitrified at the expanded blastocyst stage. Eight embryos were transferred after warming to four recipients (two embryos, each) and two of them became pregnant, The order Carnivora includes two suborders – Caniformia (dog-like species) and Feliformia (cat-like species). Similar to cows among the ungulates, the domestic dog (*Canis lupus familiaris*) and cat (*Felis catus*) are representatives of these two suborders and are highly accessible in terms of their frequent use as laboratory animals and the availability of large number of ovaries from neutered or euthanized animals. Still, despite these similarities and their being members of the same order, embryo cryopreservation and all associated technologies are highly developed for cats but lagging far behind in dogs. The domestic cat was found to be a very suitable model for other felid species, which may partially explain why things are more advanced among felids. Felines are induced ovulators (the release of LH that leads to ovulation is induced by mating) and mostly seasonal breeders. The first report on successful IVF and IVC to the blastocyst stage in a cat came in 1977 (Bowen, 1977). Eleven years later the first in-depth study on cat IVF and the first report on birth of live kittens after embryo transfer of cryopreserved, *in vivo*-derived embryos at the morula stage were published (Dresser et al., 1988; Goodrowe et al., 1988). Cryopreservation was carried out using the slow freezing technique and glycerol as cryoprotectant. However, success rate of embryo transfer was relatively low (14.4%, 17/118), most probably because all thawed embryos were transferred, regardless of their grade. Subsequently, production of offspring after transfer of *in vitro*-derived embryos from *in vivo* and *in vitro* matured oocytes and with or without post thaw culture were described (Pope et al., 1994; Wolfe & Wildt, 1996; Pope et al., 1997b; Wood & Wildt, 1997; Pope et al., 2002). Recently it was suggested that removing some of the lipids from the embryo before cryopreservation, a process known as delipidation, result in higher survival rate and higher rates of post-thaw development to morula and blastocyst stages (Tharasanit & Techakumphu, 2010).

Differences between the domestic cat and other feline species still exist and transfer of knowledge is not entirely straightforward. Still, following the success in the domestic cat, maturation and *in vitro* fertilization of oocytes from a large number of feline species was demonstrated (Johnston et al., 1991). This included tiger (*Panthera tigris*), lion (*Panthera leo*), leopard (*Panthera pardus*), jaguar (*Panthera onca*), snow leopard (*Panthera uncia*), puma (*Felis concolor*), cheetah (*Acinonyx jubatus*), clouded leopard (*Neofelis nebulosa*), bobcat (*Lynx rufus*), serval (*Felis serval*), Geoffroy's cat (*Felis geoffroyi*), Temminck's golden cat (*Felis temmincki*), and leopard cat (*Felis bengalensis*). A total of 846 oocytes were recovered from ovaries of 35 individuals from these 13 species, 508 of them were of fair to excellent quality, yet only 4 (0.8%) cleaved – one of leopard using homologous sperm and three of puma using domestic cat sperm. Matured oocytes were achieved in all but fertilization was not achieved in jaguar, cheetah, clouded leopard, bobcat and Temminck's golden cat. In another study, on puma, 6/25 recovered oocytes fertilized and five of them cleaved (Jewgenow et al., 1994). Success in IVF came at about the same time in other species, e.g. in the tiger (Donoghue et al., 1990), the Indian desert cat (*Felis silvestris ornata*) (cited in Pope, 2000) or leopard cat (*Felis bengalensis*) (Goodrowe et al., 1989). Pope (2000) also mentions IVF/ET in African wild cat (*Felis sylvestris lybica*) but pregnancy here ended with stillbirths.

Genome Banking for Vertebrates Wildlife Conservation 323

high lipid content (Reynaud et al., 2005) making their cryopreservation challenging. These multiple factors are responsible for the slow progress in ART developments in canids. Despite extensive search, the only report on embryo cryopreservation in a non-domestic canid found in the scientific literature is a few words on a trial with blue fox (*Alopex lagopus*) embryos. These were cryopreserved by slow freezing and vitrification and were later transferred to recipients. Although no live pups were achieved, two implantation sites from each of the two cryopreservation techniques were found. (Personal communication with H.

Some progress has also been reported in other carnivore families. In the Mustilidae family, a member of the caniformia suborder, some species are of commercial value, primarily in the fur industry. These include, for example, the European polecat (*Mustela putorius*) and the American mink (*Mustela vison* or *Neovision vison*). Other members in this family are listed as endangered or critically endangered species, including the black-footed ferret (*Mustela nigripes*) and the European mink (*Mustela lutreola*). The species of commercial value can thus act as models for developing reproduction technologies and for gaining needed knowledge on specific attributes of the Mustelidae family. European polecat, for example, acted as a model for the European mink and the first successful embryo cryopreservation in this family was reported in this species (Lindeberg et al., 2003). Surgically recovered *in vivo*produced European polecat embryos were cryopreserved by slow freezing and resulted, following surgical transfer, in 3/8 pregnancies and nine pups were delivers out of a total of 93 embryos transferred (9.7%). A second paper by the same group (Piltti et al., 2004) reported on the first successful embryo vitrification in carnivores. Out of 98 European polecat *in vivo*-produced embryos at the morula and blastocyst stages, 50 survived and were transferred to four recipients. Two of the recipients delivered a total of eight pups, a success rate similar to that of slow freezing (8/98; 8.2%). Further improvements came when a different vitrification technique, pipette tip, was used. Using this technique, 43.6% (44/101) of the embryos survived vitrification and resulted in live births (Sun et al., 2008). Vitrified embryos that were cultured for two or 16 h before transfer resulted in success rate (71.3% and 77.4% live births, respectively) similar to that of the control (79.3%) and significantly

Mouse was the first animal in which embryo cryopreservation was reported (Whittingham et al., 1972; Wilmut, 1972). Since then work on glires has largely concentrated on mice, rats, gerbils, hamsters and rabbits – all species in extensive laboratory use. The major cryoprotectant used for freezing embryos in this group is Me2SO. Although vitrification seem to be gradually taking the lead and many studies claim similar results to fresh controls, a recent meta-analysis found that vitrification is still inferior to fresh embryos (Manno III, 2010). It also found that a variety of covariates are associated with vitrified but not fresh embryos. These include issues such as the time lapse between hCG treatment and embryo cryopreservation, maternal age, and the time from hCG treatment to post-warming assessment. These and possibly other factors might be the result of heterogeneity of conditions of the studies included in such analysis but they can also be real factors arising from the process of cryopreservation. In rabbits, using *in vivo* produced embryos and either slow freezing (Bank & Maurer, 1974; Whittingham & Adams, 1974, 1976) or vitrification (Popelkova et al., 2009; Mocè et al., 2010), resulted in fairly high survival (up to 83%) and pregnancy (up to 92%) rates. However, rate of young born was still relatively low, in the range of 7 to 17% (Bank

higher than in embryos cultured for 32 h (25%) and 48 h (7.8%).

**3.2.1.4 Glires – rodents and lagomorphs** 

Lindeberg, cited in Farstad, 2000a).

Over the past decade or so, several reports on embryo cryopreservation in felids appeared in the scientific literature. Some investigators, using the domestic cat as a surrogate mother for frozen-thawed embryos of similar-sized wild feline species, produced offspring of ocelot (*Felis pardalis*) (Swanson, 2001) and the African wild cat (Pope et al., 2000). Transfers of frozen-thawed embryos to conspecific recipients have often failed to produce live offspring. In clouded leopard, no pregnancies were achieved with either frozen-thawed or control embryos (Pope et al., 2009). Similarly, frozen-thawed morula-stage cerval embryos failed to result in pregnancies after transfer (Pope et al., 2005). In the bobcat (*Lynx rufus*), out of three transferred embryos – two fresh and one frozen-thawed, one pregnancy (from a fresh embryo) was achieved (Miller et al., 2002). Failure, however, was not a universal phenomenon. In the ocelot (*Felis pardalis*) over 80 IVF embryos, representing 15 founders of the North American population of this species were cryopreserved for safekeeping (Swanson, 2003) and two pregnancies were established following laparoscopic transfer of frozen-thawed embryos (Swanson, 2006). IVF was also carried out in tigrina (*Leopardus tigrinus*), another South American wild felid, and the resulting embryos (n=52) were cryopreserved (Swanson et al., 2002). Regrettably, the researches failed to report on postthaw evaluation. In caracal (*Felis caracal*) from 452 recovered matured oocytes, 297 embryos were produced. Additional 16 embryos were produced following IVM of 83 oocytes. A total of 109 embryos were cryopreserved using slow freezing. Of nine recipients, three became pregnant and three kittens were delivered (Pope et al., 2006). Vitrification was also attempted in wild felids and was shown to produce superior results as compared to slow freezing. Siberian tiger (*Panthera tigris altaica*) oocytes were collected by laparoscopy from chemically stimulated ovaries. Following IVF with frozen-thawed sperm and IVC to the 2 to 4-cell stage, embryos were cryopreserved by either slow freezing or vitrified. None of the slow freezing embryos survived (0/89). From those vitrified, 46% (32/70) survived (Crichton et al., 2000; Crichton et al., 2003).

Whereas some success has been achieved in felids, situation is lagging far behind in canids and progress has been slow (Farstad, 2000a, b). Associated ART techniques such as IVM, IVF and IVC still face many difficulties and outcome is often unpredictable, most probably because *in vitro* culture media and conditions are not optimized for this group (Rodrigues & Rodrigues, 2006; Mastromonaco & King, 2007). In the vast majority of the studies, dog zygotes did not progress to the advanced embryonic developmental stages – morula and blastocyst (Rodrigues & Rodrigues, 2006). The first successful embryo cryopreservation in dogs, leading to pregnancy after ET, was reported only in 2007 (Abe et al., 2007) and pup delivery following embryo cryopreservation came two years later (Suzuki et al., 2009). This success was later repeated with *in vivo*-produced embryos using vitrification as the cryopreservation method (Abe et al., 2011). Canine females are unique in their reproductive cycle in the fact that the ovulated oocytes are still immature and their maturation may take two or more days (estimated at 48 to 60 h) while in the distal uterine horn. Also unique is the fact that luteinization and the increase in progesterone actually occur before ovulation (Reynaud et al., 2005; Chastant-Maillard et al., 2011; Concannon, 2011). The extra-follicular maturation process has proved hard to mimic and to date *in vitro* maturation and fertilization are not yet developed in dogs. The bitch anatomy makes retrieval of *in vivo*produced embryos very difficult, leading researchers to resort to a complete surgical removal of the uterus and associated structures, a procedure that limits its application. From the same reason, embryo transfer was also done surgically until the recent development of a non-surgical technique (Abe et al., 2011). Canine oocytes and early-stage embryos also have

Over the past decade or so, several reports on embryo cryopreservation in felids appeared in the scientific literature. Some investigators, using the domestic cat as a surrogate mother for frozen-thawed embryos of similar-sized wild feline species, produced offspring of ocelot (*Felis pardalis*) (Swanson, 2001) and the African wild cat (Pope et al., 2000). Transfers of frozen-thawed embryos to conspecific recipients have often failed to produce live offspring. In clouded leopard, no pregnancies were achieved with either frozen-thawed or control embryos (Pope et al., 2009). Similarly, frozen-thawed morula-stage cerval embryos failed to result in pregnancies after transfer (Pope et al., 2005). In the bobcat (*Lynx rufus*), out of three transferred embryos – two fresh and one frozen-thawed, one pregnancy (from a fresh embryo) was achieved (Miller et al., 2002). Failure, however, was not a universal phenomenon. In the ocelot (*Felis pardalis*) over 80 IVF embryos, representing 15 founders of the North American population of this species were cryopreserved for safekeeping (Swanson, 2003) and two pregnancies were established following laparoscopic transfer of frozen-thawed embryos (Swanson, 2006). IVF was also carried out in tigrina (*Leopardus tigrinus*), another South American wild felid, and the resulting embryos (n=52) were cryopreserved (Swanson et al., 2002). Regrettably, the researches failed to report on postthaw evaluation. In caracal (*Felis caracal*) from 452 recovered matured oocytes, 297 embryos were produced. Additional 16 embryos were produced following IVM of 83 oocytes. A total of 109 embryos were cryopreserved using slow freezing. Of nine recipients, three became pregnant and three kittens were delivered (Pope et al., 2006). Vitrification was also attempted in wild felids and was shown to produce superior results as compared to slow freezing. Siberian tiger (*Panthera tigris altaica*) oocytes were collected by laparoscopy from chemically stimulated ovaries. Following IVF with frozen-thawed sperm and IVC to the 2 to 4-cell stage, embryos were cryopreserved by either slow freezing or vitrified. None of the slow freezing embryos survived (0/89). From those vitrified, 46% (32/70) survived

Whereas some success has been achieved in felids, situation is lagging far behind in canids and progress has been slow (Farstad, 2000a, b). Associated ART techniques such as IVM, IVF and IVC still face many difficulties and outcome is often unpredictable, most probably because *in vitro* culture media and conditions are not optimized for this group (Rodrigues & Rodrigues, 2006; Mastromonaco & King, 2007). In the vast majority of the studies, dog zygotes did not progress to the advanced embryonic developmental stages – morula and blastocyst (Rodrigues & Rodrigues, 2006). The first successful embryo cryopreservation in dogs, leading to pregnancy after ET, was reported only in 2007 (Abe et al., 2007) and pup delivery following embryo cryopreservation came two years later (Suzuki et al., 2009). This success was later repeated with *in vivo*-produced embryos using vitrification as the cryopreservation method (Abe et al., 2011). Canine females are unique in their reproductive cycle in the fact that the ovulated oocytes are still immature and their maturation may take two or more days (estimated at 48 to 60 h) while in the distal uterine horn. Also unique is the fact that luteinization and the increase in progesterone actually occur before ovulation (Reynaud et al., 2005; Chastant-Maillard et al., 2011; Concannon, 2011). The extra-follicular maturation process has proved hard to mimic and to date *in vitro* maturation and fertilization are not yet developed in dogs. The bitch anatomy makes retrieval of *in vivo*produced embryos very difficult, leading researchers to resort to a complete surgical removal of the uterus and associated structures, a procedure that limits its application. From the same reason, embryo transfer was also done surgically until the recent development of a non-surgical technique (Abe et al., 2011). Canine oocytes and early-stage embryos also have

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

high lipid content (Reynaud et al., 2005) making their cryopreservation challenging. These multiple factors are responsible for the slow progress in ART developments in canids. Despite extensive search, the only report on embryo cryopreservation in a non-domestic canid found in the scientific literature is a few words on a trial with blue fox (*Alopex lagopus*) embryos. These were cryopreserved by slow freezing and vitrification and were later transferred to recipients. Although no live pups were achieved, two implantation sites from each of the two cryopreservation techniques were found. (Personal communication with H. Lindeberg, cited in Farstad, 2000a).

Some progress has also been reported in other carnivore families. In the Mustilidae family, a member of the caniformia suborder, some species are of commercial value, primarily in the fur industry. These include, for example, the European polecat (*Mustela putorius*) and the American mink (*Mustela vison* or *Neovision vison*). Other members in this family are listed as endangered or critically endangered species, including the black-footed ferret (*Mustela nigripes*) and the European mink (*Mustela lutreola*). The species of commercial value can thus act as models for developing reproduction technologies and for gaining needed knowledge on specific attributes of the Mustelidae family. European polecat, for example, acted as a model for the European mink and the first successful embryo cryopreservation in this family was reported in this species (Lindeberg et al., 2003). Surgically recovered *in vivo*produced European polecat embryos were cryopreserved by slow freezing and resulted, following surgical transfer, in 3/8 pregnancies and nine pups were delivers out of a total of 93 embryos transferred (9.7%). A second paper by the same group (Piltti et al., 2004) reported on the first successful embryo vitrification in carnivores. Out of 98 European polecat *in vivo*-produced embryos at the morula and blastocyst stages, 50 survived and were transferred to four recipients. Two of the recipients delivered a total of eight pups, a success rate similar to that of slow freezing (8/98; 8.2%). Further improvements came when a different vitrification technique, pipette tip, was used. Using this technique, 43.6% (44/101) of the embryos survived vitrification and resulted in live births (Sun et al., 2008). Vitrified embryos that were cultured for two or 16 h before transfer resulted in success rate (71.3% and 77.4% live births, respectively) similar to that of the control (79.3%) and significantly higher than in embryos cultured for 32 h (25%) and 48 h (7.8%).

#### **3.2.1.4 Glires – rodents and lagomorphs**

Mouse was the first animal in which embryo cryopreservation was reported (Whittingham et al., 1972; Wilmut, 1972). Since then work on glires has largely concentrated on mice, rats, gerbils, hamsters and rabbits – all species in extensive laboratory use. The major cryoprotectant used for freezing embryos in this group is Me2SO. Although vitrification seem to be gradually taking the lead and many studies claim similar results to fresh controls, a recent meta-analysis found that vitrification is still inferior to fresh embryos (Manno III, 2010). It also found that a variety of covariates are associated with vitrified but not fresh embryos. These include issues such as the time lapse between hCG treatment and embryo cryopreservation, maternal age, and the time from hCG treatment to post-warming assessment. These and possibly other factors might be the result of heterogeneity of conditions of the studies included in such analysis but they can also be real factors arising from the process of cryopreservation. In rabbits, using *in vivo* produced embryos and either slow freezing (Bank & Maurer, 1974; Whittingham & Adams, 1974, 1976) or vitrification (Popelkova et al., 2009; Mocè et al., 2010), resulted in fairly high survival (up to 83%) and pregnancy (up to 92%) rates. However, rate of young born was still relatively low, in the range of 7 to 17% (Bank

Genome Banking for Vertebrates Wildlife Conservation 325

found embryos to be normal, examination by electron microscopy revealed multiple

Only very few studies have reported attempts at cryopreservation of marine mammals oocytes and the only ones I was able to locate were on the common minke whale (*Balaenoptera acutorostrata*). These include studies on both slow freezing (Asada et al., 2000; Asada et al., 2001) and vitrification (Iwayama et al., 2005; Fujihira et al., 2006). To date, no study reporting embryo cryopreservation in cetaceans has been published (O'Brien &

Whereas embryo cryopreservation in mammals shows some success, at least in those extensively studied species, situation lagging far behind in all other vertebrates (fishes, birds, reptiles and amphibians). It is true that considerably less efforts have been invested in embryo cryopreservation in most members of these groups, but the more important cause is the different structure embryos in these vertebrates have, difference that complicates their cryopreservation. From the little that has been done in these vertebrates, the vast majority of studies were done on fish (primarily the zebrafish; *Dino rerio*) and to a lesser extent also in amphibians – the two classes with the smaller oocytes among the non-mammalian vertebrates. The ensuing discussion will therefore be primarily on fishes as representatives for these classes. When sex chromosomes are the determination method, as is the case in most vertebrates, either the male or the female can be the heterogametic sex. In mammals the male carry both X- and Y-chromosomes while the female carries two copies of Xchromosome. In birds, on the other hand, it is the female that carry the Z- and Wchromosomes while the male carries two copies of the Z-chromosome. In fishes and amphibians both systems can be found. To have both chromosomes represented, one should aim to at least preserve enough gametes of the heterogametic sex. In many of the nonmammal species this means preserving the female's gametes, which, as will be discussed here, is problematic. Several attributes differentiate oocytes in these classes from those of mammals. To start with, they are considerably larger, resulting in lower surface area to volume ratio. For example, while the diameter of human oocyte is ~120 µm or that of the mouse is ~80 µm, oocyte of the zebrafish is ~750 µm (Selman et al., 1993) or that of the marsh frog (*Rana ridibunda*) is ~1,400 µm (Kyriakopoulou-Sklavounou & Loumbourdis, 1990), oocytes of the American alligator (*Alligator mississippiensis*) are ~4,000 µm (Uribe & Guillette, 2000), those of the pink salmon (*Oncorhynchus gorbuscha*) in the range of 5,150 to 6,340 µm, and the sizes go even higher in snakes such as kingsnakes (genus: *Lampropeltis*) with diameter of about 22,000 µm (Tryon & Murphy, 1982), and birds like the Japanese quail (*Coturnix coturnix japonica*) – 17,000 to 19,000 µm (Callebaut, 1973) or the domestic chicken (*Gallus gallus domesticus*) with a diameter of about 35,000 to 40,000 µm (Schneider, 1992). The consequence of this is relatively poor water and cryoprotectant movement across the cellular membrane during chilling, freezing and thawing. The difference in size also means considerably larger volume of water to vitrify, thus greatly increasing the risk for intracellular ice formation and cell death. Fish embryos contain a large yolk compartment, enclosed in the yolk syncytial layer (YSL). The behavior of the yolk during freezing defer

damages to intracellular components.

**3.2.3 Non-mammal vertebrates** 

**3.2.2 Cetaceans** 

Robeck, 2010).

& Maurer, 1974; Whittingham & Adams, 1974, 1976). In rats both slow freezing and vitrification were attempted, with considerably better results in the latter. *In vivo* produced embryos at the two-, four- and eight-cell stages were recovered and frozen with 3.0M Me2SO. Post-thaw normal morphology recovery rate ranged between 65% and 68%. Rate of embryos carried to term, however, was low - 11% for two-cell embryos, zero for four-cell embryos and 9% for eight-cell embryos (Whittingham, 1975). In contrast, in the vitrification study, 79% (117/149) of the vitrified *in vivo* produced blastocysts were morphologically normal after warming. These were split between *in vitro* culture (n=48) and transfer to recipient rats (n=69). All cultured embryos progressed to expanded and hatched blastocysts and of the 69 embryos transferred, 41% (n=28) resulted in live pups (Kono et al., 1988). The golden hamster, also known as the Syrian hamster (*Mesocricetus auratus*), is another member of this group in frequent use as a laboratory research subject. *In vivo* produced embryos at the one- and twocell stages were flushed and vitrified by the cryoloop technique (Lane et al., 1999). Of 216 vitrified two-cell embryos, 54.2% continued development to the morula/blastocyst stage after warming. Such embryos were transferred to two recipients who delivered 6 pups. In another study, *in vivo* produced embryos at the eight-cell stage were vitrified in 250µL straws, following the technique developed for mouse embryos (Mochida et al., 2000). This study evaluated only *in vitro* development and this was fairly poor, as only two out of 37 embryos developed to the blastocyst stage. Similar to the hamster, *in vivo*-produced Mongolian gerbil (*Mesocricetus auratus*) embryos were vitrified in 250µL straws (Mochida et al., 1999). Following vitrification, 155 embryos developed to the blastocyst stage were transferred to 10 synchronized females, 3 of which became pregnant and delivered 15 pups (9.7%). In a followup study by the same group it was shown that embryos at later developmental stages (fourcell, morula and blastocyst) can also be vitrified and result in very high post-warming normal morphology (ranging between 87% and 100%) (Mochida et al., 2005). In this last study, after transfer into recipient females, 3% (4/123), 1% (1/102), 5% (4/73), and 10% (15/155) of embryos developed to full-term offspring from vitrified-warmed early two-cell embryos, late two-cell embryos, morulae, and blastocysts, respectively. The general tendency in all glires seem to be the same – post-thaw/warming *in vitro* quality of the embryos is good but when transferred to recipient females, only around 10% of transferred embryos develop to term. The study by Kono and colleagues (1988) with the reported 41% pups delivered is the exception to this rule. When vitrification was attempted, it seems to result in better outcome.

#### **3.2.1.5 Marsupials**

Marsupials are very different from eutherian mammals in many respects, attributes related to their oocytes is one of them. Their oocytes are about twice as large as those of humans (about 200 to 250 µm vs. 100 to 120 µm in humans) (Rodger et al., 1992; Breed et al., 1994). The size is probably that large because of the very large yolk sac that occupies much of the cell volume. The much larger volume and the large yolk compartment make their cryopreservation even more difficult than that of the already hard-to-cryopreserve eutherian oocytes. The alternative is to cryopreserve embryos and in that direction only a single report was found (Breed et al., 1994). In that study, *in vivo*-produced embryos of the carnivorous fat-tailed dunnart (*Sminthopsis crassicaudata*) were cryopreserved by slow freezing or vitrification. Me2SO proved to be not suitable for vitrification of embryos in this species as none of the embryos vitrified with this cryoprotectant cleaved after warming. Embryos cryopreserved by slow freezing or vitrification (with ethylene glycol as cryoprotectant) had similar cleavage rates or 17% and 18%, respectively. Even when morphological examination found embryos to be normal, examination by electron microscopy revealed multiple damages to intracellular components.

#### **3.2.2 Cetaceans**

324 Current Frontiers in Cryobiology

& Maurer, 1974; Whittingham & Adams, 1974, 1976). In rats both slow freezing and vitrification were attempted, with considerably better results in the latter. *In vivo* produced embryos at the two-, four- and eight-cell stages were recovered and frozen with 3.0M Me2SO. Post-thaw normal morphology recovery rate ranged between 65% and 68%. Rate of embryos carried to term, however, was low - 11% for two-cell embryos, zero for four-cell embryos and 9% for eight-cell embryos (Whittingham, 1975). In contrast, in the vitrification study, 79% (117/149) of the vitrified *in vivo* produced blastocysts were morphologically normal after warming. These were split between *in vitro* culture (n=48) and transfer to recipient rats (n=69). All cultured embryos progressed to expanded and hatched blastocysts and of the 69 embryos transferred, 41% (n=28) resulted in live pups (Kono et al., 1988). The golden hamster, also known as the Syrian hamster (*Mesocricetus auratus*), is another member of this group in frequent use as a laboratory research subject. *In vivo* produced embryos at the one- and twocell stages were flushed and vitrified by the cryoloop technique (Lane et al., 1999). Of 216 vitrified two-cell embryos, 54.2% continued development to the morula/blastocyst stage after warming. Such embryos were transferred to two recipients who delivered 6 pups. In another study, *in vivo* produced embryos at the eight-cell stage were vitrified in 250µL straws, following the technique developed for mouse embryos (Mochida et al., 2000). This study evaluated only *in vitro* development and this was fairly poor, as only two out of 37 embryos developed to the blastocyst stage. Similar to the hamster, *in vivo*-produced Mongolian gerbil (*Mesocricetus auratus*) embryos were vitrified in 250µL straws (Mochida et al., 1999). Following vitrification, 155 embryos developed to the blastocyst stage were transferred to 10 synchronized females, 3 of which became pregnant and delivered 15 pups (9.7%). In a followup study by the same group it was shown that embryos at later developmental stages (fourcell, morula and blastocyst) can also be vitrified and result in very high post-warming normal morphology (ranging between 87% and 100%) (Mochida et al., 2005). In this last study, after transfer into recipient females, 3% (4/123), 1% (1/102), 5% (4/73), and 10% (15/155) of embryos developed to full-term offspring from vitrified-warmed early two-cell embryos, late two-cell embryos, morulae, and blastocysts, respectively. The general tendency in all glires seem to be the same – post-thaw/warming *in vitro* quality of the embryos is good but when transferred to recipient females, only around 10% of transferred embryos develop to term. The study by Kono and colleagues (1988) with the reported 41% pups delivered is the exception to

this rule. When vitrification was attempted, it seems to result in better outcome.

Marsupials are very different from eutherian mammals in many respects, attributes related to their oocytes is one of them. Their oocytes are about twice as large as those of humans (about 200 to 250 µm vs. 100 to 120 µm in humans) (Rodger et al., 1992; Breed et al., 1994). The size is probably that large because of the very large yolk sac that occupies much of the cell volume. The much larger volume and the large yolk compartment make their cryopreservation even more difficult than that of the already hard-to-cryopreserve eutherian oocytes. The alternative is to cryopreserve embryos and in that direction only a single report was found (Breed et al., 1994). In that study, *in vivo*-produced embryos of the carnivorous fat-tailed dunnart (*Sminthopsis crassicaudata*) were cryopreserved by slow freezing or vitrification. Me2SO proved to be not suitable for vitrification of embryos in this species as none of the embryos vitrified with this cryoprotectant cleaved after warming. Embryos cryopreserved by slow freezing or vitrification (with ethylene glycol as cryoprotectant) had similar cleavage rates or 17% and 18%, respectively. Even when morphological examination

**3.2.1.5 Marsupials** 

Only very few studies have reported attempts at cryopreservation of marine mammals oocytes and the only ones I was able to locate were on the common minke whale (*Balaenoptera acutorostrata*). These include studies on both slow freezing (Asada et al., 2000; Asada et al., 2001) and vitrification (Iwayama et al., 2005; Fujihira et al., 2006). To date, no study reporting embryo cryopreservation in cetaceans has been published (O'Brien & Robeck, 2010).
