Preparation of cytoplasts

Whether using the oocyte from a domestic or a wild mammal, some fundamental criteria must be met to obtain a cytoplast suitable for cloning, such as oocyte selection, in vitro maturation, and enucleation systems (Loi et al., Reference Loi, Modlinski and Ptak2011). In general, follicular size, the oocyte collection method, and the culture environment are factors that can affect the quality of mature oocytes, and different responses to these factors can be observed in wild mammals. In some cervid species, Brahmasani et al. (Reference Brahmasani, Yelisetti, Katari, Komjeti, Lakshmikantan, Pawar and Sisinthy2013) observed that low maturation rates could probably be caused by slicing. In this method, non-competent oocytes can be recovered, as the technique can result in the recovery of structures of small diameter follicles (Rho et al., Reference Rho, Hahnel and Betteridge2001). Additionally, the quality of ovaries obtained post-mortem may have been one of the factors that reduce the quality of oocytes in these species.

Therefore, studies have shown that enriched culture medium and ovarian transport conditions may result in good results using ovaries from post-mortem animals for recovery of immature oocytes. Macías-García et al. (Reference Macías-García, González-Fernández, Matilla, Hernández, Mijares and Sánchez-Margallo2018) verified that oocytes of Iberian red deer (Cervus elaphus hispanicus) obtained from ovaries maintained for 16 h in a holding medium increased the oocyte meiotic competence. Moreover, these authors observed that the epidermal growth factor (EGF) demonstrated a differential effect depending upon oocyte grading and conditions of ovary transportation. Additionally, for ovaries derived from Hokkaido sika deer (Cervus nippon yesoensis), maturation rates of oocytes were highest when ovaries were kept for 12 h at 20–25°C, when compared with 24 h (Tulake et al., Reference Tulake, Yanagawa, Takahashi, Katagiri, Higaki, Koyama, Wang and Li2014).

Specifically, with respect to the culture environment, the requirements for both composition and maturation time should be established for the in vitro maturation of each species. In the Indian blackbuck (Antilope cervicapra), oocytes cultured in the presence of gonadotropins (follicle-stimulating hormone, FSH and luteinizing hormone, LH) showed higher rates of expansion of the cumulus oophorus (79.3%) and extrusion of the first polar body (46.1%) compared with oocytes cultured without gonadotropins (60.4% and 33.3%, respectively) (Rao et al., Reference Rao, Mahesh, Lakshmikantan, Suman, Charan and Shivaji2010). In the sika deer (Cervus nippon hortulorum), oocytes cultured in medium supplemented with fetal bovine serum (FBS), FSH, LH, cysteamine and EGF resulted in a higher maturation rate (75.4%) compared with medium without supplementation (30.1%; Yin et al., Reference Yin, Tang, Zhang, Kong, Wang, Guan and Li2013). Already, different hormonal combinations of FSH, LH and 17β-estradiol did not alter the maturation rates in oocytes derived from lions (Panthera leo; Fernandez-Gonzalez et al., Reference Fernandez-Gonzalez, Hribal, Stagegaard, Zahmel and Jewgenow2015). In the collared peccary (Pecari tajacu), we proved that oocytes need 48 h to achieve maturation instead of 24 h, according to the expansion of the cumulus cells (100% vs. 38.1%), the presence of first polar body (90.5% vs. 52.4%), and the status of the nucleus in the second metaphase (76.2% vs. 52.4%), respectively (Borges et al., Reference Borges, Santos, Queiroz Neta, Oliveira, Silva and Pereira2018c).

In addition to obtaining mature oocytes, the preparation of cytoplasts depends on the method of enucleation of these structures. The amount of ooplasm present in the reconstructed embryo is related to the enucleation technique that removes the nucleus from the oocyte. Matured oocytes can be enucleated in different ways, including squeezing the first polar body and the surrounding cytoplasm through a cleft in the zona pellucida of the oocyte (Lee et al., Reference Lee, Wirtu, Damiani, Pope, Dresser, Hwang and Bavister2003). Another method is manual removal in which zona-free oocytes are enucleated with a bisection blade that hand bisect the metaphase II chromosomes along with a small volume of the surrounding cytoplasm. Oocytes can also be aspirated using a micromanipulator at the location of the metaphase II chromosomes and the polar body via brief exposure to ultraviolet light (Pereira et al., Reference Pereira, Melo, Freitas and Salamone2015).

Selection of karyoplasts

To obtain karyoplasts appropriate for cloning, their type and age and the manipulation techniques used are important for their future reprogramming (Kim et al., Reference Kim, Jang, Oh, Yuda, Kim, Hwang, Hossein, Kim, Shin and Kang2007). Karyoplasts can be obtained from fresh or cryopreserved somatic tissues (Folch et al., Reference Folch, Cocero, Chesné, Alabart, Domínguez, Cognié, Roche, Fernández-Arias, Martí and Sánchez2009, Pan et al., Reference Pan, Zhang, Guo and Wang2014), from an adult (Moulavi et al., Reference Moulavi, Hosseini, Tanhaie-Vash, Ostadhosseini, Hosseini, Hajinasrollah, Asghari, Gourabi, Shahverdi and Vosough2017) or a fetus (Liu et al., Reference Liu, Cai, Wang, Nie, Zhang, Xu, Zhang, Lu, Wang and Poo2018), and in vivo or post-mortem (Pereira et al., Reference Pereira, Santos, Borges, Queiroz Neta, Santos and Feitosa2014). Although the recovery of these cells is not a difficult task, their processing and preservation until use in iSCNT require attention (Pereira et al., Reference Pereira, Santos, Borges, Queiroz Neta, Santos and Feitosa2014). In general, skin cells have been the most used cell type for karyoplasts (Song et al., Reference Song, Hua, Song and Zhang2007). The skin has an abundance of cells of interest that may have different efficiencies in cloning, as observed in wild buffalo (Bubalus arnee). Saini et al. (Reference Saini, Selokar, Raja, Sahare, Singla, Chauhan, Manik and Palta2015) detected that fibroblasts of this species are easier to reprogram than epithelial cells.

After harvest, cells used as nuclei donors need to be characterized with respect to their culture conditions, cryopreservation, and cell cycle synchronization (Pereira et al., Reference Pereira, Santos, Borges, Queiroz Neta, Santos and Feitosa2014). For these steps, cells are evaluated for the number of passages, nutritional requirements during in vitro culture (Santos et al., Reference Santos, Borges, Queiroz Neta, Santos, Oliveira, Silva and Pereira2016), and the damage done during cryopreservation (Song et al., Reference Song, Hua, Song and Zhang2007). Thus, karyoplasts have been established in vitro in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with FBS and growth factors (Santos et al., Reference Santos, Borges, Queiroz Neta, Santos, Oliveira, Silva and Pereira2016).

Karyoplasts have been routinely cryopreserved by slow freezing (Sharma et al., Reference Sharma, Sharma, Ahlawat, Aggarwal, Vij and Tantia2018) using a combination of dimethyl sulfoxide (DMSO), FBS, and sucrose as the cryoprotectant, as observed with Iberian lynx (Lynx pardinus, León-Quinto et al., Reference León-Quinto, Simón, Cadenas, Martínez and Serna2014). Although it is more desirable to use a somatic cell bank after tissue culture, the absence of in vitro culture conditions sometimes makes these banks unfeasible, resulting in the immediate formation of the targets for those somatic tissues (Borges et al., Reference Borges, Bezerra, Costa, Queiroz Neta, Santos, Oliveira, Silva and Pereira2017a,Reference Borges, Lima, Queiroz Neta, Santos, Oliveira, Silva and Pereirab; Queiroz Neta et al., Reference Queiroz Neta, Lira, Borges, Santos, Silva, Oliveira, Silva, Oliveira and Pereira2018). The three somatic tissue conservation techniques used for wild animals are slow-freezing cryopreservation (Mestre-Citrinovitz et al., Reference Mestre-Citrinovitz, Sestelo, Ceballos, Baranao and Saragueta2016), vitrification (Borges et al., Reference Borges, Lira, Nascimento, Queiroz Neta, Santos, Oliveira, Silva and Pereira2018a,Reference Borges, Queiroz Neta, Santos, Oliveira, Silva and Pereirab), and cooling at 4–6°C (Queiroz Neta et al., Reference Queiroz Neta, Lira, Borges, Santos, Silva, Oliveira, Silva, Oliveira and Pereira2018). In collared peccaries, we compared two techniques of vitrification and we observed that solid-surface vitrification was found to be a more efficient method for vitrifying skin tissue when compared with direct vitrification in cryovials, probably due to tissues not being involved in large amounts of cryoprotectants before passing through a drastic change in temperature during the solid-surface vitrification (Borges et al., Reference Borges, Lima, Queiroz Neta, Santos, Oliveira, Silva and Pereira2017b).

Finally, the third step in the preparation of the karyoplasts is cell synchronization in the G0/G1 stage (Gómez et al., Reference Gómez, Jenkins, Giraldo, Harris, King, Dresser and Pope2003; Yelisetti et al., Reference Yelisetti, Komjeti, Katari, Sisinthy and Brahmasani2016). In general, nuclear reprogramming is controlled by epigenetic modification. For this to occur, the somatic cells must be in G0/G1 to allow the removal of reversible epigenetic changes acquired during cell differentiation (Song et al., Reference Song, Hua, Song and Zhang2007). Therefore, cells can be subjected to different treatments for synchronization during culture. Inhibition by contact (Moulavi et al., Reference Moulavi, Hosseini, Tanhaie-Vash, Ostadhosseini, Hosseini, Hajinasrollah, Asghari, Gourabi, Shahverdi and Vosough2017), serum deprivation (Wani et al., Reference Wani, Vettical and Hong2017), and chemicals that inhibit the cell cycle (Gómez et al., Reference Gómez, Jenkins, Giraldo, Harris, King, Dresser and Pope2003) are methods used for synchronization. Serum deprivation and inhibition by contact are the most commonly used (Moulavi et al., Reference Moulavi, Hosseini, Tanhaie-Vash, Ostadhosseini, Hosseini, Hajinasrollah, Asghari, Gourabi, Shahverdi and Vosough2017).

Under high confluence or serum privation, fibroblast cells derived from the skin of adult argali (Ovis ammon) were efficiently synchronized at G0/G1; nevertheless, cells were in lower proportion in the growing stage (Pan et al., Reference Pan, Zhang, Guo and Wang2014). Authors observed that the highest proportion of cells from the African wild cat (Felis silvestris lybica) at G0/G1 was obtained by serum deprivation compared with that obtained by inhibition by contact and the inhibitor roscovitine (Gómez et al., Reference Gómez, Jenkins, Giraldo, Harris, King, Dresser and Pope2003). Leopard (Panthera pardus) skin cells treated with chemical inhibitors such as sodium butyrate have a greater propensity to undergo alterations (Yelisetti et al., Reference Yelisetti, Komjeti, Katari, Sisinthy and Brahmasani2016).

Embryonic reconstruction stages

After the transfer of the nucleus into the enucleated oocyte, the cytoplast–karyoplast complex is subjected to an electric pulse that not only induces the fusion of the somatic cell nucleus with the enucleated oocyte to form a new complex, but also promotes the release of intracellular calcium that initiates cellular activation (Pereira & Freitas, Reference Pereira and Freitas2009). In general, the successful development of a reconstructed embryo depends on the complex interactions between the cytoplast and the nuclear structure during embryonic development; failures in this interaction can cause problems during early cleavage and embryonic development (González-Grajales et al., Reference González-Grajales, Favetta, King and Mastromonaco2016).

The activation of the cytoplast–karyoplast complexes guarantees adequate embryonic reconstruction (Yamochi et al., Reference Yamochi, Kida, Oh, Ohta, Amano, Anzai, Kato, Kishigami, Mitani and Matsumoto2013). Because the iSCNT technique reprograms the nucleus of a somatic cell of one species using the oocyte cytoplasm of another species, it is essential that the activation protocol be able to activate the reconstructed embryo (Zhao et al., Reference Zhao, Ouyang, Nan, Lei, Song, Sun and Chen2006). Physiologically, a mammalian oocyte is activated during fusion with a sperm, releasing meiotic cell cycle arrest and enabling the resumption of the oocyte meiotic cell cycle (Sparman et al., Reference Sparman, Tachibana and Mitalipov2010). Therefore, a well developed protocol allows a high rate of blastocyst formation by promoting good embryonic development through activation.

Activation protocols, including physical methods such as electrical pulses and alteration of osmolarity, and chemical methods such as calcium-mobilizing compounds like strontium chloride, ionomycin, and ethanol, to promote the initial release of calcium have been evaluated in different species, as sika deer (Yin et al., Reference Yin, Tang, Zhang, Kong, Wang, Guan and Li2013), alpaca (Vicugna pacos) and llama (Lama glama, Ruiz et al., Reference Ruiz, Landeo, Mendoza, Correa, Silva and Ratto2015), with blastocyst rates of 32.4%, 22.5% and 18.7%, respectively. In general, calcium mobilizers are used in combination with kinase protein inhibitors or protein synthesizers such as cycloheximide and 6-dimethylaminopurine (6-DMAP). In addition, a cytostatic factor inactivator and microfilament inhibitor such as cytochalasin B are used to prevent extrusion of the second polar body and maintain the diploidy of the presumed embryo (Ruiz et al., Reference Ruiz, Landeo, Mendoza, Correa, Silva and Ratto2015).

For red deer (Cervus elaphus), electrical activation before chemical activation with ionomycin and 6-DMAP was efficient for the production of clone embryos (32–44%), obtaining genetically healthy calves (Berg et al., Reference Berg, Li, Asher, Wells and Oback2007). Nevertheless, the same protocol resulted in a low developmental rate (5.7%) of activated oocytes in swamp deer and 0.0% embryos in spotted deer, sambar deer, and brow-antlered deer after oocyte parthenogenetic activation (Brahmasani et al., Reference Brahmasani, Yelisetti, Katari, Komjeti, Lakshmikantan, Pawar and Sisinthy2013). Blackbuck (Antilope cervicapra) oocytes activated with ionomycin and 6-DMAP resulted in 58% cleaved embryos and 13% blastocysts (Rao et al., Reference Rao, Mahesh, Lakshmikantan, Suman, Charan and Shivaji2010). Therefore, the artificial activation method (chemical, electrical protocols or your combination) can result in different responses among species. In this sense, it is necessary to evaluate the type of artificial activation that promotes the best rates of embryonic development in the species of interest.

In vitro culture systems are essential for early embryonic development and nuclear reprogramming (Gómez et al., Reference Gómez, Pope, Kutner, Ricks, Lyons, Ruhe, Dumas, Lyons, López and Dresser2008; Pereira et al., Reference Pereira, Feltrin, Almeida, Carneiro, Avelar, Alcântara Neto, Sousa, Melo, Moura, Teixeira, Bertolini, Freitas and Bertolini2013). Choosing the appropriate culture medium for each species is considered the initial step in proper embryonic development (Zhao et al., Reference Zhao, Ouyang, Nan, Lei, Song, Sun and Chen2006). Lee et al. (Reference Lee, Wirtu, Damiani, Pope, Dresser, Hwang and Bavister2003) used somatic cells of the mountain bongo (Tragelaphus eurycerus isaaci) and domestic cow (Bos taurus) oocytes and observed that a chemically defined, protein-free medium of TCM199 supplemented with FBS supported embryonic development. Nonetheless, there is no one culture medium suitable for all species that allows better embryonic development for a given species under study.

Finally, the effect of epigenetic reprogramming is a very relevant factor in the success of iSCNT (Gómez et al., Reference Gómez, Pope, Kutner, Ricks, Lyons, Ruhe, Dumas, Lyons, López and Dresser2008). Some epigenetic markers were characterized with respect to their function during embryonic reprogramming and their influence on the chromatin structure from post-translational modifications (Song et al., Reference Song, Hua, Song and Zhang2007). The overall level of the acetylation of histone H3 at lysine 18 (H3K18ac) and trimethylation of histone H3 at lysine 27 (H3K27me3), and the expression level of some important apoptosis proteins (caspase 3 and caspase 7), and p53 were evaluated. The hyperacetylated state of histones is associated with transcriptionally active domains, while the hypoacetylated state is associated mainly with silenced chromatin regions of histone acetyl transferases and histone deacetylases. The methylation pattern of the DNA is determined by DNA methyltransferases. OCT3/4, NANOG, and CDX2 are very important because of their close association with pluripotency and early embryonic development (Saini et al., Reference Saini, Selokar, Raja, Sahare, Singla, Chauhan, Manik and Palta2015).

Carnivores

Some works have shown the advances achieved by iSCNT in wild canine species. These species, including the grey wolf (Canis lupus), have gradually become endangered or extinct. Therefore, in 2007, with the goal of canid conservation, Kim et al. (Reference Kim, Jang, Oh, Yuda, Kim, Hwang, Hossein, Kim, Shin and Kang2007) cultured fibroblasts derived from the ear of an adult female grey wolf that were then used as donor cells of nuclei. Using domestic canine oocytes, the authors produced a pregnancy with cloned embryos of two genetic identities of the cloned wolves, but there were no births. In 2008, Oh et al. (Reference Oh, Kim, Jang, Kim, Hong, Park, Park, Park, Sohn and Kim2008) obtained three wolf pups from cloned embryos using cells obtained from a male grey wolf 6 h after death and domestic canine oocytes. These studies demonstrated the successful cloning of endangered wild canines.

In felid species, the main oocyte source has been the domestic cat. In species from the Felidae subfamily, some progress has been achieved. Therefore, synchronized nuclei were donated by the African wild cat and transferred to enucleated domestic cat oocytes resulting in a high rate of blastocyst formation but no pregnancies (Gómez et al., Reference Gómez, Jenkins, Giraldo, Harris, King, Dresser and Pope2003). In another study in which embryos were constructed using somatic cells derived from the African wild cat and domestic cat oocytes, 75% of the embryos developed to term and 25% underwent fetal resorption or abortion (Gómez et al., Reference Gómez, Pope, Giraldo, Lyons, Harris, King, Cole, Godke and Dresser2004). Of the 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are currently alive and healthy. Additionally, some studies on wild felids have shown the establishment of somatic resource banks. There is a bank with somatic samples of 69 individual Iberian lynx, considered the most endangered felid in the world, with the aim of future cloning (León-Quinto et al., Reference León-Quinto, Simon, Cadenas, Jones, Martinez-Hernandez, Moreno, Vargas, Martinez-Hernandez and Soria2009; Reference León-Quinto, Simón, Cadenas, Martínez and Serna2014).

In addition, works on the cheetah (Acinonyx jubatus), a species of the Pantherinae subfamily, have been performed in South America and Asia. Somatic cells from a cheetah raised in South America were transferred to domestic cat oocytes, and, after embryo aggregation during in vitro culture, high blastocyst formation rates were obtained (16.7–28.3%) (Moro et al., Reference Moro, Hiriart, Buemo, Jarazo, Sestelo, Veraguas, Rodriguez-Alvarez and Salamone2015). Moulavi et al. (Reference Moulavi, Hosseini, Tanhaie-Vash, Ostadhosseini, Hosseini, Hajinasrollah, Asghari, Gourabi, Shahverdi and Vosough2017) used non-viable frozen cells derived from frozen tissue from an Asiatic cheetah (Acinonyx jubatus venaticus) and in vitro-matured domestic cat oocytes and obtained morula rates of 5.9%. Although no blastocyst was obtained, this study demonstrated that enucleated cat oocytes can partially remodel and reactivate nonviable nuclei of the Asiatic cheetah and support its reprogramming back to the embryonic stage.

Ungulates

Some studies with ungulates have been performed with significant success, especially for species already extinct. The first animal derived from an extinct subspecies was obtained using fibroblasts from skin biopsies collected before the death of the last female Capra pyrenaica pyrenaica. After a year under cryopreservation, these cells were used as karyoplasts and fused with the cytoplasts of a domestic goat to reconstruct embryos. The rate of cleaved embryos after 36 h was 47.3%, of which 65.5% were transferred. Five recipients were pregnant at 45 days but only one pregnancy went to term. Unfortunately, a few minutes after birth the animal died from pulmonary complications (Folch et al., Reference Folch, Cocero, Chesné, Alabart, Domínguez, Cognié, Roche, Fernández-Arias, Martí and Sánchez2009).

Experiments were carried out with wild yak (Bros grunniens) with the goal of evaluating the parameters that affect the success of iSCNT (Li et al., Reference Li, Dai, Du, Zhao, Wang, Wang, Liu, Li and Li2007). Fibroblasts and cumulus cells were used as donor cells, but the cell type and different ages were found to have no significant effect on iSCNT.

In 2017, the birth of a Bactrian camel cloned by iSCNT was first reported (Wani et al., Reference Wani, Vettical and Hong2017). The fibroblasts used to donate nuclei were obtained from ear skin biopsy samples from an adult male Bactrian camel (Camelus bactrianus) and the cytoplast of dromedary camel (Camelus dromedaries) was the oocyte recipient. Twenty-six blastocysts were transferred to 23 synchronized dromedary recipients yielding five pregnancies with one going to term. This work has great importance because the Bactrian camel is the eighth most endangered large mammal on Earth.

Finally, the woolly mammoth (Mammuthus primigenius) is perhaps the one wild mammal of the ungulates whose cloning arouses the greatest interest. This animal became extinct about 10,000 years ago. However, epithelial and muscular cells from 14,000–15,000-year-old mammoth tissues were cryopreserved, with the goal of producing embryos of this species (Kato et al., Reference Kato, Anzai, Mitani, Morita, Nishiyama, Nakao, Kondo, Lazarev, Ohtani and Shibata2009). In this study, the authors injected cell nucleus-like structures into mature mouse enucleated oocytes; however, the oocytes did not form pronuclear-like structures at 7 h after injection.

Other species

The ability of bovine enucleated oocytes to support dedifferentiation of nuclei from monkey fibroblasts in interspecies cloned monkey embryos has been observed (Lorthongpanich et al., Reference Lorthongpanich, Laowtammathron, Chan, Ketudat-Cairns and Parnpai2008). These embryos were cultured in conditions different from the medium used for cattle with monkey-specific alterations, but the embryos were not able to develop past 16 cells under any culture condition. Nevertheless, OCT-4 was detected, demonstrating the ability of bovine ooplasm to support dedifferentiation but not embryonic development. Therefore, the culture medium promotes dedifferentiation but is not able to support complete embryonic development (Lorthongpanich et al., Reference Lorthongpanich, Laowtammathron, Chan, Ketudat-Cairns and Parnpai2008). In another work that used porcine cytoplasts and donor cells from a rhesus monkey, it was possible to obtain blastocysts despite the low rate (2.04%) (Zhu et al., Reference Zhu, Kang, Li, Jin, Hong, Jin, Guo, Gao, Yan and Yin2014). Although being a SCNT study, the cloning of cynomolgus monkeys (Macaca fascicularis) is cited here because of recent advances in this species. Thus, in a study on cynomolgus monkeys using SCNT, Liu et al. (Reference Liu, Cai, Wang, Nie, Zhang, Xu, Zhang, Lu, Wang and Poo2018) applied histone demethylase Kdm4d mRNA and histone deacetylase inhibitor trichostatin A after activation. Embryonic development improved followed by a greater number of pregnancies, which resulted in the birth of two monkeys via the SCNT technique using fetal fibroblasts and oocytes of cynomolgus monkeys.

With respect to aquatic mammals, a study performed on the minke whale (Balaenoptera bonaerensis) compared different conditions of iSCNT, including the ability of porcine and bovine ooplasms to produce reconstructed embryos and the effects of different donor cell types (viable or nonviable cells) on whale SCNT embryos (Ikumi et al., Reference Ikumi, Sawai, Takeuchi, Iwayama, Ishikawa, Ohsumi and Fukui2004). The authors concluded that whale iSCNT embryos can develop to at least the four-cell stage, regardless of the survivability of the donor cells and the porcine or bovine ooplasm.