Cloned Porcine Embryos can Maintain Developmental Ability after Cryopreservation at the Morula Stage

Abstract

Abstract. The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/ 635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer. Key words: Pig cloning, Somatic cell nuclear transfer, Vitrification (J. Reprod. Dev. 57: [312][313][314][315][316] 2011) n recent years, pigs have often been used as large laboratory animals in biomedical research [1][2][3][4][5][6][7], and genetically engineered pigs are highly desired in rapidly advancing research areas, such as regenerative medicine, organ transplantation, analysis of intractable genetic disorders and stem cell therapy [for review, see 7]. For the production of genetically engineered pigs, including gene knockout pigs, somatic cell cloning is a key technology [8][9][10][11][12][13][14][15]. Therefore, if a reliable cryopreservation technique for cloned embryos can be established, the production and application of various specially designed pigs will advance further.We conducted the present study to elucidate the conditions that could achieve a high survival rate of cloned porcine embryos after cryopreservation in a stable manner. We previously showed that, for the cryopreservation of porcine IVM-derived embryos, it is effective to combine vitrification and delipation, a process for reducing the amount of lipid droplets in the embryonic cytoplasm [16,17]. We have also confirmed in our previous studies that delipation of porcine in vitro-produced embryos at the morula stage is less injurious than when done at the early cleavage stages or the blastocyst stage [16][17][18][19]. In addition, it was shown that vitrification of delipated porcine embryos at the morula stage results in a high postwarming survival rate [16,18,19]. In conjunction with these studies, we carried out a study of the vitrification of porcine cloned embryos at the morula stage. The overall efficiency of cloned pig production by SCNT with cryopreservation (i.e., how many cloned piglets could be produced from all of the SCNT embryos prepared via cryopreservation) was clarified.In the cryopreservation experiment, the SCNT embryos ...

Hollow Fiber Vitrification: A Novel Method for Vitrifying Multiple Embryos in a Single Device

“…Porcine embryos are known to be highly cryosensitive [37,38], and IVM-derived porcine morulae are even more cryosensitive than in vivo derived embryos. To date, successful application of cryopreservation with IVM-derived porcine morulae has required treating the embryos to increase their cryotolerance by removing cytoplasmic lipid droplets, i.e., delipation [30,39]. By contrast, in this study, we were able to attain very high viability in the vitrification of IVM-derived porcine morulae without delipation.…”

Advancing Pig Cloning Technologies Towards Application in Regenerative Medicine

“…Nakano et al. (2011b) reported that cloned piglets could be produced from cloned embryos that had been cryopreserved using a vitrification method, with a production efficiency equivalent to that for non‐vitrified embryos.…”

“…A solution to this difficulty would be to cryopreserve somatic cell cloned pig embryos; as pig embryos are notoriously sensitive to damage during cryopreservation, improvements in the methodology are required to increase the production efficiency of cloned pigs. Nakano et al (2011b) reported that cloned piglets could be produced from cloned embryos that had been cryopreserved using a vitrification method, with a production efficiency equivalent to that for non-vitrified embryos. In this study, the cloned embryos were vitrified at the morula stage following the removal of cytoplasmic lipid droplets (delipation) (Nagashima et al 1995).…”

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

“…Sufficient time in equilibration solution would allow the blastocoelic contents to be suffused by the cryoprotectants, thus reducing the risk of ice crystal formation, but with an increase in toxic exposure (Li et al., ). In response to this, a number of studies describe carrying out the entire vitrification procedure at room temperature, which has been proposed to reduce the embryo's metabolic rate and thus lower the toxic effects of the cryoprotectants on embryos (Kuwayama, Ieda, Zhang, & Kato, ; Maehara et al., ; Nakano et al., ).…”

“…While the cryopreservation of sheep and cattle embryos is routinely performed and offered commercially (Mapletoft & Hasler, 2005;Martinez et al, 2006), a reliable method for vitrifying porcine embryos still seems far off due to the low post-thaw survival rates typically achieved. The two main reasons for these poor survival rates are the suboptimal conditions of in vitro culture (IVC), which results in lower quality embryos (Grupen, 2014), and the endemically high lipid content of porcine oocytes and embryos, which impedes the freezing process (Nagashima, Kashiwazaki, Ashman, Grupen, & Nettle, 1995;Nakano et al, 2011;Yoshioka, Suzuki, Tanaka, Anas, & Iwamura, 2002). Over the past decade, vitrification protocols have been developed with varying success to overcome the porcine embryo's extreme sensitivity to cooling.…”