Blastocysts cloned from the Putian Black pig ear tissues frozen without cryoprotectant at −80 and −196 degrees Celsius for 3 yrs

The Putian Black pig, as one of elite cultivars of endemic species in China, has been on the verge of extinction and urgently needs protection. Somatic cell nuclear transfer (SCNT) and noncryoprotected frozen tissue technology have successfully resurrected several mammalian species. Therefore, this study explored the primary feasibility of conserving this breed using a combination of both technologies. Skin tissues obtained from the ears of adult Putian Black boars were frozen without cryoprotectant at −20, −80, or −196 °C and stored for 3 yrs. Primary cell culture, passage and subculture were performed on frozen samples after being rapidly thawed at 39 °C and on fresh pig ear tissues (control). Cloned embryos were reconstructed using fibroblasts (from frozen and fresh tissues) with enucleated oocytes. Live cell lines were obtained from tissues frozen at −80 and at −196 °C and appeared to have normal proliferative activity after passage; furthermore, they directed cloned embryos to develop to the blastocyst stage after nuclear transfer. We concluded that the population of Putian Black pig might be increased in the future by transferring cloned blastocysts into synchronized recipient pigs.