Fibroblast Cell Line Establishment And Cryopreservation And Interspecies Embryos Reconstruction In Red Panda (Ailurus Fulgens)

The evolutionary history of the red panda (Ailurus Fulgens) plays a pivotal role on high-level phylogeny of the arctoid carnivoran mammals. Because of environmental factors, the red panda has become endangered. For the first time, the present study was conducted to establish and cryopreserve the fibroblast cells, and then the interspecies embryos were reconstructed with this fibroblast cells as somatic cell donors and enucleated rabbit oocytes as recipients.The results showed that an efficient culture and cryopreservation system was established. Briefly, the fibroblast cells could be well cultured by traditional ear tissue piece culture. For original passage culture, the medium could be DMEM (low glucose) supplemented with 20% fetal bovine serum (FBS). The adhesion reached 100% after 96 h. For subculture, the medium could be DMEM (low glucose) supplemented with 10% FBS. The curve of growth at 3rd passage said normal. The fibroblast cells were subcultured to tenth passage with normal morphology. For cryopreservation, 20% FBS and 8% dimethyl sulfoxide (DMSO) could be the cryoprotectants. When the fibroblasts were cryopreserved, the cells could be equilibrated at 4°C 30 min, at -80°C overnight, and then finally plunged into liquid nitrogen.In order to get meiotically mature oocytes the rabbits were superovulated and the metaphase II (MII) oocytes were collected from oviduct. The results showed that the treatment of 120 IU eCG and 180 IU hCG caused better effect. And 16.3 oocytes per rabbit were obtained, and 97.3% of them were at MII.These oocytes were enucleated as the recipients while the frozen/thawed fibroblast cells as donors. The interspecies embryos were reconstructed by microinjection. The electrofusion used direct currency of 140 v/mm, 80μs/2s. The activation was performed in 5μM ionomycin for 4-5 min, 2 mM 6-dimethylaminopurine (6-DMAP) for 4 h. The 194 reconstructed embryos were obtained after fusion and activation, and the cleavage rate was 70.61%. Among the zygotes, 46.72% developed to morulea and 22.63% to blastocysts. The control was designed with the same nuclear transfer protocol but without fusion. The cleavage and morulea rate was significantly lower (P<0.01). The results indicate that rabbit oocyte cytoplasm could support the reprogramming of highly differentiated red panda somatic cells.