Cloned Asian Elephant (Elephas maximus) Embryos Reconstructed from Rabbit Recipient Oocytes

Abstract

The information about reproductive biology, especially embryo development of Asian elephant (Elephas maximus) generated from naturally fertilization is lacking. In the present study, somatic cell nuclear transfer (SCNT) was applied as an alternative way to produce the elephant embryos. The fibroblasts derived from ear skin of Asian elephant and rabbit were used as the donor cells and rabbit oocytes were used as the recipient cytoplasm. The objectives of the study were 1) to find the optimal conditions for fusion and activation by using electrical pulses (experiment I) and 2) to investigate the in vitro development of cloned Asian elephant in comparison to clone rabbit embryos (experiment II). Enucleation was accomplished by aspiration of the first polar body and the metaphase II
plate together with a small amount of cytoplasm. The donor cells were transferred into the perivitelline space of the enucleated oocytes. In experiment I, sixty-one of elephant-rabbit reconstructed units were fused by electrical pulses E1 (3.2 kV/cm, 3 pulses, 20 μs) and sixty-nine units were fused by E2 (2.0 kV/cm, 2 pulses, 20 μs) in 0.3 M mannitol
with 0.1 mM Ca2+ and Mg2+. The fused units were activated by using the same electrical pulses and incubated in activation medium for 1 h. Subsequently, the activated embryos were cultured in B2 medium containing 2.5% fetal calf serum and the developmental rate was observed daily for 7 days. The results showed that the fusion and
cleavage rates of elephant-rabbit cloned embryos fused and activated by E1 were significantly higher than E2 (p<0.05). Electrical pluses program E1 was selected for further investigation in experiment II. The fusion and
activation rated of elephant-rabbit units displayed significantly higher than rabbit-rabbit units (p<0.05). However, by comparison of cloned embryo development between elephant- and rabbit-rabbit units, the development from cleavage throughout the blastocyst stage of elephant-rabbit cloned units was similar to those of rabbit-rabbit units. In conclusion, fusion and activation protocol of E1 is suitable for elephant-rabbit SCNT and the elephant nuclei could be reprogrammed and developed to blastocyst stage in enucleated rabbit oocytes. The present study provides the
fundamental knowledge for further investigation of conservation and therapeutic aims, including cloned elephant embryo development in vivo after transfer, rescuing valuable elephant and establishment of elephant embryonic stem cells.