59 A PRELIMINARY STUDY OF THE IN VITRO DEVELOPMENT OF ASIAN ELEPHANT, CLONED EMBRYOS, RECONSTRUCTED USING A RABBIT RECIPIENT OOCYTE

Abstract

Interspecies nuclear transfer is an important tool for studying the interaction between the cytoplasm of one cell and the donor nucleus of another (Chen et al. 2002 Biol. Reprod. 67, 637–642). The aim of this experiment was to investigate the possibility of developing in vitro an asian elephant cloned embryo using a rabbit recipient oocyte. The elephant donor cells were obtained from the ear skin of a stillborn Asian elephant (Elephus maximus) and the in vivo-matured recipient oocytes were obtained from FSH-stimulated New Zealand White doe rabbits. Enucleation was accomplished by aspiration of the first polar body and the metaphase II plate together with a small amount of cytoplasm. Successful enucleation was confirmed by UV examination after staining with 5 μg mL⁻¹ Hoechst 33342. The donor cells were introduced into the perivitelline space of the enucleated oocytes immediately after enucleation. The elephant-rabbit reconstructed embryos were fused in 0.3 M manitol with 0.1 mM Ca²⁺ and Mg²⁺ using two types of electrical pulses: E1 (n = 61): 3.2 kV/cm, 3 pulses, 20 μs (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369); E2 (n = 69): 2.0 kV/cm, 2 pulses, 20 μs (Chen et al. 2002 Biol. Reprod. 67, 637–642). The fused embryos were activated 1 h after fusion by electrical pulses to those used for fusion and then incubated in 5 μg mL⁻¹ cyclohexamide and 2 mM 6-DMAP for 1 h. Subsequently, the activated embryos were cultured in B2 medium containing 2.5% fetal calf serum. The developmental rate was observed every 24 h for 7 days and the differences in the percentages of embryos developing to a particular stage were determined by chi-square analysis. The results showed that the fusion and cleavage rates of elephant-rabbit cloned embryos fused and activated by E1 were significantly higher than for E2 (P < 0.05; see Table 1). Compared with rabbit-rabbit cloned embryos using adult skin fibroblast as a donor cell and E1 for both fusion and electrical activation, we found that the cleavage and blastocyst rates of elephant-rabbit cloned embryos was higher than for the rabbit-rabbit ones (65% (28/43) versus 58% (28/48) and 7% (3/43) versus 4% (2/48) respectively). Results from this study showed that either of the electrical pulses, 3.2 kV/cm, 3 pulses, 20 μs or 2.0 kV/cm, 2 pulses, 20 μs, can be used to fuse elephant somatic cells to rabbit ooplasm and the rabbit oocytes can be served as recipient oocytes to support the development of elephant cloned embryos up to the blastocyst stage.

Table 1.
Developmental rate of elephant–rabbit cloned embryos after being fused by different electrical pulses

This work was supported by Rajadapisek Sompoj Fund, Chulalongkorn University.