Interspecies nuclear transfer using fibroblasts from leopard, tiger, and lion ear piece collected postmortem as donor cells and rabbit oocytes as recipients.

Abstract

Skin fibroblast cells were obtained from a small piece of an ear of leopard, lion, and tiger collected postmortem and attempts were made to synchronize the skin fibroblasts at G0/G1 of cell cycle using three different approaches. Efficiency of the approaches was tested following interspecies nuclear transfer with rabbit oocytes as recipient cytoplasm. Fluorescence-activated cell sorting revealed that the proportion of G0/G1 cells increased significantly (P < 0.05) when cells subjected to serum starvation, contact inhibition, and 3mM sodium butyrate (NaBu) treatment when compared with cycling cells. However, 3mM NaBu treatment caused alterations in cell morphology and increase in dead cells. Thus, interspecies nuclear transfer was carried out using fibroblast cells subjected to contact inhibition for 72h, serum starvation for 48h, and cells treated with 1.0mM NaBu for 48h. The fusion rates, the proportion of fused couplets that cleaved to two-cell and developed to blastocyst, were highest in all three species when the donor cells were treated with 1.0mM NaBu for 48h. But, the blastocyst percentage of interspecies nuclear embryos (5-6%) was significantly lower when compared with rabbit-rabbit nuclear transfer embryos (22.9%). In conclusion, fibroblast cells of leopard, lion, and tiger were successfully synchronized and used for the development of blastocysts using rabbit oocytes as recipient cytoplasm.