Abstract: The hand-made cloning (HMC) technique describes a simplified nuclear transfer process without the need for micromanipulators. This study was conducted to investigate the effect of different enucleation,fusion methods and donor cell types on the efficiency of HMC in buffalo. The results indicated that chemically assisted enucleation with cytochalasin b (CB) showed similar enucleation efficiency to Hoechst 33342 staining enucleation;the fusion rate of one-step method was significantly higher than two-steps method(P＜0.01),while the developmental abilities of the couplets reconstructed by the two methods were no significant different from each other(P＞0.05).The fusion rate of HMC couplets derived from ear fibroblasts was significantly lower than that of couplets from cumulus cells (P＜0.01),while the cleavage and blastocyst rates of the embryos from the two cell types were not different from each other(P＞0.05). The study showed that CB assisted enucleation,one-step fusion,cumulus cells can be adopted for HMC in buffalo to improve the efficiency of cloning.

Key words: buffalo; HMC; enucleation; fusion; donor cells