Abstract: This study was conducted to optimize the culture and cryopreservation conditions for buffalo hand-made clone (HMC) embryos. Microdrops (MD), well of the wells (WOW), flat surface (FS) were adopted to culture HMC reconstructed embryos;and 40% EG, 25% EG (ethylene glycol, EG) + 25% DMSO (dimethylsulphoxide, DMSO) and 20% EG + 20% DMSO + 0.5 mol/L sucrose were adopted to vitrify HMC blastocysts. Furthermore, the efficiency of HMC and traditional somatic cell nuclear transfer (SCNT) were also compared. The results showed that the cleavage rate of embryos cultured in WOW was higher than those cultured on FS (P<0.05) and in MD(P<0.01);the blastocyst rate of embryos derived from WOW system (40.0%) was also higher than those derived from FS (19.8%) and MD (8.3%) systems (P<0.01);the cryosurvival rate of blastocysts vitrified with 20% EG + 20% DMSO + 0.5 mol/L sucrose was higher than those vitrified with 40% EG (P<0.01);both the fusion rate and blasotycst rate of HMC reconstructed embryos were higher than those of SCNT reconstructed embryos (P<0.05;P<0.01), while the cryotolerances of HMC and SCNT blastocysts were not different from each other. These results indicated that HMC could be an alternative to traditional SCNT in buffalo, WOW was most suitable for culture of HMC embryos, and vitrification of HMC blastocysts with 20% EG＋20% DMSO＋0.5 mol/L sucrose could get a reasonable cryosurvival rate.

Key words: buffalo; hand-made clone embryos; culture; cryopreservation