Effect of post-fusion holding time, orientation and position of somatic cell-cytoplasts during electrofusion on the development of handmade cloned embryos in buffalo (Bubalus bubalis)

The present study was conducted primarily to optimize electrofusion conditions for efficient production of zona-free nuclear transfer embryos in buffalos (Bubalus bubalis). We found that 4V AC current for proper triplet alignment and single step fusion method, using a single DC pulse of 3.36 kV/cm for 4-μs duration, produced the most convincing results for efficient reconstitution of zona-free cloned embryos. Lysis rate was very high (84.28 ± 2.59%) when triplets were in physical contact with negative electrode after applying DC current, however, cleavage rate and blastocyst rate were found to be similar when the triplets were not in physical contact with either positive or negative electrodes or when they were in physical contact with the positive electrode. Significant improvement in blastocyst production was observed when the somatic cell faced the positive electrode than when it faced the negative electrode (39.17 ± 2.74% vs. 25.91 ± 2.00%, respectively) during electrofusion. Similarly, the blastocyst rate (52.0 ± 3.4%) was found to be significantly higher when reconstructed embryos were activated 6 h post electrofusion as compared to 0, 2, 4 and 8 h (16.04 ± 6.3%; 18.36 ± 1.4%; 22.44 ± 3.7% and 30.02 ± 4.6%, respectively). This study establishes the application of zona-free nuclear transfer procedures for the production of handmade cloned buffalo embryos through optimization of electrofusion parameters and post fusion holding time for enhancing their preimplantation development.