Micromanipulation and cloning studies on buffalo oocytes and embryos using nucleus transfer.

An investigation for testing the viability of production of cloned buffalo embryos through nucleus transfer has been made. Matured buffalo oocytes, after zona cutting to an extent of 60 degrees near polar body, were enucleated using a new approach. Instead of aspirating the cytoplasm contents in a pipette, the half of cytoplasm of oocyte was pushed out, thereby also taking away the nuclear material of the oocyte, leaving the demi-oocyte with the zona pellucida enucleated. The absence of fluorescence confirmed the success of the enucleating process. For enucleating, the oocytes which had intact plasma membrane were eligible for bisectioning. There was no significant difference in oocytes having intact membrane among grade I (33.9%) and grade II (31.4%) oocytes, whereas lower percentage of grade III oocytes had a very low percentage having intact plasma membranes (8.5%). The hours of maturation for 32, 37 and 42 did not influence the per cent oocytes which had intact membranes. All the bisected or demi-oocytes tested with fluorescence screening yielded successful enucleation in 88.2% demi-oocytes. The temporal effect of three maturation hours of 32, 37, and 42 hr; two electrical pulse numbers of 2 and 3 pulses and two magnitudes of electric pulses of 15 and 20 V were studied for their effect on the percentage of successful fusion of demi-oocyte blastomere complexes and the rate of complexes undergoing cleavages. The time period for which the oocytes were subjected to the process of maturation significantly affected the per cent fusions and per cent cleavage of the demi-oocyte blastomere complexes and 32 hr maturation yielded less fusions (38.5%) compared to maturation for 37 and 42 h (53.2 and 57.8%, respectively). The treatment of either 2 or 3 electrical pulse numbers resulted in significantly different fusion (45.6 and 54.1%) as well as cleavage rates (18.2 and 26.1%) of demi-oocyte-blastomere complexes electrofused. The treatment of two levels of magnitude of 15 and 20 V of an electric current resulted in similar per cent fusion (48.0 and 51.6%) and cleavage rates (21.0 and 23.2%). Fortified TCM with either 10 or 20% FBS for culturing freshly electrofused complexes for 1 hr did not differ significantly with respect to per cent complexes fused and cleaved, giving a fusion rate of 46.2 and 53.8% and cleavage rate of 21.2 and 23.2% for 10% and 20% FBS, respectively. Production of cloned embryos through the process of nuclear transfer has been accomplished. The successful cleavages of the nuclear transferred oocytes demonstrated the viability of enucleation procedures of the oocytes and technology implementation of electrofusion in buffalo oocytes.