Tan Jinghe;Zhou Qi;Zhang Qiuming;He Guixin;Liu Ruixiang;Xu Libin;Yang Chanyun;Qin Pcngchun

;Northeast Agriculture University Harbin, 150030 PRC

nuclear transplantation;electrofusion;electrical activation;oocyte;embryo;mouse;rabbit;sheep

A series of experiments were conducted to study the major procedures in nuclear transplantation such as oocyte enuclcation and activation, electrofusion and developncnt of the nuclear transplant embryos in the mouse, rabbits and sheep. The important results are as follows: 1. In the mouse, only 35% of the oocytes collected 15~ 16 h after hCG had a notable first polar body (FPb) and those without FPb were enucleated by removing cytoplasm from the PVS—wider side and the enucleation rate was similar tothat in the oocytes with FPb, and the enuclcation rate of removing 1 / 3 cytoplasm was remarkably higher than that of removing 1/4 cytoplasm. 2. Among the three fusion media tested, mannitol and sucrose solutions produced better results than M_2 in electrofusion of mouse 2-cell embryos. Under favorable pulse conditions, the osmotic pressure of fusion medium had no motable effect on electrofusion, but as the conditions became so unfavorable that some embryos began to lyse, the fusion rates in hypcrtonicmannitol solution were significantly higher than those in isotonic or hypotonic solutions. A wide range of pulse strengths (0.31~2.04 kv/ cm) and durations(10~ 1280 mu s) were used and 100% of fusion were obtained in many cases. Optimal pulse durationswere plotted for field strengths to obtain high fusion rates (96%— 100%) in mouse2-cell embryos. 3. With one pulse of 0.45 kv / cm, satisfactory results of mouse oocyte activation were obtained only when the duration Increased to 160 ^s or longer. The activation rate increased as the oocytes got older. Some of the oocytes ar. rested at metaphase IH after electrical stimulation and their proportion to the number of oocytes not activated increased with egg age. 4. 10% and 31% of the nuclear transplant embryos developed to morula or blastocyst stage in sheep and rabbits, respectively, with Chinese-made hormones and chemicals.