Spherical Sperm In Mice Embryo Preparation And Training Improvements

Round spermatids have been used as substitute gametes in basic reproductive research and in infertility clinics. The embryos of round spermatids and oocytes had been obtained with the assistant of micromanipulator, but the appearance of hand-made cloning technique made it unnecessary in nuclear transfer technique. Spermatids and zona-free oocytes were fused by handmade cloning in this experiment and the fused zona-free embryos were cultured to morula/blastocyst phase.Different kinds of wells made by different needles and different culture mediums were used to culture mouse zona-free fertilized eggs. The best conditions were chose to establish the optimized culture system for the zona-free embryos; Different parthenogenetic activation methods were used to conform the most suitable conditions for zona-free oocytes, thus to establish the best conditions of the parthenogenetic activation of the round spermatid and zona-free egg pairs; Different voltages were taken in order to improve the construction efficiency of round spermatids and zona-free oocytes. The main results of this paper were as follows:1) In order to optimize the WOW system, the metal needle, the glass needle and the embryo aggregation needle were used to make wells respectively. Blastocyst development rate in the wells made by embryo aggregation needle was significantly higher than that in the wells made by the metal needle and the glass needle(55.61% vs 44.38%,43.16%). But there were no significant difference of the rates of cleavage and the 4-cell among the three groups. The results showed that the rate of blastocyst was significantly high in the wells made by embryo aggregation needle.Fertilized eggs were cultured in the wells made by embryo aggregation needle with CZB, M16 and KSOM culture medium respectively. The rates of cleavage, 4-cell and blastocyst were not significantly different with each other.2) The developmental potential of zona-free oocytes activated by different concentrations of strontium chloride was investigated. In this experiment, zona-free oocytes were activated by 2 mmol/L, 4 mmol/L, 10 mmol/L SrCl₂ supplemented with 5μg/ml CB respectively. The rate of cleavage (13.22%,12.62%,16.54%) and death were not remarkably different, and all the activated oocytes could not develop to blastocyst in this experiment.The concentration of 10mmol/L SrCl₂ was chose for the next experiment, and the treatment time was extended to 3h, 4h or 5h. The death rate was not significantly different after different times. The cleavage rate of 3h group was significant higher compared with that of 4h and 5h (61.40% vs 49.12%,47.95%), whereas the rate of morula/blastocyst of 5h was remarkable higher (23.29% vs 10.53%,14.04%) than 3h and 4h. These results showed that 3h was the best period to activate the zona-free oocytes, and the development rate of post-parthenogenetic embryos can be significantly increased when embryos were cultured in strontium for 5h.3) Electrofusion was used to fuse round spermatids and zona-free oocytes. In this experiment, fusion was taken under a single direct current pulse of 60 v/mm, 80v/mm and 100v/mm respectively, and the width of the voltages was 10μs. The results showed that the fusion rate of 60v/mm was significantly higher than 80v/mm and 100v/mm (50.00% vs 38.55% and 39.59%). The rates of cleavage and morula/blastocyst were not remarkably different among different voltages. So 60v/mmol/L was the best voltage to fuse the round spermatids and the zona-free oocytes.