| This study focused on the pretreatments of sperm and sperm injection methods to optimize experiment condition, improve operation efficiency and get higher success rate of bovine cloned embryos derived from sperm. The results were shown as follows.1. In order to improve the survival rate of bovine embryos, found that the survival rate of one-step (enucleation of oocytes followed by sperm injection) was significantly higher than two-step(enucleation followed by repairation, then perform sperm injection) (86.7% Vs 51.7%), suggested that the one-step can improve the embryo survival rate.2. The effect of different methods of sperm pretreatment on cloned embryos derived from bovine sperms. DTT processing and DTT with tail treatment methods can significantly increase the cleavage rate and the blastocyst rate of sperm reconstructed embryos compare with only tail treatment, suggested that DTT processing can promote sperm nucleus decondensation significantly and development of sperm reconstructed embryo could be greatly promoted3. Effects of different concentrations of DTT on sperm were compared, we found 20mMol/L DTT treatment contribute to highest death rate of sperm (25.5%), and even 2mMol/L and 5mMol/L DTT also could make sperm dead comparing with the control group, suggested that higher concentration of DTT could cause higher death rate of sperm.4. Effect of different concentrations DTT on sperm head was observed by electron microscope. With the increase of concentration of DTT, the sperm head obviously dilat, and tail of spermatozoon become bend, suggested that the DTT not only effect on protamine, to reduce the density of nucleus,but also in the plasma membrane sperm tail, weak occurred bend. |
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