Isolation and cryopreservation techniques of Pseudorasbora parva blastomeres

To study the isolation and suitable cryopreservation of Pseudorasbora parva blastomeres， comparison experiments were conducted in taking off the egg membrane of P. parva blastula with different concentrations of trypsase at 25℃， and in cryopreservation for P. parva blastomeres with different protectants and at different temperatures. Results showed that the blastula stage of P. parva was achieved in 120 min after fertilization， under the condition of microstream at 20℃. It took 12-18 min to take off the egg membrane with 0.25% trypsase at 25℃， furthermore， the survival rate of isolated blastomeres was above 90%. 10% DMSO+DMEM was used as a tprotectant at -80℃， the blastomeres survival rate was above 50% after 8 d； at -196℃， the survival rate was above 70% after 8 d， and the survival rate was also above 60% after 128 d. The result indicated that better condition for taking off the egg membrane was treatment with 0.25% trypsase for 12-18 min at 25℃. Higher blastomeres survival rate（above 60%）could be maintained at -196℃ and 10% DMSO+DMEM.