Abstract：Marbled flounder (Pseudopleuronectes yokohamae) is an important natural fishing and aquaculture object in the flounder fish. The number of germplasm resources has been decreased, due to factors such as changed ecological environment, artificial overfishing and so on. So research the sperm cryopreservation technology which is of great significance for germplasm resources. In present study, the series of experiments for sperm diluents, cryoprotectant, the suitable salinity of seawater and frozen semen artificial insemination were selected using the mature male marbled flounder. The experiment results were analyzed by One-way ANOVA and Student-Newman-Keuls from SPSS 19.0. Figures made with Origin 8.0 software. The results indicated that the antifreeze effect of sperm cryopreservation was good, using MFs-3 (8 g/L NaCl+0.65 g/L KCl+15 g/L glucose) with 20% PG and EG respectively, which the sperm motility, fast moving time and life time was (95.26+0.39)%, (46.00+1.00) s, (124.33+4.04) s and (95.15+0.41)%, (45.67+0.58) s, (124.00+3.00) s, respectively. It indicated the diluent MFs-3 suits for marbled flounder sperm cryopreservation, and the best cryoprotectants were PG and EG. In the experiment of selecting of the suitable salinity of seawater, the frozen sperm were activated with 10‰~50‰ seawater, showed that the sperm motility was the highest at 30‰ and there was no significant difference compared with fresh sperm but there was significant difference between others and fresh sperm (P＜0.05). And sperm motility has been gradually increased from (86.13+1.44)% to (95.07+0.69)% when the salinity is 16.7‰~30‰; Sperm vitality has been gradually decreased from (95.07+ 0.69)% to (84.09+2.61) % with 30‰~45‰ sea water. Lower than 16.7‰ and higher than 45‰ of sea water, sperm was almost no movement. The thawed sperm with PG and EG cryopreservation and marbled flounder eggs made fertilization experiments, fertilization rate and hatching rate was (80.08+0.68)%, (77.44+1.76)% and (80.17+0.45)%, (77.92+1.33)%, respectively, and there was no significant difference compared with fresh sperm. It was also used computer aided sperm analysis (CASA) to analyze and test for motion parameters from frozen sperm sample in MSs+20% PG and MSs+20% EG including curvilinear velocity (VCL), straight line velocity (VSL), beat cross frequency (BCF), linearity (LIN) and straightness (STR) and so on, showed that there was no significant difference between them. In present study, the sperm from marble flounder could be effective cryopreserved with MSs-3+20% PG and MSs-3+20% EG. This study has been successfully preserved marbled flounder sperm with 20% MFs-3+PG and MFs-3+20% EG at -196 ℃. And the frozen sperm motility, fast moving time and life time was no significance with the fresh sperm. The technology had preserved the marbled flounder sperm cryopreservation, which has set a solid foundation for artificial cross breeding and genetic resources center. The research result firstly provides important the theory and technology basis.
