Seven-band grouper Epinephelus septemfasciatus is a protogynous hermaphrodite. The male individuals' number is more less than the female one. Thus, it is necessary for artificial reproduction and crossbreeding to research the sperm cryopreservation of sex-reversed seven-band grouper. In present study, the spermatozoa of sex-reversed immature fish were frozen using the different cryopreserving solutions for cryopreservation. The several factors that may affect the freezing survival rate of seven-band grouper spermatozoa such as the spermatozoa diluent, the concentration and composition of cryoprotective agent have been studied. The results showed that ES1-3 (60g/L glucose+10g/L NaCl+0.5g/L NaHCO3) was significantly better as a diluent compared with MPRS, TS-2 and other series of diluent ES1. The further experiment revealed that the optimal cryoprotectants were 10% dimethyl sulfoxide (DMSO) or 10% 1,2-propylene glycol (PG) with the post-thaw sperm motility was 76.67±0.00% and 75.00±5.00%, respectively. In addition, salinity of seawater is an important motility stimulator because that the highest motility of 96.00±1.73% was obtained at salinity 30‰. In crossbreeding test with fresh unfertilized eggs of kelp grouper Epinephelus moara, the ratio of fertilization and hatchability had no significant differences between the cryopreserved sperm and fresh sperm of seven-band grouper. It is suggested that the frozen sperm of seven-band grouper could be applied in artificial reproduction and crossbreeding.
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