The Germplasm Preservation Of Spermatozoa From Epinephelus Malabaricus

Epinephelus malabaricus is the one valuable economic mariculture species in southeast coast of China. Currently, researches on the biology and artificial propagation technology of the fish were quite proven. But the germplasm preservation of the fish spermatozoa is not reported. The spermatozoa motility, the ultrastructure and the variation before and after the cryopreservation, the method of cryopreservation were investigated in this paper. It provided the theoretical basis for the spermatozoa cryopreservation and the establishment of frozen sperm pool in E.malabaricus.The effect of salinity and pH value on the spermatozoa motility of E. malabaricus was studied by the gradient settings of them, respectively. The ultrastructure and the variation before and after the cryopreservation were observed and compared. The results are shown below:(1) The optimum range of salinity for the spermatozoa motility is 15-35. The maximum longevity was 26min, which appeared at the salinity of 15, and the optimum range of pH value is 6.5-8.0. (2) The transmission electron microscope (TEM) result showed that the normal sperm was consisting of the head, mid-piece and tail. Its head shaped in round and ellipse, whose diameter was about 1.8μm, the mid-piece was obscure while the mitochondria are observed, and the slender flagellum was about 15μm, whose central structure was axoneme with a typical structure of the "9+2"model. (3) The ultrastructure of sperm was damaged by the ultra-low temperature treatment obviously. It mainly presented in the membrane crimpled, cytoplasm leakage, mitochondria swelling or broken and tail cracked.Six different extenders solutions (TS-19, TS-2, Hank’s, MPRS, Cortland and 0.75% NaCl) were chosen for diluent, and three (10%-25% dimethyl sulphoxide, glycerol, methanol) for the cryoprotectants were used in the cryopreservation of spermatozoa of E.malabaricus. The results were shown below:(1) Compared with other diluents, the Hank’s solution was the optimum one to apply the cryopreservation spermatozoa of E.malabaricus. (2) The highest survival rate of frozen-thawed semen was more than 60%, which the optimum combination of freeze protection to the cryopreservation spermatozoa of E.malabaricus was the Hank’s solution and 10%-15% DMSO solution. But all spermatozoa were not activated by using any concentration of MeOH. (3) When the 10%-25% glycerol solution was chosen as cryoprotectant, the spermatozoa of E.malabaricus could be activated to the normal movement without nature seawater, and the effect was similar to natural seawater before the cryopreservation. But the spermatozoa motility decreased rapidly with seawater added, the same phenomenon exists after the cryopreservation. (4) To gain the better survival rate of frozen-thawed semen, the optimal collection period of cryopreservation semen sampling of E.malabaricus was 1 day before the spermiation.