Sperm of the giant grouper: cryopreservation, physiological and morphological analysis and application in hybridizations with red-spotted grouper.

In order to develop excellent germplasm resources for giant grouper (Epinephelus lanceolatus), cryopreservation of giant grouper sperm was examined in the present study. Firstly, 13 kinds of sperm dilution (ELS1-3, EM1-2, TS-2, MPRS, ELRS0-6) were prepared with physiological salt, sucrose, glucose and fetal bovine serum. The physiological parameters of ELRS3 (ratio of fast motion, ratio of slow motion, time of fast motion, time of slow motion, lifespan and motility) and ELS3 (sperm ratio of slow motion, time of slow motion and motility) were significantly higher than those of the other dilutions (P < 0.05). Secondly, after adding 15% DMSO and 10% FBS to ELRS3 and ELS3, most physiological parameters of frozen sperm were also significantly higher than the other gradients (P < 0.05), and sperm motility was as high as 63.68 ± 4.16% to74.75 ± 12.71% (fresh sperm motility, 80.70 ± 1.37% to 80.71 ± 1.49%). Mixed with the above dilutions, a final volume of 105 ml semen was cryopreserved. Finally, the sperm of giant grouper cryopreserved with cryoprotectants (ELRS3 + 15% DMSO + 10% FBS) was used for electron-microscopic observation and crossbreeding with red-spotted groupers (Epinephelus akaara). The electron-microscopic observation revealed that part of the frozen-thawed sperm was cryodamaged, e.g., flagellum fracturing and mitochondria falling out, while the ultrastructure of sperm membrane, mitochondria and flagellum remained intact. Also, the fertilization and hatchability rates of giant grouper frozen sperm and red-spotted grouper eggs were as high as 94.56% and 75.56%, respectively. Thus, a technique for cryopreservation of giant grouper sperm was successfully developed and applied to crossbreeding with red-spotted grouper eggs.

Sperm cryopreservation of sex-reversed seven-band grouper, Epinephelus septemfasciatus.

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.