Long-term cryopreservation of sperm from Mandarin fish Siniperca chuatsi

In order to develop cryopreservation techniques for long-term preserving the sperm of Mandarin fish Siniperca chuatsi, we examined the effects of various extender and cryopreservation on post-thaw motility. We found the optimal freezing procedures for the Mandarin fish sperm is diluting the semen in D-15 extender, chilling it to 4 °C, adding ME2SO to a final concentration of 10% (v/v), then transferring the semen in cryotubes, holding the cryotubes for 10 min at 6 cm (about −180 °C) above the surface of liquid nitrogen, for 5 min on the surface of liquid nitrogen, and finally plunged into liquid nitrogen. After thawed at 37 °C for 60 s, the sperm had the highest post-thaw motility (96.00 ± 1.73%). The optimal fertilization procedures for the frozen sperm is mixing the eggs with sperm, then adding 1 ml of swimming medium (SM = 45 mM NaCl + 5 mM KCl + 20 mM Tris–HCl, pH 8.0) immediately. At the sperm/egg ratio of 100,000:1, the fertilization rate and the hatching rate of the frozen sperm cryopreserved for 1 week or 1 year in liquid nitrogen (66.01 ± 5.14% and 54.76 ± 4.40% & 62.97 ± 14.28% and 52.58 ± 11.17%) were similar to that of fresh sperm (69.42 ± 8.11% and 59.82 ± 5.27%) (p > 0.05). This is the first report that the Mandarin fish (S. chuatsi) sperm can successfully fertilized eggs after long-term cryopreservation.