Study On Sperm Cryopreservation Technology Of Two Important Fresh Water Fish

Cryopreservation of fish gametes plays an important role in the preservation of seed resource, cryobiology, genetics breeding and aquaculture. This paper dealt with the factors affecting cryopreservation of two important fresh water fish sperm.Yellow catfish (Pseudobagrus fulvidraco) is a candidate freshwater fish for aquaculture in China with its high consumer demand. The aim of this study was to examine the possibility of long storage of the sperm of yellow catfish by cryopreservation in liquid nitrogen. Experiments were designed to investigate the effects of the different combination of three extenders (Ringer extender, Kurokura-1extender and D-15extender) and three cryoprotectants (DMSO, Glycerol and Methanol) on the cryopreservation of yellow catfish sperm. Post-thaw sperm motility, fertilization and hatching rate were detected to evaluate the reliability of sperm cryopreservation. The results demonstrated that Ringer extender and10%methanol was the best combination for protecting the sperm during freezing in liquid nitrogen by a three-step method and thawing in a water bath at37℃for60s. In this combination for cryopreservation, sperm maintained the highest post-thaw motility (65±5)%, fertilization (90.47±3.67)%and hatching rate (88±4)%. And more interestingly, the fertilization and hatching rate were similar to those of fresh sperm (97.55±2.74)%and (92±5)%. Frozen-thawed sperm samples were able to successfully fertilize fresh eggs. Successful sperm cryopreservation techniques for yellow catfish have been developed for hatchery purpose.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-15extender, chilling it to4℃, adding DMSO to a final concentration of10%, then transferring the semen in cryotubes, holding the cryotubes for10min at6cm (about-180℃) above the surface of liquid nitrogen, for5 min on the surface of liquid nitrogen, and finally plunged into liquid nitrogen. After thawed at37℃for60s, 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 one milliliter of swimming medium (SM:45mM NaCl+5mM KC1+20mM Tris-HCl, pH8.0) immediately. At the sperm/egg ratio of100,000:1, the fertilization rate and the hatching rate of the frozen sperm cryopreserved for1week or1year in liquid nitrogen (66.01±5.14%and54.76±4.40%&62.97±14.28%and52.58±11.17%) were similar to that of fresh sperm (69.42±8.11%and59.82±5.27%)(P>0.05). This is the first report that the Mandarin fish (Siniperca chuatsi) sperm can successfully fertilized eggs after long-term cryopreservation.