Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants, antioxidant, membrane stabilizer, equilibration time and dilution ratio on sperm motility and fertility

摘要

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5, 10, 15 and 20. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10mgmL(-1)) and ascorbic acid (0, 2.5, 5 and 10UmL(-1)), on the post-thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10mgmL(-1) BSA with selected cryoprotectants (15 MeOH and 10 DMSO) for 5min. After a month of storage in liquid nitrogen, post-thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15 and DMSO 10 were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post-thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20min) (P>0.05) although higher equilibration times than 20min resulted low post-thaw motility (P0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10mgmL(-1) BSA significantly improved post-thaw motility (P0.05). The results of experiment 3 showed that the highest fertilization (30.2 +/- 5.75) and hatching rates (28.2 +/- 5.25) were observed when samples were frozen with 15 MeOH (P>0.05). Our study indicates that the use of mT extender consisting of 10mgmL(-1) BSA in 15 MeOH diluted with sperm at 1:1 ratio for 5min can be recommended cryopreservation method for Persian sturgeon sperm.