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

Abstract

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 60 min) 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 10 mg mL À1 ) and ascorbic acid (0, 2.5, 5 and 10 U mL À1 ), on the post-thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL À1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. 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, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post-thaw motility (P < 0.05). The motility of frozen-thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL À1 BSA significantly improved post-thaw motility (P < 0.05), but ascorbic acid did not improve post-thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 AE 5.75) and hatching rates (28.2 AE 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 10 mg mL À1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.

Oxidative stress and use of antioxidants in fish semen cryopreservation

“…Although ascorbic acid is the principal non-enzymatic antioxidant in sperm cells of salmonids, supplementation with this molecule does not appear to be related to improvements in post-thaw sperm quality in this group of fish (Kutluyer et al 2014). Likewise, no improvement was found in the sperm quality of Persian sturgeon (Acipenser persicus) (Shaluei et al 2017) or of gilthead seabream and European seabass supplemented at concentrations of 1 and 10 mM (Cabrita et al 2011). On the contrary, supplementation with ascorbic acid in the cryopreservation of semen of other species has been found to promote principally sperm motility after thawing.…”

“…Likewise, no improvement was found in the sperm quality of Persian sturgeon ( Acipenser persicus ) (Shaluei et al . 2017) or of gilthead seabream and European seabass supplemented at concentrations of 1 and 10 m m (Cabrita et al . 2011).…”

Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation

“…For cryopreservation of Brazilian flounder and starry flounder, a dilution ratio of 1:3 has been effectively applied and dilution ratio between the 1:1 and 1:10 showed higher MSR in cryopreservation of stone flounder [ 16 , 18 , 29 , 37 ]. The duration of motility in Persian sturgeon ( Acipenser persicus ) sperm is highest with dilution ratios from 1:1 to 1:2 [ 44 ]. Although the optimal dilution ratio for cryopreserved fish sperm is species-specific and ranges from 1:1 to 1:20 [ 5 ], a higher dilution ratio generally reduces the percentage of motile sperm, SAI, and sperm survival rate during the dilution process.…”

“…The optimal freezing/thawing rate differs greatly among species, such as Atlantic halibut and summer flounder, with its efficiency linked to the process of dehydration, protection of intracellular ice formation during freezing and recrystallization during thawing [ 15 , 20 , 44 , 45 ]. In the present study, the estimated optimal freezing rate was in the range −1 to −5 °C/min, indicating that these freezing rates were better able to cover sperm cells from intracellular ice crystal formation during the milt cryopreservation process than other rates tested.…”

Motility of sea urchin Paracentrotus lividus spermatozoa in the post-activation phase

“…Interestingly, this dilution rate is 10‐ to 125‐fold higher than that reported by other authors (Gallego et al., ; Mita et al., ; Ohtake et al., ), despite the fact that the rate of oxygen consumption has been found to increase with dilution and low sperm concentrations are known to reduce semen lifespan (Kazama & Hino, ; Levitan, Sewell & Chia, ). On the other hand, our dilution buffer contains BSA, one of the most common antioxidants used in culture media for mammalian sperm (Aitken et al., ; Yanagimachi, ), which has also been found to improve post‐thaw motility in cryopreserved sturgeon sperm (Shaluei, Sadeghi & Zadmajid, ). Therefore, it could be supposed that even in very small amounts (at a 20‐fold lower concentration), it offers sperm cells some protection from ROS during incubation.…”