Abstract
The marbled flounder (Pseudopleuronectes yokohamae) is a commercial flatfish in East Asia. The aim of this study was to improve its sperm cryopreservation protocol based on the vitality assessment of 7-day and 1-year cryopreserved sperm. Four extenders (extender-1: sucrose solution; extender-2: glucose solution; extender-3: fish Ringer's solution; and extender-4: modified fish Ringer's solution) were tested with a combination of five cryoprotectants (CPAs) (dimethyl sulfoxide: Me2SO; glycerol: GLY; ethylene glycol: EG; propylene glycol: PG; and methanol: MeOH) at four different concentrations (5, 10, 12, and 15%). Fluorescent technique was applied to detect the plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and DNA integrity of fresh and cryopreserved sperm specimens. Fresh sperm was diluted at a ratio of 1:2 (sperm:extender). Post-thaw motility of sperm cryopreserved using 15% Me2SO along with either extender-1 (86.0 ± 5.2%) or extender-2 (85.7 ± 7.1%) was similar (p > 0.05) to that of fresh sperm. Sperm cryopreserved using 12% GLY combined with extender-1 (83.67 ± 6.7%) or extender-2 (83.3 ± 4.7%) showed a similar motility to those cryopreserved with 15% Me2SO, but significantly lower from fresh sperm. The type of straw (0.25 or 0.50 mL) did not show any significant difference (p > 0.05) in post-thaw sperm motility. The highest values of PMI and MMP were observed for 7-day cryopreserved sperm using extender-1 in combination with 15% Me2SO (91.0 ± 2.9% and 90.0 ± 2.0%, respectively) or 12% GLY (90.0 ± 1.3% and 90.0 ± 4.6%, respectively). These results were similar to those of fresh sperm (95.3 ± 2.1% and 92.9 ± 2.5%, respectively). PMI and MMP of 1-year cryopreserved sperm using extender-1 in combination with 15% Me2SO (90.3 ± 2.5% and 89.3 ± 2.1%, respectively) or 12% GLY (90.0 ± 4.4% and 88.7 ± 2.2%, respectively) were significantly similar (p > 0.05) to those of fresh sperm. Sperm DNA integrity did not reveal any significant difference (p > 0.05) between fresh and cryopreserved (7-day and 1-year) sperm. Based on the assessed sperm vitality indicators, a cryopreservation protocol using extender-1 in combination with 15% Me2SO or 12% GLY has potential for hatchery as well as to create a germplasm bank.
Highlights
The marbled flounder (Pseudopleuronectes yokohamae) is a commercially important species in Korea (Park et al, 2016), China (Liu et al, 2015), and Japan (Kusakabe et al, 2017)
The present study showed a significant difference in the post-thaw sperm motility between two dilution ratios (1:2 and 1:4), similar to the results reported for sperm of H. hippoglossus (Ding et al, 2011) and Paralichthys dentatus (Brown et al, 2012)
The present findings suggest that the absence of sugar in extender-3 might be responsible for the reduced motility of postthaw sperm, a positive impact of sugar-based extenders on the sperm cryopreservation has been described in the previous section because such extender can act as a non-penetrating CPA (Rusco et al, 2019)
Summary
The marbled flounder (Pseudopleuronectes yokohamae) is a commercially important species in Korea (Park et al, 2016), China (Liu et al, 2015), and Japan (Kusakabe et al, 2017). Sperm cryopreservation is an important method of storing genetic materials (Nahiduzzaman et al, 2011) and providing a constant supply of sperm (Kim et al, 2020a). This technique provides a stable source of sperm for artificial fertilization, selective breeding (Viveiros et al, 2012), and rapid sperm transfer among hatcheries (Xin et al, 2018a; Cejko et al, 2020). Evaluation of the post-thaw vitality of the long-term cryopreserved sperm is needed for storage and commercial applications (Kim et al, 2020a)
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