AUTHOR=Hossen Shaharior , Kim Soo Cheol , Cho Yusin , Kho Kang Hee 

TITLE=Vital Analysis of Cryopreserved Sperm of Marbled Flounder, Pseudopleuronectes yokohamae

JOURNAL=Frontiers in Physiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2021.696737

DOI=10.3389/fphys.2021.696737

ISSN=1664-042X

ABSTRACT=<p>The marbled flounder (<italic>Pseudopleuronectes yokohamae</italic>) 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: Me<sub>2</sub>SO; 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% Me<sub>2</sub>SO along with either extender-1 (86.0 ± 5.2%) or extender-2 (85.7 ± 7.1%) was similar (<italic>p</italic> &gt; 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% Me<sub>2</sub>SO, but significantly lower from fresh sperm. The type of straw (0.25 or 0.50 mL) did not show any significant difference (<italic>p</italic> &gt; 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% Me<sub>2</sub>SO (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% Me<sub>2</sub>SO (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 (<italic>p</italic> &gt; 0.05) to those of fresh sperm. Sperm DNA integrity did not reveal any significant difference (<italic>p</italic> &gt; 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% Me<sub>2</sub>SO or 12% GLY has potential for hatchery as well as to create a germplasm bank.</p>