Pacific abalone, Haliotis discus hannai, is a high commercial seafood in South-East Asia. The aim of the present study was to determine effects of cryopreservation on gene expression and post thaw sperm quality of Pacific abalone. Two ions, Na+ (459.1 ± 3.1 mM) and Cl– (515.9 ± 1.1 mM), were predominant in the seminal plasma (pH: 6.8 ± 0.1; osmolarity: 1,126 ± 3 mOsmL–1). Cryopreservation reduced mRNA expression levels of protein kinase A (PKA-C) and heat shock proteins (HSP70 and HSP90) genes in sperm. Fluorescent technique was used to compare morphological defects, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and DNA integrity of sperm cryopreserved with five different cryopreservation solutions (8% Me2SO, 8% EG, 6% PG, 2% GLY, and 2% MeOH). Droplet in tail and coiled tail defects was not observed for sperm cryopreserved with 8% Me2SO or 2% GLY. Sperm cryopreserved with 8% Me2SO showed improved DNA integrity and lower cryodamage than sperm cryopreserved with other cryoprotectants. Sperm to egg ratio of 10,000:1 was found to be the most suitable ratio for in vitro fertilization among different ratios tested. The fertilization rate of sperm cryopreserved with 8% Me2SO was not significantly (p > 0.05) different from that of sperm cryopreserved with 2% GLY. DNA fragmentation showed strongly negative relationships with sperm quality parameters. Sperm cryopreserved with 8% Me2SO showed higher post thaw quality and mRNA expression of sperm motility associated gene than those cryopreserved with other cryoprotectants. The present research suggests to use 8% Me2SO for cryopreservation of Pacific abalone sperm as well as for hatchery production.

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Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII. A combination of 10 µg/mL AFPIII with 8% DMSO resulted in the highest post-thaw motility, showing AI of 60.1 ± 3.9%, PMI of 67.2 ± 4.0%, and MMP of 59.1 ± 4.3%. DNA integrity of sperm cryopreserved using 10 µg/mL AFPIII combined with 8% DMSO was not significantly (p > 0.05) different from that of fresh sperm. Cryopreservation using a combination of AFPIII with 8% DMSO improved fertilization and hatching rates of sperm compared to that of cryopreservation without supplementation of 10 µg/mL AFPIII. Sperm cryopreserved using AFPIII showed higher mRNA abundance levels of HSP90 than those cryopreserved without AFPIII. Results of the present study suggest that 10 µg/mL AFPIII combined with 8% DMSO can be used for large scale cryopreservation of Pacific abalone sperm and for hatchery production.

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.