Cryopreservation of Roughscale Sole (Clidoderma asperrimum) Sperm: Effects of Cryoprotectant, Diluent, Dilution Ratio, and Thawing Temperature

SIMPLE SUMMARY: The roughscale sole, Clidoderma asperrimum, is found in the East and West Seas of Korea, waters north of Hokkaido in Japan, as well as the East China Sea, the East Pacific, and the Canadian Maritimes. In 2021, this fish was categorized as an endangered species. Thus, there is a need to maintain gametes by freezing sperm. This study investigated the impacts of the cryoprotective agent, diluent, dilution ratio, and thawing temperature on the cryopreservation of fish sperm. In this investigation, sperm dilution 1:1 with a mixture of 10% dimethyl sulfoxide + Stein’s solution and thawing at 10 °C provided the most effective DNA damage prevention. These results support the development of a roughscale sole sperm cryopreservation procedure. ABSTRACT: The roughscale sole, Clidoderma asperrimum is categorized as an endangered species. Sperm freezing is essential for preserving gametes. This study examined the CPA concentration, diluent, dilution ratio, and thawing temperature to design a sperm cryopreservation protocol for roughscale sole. The variables examined included sperm motility and kinematics, cell survival, fertilization, and DNA fragmentation. Sperm motility parameters were assessed via computer-assisted sperm analysis using a CEROS II instrument. Cell survival rate and DNA damage were assessed using the Cell Counting Kit-8 and single-cell gel electrophoresis assay, respectively. Sperm preservation was tested using several CPAs, including ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and methanol. The diluents tested were 300 mM sucrose, 300 mM glucose, Stein’s solution, Ringer’s solution, and Hank’s solution. The optimal conditions for sperm cryopreservation were 10% DMSO + Stein’s solution. After thawing, sperm motility was highest with a 1:1 dilution ratio (sperm to CPA + diluent), at 69.20 ± 0.32%; thawing at 10 °C was optimal for post-thaw motility (72.03 ± 0.95%). The highest fertilization rate (40.00 ± 1.22%) was obtained using DMSO. The fresh sperm had the lowest tail DNA, followed by 10% DMSO + Stein’s solution. The developed cryopreservation methods can be used in roughscale sole hatcheries.