Short-term storage of sperm is a useful method in the reproductive management of fish spermatozoa; however no information is available on spotted halibut, Verasper variegatus sperm. One of the important parameters in analyzing sperm quality is sperm motility. This study aimed to optimize a short-term storage protocol for spotted halibut sperm, by investigating the effects of different diluents, storage temperatures, dilution ratios, and evaluating the effect of the addition of antibiotics, and antioxidants on sperm motility and cell survival rate of spotted halibut sperm by computer-assisted sperm analysis (CASA) and Cell Counting Kit-8, respectively. All the parameters tested in this study were determined using one-way ANOVA and evaluated using a multiple range test with Duncan (p > 0.05).
Various diluents were evaluated, such as marine fish Ringer’s solution (MFRS), Stein’s solution, 300 mM sucrose, and 300 mM glucose. The spotted halibut sperm was stored at 0?, 2?, 4?, and 6?, diluted at 1:1 (sperm: diluent), 1:2, 1:4, and 1:10. Different antibiotics such as neomycin and gentamycin at concentrations of 100, 200, 400, and 800 ppm, variations of antioxidants including Mito-TEMPO (0, 25, 50, 75, 100, 125, 150, 175, and 200 µM), reduced glutathione (0, 2, 4, 6, 8 and 10 mM), and trehalose (0, 50, 100, 150, 200, 250 mM) were tested for sperm preservation. In this study, sperm motility and cell survival rate were evaluated every 5 days. The results showed that sperm motility was affected significantly by diluent, storage temperature, dilution ratio, antibiotic, and antioxidant (P < 0.05). The most effective condition for cold storage of spotted halibut sperm was Stein’s solution as a diluent in a dilution ratio of 1:4 at 2? in which the preserved sperm-maintained motility for 30 days. The best antibiotic concentration was 800 ppm of gentamycin and neomycin and the preserved sperm retained motility for 60 days. The combination of neomycin 800 ppm with 250 mM trehalose as antioxidant showed a spermatozoa motility> 43% for 60 days. The short-term storage protocols obtained in this study will be useful in spotted halibut hatcheries.