Effect of antioxidants in cryopreservation media on spotted halibut (Verasfer variegatus) sperm quality during cryopreservation

In fish hatcheries, sperm cryopreservation is a helpful tool for managing broodstock and artificial fertilization. However, cold shock can cause sperm injury resulting in decreasing sperm motility, low cell viability, structural damage to the plasma membrane and DNA fragmentation. This parameters are critical factor for the assessment of cryopreserved sperm for commercial scale application. In recent years, antioxidants have been increasingly useful for treating physiological issues in fish, including in reproduction and cryopreservation procedures. The purpose of this study was to examine the effects of supplementation of various antioxidants, including trehalose, reduced glutathione (GSH), Mito-TEMPO (MT), and rosmarinic acid (RA), during fish sperm cryopreservation on sperm motility, cell survival rate, and DNA damage. One-way ANOVA and Duncan's multiple range tests (p > 0.05) were used to compare the parameters. Sperm from adult fish were cryopreserved in 10% DMSO with 300 mM sucrose (1:1) supplemented with different concentrations of trehalose (0, 50, 100, 150, 200, and 250 mM), GSH (0, 2, 4, 6, 8, and 10 mM), MT (0, 25, 50, 75, 100, 125, 150, 175, and 200 μM), or RA (0, 25, 50, 75, and 100 μM). Compared with the control groups, antioxidant supplementation in sperm was more efficient in increasing post-thaw motility, maintaining cell survival rates, and inhibiting the increase in DNA damage that occurs during non-activated sperm during storage. The results demonstrated that the post-thaw motility of sperm was higher in 8 mM GSH (85.71 ± 0.04%), followed by 6 mM GSH (82.97 ± 1.30%), 75 and 25 μM RA (82.93 ± 0.93% and 82.76 ± 1.42%, respectively) and 100 mM trehalose (80.39 ± 0.61%) with not statistically different. The cell survival rate, VCL, and VSL of frozen–thawed sperm were higher in 100 mM trehalose resulted 84.22 ± 2.24%, 114.72 ± 2.00 μm/s, and 95.49 ± 0.70 μm/s, respectively. Mito-TEMPO at concentrations 100 μM resulted lower percentages of tail DNA (1.60 ± 0.08%) with not statistically different with 100 mM trehalose. In conclusion, the present study indicated that antioxidant 100 mM trehalose provided the most pronounced protective effect in improving frozen-thawed quality of frozen–thawed spotted halibut sperm. •Increase in sperm motility and cell survival rate in frozen sperm of spotted halibut.•Decrease in DNA damage in frozen sperm of spotted halibut.•This is the first time Mito-TEMPO and rosmarinic acid have been added to cryomedia for