CRYOPRESERVATION OF SPOTTED WOLFFISH (Anarhichas minor) SPERM EFFECTS OF EXTENDER SUPPLEMENTED WITH ASCORBIC ACID AND SUPEROXIDE DISMUTASE ON SPERM QUALITY

Abstract

The spotted wolffish (Anarhichas minor) holds considerable promise for cold-water aquaculture due to its high fillet yield, docile behaviour, and resistance to disease. However, reproductive constraints—including limited sperm motility, low ejaculate volume, and narrow spawning windows—impede large-scale hatchery production. Cryopreservation offers a strategic solution for overcoming these limitations by enabling long-term storage and asynchronous fertilisation. This study investigated whether supplementation with two antioxidants—ascorbic acid (a non-enzymatic antioxidant) and superoxide dismutase (an enzymatic antioxidant)—could mitigate oxidative damage incurred during cryopreservation, thereby improving post-thaw sperm quality.

Spotted wolffish sperm were cryopreserved in a modified Scophthalmus maximus extender using three concentrations of each antioxidant: 0.5 mmol (low), 1.0 mmol (medium), and 1.5 mmol (high) for ascorbic acid, and 250 U/mL, 500 U/mL, and 750 U/mL for superoxide dismutase. Fresh and cryopreserved control groups (without antioxidants) were included for comparison. Freezing was performed using 0.5 mL straws exposed to nitrogen vapour, then submerged in liquid nitrogen. Thawing was standardised at 4°C for 1.5 minutes. Sperm quality assessments included total motility and kinematic parameters via Computer-Assisted Sperm Analysis (CASA), membrane viability using flow cytometry (SYBR-14/PI staining), and DNA fragmentation via the Halo Assay.

The results indicated that antioxidant efficacy was concentration-dependent and varied between parameters. Ascorbic acid at 1.0 mmol yielded the most consistent improvements, significantly enhancing total motility (p = 0.0148), curvilinear velocity (VCL; p = 0.0143), and straight-line velocity (VSL; p = 0.0097), while also reducing DNA fragmentation (p = 0.036). Viability, as measured by live cell percentage, was significantly higher in the low-dose ascorbic acid group (p = 0.0009), with moderate improvements observed in the medium-dose group. In contrast, superoxide dismutase at 500 U/mL showed non-significant yet numerically favourable effects on motility and viability, although no concentrations of SOD produced statistically significant enhancements across evaluated parameters. High-dose treatments of both antioxidants either plateaued or induced marginal declines in quality, suggesting a narrow optimal range for antioxidant efficacy.

Sperm concentration post-thaw did not differ significantly between treatments and was primarily influenced by individual variation rather than antioxidant supplementation. Linearity of sperm movement also remained unaffected by any antioxidant treatment (p < 0.05). The overall findings point to a beneficial role of moderate antioxidant dosing, particularly with ascorbic acid, in preserving functional sperm attributes post-thaw.

From an applied perspective, these results carry important implications for aquaculture development and broodstock management. Improved cryopreservation protocols will support selective breeding, enhance hatchery scalability by decoupling gamete collection from spawning seasonality, and facilitate the commercialization of spotted wolffish—a species with substantial export and nutritional value. Furthermore, robust cryobanking methods contribute to ex-situ conservation and genetic resource management by safeguarding male genetic diversity.

This work highlights the critical role of redox homeostasis in sperm cryopreservation and demonstrates the utility of integrating CASA, flow cytometry, and chromatin integrity assays to evaluate post-thaw functionality. Future research should build upon these findings by exploring synergistic antioxidant combinations, validating fertilization outcomes in vitro and in vivo, and examining epigenetic impacts on offspring. Ultimately, the adoption of refined, species-specific cryopreservation strategies will strengthen cold-water aquaculture and bring under-utilized species like the spotted wolffish into mainstream production and international markets.

The spotted wolffish (Anarhichas minor) holds considerable promise for cold-water aquaculture due to its high fillet yield, docile behaviour, and resistance to disease. However, reproductive constraints—including limited sperm motility, low ejaculate volume, and narrow spawning windows—impede large-scale hatchery production. Cryopreservation offers a strategic solution for overcoming these limitations by enabling long-term storage and asynchronous fertilisation. This study investigated whether supplementation with two antioxidants—ascorbic acid (a non-enzymatic antioxidant) and superoxide dismutase (an enzymatic antioxidant)—could mitigate oxidative damage incurred during cryopreservation, thereby improving post-thaw sperm quality.

Spotted wolffish sperm were cryopreserved in a modified Scophthalmus maximus extender using three concentrations of each antioxidant: 0.5 mmol (low), 1.0 mmol (medium), and 1.5 mmol (high) for ascorbic acid, and 250 U/mL, 500 U/mL, and 750 U/mL for superoxide dismutase. Fresh and cryopreserved control groups (without antioxidants) were included for comparison. Freezing was performed using 0.5 mL straws exposed to nitrogen vapour, then submerged in liquid nitrogen. Thawing was standardised at 4°C for 1.5 minutes. Sperm quality assessments included total motility and kinematic parameters via Computer-Assisted Sperm Analysis (CASA), membrane viability using flow cytometry (SYBR-14/PI staining), and DNA fragmentation via the Halo Assay.

The results indicated that antioxidant efficacy was concentration-dependent and varied between parameters. Ascorbic acid at 1.0 mmol yielded the most consistent improvements, significantly enhancing total motility (p = 0.0148), curvilinear velocity (VCL; p = 0.0143), and straight-line velocity (VSL; p = 0.0097), while also reducing DNA fragmentation (p = 0.036). Viability, as measured by live cell percentage, was significantly higher in the low-dose ascorbic acid group (p = 0.0009), with moderate improvements observed in the medium-dose group. In contrast, superoxide dismutase at 500 U/mL showed non-significant yet numerically favourable effects on motility and viability, although no concentrations of SOD produced statistically significant enhancements across evaluated parameters. High-dose treatments of both antioxidants either plateaued or induced marginal declines in quality, suggesting a narrow optimal range for antioxidant efficacy.

Sperm concentration post-thaw did not differ significantly between treatments and was primarily influenced by individual variation rather than antioxidant supplementation. Linearity of sperm movement also remained unaffected by any antioxidant treatment (p < 0.05). The overall findings point to a beneficial role of moderate antioxidant dosing, particularly with ascorbic acid, in preserving functional sperm attributes post-thaw.

From an applied perspective, these results carry important implications for aquaculture development and broodstock management. Improved cryopreservation protocols will support selective breeding, enhance hatchery scalability by decoupling gamete collection from spawning seasonality, and facilitate the commercialization of spotted wolffish—a species with substantial export and nutritional value. Furthermore, robust cryobanking methods contribute to ex-situ conservation and genetic resource management by safeguarding male genetic diversity.

This work highlights the critical role of redox homeostasis in sperm cryopreservation and demonstrates the utility of integrating CASA, flow cytometry, and chromatin integrity assays to evaluate post-thaw functionality. Future research should build upon these findings by exploring synergistic antioxidant combinations, validating fertilization outcomes in vitro and in vivo, and examining epigenetic impacts on offspring. Ultimately, the adoption of refined, species-specific cryopreservation strategies will strengthen cold-water aquaculture and bring under-utilized species like the spotted wolffish into mainstream production and international markets.