Intermediate-Term Storage of Spotted Halibut (Verasper variegatus) Sperm: Effects of Storage Methods, Extenders Supplemented with Antibiotics and Antioxidants on Sperm Quality

Abstract

Intermediate-term preservation of sperm assists the reproductive management of fish spermatozoa; however, no information is available on sperm of the spotted halibut, Verasper variegatus. We aimed to identify the optimum diluents, temperatures, dilution ratios, antibiotics, and antioxidants for sperm motility and cell viability. The diluents evaluated were marine fish Ringer’s solution (MFRS), Stein’s solution, 300 mM sucrose, and 300 mM glucose (diluted 1:1 [sperm: diluent], 1:2, 1:4, and 1:10 and stored at 0, 2, 4, and 6 °C). Neomycin and gentamycin (100, 200, 400, and 800 mg/L) and antioxidants (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, and 250 mM]) were assessed in terms of sperm preservation. The most effective condition for cold storage of spotted halibut sperm was Stein’s solution at a dilution ratio of 1:4 at 2 °C, with a combination of neomycin 800 mg/L and 250 mM trehalose that showed spermatozoa motility of >43% after 60 days. These storage conditions will be valuable for spotted halibut hatcheries.

The Increasing Role of Short-Term Sperm Storage and Cryopreservation in Conserving Threatened Amphibian Species

“…For example, Rahimi et al [ 99 ] observed that sperm motility was higher in Caspian brown trout ( S. trutta caspius ) sperm samples stored in storage medium with antibiotics added compared to the control group after three days of storage. Similarly, Zidni et al [ 77 ] found that the addition of antibiotics resulted in improved sperm motility and viability in spotted halibut ( V. variegatus ), with sperm successfully stored for 60 days with antibiotic treatment compared to only 30 days in untreated suspensions. Based on the beneficial effects across species, treatment of semen with antibiotics is considered to be a critical component of prolonging the viability of sperm for aquaculture [ 72 ].…”

“…Temperatures ranging from 0 to 5 °C are most commonly used for the short-term storage of amphibian sperm, and this taxon has a high tolerance to cold shock [ 49 , 72 ]. Low temperatures aid in reducing the metabolic rate and energy consumption of sperm as well as reducing bacterial growth [ 71 , 76 , 77 ]. The sperm of several toad species ( Anaxyrus americanus , A. baxteri , A. fowleri , and A. boreas ) have been reported to lose motility significantly less rapidly when immediately placed in cold storage at 4 °C compared to storage at room temperature [ 49 ].…”

“…The addition of antibiotics to control bacteria in sperm samples is routinely used in humans, livestock, and aquaculture species [ 96 , 97 ]. In fish, penicillin, streptomycin, erythromycin, gentamicin, and neomycin are the most commonly used antibiotics [ 72 , 77 ]. The addition of antibiotics to fish semen has been reported to improve sperm quality (motility, velocity and viability) and longevity during short-term storage in species including common carp ( C. carpio ) [ 91 ], Atlantic cod ( Gadus morhua ), haddock ( Melanogrammus aeglefinus ) [ 98 ], Atlantic salmon ( S. salar ) [ 90 ], African catfish ( Clarias gariepinus ) [ 92 ], Caspian brown trout ( Salmo trutta caspius ) [ 99 ], Nile tilapia ( Oreochromis niloticus ) [ 96 ], spotted halibut ( Verasper variegatus ) [ 77 ], and other commercially valuable fish species [ 72 ].…”

“…In fish, penicillin, streptomycin, erythromycin, gentamicin, and neomycin are the most commonly used antibiotics [ 72 , 77 ]. The addition of antibiotics to fish semen has been reported to improve sperm quality (motility, velocity and viability) and longevity during short-term storage in species including common carp ( C. carpio ) [ 91 ], Atlantic cod ( Gadus morhua ), haddock ( Melanogrammus aeglefinus ) [ 98 ], Atlantic salmon ( S. salar ) [ 90 ], African catfish ( Clarias gariepinus ) [ 92 ], Caspian brown trout ( Salmo trutta caspius ) [ 99 ], Nile tilapia ( Oreochromis niloticus ) [ 96 ], spotted halibut ( Verasper variegatus ) [ 77 ], and other commercially valuable fish species [ 72 ]. For example, Rahimi et al [ 99 ] observed that sperm motility was higher in Caspian brown trout ( S. trutta caspius ) sperm samples stored in storage medium with antibiotics added compared to the control group after three days of storage.…”

“…Furthermore, in another study in coho salmon ( O. kisutch ), BHT, an antioxidant with additional antiviral and antibiotic properties, was supplemented in the semen at a concentration of either 1 or 2 mM, with a concentration of 2 mM found to significantly improve sperm motility after three days of storage [ 125 ]. Additionally, supplementing the storage medium of spotted halibut ( V. variegatus ) with the antioxidants mito-TEMPO, trehalose and glutathione improved sperm motility and viability during storage [ 77 ]. Overall, these studies conclude that supplementing the storage medium with antioxidants is a way to improve the storage of fish sperm by minimising oxidative stress.…”