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

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.


Introduction
The spotted halibut (Verasper variegatus) is categorized is an endangered species in South Korea, Japan, and China [1]. Therefore, a reproductive aquaculture plan is needed to protect this species. Gamete conservation is important for fish reproduction in the aquaculture industry [2]. Short-term storage is often used in gamete conservation because it is simpler, easier, and can be implemented in hatcheries [3]. Such short-term storage enables a variety of genetic enhancement initiatives, such as cryopreservation, artificial insemination, and hybridization [4]. In practice, cold storage of sperm promotes egg production in hatcheries because it is a low-cost and easy means of overcoming the insufficient number of males. The effect of short-term storage of fish sperm has been evaluated in marine fish species such as olive flounder (Paralichthys olivaceus) [5], marbled sole (Limanda yokohamae), brown sole (Limanda herzensteini) [6], starry flounder (Platichthys stellatus) [7], yellow croaker (Larimichthys polyactis) [8], and giant grouper (Epinephelus lanceolatus) [9]. However, to our knowledge, no study has evaluated the influence of short-term preservation of spotted halibut sperm on sperm function.
The successful storage of fish sperm depends on numerous factors, including extenders, dilution ratio, temperature, and antibiotics [5,7]. During storage, sperm motility, fertilization capacity, cell viability, and energy availability for sperm activation must be maintained [10]. The storage temperature has an important impact on the in vitro survivability of fish gametes. However, species have different temperature tolerances. The storage of the sperm of some fish is enhanced with antibiotics and antioxidants. The success of short-term sperm storage is based on motility and cell viability and is hampered by infection or bacterial development in the sperm volume and/or sperm solution [11]. This reduces fertilizing potential, cell quality, and viability, endangering germplasm [12]. Antibiotics-e.g., penicillin, gentamicin, streptomycin, and neomycin-are used in semen cold storage to prevent these issues. However, the type and effective dose vary depending on the fish species [8].
Storage itself and cell metabolism create free radicals, which damage polyunsaturated fatty acids in the cell membrane and disrupt sperm DNA [13]. An antioxidant is a nucleophilic constituent that inhibits the harmful effects of an oxidative cellular environment. Antioxidants preserve the viability of sperm cells during storage by reducing, stopping, or preventing free-radical processes [14]. Antioxidants have been used to alleviate several fish health problems, such as reproduction and freezing fish spermatozoa [15]. Antioxidants have different effects based on their type and concentration [16].
We tested the effects of diluents on spotted-halibut sperm quality during short-term storage. We also evaluated the influence of sperm diluent ratio, sperm storage temperature, antibiotics, and antioxidants on sperm quality.

Broodstock and Collection of Gamates
The research was performed from January to May 2021. In the Marine Seed Hatchery in Yeosu-si, Jeollanam-do, South Korea, 12 mature male spotted-halibut broodstock of average length 36.06 ± 0.28 cm and average weight 373.4 ± 4.4 g were maintained in seawater at a temperature of 10-12.5 • C, salinity of 30.1 ± 1.4 Practical Salinity Units (PSU), dissolved oxygen of 7.4 mg/L, and pH of 7.8. During the experiment, the fish were twice-daily recipients of commercial floatable feed (Merk, Super Plus 7S, Uiryeong, South Korea). For milt collection, the fish were sedated using 50 mg/L 2-phenoxyethanol. Samples of sperm were obtained by stripping and promptly collected using 1.0 mL plastic syringes (without needle). Care was taken to avoid contaminating sperm with water, urine, blood, or feces. The sperm was transferred to 1.5 mL microtubes in an icebox (constant temperature of 4 • C) and immediately transported to the Marine Aquaculture Laboratory of Mokpo National University, South Korea for assessment. Only sperm of motility > 80% by microscopy were used in this study.

Sperm Motility, Duration Motility, and Cell Viability
Computer-assisted sperm analysis was conducted using the CEROS II instrument (Hamilton Thorne, Inc., Beverly, MA, USA) equipped with a Zeiss Axiolab 5 microscope at 10× magnification, including a CM-040GE camera with 0.4-megapixel resolution at 60 frames per second (JAI, Tokyo, Japan). Each sample was diluted in a ratio of 1:200 with artificial seawater (27 g of NaCl, 0.5 g of KCl, 1.2 g of CaCl 2 , 4.6 g of MgCl 2 , and 0.5 g of NaHCO 3 per liter of distilled water kept at 4 • C), placed into a Leja slide 20 µm (IMV Technologies, France), and tested at 10 • C of room temperature. The motion characteristics investigated were the percentage of moving sperm (motility); path velocity (VAP), quantifies the speed of the sperm head along its average spatial path; curvilinear velocity (VCL), the average speed measured along the actual point to point path taken by the cell; straight-line velocity (VSL), the average velocity of a sperm head along the straight line between its initial and final detected places; percentage straightness (STR), calculated as VSL/VAP × 100; and percentage linearity (LIN), which is the linearity of the curvilinear trajectory as VSL/VCL × 100 [17]. The duration of motility was assessed by measuring the period between spermatozoa activation and their full cessation of activity.
The viability of sperm was evaluated using the Cell Counting Kit-8 (CCK-8) test (Bimake, Houston, TX, USA) as described previously [17]. At a volume ratio of 1:10, CCK-8 reagent was directly applied to the cells in a culture medium. Initially, 100 µL of cell suspension were added to each well of 96-well plates. The cells and reagent (10 µL per well) were incubated for 1-4 h until an orange color formed. Using a microplate reader, the absorbance at 450 nm was determined (Spectra Max 190, Molecular Devices, San Jose, CA, USA). The amount of formazan (orange dye) produced by dehydrogenase activity indicates the number of live cells. White sperm were deemed non-viable while orange sperm were deemed viable.

Effect of Temperature
Sperm samples were diluted 1:2 (sperm: Stein's solution), loaded in 1.5 mL microfuge tubes, and kept at 0, 2, 4, or 6 • C in a refrigerator. Three replicates were performed. Sperm motility, kinematic characteristics, duration motility, and viability were evaluated at 5-day intervals until spermatozoa ceased movement.

Effect of Antibiotics
Sperm were diluted at 1:4 (sperm: Stain's solution) in triplicate, neomycin and gentamycin (100, 200, 400, and 800 mg/L) were added, and the samples were stored in a refrigerator at 2 • C. Sperm motility, kinematic parameters, duration motility, and viability were evaluated at 5-day intervals until spermatozoa creased movement. . Three replicates of each treatment were conducted, and samples were refrigerated at 2 • C. Sperm motility and viability were evaluated at 5-day intervals until spermatozoa ceased movement.

Statistical Analysis
The variables are presented as means and standard errors, and the percentage data was normalized prior to analysis via arcsine square root transformation. For the analysis, the SPSS version 23 software was used. One-way ANOVA and Duncan's multiple-range test (p > 0.05) were selected for statistical evaluation.

Mito-TEMPO
The effects of the indicated concentrations of Mito-TEMPO (MT) in combination with antibiotics (800 mg/L gentamicin or neomycin) on sperm motility and viability are shown in Figure 6. Sperm quality was dose-dependently increased by MT in combination with neomycin. By contrast, sperm quality decreased with increasing MT concentration in combination with gentamicin. Sperm motility and cell viability were maximally increased by 200 µM MT in combination with neomycin (42.30 ± 2.94% and 40.69 ± 3.09%, respectively), followed by 175 µM MT (40.30 ± 1.79% and 37.80 ± 2.38%; p > 0.05). The lowest sperm quality resulted from 25 µM MT in combination with neomycin. In the presence of 800 mg/L gentamicin, maximum sperm motility and viability were obtained using 150 µM MT (39.65 ± 1.92% and 29.20 ± 1.88%, respectively), followed by 125 µM MT (36.37 ± 2.40% and 26.05 ± 2.23%) (p > 0.05). The lowest sperm quality resulted from 200 µM MT in combination with gentamicin.

Discussion
Short-term storage of sperm at lower temperatures may promote brood stock upgradation at distantly located hatcheries. This is the first study to develop a storage protocol for spotted halibut sperm. The implementation of sperm short-term storage methods for endangered or threatened fish is an effective conservation strategy for these species. In addition, the short storage of sperm can support artificial fertilization in fish. Sperm motility, kinematic parameters, and viability are important aspects of sperm quality, particularly if the fertilization rate cannot be evaluate because of a lack of mature eggs [7,18].
In sperm storage, the extender, which has a composition similar to seminal plasma, maintains sperm function and extends lifespan [19]. The selection of an appropriate extender is crucial for the successful cold storage of fish sperm [7]. In this study, Stein's solution, was the most effective extender for the cold storage of spotted halibut sperm. Indeed, similar findings have been reported for olive flounder (Paralichthys olivaceus) [5], brown sole (Pleuronectes herzensteini) [6], marbled sole (Limanda yokohamae), and starry flounder (Platichthyes stellatus) [7]. In this study, MFRS, 300 mM sucrose, and 300 mM glucose reduced sperm quality. However, MFRS was effective for the short-term storage of the sperm of tiger puffer (Takifugu rubripes) [20], yellowtail tetra (Astyanax altiparanae) [21], yellow drum (Nibea albiflora) [22], and Scapharca broughtonii [22]. A sucrose base increased the quality of the sperm of effective rainbow smelt (Osmerus mordax) [23] and glucose that of meagre (Argyrosomus regius) [24]. Extender effectiveness differs by fish species. Fish sperm diluents are dependent on the chemical composition of seminal plasma because sperm must be kept stationary to avoid energy depletion [25]. The extender solution should also provide the nutrients and energy sperm needed to survive during storage. Stein's solution contains NaCl, KCl, egg yolk, NaHCO 3 , and C 6 H 12 O 6 , which exert a buffering effect, tonicity, prevent sperm activation, and protect the membrane, respectively, during cold storage [26]. The medium, dilution ratio (sperm: medium), storage temperature, and antibiotics and antioxidants are determinants of sperm quality after storage.
Low temperatures (approaching 0 • C) are typically advised because they decrease the metabolic rate and energy consumption of cells without altering their structural integrity [42,43]. However, for practical reasons, most studies have used 4 • C, the typical temperature of domestic refrigerators. In this study, cold-stored spotted halibut sperm remained motile for 30 days at 2 • C, 20 days at 0 • C, and 10 days at 4 and 6 • C. The most optimum cold storage temperatures were 2 • C for starry flounder (Platichthys stellatusi) [7] and 2.5 • C for yellowtail tetra (Astyanax altiparanae) [21]. Due to the cold shock resistance of aquatic species sperm, the optimal temperature for sperm storage is 0-4 • C. Low temperatures reduce sperm metabolism and prevent bacterial growth [21,25]. Nile tilapia and channel catfish (Ictalurus punctatus) sperm should be stored at 0-4 • C [44,45].
Antibiotics-typically penicillin, erythromycin, gentamicin, streptomycin, and neomycin-are added to prevent bacterial contamination of the stored sperm [46,47]. In this study, sperm motility, velocity, and viability were maintained until day 60 compared to day 30 in the antibiotic-free group. Gentamicin and neomycin at 800 mg/L increased sperm motility, velocity, and viability. The optimum antibiotic concentration varies among fish species. The application of 600 mg/L gentamicin and 200 mg/L neomycin increased the sperm quality of yellow croaker (Larimichthys polyactis) [18] and 500 and 750 µg/mL gentamicin for Nile tilapia [12]. In the cold storage of Caspian brown trout (Salmo trutta caspius) sperm, penicillin and streptomycin at 500 IU/mL increased sperm motility and viability [48]. Similarly, during sperm storage of the common frog (Rana temporaria), sperm quality increased with increasing gentamicin concentration [49]. In Atlantic salmon, antibiotics enhanced fertilization capacity [50]. According to Jenkins & Tiersch [51], cold storage decreased the quality of channel catfish (Ictalurus punctatus) sperm because of contamination by a Pseudomonas sp. Therefore, antibiotics may enhance stored sperm quality by suppressing microbial growth and/or activity.
During the cold storage of sperm, antioxidants inhibit free-radical reactions. In this study, 250 mM trehalose with neomycin increased the motility and viability of spotted halibut sperm during storage for 60 days. Indeed, higher concentrations of trehalose further increased the sperm quality. By contrast, in the presence of gentamicin, a high trehalose concentration reduced sperm motility and viability. Trehalose was useful for the cryopreservation of rainbow trout [52], whitefish [53], seabream (Pagrus major) [54], and Amazonian catfish (Leiarius marmoratus) [55] sperm. Trehalose reduces intracellular ice crystal formation and cryoinjuries caused by freezing by boosting the level of GSH and decreasing lipid peroxidation [56]. In addition, 200 µM MT + neomycin increased sperm motility and viability in a dose-dependent manner. In combination with gentamicin, a high concentration of MT reduced sperm motility and viability. MT at 100 µM was optimal for cryopreservation of spotted halibut sperm [14]. MT reportedly enhances the cryopreservation of testicular tissue of several mammalian species. In addition, MT reduces DNA damage and oxidative stress in variety of cell types [57]. MT maintains sperm function and energy homeostasis via oxidative phosphorylation and ATP synthesis [58]. In rooster semen storage, MT at 5 and 50 µM preserved sperm quality [59]. In this study, 2 and 4 mM RG in the presence of gentamicin or neomycin prolonged sperm viability and motility until 60 days. However, higher concentrations of RG decreased sperm quality. RG participates in antioxidant activity as a substrate for glutathione peroxidase [60]. RG at 2, 4, and 6 mM enhanced the cryopreservation of tambaqui (Colossoma macropomum) sperm [61]. RG is also used in the preservation of mammalian sperm. The fertility of cryopreserved rooster sperm was increased by 2 and 4 mM RG [62]. The application of 2 mM RG enhanced boar sperm cryopreservation [63], and 2 and 5 mM RG promoted cryopreservation of ram spermatozoa [64]. RG at 10 mM prevented DNA damage in cryopreserved dog sperm [65].

Conclusions
Optimal conditions for the storage of spotted halibut sperm were Stein's solution (extender), a 1:4 sperm: extender dilution ratio, 800 mg/L neomycin, 250 mM trehalose, and a temperature of 2 • C. Data Availability Statement: All of the data is contained within the article.