1 INTRODUCTION

Many studies have shown that reactive oxygen species (ROS) are produced during the process of sperm storage at low temperatures such as 4℃ or sperm cryopreservation (Peris et al., 2007; Dominguez-Rebolledo et al., 2008; Li et al., 2010; Gadea et al., 2011) . These ROS often cause oxidative damage to the sperm membrane integrity and mitochondrial function (Liu et al., 2014) . The most common oxidative damage to the sperm membrane is lipid peroxidation, which manifests as a reduction in sperm viability and fertilizing ability (Baumber et al., 2000) . The level of lipid peroxidation is often represented by the malondialdehyde (MDA) content (Aitken et al., 1989) .

The antioxidant system of milt could play an important role in preventing oxidative damage (Bell et al., 1993) ; oxidant defensive enzymes in the milt include superoxidative dismutase (SOD) , catalase (CAT) , glutathione peroxidase (GPx) , and glutathione reductase (Gr) . Under normal conditions, there is a balance in spermatozoa between ROS production and ROS elimination by this antioxidant system.

When spermatozoa are stored at a low temperature or cryopreserved, the function of this antioxidant system is suppressed to protect spermatozoa from ROS (Lahnsteiner et al., 2010) , resulting in a reduction in sperm viability and fertilizing ability. Therefore, techniques for improving low temperature storage or cryopreservation of spermatozoa have been investigated. A great variety of antioxidant substances, including trehalose, enzymes, vitamins, taurine, and other free radical scavengers, have been evaluated in sperm cryopreservation (Peña et al., 2003; Bucak et al., 2007; Hagedorn et al., 2012; Gadea et al., 2013) , with their effects reported to be species-specific (Cabrita et al., 2011) .

The antioxidant system and sperm characteristics of brown trout (Salmo trutta fario) (Lahnsteiner et al., 2010) , common carp (Cyprinus carpio) , brook trout (Salvelinus fontinalis) (Shaliutina-Kolešová et al., 2013) , and the Atlantic cod (Gadus morhua L .) (Butts et al., 2011; Flannery et al., 2013) have been well established. In contrast, limited knowledge is available regarding the Pacific cod (Gadus macrocephalus) , although spermatozoa chilled storage and cryopreservation could substantially improve the efficiency of its hatchery production and genetic improvement programs. The Pacific cod is an economically important marine species globally. It is very delicious and nutritious, and can be used fresh, thus making it popular with consumers.

The aim of this study was to determine the influence of milt cold storage and cryopreservation on Pacific cod sperm characteristics, antioxidant enzyme activities, and lipid peroxidation. It was anticipated that this study would enhance our understanding of oxidative damage to Pacific cod spermatozoa during cold storage and cryopreservation, and further improve the spermatozoa cryopreservation technology in this species.

2 MATERIAL AND METHOD 2.1 Sperm collection

Naturally mature male Pacific cod (3-4 years old; 1800-2 500 g; 50-70 cm length) were maintained in the Haifu Hatchery (Rizhao, Shandong Province, China) during the spawning season in February 2014. They were fed on small miscellaneous fishes and kept in indoor circular tanks (30 m 3) supplied with flowthrough seawater, at an ambient temperature of 3-8℃, salinity of 29-30, and dissolved oxygen ≥5 mg/mL. The males used in this study were chosen randomly; milt from each male was collected into a 50-mL centrifuge tube by applying gentle pressure on the abdomen. The sperm were transported on ice to the laboratory as soon as possible. A Nikon-YS-100 light microscope (Nikon Corporation, Tokyo, Japan) was used to assess sperm motility at 200× magnification. Sperm with motility >90% were selected for use in the subsequent experiments.

2.2 Experimental design

Two experiments were conducted to evaluate the effects of: 1) chilled storage, and 2) cryopreservation. In the first experiment, the milt samples were divided into two groups and assessed without any treatment as control samples, or after 12 h storage at 4℃. In the second experiment, five treatments were conducted: a) fresh milt (control) , b) milt cryopreserved without extender and cryoprotectant (CPA) (negative control) , c) milt cryopreserved in 12% propylene glycol (PG) , d) milt cryopreserved in 12% PG without seminal plasma (produced by centrifuging at 3 000 × g at 4℃ prior to cryopreservation) , and e) milt cryopreserved in 12% PG+0.1 mol/L trehalose. The milt was mixed with extender+CPA at a ratio of 1:3 (v/v) . The extender ingredients and final concentrations were 8 g/L NaCl, 0.4 g/L KCl, 0.14 g/L CaCl₂, 0.1 g/L MgSO₄ ∙7H₂O, 0.1 g/L MgCl₂ ∙6H₂O, 0.12 g/L Na₂HPO₄ ∙12H₂O, 1 g/L glucose, 0.35 g/L NaHCO₃, and 0.06 g/L KH₂PO₄ . The cryopreservation was carried out using a programmable freezer (Kryo-360- 1.7, Planer Plc. Middlesex, UK) . The samples were first maintained at 0℃ for 5 min, and then frozen to -150℃ at a cooling rate of -20℃/min before being plunged into liquid nitrogen for storage, as described by Liu et al. (2007) .

2.3 Analyses of sperm motion parameters

A computer-assisted sperm analysis system (CASA, Tsinghua Tongfang Inc., Beijing, China) , was used to assess sperm motion parameters including percentage of motile sperm, average path velocity (VAP; μm/s) , curvilinear velocity (VCL; μm/s) , and straight line velocity (VSL; μm/s) . According to the VAP, sperm were divided into four classes: sperm with VAP greater than 100 μm/s were defined as A class, sperm with VAP 50-100 μm/s as B class, sperm with VAP 20-49 μm/s as C class, and sperm with VAP less than 20 μm/s as D class. The sperm of the control, 4℃ storage, and negative control samples were activated at a milt or milt plus extender and CPA to natural seawater ratio of 1:1 000 (v/v) , while other treatments were activated at a ratio of 1:250. Prior to activation, bovine serum albumin (BSA) was added to seawater (5% v/v) to prevent the spermatozoa from sticking to the glass slides (Rouxel et al., 2008; Butts et al., 2010) . The milt and seawater with BSA was then gently mixed. Twenty microliters of activated sperm were placed on a glass slide and observed under the negative phase-contrast objective of a Nikon-YS-100 compound microscope (Nikon Corporation, Tokyo, Japan) at 200× magnification. Sperm motion parameters were examined with 30 S after activation in three visual fields. Each treatment had three replicates, and each replicate was evaluated three times.

2.4 Biochemical assays

Biochemical assays were performed immediately after collection. The milt from each treatment was centrifuged at 3 000 × g for 10 min at 4℃, then the supernatant was collected and kept at 4℃ until enzyme analyses. The pellet was washed three times with ice-cooled Hanks solution. The pellet was then incubated with 1 mL 0.2% Triton X-100 for 30 min at 4℃. The SOD, CAT, Gr, and GPx activities, and MDA content were analyzed according to the manufacturer’s instructions provided in their respective analysis kits (Nanjing Jiancheng Biological Engineering Research Institute, Nanjing, China) . The enzyme activity was expressed as units per mg of protein (U/mg prot) .

2.5 Statistical analysis

Statistical analysis was carried out using SPSS 19.0 for Windows (SPSS Inc. Chicago, IL, USA) , and data were expressed as mean±SD. One-way ANOVA was used to assess the effect of different treatments on sperm motion parameters, activities of SOD, CAT, GPx, and Gr, and MDA content. The SNK test was applied when significance was identified (P＜0.05) . The arcsine square root transformation was performed on the raw data prior to the ANOVA analysis.

3 RESULT 3.1 Effects of chilled storage and cryopreservation on sperm motion parameters

The motility of sperm in the control samples (87.67%±2.56%) was significantly higher than after 12 h storage at 4℃ (Fig. 1a) and frozen-thawed treatment (from 0 to 63.21%±2.43%) . The motility of sperm in the negative control samples was nearly zero, the lowest among all the treatments (Fig. 1b) .

The VAP, VCL, and VSL in the control samples were significantly higher than those in the treatment samples (Fig. 2a, b) . In the treatments, the VAP of the sperm cryopreserved in 12% PG+0.1 mol/L trehalose was significantly higher than in the other treatments (P＜0.05) , whereas there was no significant difference in VCL and VSL among the treatments (P >0.05) .

The sperm classifications according to the VAP values are presented in Fig. 3. After 12-h storage at 4℃, the percentage of class A sperm was significantly reduced (P＜0.05, Fig. 3a) . In the cryopreservation experiment, the percentage of class A sperm in the control samples (51.75%±1.81%) was significantly higher than in those treated by cryopreservation, followed by the milt cryopreserved in 12% PG milt+0.1 mol/L trehalose (7.33%±1.20%) and in 12% PG (0.91%±0.33%) , the milt in 12% PG without seminal plasma (0.36%±0.02%) , and the negative control (0) .

3.2 Effects of chilled storage and cryopreservation on superoxide dismutase activity in spermatozoa and seminal plasma

In the chilled storage experiment, the spermatozoa SOD activity of the samples stored at 4℃ (160.78±10.42 U/mg prot) was significantly lower than that in the control samples (284.60±16.01 U/mg prot, Fig. 4a) .

In the cryopreservation experiment, the highest spermatozoa SOD activity was found in the control samples (284.60±16.01 U/mg prot) and the milt cryopreserved in 12% PG+0.1 mol/L trehalose (270.81±45.79 U/mg prot) , followed by the milt in 12% PG without seminal plasma (212.6±20.96 U/mg prot) and the milt in 12% PG (165.49±45.62 U/mg prot) . The lowest SOD activity was detected in the negative control (milt cryopreserved without CPA; 25.92±1.66 U/mg prot, Fig. 5a) .

The highest seminal plasma SOD activity was found in the negative control samples (181.95±5.78 U/mg prot) , whereas the lowest was in the control samples (17.14±0.24 U/mg prot) . There was no significant difference in the seminal plasma SOD activity between the milt cryopreserved in 12% PG+0.1 mol/L trehalose and that in 12% PG (P >0.05, Fig. 5a) .

3.3 Effects of chilled storage and cryopreservation on catalase activity in spermatozoa and seminal plasma

In the chilled storage experiment, the spermatozoa CAT activity in treated samples (0.83±0.17 U/mg prot) was significantly lower than in the control samples (1.85±0.21 U/mg, Fig. 4b) .

In the cryopreservation experiment, the spermatozoa CAT activity was the highest in the control samples (1.85±0.21 U/mg prot) , followed by the 12% PG+0.1 mol/L trehalose samples (0.99±0.085 U/mg prot) , 12% PG without seminal plasma milt samples (0.55±0.14 U/mg prot) , 12% PG milt samples (0.3±0.02 U/mg prot) , and the negative control samples (0.05±0.014 U/mg prot, Fig. 5b) . In contrast, the seminal plasma CAT activity was highest in the negative control samples (1.22±0.12 U/mg prot) , and lowest in the control samples (0.18±0.018 U/mg prot, Fig. 5b) .

3.4 Effects of chilled storage and cryopreservation on glutathione peroxidase activity in spermatozoa and seminal plasma

In the chilled storage experiment, the spermatozoa GPx activity in the treated samples (102.48±4.1 U/mg prot) was significantly lower than that in the control samples (250.32±19.85 U/mg prot, Fig. 4c) .

In the cryopreservation experiment, the spermatozoa GPx activity was highest in the control samples (250.32±19.85 U/mg prot) , followed by the 12% PG+0.1 mol/L trehalose samples (57.20±4 U/mg prot) , the negative control samples (42.96±9.94 U/mg prot) , and the 12% PG milt samples (35.31±1.97 U/mg prot, Fig. 5c) .

The seminal plasma GPx activity in the cryopreservation samples was significantly higher than in the control samples (Fig. 5c) .

3.5 Effects of chilled storage and cryopreservation on glutathione reductase activity in spermatozoa and seminal plasma

In the chilled storage experiment, the spermatozoa Gr activity in the chilled storage samples (168.95±19.28 U/mg prot) was significantly higher than in the control samples (67.37±17.27 U/mg prot, Fig. 4d) .

The spermatozoa Gr activity in the samples cryopreserved with a CPA were similar, ranging from 102.32±11.4 to 109.34±8.53 U/mg prot, and were significantly higher than that in the control samples (67.37±17.27 U/mg prot) . The spermatozoa Gr activity in the control samples was significantly higher than in the negative control samples (34.01± 2.84 U/mg prot, Fig. 5d) .

The seminal plasma Gr activity in the negative control samples was 41.34±10.33 U/mg prot, which was significantly higher than in the other treatments and the control samples (Fig. 5d) .

3.6 Effects of chilled storage and cryopreservation on malondialdehyde content in spermatozoa and seminal plasma

In the chilled storage experiment, the spermatozoa MDA content in the milt stored at 4℃ was significantly higher than in the control samples (Fig. 6a) .

In the cryopreservation experiment, the spermatozoa MDA content in the negative control samples was 4.86±1.7 U/mg prot, which was significantly higher than in the other treatments and the control samples (ranging from 2.12±1.39 to 2.39±0.56 U/mg prot, Fig. 6b) .

The seminal plasma MDA content was highest in the negative control samples (4.06±0.48 U/mg prot) . There was no significant difference in the seminal plasma MDA content between the control samples, milt cryopreserved in 12% PG+0.1 mol/L trehalose, and milt cryopreserved in 12% PG (P >0.05) .

4 DISCUSSION

Motility and velocity (VAP, VCL and VSL) have been used as sperm quality indicators in many species, including species closely related to the Pacific cod such as the Atlantic cod (Butts et al., 2011) . The present study showed that the Pacific cod sperm VCL and VSL significantly declined after chilled storage or cryopreservation in comparison with controls. These results are in agreement with previous reports on Atlantic cod where sperm cryopreservation had a significant negative effect on VCL (Butts et al., 2010, 2011) . In the present study, the addition of trehalose in CPA could improve the sperm quality as the VAP in the milt cryopreserved with 12% PG+0.1 mol/L trehalose was significantly higher than in that cryopreserved with PG only; a similar phenomenon has also been reported in other species (Chen et al., 1993; Chhillar et al., 2012; Liu et al., 2014) .

The decrease in sperm motility, velocity and fertilization capacity after refrigeration or cryopreservation is generally considered to be due to the accumulation of ROS (Ozkavukcu et al., 2008; Zribi et al., 2010) . Because antioxidants such as trehalose may neutralize the ROS and maintain the balance of ROS production and scavenging during the cryopreservation (Liu et al., 2014) , the addition of these chemicals could therefore improve the postthaw sperm quality.

Previous studies have reported that the sperm antioxidant enzyme system could play an important role in preventing plasma membrane lipid peroxidation (Lahnsteiner et al., 2010; Shiva et al., 2011) and oxidative damage (Bell et al., 1993) . The current study evaluated the SOD, CAT, GPx and Gr activities in Pacific cod spermatozoa and seminal plasma. The activities of the first three enzymes showed a similar pattern of change after chilled storage or cryopreservation: the enzyme activity declined in the sperm cells and increased in the seminal plasma. This could be due to the sperm membrane damage caused by lipid peroxidation, leading to the leakage of enzymes into the seminal plasma. A similar decrease in the post-thaw sperm antioxidant enzyme activities has also been found in humans (Shiva et al., 2011) and fowl (Partyka et al., 2012) ; these studies indicated that the damaged plasma membrane was more susceptible to perioxidative damage, accompanied by the loss of antioxidant enzymes from the sperm cells. The Gr activity, on the other hand, showed the opposite tendency. This result was consistent with the findings of Huang et al. (2014) and Yan et al. (2008) in other species.

The increased spermatozoa lipid peroxidation after storage at 4℃ and cryopreservation without CPA found in the current study suggests that the extender, CPA or their combination could effectively reduce this reaction. However, the sperm lipid peroxidation in stallions did not increase significantly during chilled storage of milt (Kankofer et al., 2005) . This difference in results may be due to species specificity.

5 CONCLUSION

The aim of this study was to evaluate the influence of chilled storage and cryopreservation on Pacific cod sperm quality by evaluating sperm characteristics, antioxidant enzyme activities, and lipid peroxidation. The activities of SOD, CAT, and GPx were significantly decreased in spermatozoa and increased in seminal plasma after chilled storage and cryopreservation. In addition, these changes in antioxidant enzyme activities also contributed to the reduced post-thaw sperm motility and velocity. Further studies are required to determine the cell protection mechanism used by the oxidant defensive enzymes during cryopreservation or chilled storage.