Antioxidant systems of brown trout (Salmo trutta f. fario) semen

Animal Reproduction Science 119 (2010) 314–321

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Antioxidant systems of brown trout (Salmo trutta f. fario) semen Franz Lahnsteiner a,∗ , Nabil Mansour a , Kristjan Plaetzer b a b

Department of Organismic Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria

a r t i c l e

i n f o

Article history: Received 20 August 2009 Received in revised form 21 January 2010 Accepted 22 January 2010 Available online 1 February 2010 Keywords: Spermatozoa Antioxidants Lipid peroxidation Sperm motility Sperm membrane integrity Brown trout Salmo trutta f. fario

a b s t r a c t The present study characterizes the antioxidant systems of brown trout, Salmo trutta, semen as supplementation of semen dilution media with antioxidants can be beneficial to improve techniques for semen storage and cryopreservation. Antioxidants and oxidant defensive enzymes of spermatozoa and seminal plasma were analyzed. To determine whether antioxidants and oxidant defensive enzymes have an effect on sperm functionality, in vitro experiments were performed. Selected antioxidants and oxidant defensive enzymes were added to sperm motility-inhibiting saline solution and their effects on sperm viability (motility when activated, membrane integrity, and lipid peroxidation) were measured. In seminal plasma and spermatozoa the enzymes catalase, glutathione reductase, methionine sulfoxide reductase, peroxidase, and superoxide dismutase and the metabolites ascorbic acid, glutathione, methionine, tocopherol, and uric acid were detected. Of the enzymes superoxide dismutase had the highest activity, of the metabolites uric acid occurred in highest concentrations. During in vitro incubation uric acid and catalase increased the sperm motility, sperm membrane integrity, and decreased the sperm lipid peroxidation in comparison to the control. However, catalase was effective only at an activity much higher than that occurring in seminal plasma. Reduced methionine increased the sperm motility and sperm membrane integrity and oxidized methionine the motility. However, neither reduced nor oxidized methionine decreased the sperm membrane lipid peroxidation. It is concluded, that uric acid is the main antioxidant of brown trout semen. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Antioxidants are molecules removing free radicals and inhibiting oxidation reactions by being oxidized themselves. Low levels of antioxidants or inhibition of antioxidant enzymes causes oxidative stress and may damage or kill cells. In mammals the effect of reactive oxygen species (ROS) on spermatozoa is well characterized (Alvarez and Storey, 1989; Sikka et al., 1995; Tramer et al., 1998; Sikka, 2004). The production of ROS by spermatozoa is a normal physiological process, but generation of ROS

∗ Corresponding author. Tel.: +43 662 8044 5630; fax: +43 662 8044 5698. E-mail address: [email protected] (F. Lahnsteiner). 0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2010.01.010

can also be induced by leukocyte contamination (Aitken et al., 1992), and by abnormal spermatozoa with excess residual cytoplasm (Kessopoulou et al., 1992). Spermatozoa are particularly susceptible to peroxidative damage, because most of their cytoplasm is removed during the final stages of spermatogenesis (Alvarez et al., 1987) and they have almost no enzymes, which are involved in the protection from peroxidative damage induced by ROS (Sikka, 2004). Further, the plasma membranes of spermatozoa contain large amounts of unsaturated fatty acids, which are very sensitive to free radical attack (Alvarez et al., 1987; Sikka, 2004). Therefore, ROS may cause lipid peroxidation of sperm cell membranes, damage of midpiece, axonemal structure, and DNA, malfunctions of capacitation and acrosomal reaction, loss of motility, and infertility (Sikka et al., 1995; Tramer et al., 1998).

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While the antioxidant systems in semen of mammals have been well characterized in several studies (Alvarez and Storey, 1989; Zini et al., 1993; Bauchè et al., 1994; Gu and Hecht, 1996), only little knowledge is available about the antioxidant systems of semen of teleost fish. According to Sasaki et al. (1996) the antioxidant potential of seminal plasma is attributed to the low molecular weight fraction, while Liu et al. (1995) suggest that spermatozoal antioxidative protection is related to seminal plasma proteins. Ascorbic acid seems to be an important semen antioxidant as its concentration is higher in rainbow trout seminal plasma than blood plasma (Ciereszko and Dabrowski, 1995). Feeding rainbow trout and grass carp with ascorbic acid or ascorbyl monophosphate containing diets resulted in increased sperm motility or fertility (Metwally and Fouad, 2009). Also ␣-tocopherol may be an antioxidant of teleost semen as ␣-tocopherol containing diets decreased sperm lipid peroxidation and increased the antioxidant potential of the seminal plasma in Arctic char (Salvelinus alpinus) (Mansour et al., 2006). Uric acid, a strong reducing agent, occurs in high concentrations in seminal plasma of rainbow trout (Oncorhynchus mykiss), yellow perch (Perca flavescens), muskellunge (Esox masquinongy), Northern pike (Esox lucius), carp koi (Cyprinus carpio), bream (Abramis brama), and tench (Tinca tinca) and could have antioxidant function, too (Ciereszko et al., 1999). To obtain more information about the antioxidant systems in semen of teleost fish the present study was conducted. Antioxidants (ascorbic acid, carnitine, carotine, glutathione, methionine, tocopherol, and uric acid) and oxidant defensive enzymes (catalase, gluthatione reductase, peroxidase, superoxide dismutase) of brown trout (Salmo trutta f. fario) spermatozoa and seminal plasma were investigated in fresh and in stored semen. To determine whether antioxidants and oxidant defensive enzymes have an effect on sperm functionality, in vitro experiments were performed. Antioxidants (ascorbic acid, carnitine, carotene, reduced and oxidized glutathione, reduced and oxidized methionine, ␣-tocopherol, uric acid, ZnCl2 ) and oxidant defensive enzymes (catalase, peroxidase, superoxide dismutase, gluathione reductase) were added to sperm motility-inhibiting saline solution and their effects on sperm motility, sperm membrane integrity, and sperm lipid peroxidation were measured. 2. Materials and methods 2.1. Collection of semen Brown trout (S. trutta f. fario) semen was obtained from the fish farm Kreuzstein in Upper Austria. Best possible accommodation and care was given to the animals used in the research and delivered by professionally trained staff who are committed to a culture of care. Semen was stripped by abdominal massage, collected into glass vials and stored on ice until use. No special approval by an ethics committee is necessary for this procedure of semen collection. Sperm motility of the samples was evaluated by subjective estimations and semen samples with a progressive motility rate < 50% were excluded from the experiment. The semen

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was centrifuged at 300 × g for 10 min at 4 ◦ C to separate seminal fluid and spermatozoa. The supernatant seminal fluid was centrifuged a second time under similar conditions to exclude possible contamination with spermatozoa. Using this procedure no cells remained in the supernatant as determined in previous, unpublished investigations. The spermatozoa were diluted in sperm motility-inhibiting saline solution (SMIS) (103 mmol/l NaCl, 40 mmol/l KCl, 1 mmol/l CaCl2 , 0.8 mmol/l MgSO2 , 20 mmol/l tris [pH 7.8]—Lahnsteiner et al., 1999) and centrifuged a second time to remove remnants of seminal fluid. Finally, spermatozoa were diluted in SMIS to obtain a sperm density of circa 1 × 109 spermatozoa/ml. 2.2. Analysis of potential antioxidants in spermatozoa and seminal fluid Semen from 8 different brown trout samples was incubated at a temperature of 4 ◦ C for 48 h (as after 48 h the sperm motility which could be activated was <10%). From fresh (0 h) and incubated semen two subsamples were taken, respectively. They were centrifuged at 300 × g for 10 min at 4 ◦ C. Thereafter, the supernatant seminal plasma was taken from each subsample. One supernatant was frozen at −70 ◦ C for subsequent analysis of enzyme activities. The other supernatant was diluted in 3 mol/l perchloric acid at a ratio of 1:1 for denaturation of enzymes and analysis of antioxidant and peroxides concentrations. The two sperm pellets were processed in the following way: one pellet was diluted in 500-␮l 0.1 mol/l tris buffer pH 7.8 for extraction of enzymes and the other pellet in 3 mol/l perchloric acid for extraction of metabolites. The sperm extracts were frozen at −70 ◦ C. No cryoprotectants were added to the enzyme extracts to prevent potential freeze damage of enzyme activity. For seminal plasma enzymes of Salmonidae no decrease in enzyme activity due to freezing has been detected until now (unpublished data) in contrast to seminal plasma enzymes of mammalian semen (Upreti et al., 1998). Sperm extracts were homogenized after thawing and centrifuged at 1000 × g for 10 min at 4 ◦ C. The perchloric acid extracts were neutralized using 1 mol/l KOH before analysis. The concentrations of peroxides, ascorbic acid, carotene, carnithine, gluthatione, methionine, and uric acid and the activities of catalase, glutathione reductase, superoxide dismutase, and peroxidase in spermatozoa and seminal plasma were measured with routine enzymatic assays as described in Bergmeyer (1985). Methionine sulfoxide reductase was determined with a colorimetric method of Sagher et al. (2006) based on the reduction of DABS (4-N,N-dimethylaminoazobenzene4-sulfonyl chloride)-methionine sulfoxide to DABS-lmethionine. The assay mixture contained 100 mmol/l tris buffer (pH 7.4), 15 mmol/l dithiothreitol, 12 ␮mol/l thionein, and 290 ␮mol/l DABS-methionine sulfoxide as substrate. Incubation time was 60 min. Reactions were terminated with 1 mol/l sodium acetate (pH 6.0) followed by acetonitrile and the formed DABS-l-methionine was extracted into benzene for photometric determination. All enzymatic assays were performed at 20 ◦ C. Methionine was analyzed with thin layer chromatography (TLC) using sil-

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ica gel plates as stationary phase and a 4:1 (v/v) mixture of phenol and 0.06 borate buffer (pH 9.3) as mobile phase. Plates were stained with a 0.5% ninhydrin solution in 96% ethanol. Methionine was identified based on the RF value (migration distance of spot/migration distance of frontline 100×) and quantified based on the size and color intensity of the spots in comparison to an appropriate control using a ImageJ 1.38x program. Tocopherol and carotene were also analyzed with TLC. The samples were saponificated in a mixture of 1.2% ascorbic acid and 2% potassium hydroxide (final concentration) in 90% ethanol at 100 ◦ C for 30 min. Tocopherol was extracted with benzene and analyzed on silica gel plates using benzene–ethanol (99:1, v/v) as solvent. Tocopherol and carotene were visualized with a 5% antimony chloride solution in chloroform and quantified using appropriate standards and the methods described above for methionine. 2.3. Influence of antioxidants and oxidant defensive enzymes on sperm motility, sperm membrane integrity, and sperm lipid peroxidation Washed spermatozoa from 8 semen samples were incubated in SMIS containing different types of potential antioxidants and oxidant defensive enzymes (see below) for 72 h and 120 h at 4 ◦ C and at a cell density of 5–7 × 107 cells/ml. Ascorbic acid (0.5, 1.0, 2.0 mmol/l), carnitine (0.5, 1.0, 2.0 mmol/l), carotene (0.1, 0.5, 1 mmol/l), reduced and oxidized glutathione (0.5, 1.0, 2.0 mmol/l, respectively), reduced methionine and oxidized methionine (=methionine sulfoxide) (0.5, 1.0, 2.0 mmol/l, respectively), tocopherol (0.5, 1.0, 2.0 mmol/l), uric acid (0.25, 0.5 mmol/l), ZnCl2 (0.1, 0.5, 1.0 mmol/l), catalase (150 U/l, 500 U/l), glutathione reductase (150 U/l, 500 U/l), and peroxidase (150 U/l, 500 U/l) were tested. Concentrations and enzyme activities derived from preliminary standardization experiments and were non-toxic during short-term incubation for 30 min. The pH of the solutions was checked and adjusted to 7.8 when necessary. Quantities of 200 ␮l sperm suspensions were stored in 1.5 ml Eppendorf tubes at 4 ◦ C and the sperm suspensions were mixed thoroughly before analysis. The effect of the described compounds was tested separately on the sperm motility which could be activated in sperm motility activating saline solution (composition see below) after 72 and 120 h. After 72 h the sperm membrane integrity and – in selected samples – the sperm lipid peroxidation were also investigated. 2.4. Sperm lipid peroxidation test Sperm lipid peroxidation tests were performed for spermatozoa incubated in SMIS, in seminal plasma, and in SMIS containing 0.5 mmol/l uric acid, 1.0 mmol/l methionine, or 0.5 mmol carnithine to determine whether seminal plasma and the different test substances acted as antioxidants or influenced sperm physiology and metabolism in different ways. Additionally, seminal plasma molecular weight fractions were tested. Seminal plasma from 3 individuals was pooled and fractions were prepared with molecular weight filters with an exclusion size of 100 kDa and 50 kDa. The

filtrate and the concentrate (supernatant) were used. The concentrate was re-diluted to the original volume with SMIS. Five semen samples were incubated in the different solutions under similar conditions as described under Section 2.2 for 72 h, however, higher volumes of 6 ml sperm suspensions were used. The sperm lipid peroxidation test was performed with the thiobarbituric acid (TBA) reaction according to the method of Aitken et al. (1993). After incubation was terminated the lipid peroxidation was challenged in the sperm suspensions with ferrous sulfate (0.04 mmol/l, final concentration) and ascorbic acid (0.2 mmol/l, final concentration) according to Haberland et al. (1996). Then the sperm suspensions were incubated in a water bath at 37 ◦ C for 3 h. After 60, 120, and 180 min, 125 ␮l aliquots were removed and added to 300 ␮l of TBA reaction mixture (1.0 ml of 7% sodium dodecyl sulfate, 10.0 ml of 0.1 mol/l HCl, 1.5 ml of 10% phosphotungstic acid, 5.0 ml of 0.67% TBA, and 0.5 ml of 0.2 mmol/l butylated hydroxytoluene). The reaction mixtures were heated to 100 ◦ C for 30 min, cooled, and the developed color was extracted in 1.5 ml n-butanol. The butanol layer was collected and the color corresponding to the TBARS level was measured at excitation and emission wave lengths of 532 and 553 nm, respectively, using a spectrofluorometer (Hitachi F-4500 Flourescence Spectrophotometer). As standard, 1,1,3,3tetramethoxypropane (Sigma Chemical Co.) was used. Concentration of TBARS is reported as area under the curve for the investigated time interval (60–180 min) (Davis, 2002). 2.5. Measurement of sperm motility Motility investigations were performed at 4 ◦ C using a 60 mmol/l NaHCO3 and 20 mmol/l glycine solution (pH 9) for motility activation (Lahnsteiner et al., 1999) and computer assisted cell motility analysis for measurements. Sperm motility was activated in a Makler investigation chamber (depth 10 ␮m, volume of 20 ␮l). Hundred ␮l water and 2 ␮l sperm suspension were mixed in the chamber. The chamber was closed with a cover slip and transferred into an inverse phase contrast microscope where the motility was recorded at 200-fold magnification. 10 ± 1 s after activation, sperm motility was measured using a CellTrack Version 1.5. (Motion Analysis Corporation) cell motility analysis program. The setup derived from preliminary, still unpublished standardization procedures. For edge detection the binary smoothed edges detection method was used, to calculate the metrics the neighbor hood size of particles was set to 18 pixels, the minimum object size to 4 pixels and the maximum object size to 90 pixels. For tracking of sperm paths 25 pixels per frame were allowed as maximum object speed. About 100 spermatozoa were analyzed from each sample and sperm motility was classified as follows: spermatozoa with an velocity < 5 ␮m/s were defined as immotile, spermatozoa with a velocity of 5–20 ␮m/s as locally motile, spermatozoa with a velocity > 20 ␮m/s as motile. The motile spermatozoa were classified into linear, non-linear and circular based on the rate of change of direction. Spermatozoa with a rate of change of direction (RCD) < 250◦ s−1 were con-

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sidered as linear motile, spermatozoa with a RCD from 250 to 900◦ s−1 as circular motile, and spermatozoa with a RCD > 900◦ s−1 as non-linear motile. 2.6. Sperm membrane integrity Sperm membrane integrity was determined according to a modified procedure of Garner et al. (1986). Briefly, 50 ␮l diluted semen was mixed with 1 ␮l of a 6-carboxyfluorescein diacetate (CFDA) solution (0.4% in dimethylsulfoxide) in an Eppendorf tube on ice. After 4 min, 3 ␮l of popidium iodide (0.8% in distilled water) was added and mixed well. One min thereafter, 20 ␮l stained semen was pipetted on a slide, covered with a cover slip and transferred to a fluorescence microscope. Using 400-fold magnification about 100 spermatozoa were photographed from each trial and the number of red spermatozoa with damaged membranes and the number of green spermatozoa with intact membranes were counted from the micrographs. 2.7. Statistics Data are presented as mean ± standard deviation. For statistical procedures percentage data were transformed √ by angular transformation (arcsin data) and metrical data by log transformation to reach the assumptions of normal distribution. To determine if the sperm viability parameters (motility, membrane integrity, lipid peroxidation) differed between the treatments and between the tested storage periods analysis of variance (ANOVA) was used with the viability parameters as dependent variables and the storage time as independent variable. Dunnett’s T3 test was used as multiple comparison posthoc test. For pair wise comparison of analyte values the student t-test was applied.

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3. Results 3.1. Concentrations of peroxides and antioxidants and activities of oxidant defensive enzymes in spermatozoa and seminal fluid When spermatozoa were stored in seminal plasma for 48 h at 4 ◦ C, the concentrations of peroxides increased significantly in seminal plasma and spermatozoa (Table 1). Seminal plasma and spermatozoa contained ascorbic acid, glutathione, methionine, tocopherol, and uric acid, the last at the highest concentrations (Table 1). The concentrations of these substances were constant during 48 h of storage (Table 1). Carotene and carnitine were not detected. The activities of catalase, peroxidase, glutathione reductase, and methionine sulfoxide reductase were low and fluctuating in seminal plasma and spermatozoa (Table 2). Superoxide dismutase activities were higher and more constant (Table 2). The activities of these enzymes did not change in seminal plasma and in spermatozoa during storage. 3.2. Effect of potential antioxidants on sperm motility and sperm membrane integrity Spermatozoa were incubated in SMIS containing different types of antioxidants and oxidant defensive enzymes to determine their effect on sperm viability. 3.2.1. Effect after 72 h incubation period The results are shown in Table 3. Ascorbic acid in concentrations >0.5 mmol/l decreased the sperm motility rate (% MOT) and the sperm swimming velocity (VEL), when activated, and the percentage of spermatozoa with intact membranes (% INTEG) in comparison to the control. Also

Table 1 Concentrations of peroxides and antioxidants in brown trout seminal plasma and spermatozoa. Data are mean ± standard deviation (n = 8). Asterisks indicate significant differences for spermatozoal and seminal plasma concentrations between 0 h and 48 h incubation (P < 0.01). n.d. = not detected. Metabolites

Peroxides Uric acid Glutathione Ascorbic acid Methionine Tocopherol

Seminal plasma (␮mol/l)

Spermatozoa (␮mol/100 mg protein)

0h

48 h

0h

48 h

876 ± 146 96.3 ± 33.9 5.8 ± 1.9 21.2 ± 19.1 32.8 ± 9.9 <0.05

1316 ± 450* 97.5 ± 52.3 6.3 ± 2.7 24.3 ± 22.4 26.7 ± 11.2 <0.05

66 ± 7 34.2 ± 8.2 1.0 ± 0.2 8.3 ± 8.5 n.d. <0.05

99 ± 5* 30.1 ± 8.2 1.1 ± 0.4 7.6 ± 7.7 n.d. <0.05

*The significance of asterisk is P < 0.01. Table 2 Activities of oxidant defensive enzymes in brown trout seminal plasma and spermatozoa. Data are mean ± standard deviation (n = 8). For enzyme activities in seminal plasma and spermatozoa no significant differences were found between 0 h and 48 h incubation (P > 0.01). Seminal plasma enzyme activities are expressed in ␮mol/min/l, spermatozoal enzyme activities in ␮mol/min/100 mg protein with exception of superoxide dismutase. For this enzyme activity is expressed in units whereby 1 unit inhibits the rate of reduction of cytochrome c by 50% in a coupled system, using xanthine and xanthine oxidase. Parameter

Seminal plasma 0h

Catalase Glutathione reductase Superoxide dismutase Peroxidase Methionine sulfoxide reductase

0.79 22.6 1142 0.17 0.022

Spermatozoa 48 h

± ± ± ± ±

0.57 13.5 232 0.12 0.018

0.79 19.2 1122 0.16 0.023

0h ± ± ± ± ±

0.57 18.7 129 0.11 0.017

0.017 0.662 23.19 0.66 0.003

48 h ± ± ± ± ±

0.009 0.618 8.30 0.79 0.004

0.014 0.575 23.21 0.67 0.003

± ± ± ± ±

0.009 0.470 9.32 0.68 0.003

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Table 3 Influence of antioxidants on viability of brown trout spermatozoa. Spermatozoa were incubated in SMIS containing the different types of antioxidants and viability parameters were examined after 72 and 120 h. Data are mean standard deviation (n = 6), data within a column superscripted by different letters are significantly different (P < 0.01). n.i. = not investigated; oxid. = oxidized form; red, reduced form. Membrane integrity (%) After 72 h incubation Control

Motility rate (%) Locally

Velocity (␮m/s)

Progressive

Motility pattern (%) Linear

Non-linear

Circular

27.7 ± 19.8a

46.2 ± 8.4 a

91.8 ± 12.4a

34.3 ± 9.8a

Ascorbic acid, 0.5 mmol/l 1.0 mmol/l 2.0 mmol/l

b

45.3 ± 12.8 42.7 ± 10.1b 41.1 ± 15.9b

a

24.5 ± 11.9 26.2 ± 13.8a 18.4 ± 11.9a

b,d

30.7 ± 6.9 16.3 ± 11.7c 10.5 ± 9.9c,e

68.1 ± 13.3 55.7 ± 22.4b,c 48.4 ± 19.5c

53.2 ± 19.6 52.4 ± 11.2b 49.3 ± 21.4b

29.3 ± 15.1 30.4 ± 18.8a,c 26.5 ± 18.3a

17.5 ± 14.0b 17.3.2 ± 15.4b 24.2 ± 11.6b

Carnitine, 0.5 mmol/l 1.0 mmol/l 2.0 mmol/l

67.2 ± 8.6a 51.4 ± 7.5b 44.4 ± 10.2b

25.5 ± 12.3a 28.5 ± 8.8a 32.3 ± 14.3a

48.4 ± 6.2a 23.4 ± 6.7b 10.3 ± 8.9c,e

89.9 ± 14.7a 67.6 ± 13.4b 57.3 ± 18.1b,c

38.0 ± 12.1a 14.1 ± 7.5c 13.0 ± 12.2c

24.1 ± 9.4a 64.3 ± 39.7b 59.4 ± 36.4b

37.9 ± 14.4a 21.6 ± 9.1b 27.6 ± 14.3b

Carotene, 0.1 mmol/l 0.5 mmol/l 1.0 mmol/l

49.6 ± 74.3b 31.4 ± 11.5c 31.0 ± 7.0c

27.8 ± 7.1a 23.4 ± 9.6a 24.3 ± 11.1a

35.3 ± 7.2d 5.0 ± 9.0e 8.4 ± 8.2e

71.9 ± 14.0b 47.0 ± 24.4c 47.3 ± 28.1c

37.2 ± 17.2a 33.5 ± 22.1a 36.3 ± 23.5a

25.1 ± 25.6a 29.1 ± 15.0a,c 24.8 ± 21.3a

37.7 ± 8.2a 37.4 ± 21.4a 38.9 ± 22.1a

Glutathione oxid., 0.5 mmol/l 1.0 mmol/l 2.0 mmol/l

62.2 ± 8.3a 69.1 ± 7.7a 69.1 ± 7.7a

26.4 ± 12.7a 42.4 ± 13.3b 61.9 ± 9.5c

48.5 ± 9.9a 22.6 ± 5.2b 12.5 ± 4.3c

90.0 ± 15.8a 73.2 ± 19.7b 60.2 ± 13.1b

39.1 ± 13.4a 37.1 ± 28.3a 40.0 ± 21.5a

26.5 ± 12.4a 54.6 ± 38.4b 50.1 ± 19.3b

34.4 ± 14.7a 8.3 ± 8.4b,c 9.9 ± 8.0b,c

Glutathione red., 0.5 mmol/l 1.0 mmol/l 2.0 mmol/l

64.9 ± 8.2a 68.5 ± 14.5a 64.4 ± 8.7a

29.4 ± 5.3a 39.9 ± 12.5b 46.4 ± 7.8b

42.3 ± 9.5a 22.5 ± 8.7b 20.3 ± 11.7b

87.6 ± 9.3a 63.4 ± 9.8b 49.4 ± 12.7c

30.2 ± 8.4a 39.2 ± 11.4a 40.3 ± 23.4a

28.6 ± 9.5a 26.4 ± 18.4a 20.8 ± 16.5a

41.2 ± 8.3a 34.4 ± 12.2a 38.9 ± 21.9a

Methionine oxid., 05 mmol/l 1.0 mmol/l 2.0 mmol/l

62.0 ± 12.4a 77.5 ± 13.0d 78.4 ± 10.9d

22.1 ± 8.5a 20.6 ± 11.6a 22.3 ± 14.0a

48.9 ± 6.5a 61.9 ± 18.6f 64.1 ± 13.2f

94.2 ± 8.3a 101.9 ± 14.5a 104.1 ± 16.2a

32.5 ± 8.7a 57.8 ± 9.7b 55.0 ± 11.4b

20.4 ± 8.6a 35.6 ± 8.6c 39.1 ± 12.4c

47.1 ± 16.2a 6.7 ± 5.5c 5.9 ± 3.1c

Methionine red., 0.5 mmol/l 1.0 mmol/l 2.0 mmol/l

79.5 ± 8.2d 63.0 ± 7.2a 50.5 ± 12.3b

21.4 ± 12.1a 27.4 ± 14.2a 33.8 ± 10.5a

61.6 ± 28.6f 45.9 ± 7.7a 24.4 ± 5.9b

95.7 ± 14.4a 94.6 ± 10.5a 90.3 ± 11.3a

62.4 ± 12.5b 35.7 ± 8.9a 39.1 ± 15.7a

20.5 ± 7.0a 22.6 ± 7.5a 15.4 ± 5.6a

17.1 ± 6.9b 41.7 ± 9.3a 45.5 ± 10.6a

Uric acid, 0.25 mmol/l 0.50 mmol/l

85.2 ± 9.3d 83.1 ± 8.6d

29.7 ± 9.0a 24.3 ± 10.2a

58.2 ± 7.2f 61.4 ± 6.3f

34.6 ± 4.7a 36.5 ± 8.3a

24.8 ± 14.6a 28.2 ± 12.2a

40.6 ± 14.0a 35.3 ± 8.4a

ZnCl2 , 0.1 mmol/l

28.8 ± 12.5c

After 120 h incubation Control Methionine oxid., 1.0 mmol/l Methionine red., 0.5 mmol/l Uric acid, 0.25 mmol/l Uric acid, 0.5 mmol/l

63.5 ± 6.4a

n.i. n.i. n.i. n.i.

0d

29.9 ± 8.0a 23.3 ± 9.9a 30.3 ± 8.9a 13.1 ± 11.5e 19.7 ± 5.7e

0g

118.9 ± 11.8d 122.49 ± 13.2d 0e

24.7 ± 8.8b 47.7 ± 8.1a 37.2 ± 10.0d 63.1 ± 9.3f 55.5 ± 4.4f

the percentage of linear spermatozoa (% LIN) was increased, while the percentage of circular spermatozoa (% CIRC) was decreased. Carnitine concentrations of 0.5 mmol/l had no effect on the evaluated sperm viability parameters (Table 3). Higher concentrations of >1.0 mmol/l had a negative effect on % MOT, VEL, and % INTEG. Carnitine concentrations of 1.0 mmol/l affected also the motility pattern as the percentage of non-linear spermatozoa (% NONLIN) was increased and the % LIN and % CIRC decreased. Carotene had a negative effect on % MOT, VEL, and % INTEG in all tested concentrations (0.1–1 mmol/l). Reduced and oxidized glutathione (0.5 mmol/l, respectively) had no effect on the sperm viability, >1.0 mmol/l increased the percentage of locally motile spermatozoa (% LOC) and decreased % MOT and VEL. Further, 1.0 and 2.0 mmol/l oxidized glutathione increased % NONLIN and decreased % CIRC. Reduced methionine in a concentration of 0.5 mmol/l increased % MOT and % INTEG. It changed also the sperm motility pattern as % LIN was increased and % CIRC was decreased. Reduced methionine in a concentration of

b

91.1 ± 4.5a 95.4 ± 30.1a 95.6 ± 37.0a 130.0 ± 10.0d 110.3 ± 7.3a

21.3 ± 4.2a b

0c

45.2 ± 26.4a 45.9 ± 37.5a 46.7 ± 17.6a 42.7 ± 12.2a 42.3 ± 24.6a

44.4 ± 9.6a a,c

0d

23.7 ± 12.6a 21.6 ± 4.4a 26.3 ± 7.1a 26.1 ± 15.0a 22.8 ± 7.0a

0d

31.3 ± 22.1a 32.5 ± 33.4a 27.0 ± 11.0a 31.2 ± 6.3a 34.9 ± 17.8a

1.0 mmol/l had no effect, 2 mmol/l reduced methionine had a negative effect as it decreased % MOT and % INTEG. In a concentration of 0.5 mmol/l oxidized methionine had no effect on the sperm viability. Higher concentrations of 1.0 and 2.0 mmol/l had positive effects as they increased % MOT and % INTEG (Table 3). Oxidized methionine concentrations of 1.0 and 2.0 mmol/l also changed the sperm motility pattern as they increased % LIN and % NONLIN and decreased % CIRC. Uric acid (0.25 and 0.5 mmol/l) increased % MOT, VEL and % INTEG. Tocopherol had no effect on sperm viability parameters in all tested concentrations (0.5–2.0 mmol/l) and therefore the data are not shown. ZnCl2 had a negative effect on % MOT and % INTEG in all tested concentrations. The results for 0.1 mmol/l are shown in Table 3, data for 0.5 and 1.0 mmol/l are not shown as 100% of the spermatozoa were immotile. 3.2.2. Effect after 120 h incubation period After 120 h only those treatments were examined which had had a positive effect after incubation periods of 72 h.

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Table 4 Influence of oxidant defensive enzymes on viability of brown trout spermatozoa. Spermatozoa were incubated in SMIS together with the enzymes and viability parameters were examined after 72 and 120 min. Data are mean standard deviation (n = 6), data within a column superscripted by different letters are significantly different (P < 0.01). n.i. = not investigated. Membrane integrity (%) Motility rate (%) Locally

Progressive

After 72 h incubation Control Catalase, 500 U/l Catalase, 150 U/l Peroxidase, 500 U/l Peroxidase, 150 U/l Gluthatione reductase, 500 U/l Gluthatione reductase, 150 U/l Superoxide dismutase, 1000 U/l Superoxide dismutase, 1500 U/l

54.2 ± 14.4a 66.0 ± 12.9a 95.3 ± 10.5b 49.3 ± 14.4a 63.4 ± 12.5a 56.1 ± 12.5a 54.6 ± 11.1a 55.2 ± 16.9a 51.8 ± 14.9a

17.1 25.6 20.9 24.1 22.3 27.4 25.4 15.3 20.8

After 120 h incubation Control Catalase, 500 U/l Catalase, 150 U/l

n.i. n.i. n.i.

17.4 ± 6.9a 16.8 ± 9.6a 14.4 ± 7.8a

± ± ± ± ± ± ± ± ±

Velocity (␮m/s) Motility pattern (%)

8.1a 6.3a 5.7a 8.4a 12.7a 6.0a 5.1a 9.3a 6.0a

The results are shown in Table 3. Oxidized methionine in concentrations of 1.0 and 2.0 mmol/l, 0.5 mmol/l reduced methionine, and 0.25–0.5 mmol/l uric acid significantly improved % MOT (Table 3); 0.25 mmol/l uric acid increased also the swimming velocity. Further, in the uric acid treatment % LOC was significantly reduced in comparison to the other treatments (Table 3). After 120 h the motility pattern was similar for the control and for all treatments. % INTEG was not investigated after 120 h. 3.3. Effect of oxidant defensive enzymes on sperm motility and sperm membrane integrity Incubation of spermatozoa in SMIS containing 150 U/l catalase activity increased % MOT, when activated after 72 h, and % INTEG in comparison to the control (Table 4). The positive effect of 150 U/l catalase activities was also observed after 120 h (Table 4). Catalase activities of 500 U/l had no effect after 72 h of incubation (Table 4). After 120 h 500 U/l catalase activities increased VEL in comparison to the control, however they had no significant effect on % MOT (Table 4). Peroxidase and glutathione reductase had no positive effect after 72 h of incubation (Table 4). At the lower enzyme activity (150 U/l) the evaluated sperm parameters were similar to the control, at the higher enzyme activity (500 U/l) % MOT and VEL were decreased in comparison to the control (Table 4). Superoxide dismutase which was investigated at activities similar to those occurring in seminal plasma had no effect. The glutathione reductase, superoxide dismutase, and peroxidase treatments were not examined after 120 h. 3.4. Lipid peroxidation assay When spermatozoa were incubated for 72 h in seminal plasma or SMIS and lipid peroxidation was challenged thereafter with ferrous sulfate and ascorbic acid the concentrations of thiobarbituric acid reactive substances (TBARS) were significantly lower for spermatozoa incubated in seminal plasma than for those incubated in SMIS (Table 5). For spermatozoa incubated in SMIS containing

44.0 38.3 74.1 31.9 38.3 21.4 43.8 42.3 38.3

± ± ± ± ± ± ± ± ±

8.7a 10.5a 9.7b 6.1c 10.4a,c 4.8d 8.1a 15.3a 12.4a,c

19.9 ± 8.8d 28.4 ± 7.1c 36.2 ± 9.0a,c

Linear 110.8 104.6 109.0 77.5 98.8 81.8 103.4 109.5 112.8

± ± ± ± ± ± ± ± ±

10.3a 19.3a 9.1a 9.6b 12.6a 10.3b 10.4a 15.4a 22.0a

75.7 ± 21.3b 96.8 ± 11.5a 109.9 ± 12.7a

32.9 31.0 43.4 39.3 32.0 40.6 41.4 33.2 32.2

± ± ± ± ± ± ± ± ±

Non-linear 12.8a 11.4a 14.5a 13.3a 11.2a 10.7a 3.1a 16.6a 14.7a

32.4 32.4 28.7 31.6 36.0 29.8 30.7 27.2 34.4

± ± ± ± ± ± ± ± ±

20.4a 13.6a 15.1a 10.1a 19.0a 16.3a 7.2a 17.4a 21.9a

Circular 34.7 36.6 27.9 29.1 32.0 29.6 27.9 40.6 33.3

± ± ± ± ± ± ± ± ±

31.3a 14.0a 6.7a 21.4a 8.9a 22.0a 6.2a 23.1a 19.1a

27.0 ± 13.4a 41.8 ± 13.0a 31.2 ± 14.5a 33.3 ± 17.1a 37.5 ± 9.5a 29.2 ± 12.0a 31.7 ± 14.8a 38.8 ± 12.2a 25.5 ± 16.3a

Table 5 Lipid peroxidation (expressed as area under the curve concentration of thiobarbituric acid reactive substances) of brown trout spermatozoa in SMIS, seminal plasma, seminal plasma molecular weight fractions, and in the presence of different antioxidants. Spermatozoa were incubated for 72 h in the different solutions and lipid peroxidation was challenged with ferrous sulfate and ascorbic acid for 60–180 min. Data are mean standard deviation, those belonging to the same experiment and superscripted by different letters are significantly different (P < 0.01). nmol of TBARS/1012 sperm Experiment 1 SIMIS Seminal plasma

4825 ± 880a 2087 ± 506b

Experiment 2 SMIS Seminal plasma Uric acid, 0.5 mmol/l Reduced methionine, 0.5 mmol/l Oxidized methionine, 1.0 mmol/l Carnitine, 0.5 mmol/l

4351 ± 739a 1949 ± 592b 2190 ± 423b 4611 ± 933a 3818 ± 628a 4304 ± 393a

Experiment 3 Seminal plasma >100 kDa >50 kDa <100 kDa <50 kDa

2355 ± 257a 2441 ± 437a 2636 ± 569a 2679 ± 814a 2370 ± 309a

0.5 mmol/l uric acid the concentrations of TBARS were lower than in SMIS (Table 5). In SMIS containing 1.0 mmol/l oxidized methionine (=methionine sulfoxide) the concentrations of TBARS were non-significantly decreased in comparison to the control. Reduced methionine and carnitine (0.5 mmol/l) had no effect (Table 5). Between the different seminal plasma molecular weight fractions (<100 kDa, <50 kDa, >100 kDa, >50 kDa) there was no significant difference in the concentration of TBARS and the concentrations were similar to untreated seminal plasma (Table 5). 4. Discussion During in vitro storage the concentrations of peroxides increased in brown trout seminal plasma and sperma-

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tozoa indicating the generation of free reactive oxygen species (ROS). In mammalians several negative effects of ROS on sperm viability have been recorded. They may cause lipid peroxidation of sperm cell membranes, damage of midpiece and axonemal structure, malfunctions of capacitation and acrosome reaction, loss of motility, and infertility (Sikka et al., 1995; Tramer et al., 1998). Therefore, it may be concluded that ROS are also harmful and a limiting factor for viability of brown trout spermatozoa. The conducted experiments demonstrate also that brown trout seminal plasma has antioxidative functions as the sperm lipid peroxidation was lower in seminal plasma than in SMIS. Sperm lipid peroxidation was similar in the tested molecular weight fractions of the seminal plasma indicating that several components of the seminal plasma contribute to antioxidant protection. Antioxidative functions for the seminal plasma of teleost fish have been proposed in earlier studies (Ciereszko et al., 1999; Ciereszko and Dabrowski, 2000), but the present study is the first demonstration of this. In the seminal plasma of mammals effective antioxidant systems have been described in several studies (Alvarez and Storey, 1989; Zini et al., 1993; Bauchè et al., 1994; Gu and Hecht, 1996). Biochemical analysis revealed that brown trout seminal plasma and spermatozoa contained different enzymes and metabolites which might play a role in antioxidative protection. These were the oxidant defensive enzymes catalase, glutathione reductase, methionine sulfoxide reductase, peroxidase, and superoxide dismutase and the antioxidants ascorbic acid, glutathione, methionine, tocopherol, and uric acid. However, the activities of enzymes were low and fluctuating with exception of superoxide dismutase. From the antioxidants uric acid was found in highest concentrations and in an approximately similar range as detected in an earlier study (Ciereszko et al., 1999). Therefore, it might be concluded that superoxide dismutase and uric acid play a major role in antioxidative protection of brown trout spermatozoa under in vivo conditions. To examine how different antioxidants and oxidant defensive enzymes affect the sperm viability in vitro experiments were performed, whereby spermatozoa were incubated together with the test substances in SMIS. The experiments revealed very clearly that uric acid and catalase have a positive effect on sperm viability. Uric acid increased the percentage of spermatozoa with intact membranes, the sperm motility rate and swimming velocity, when activated, and decreased the sperm membrane lipid peroxidation. These results demonstrate that uric acid most probably is the main antioxidant of brown trout semen. Uric acid is a strong reducing agent and therefore a potent antioxidant. It is produced by xanthine oxidase from xanthine and hypoxanthine, which in turn are produced from purine (Berg et al., 2006). Catalase increased the percentage of spermatozoa with intact membranes, and the sperm motility rate and swimming velocity which could be activated. However, the enzyme activities necessary to obtain the observed effects were much higher than those occurring in vivo in seminal plasma and spermatozoa indicating that catalase has probably no importance under in vivo conditions.

In the in vitro incubation experiments also reduced methionine and oxidized methionine had positive effects: reduced methionine increased the percentage of spermatozoa with intact membranes and the motility rate when activated after 72 h and 96 h. Oxidized methionine had no effect in comparison to the control after 72 h but a positive effect on the motility rate after 96 h. As methionine sulfoxide reductase was present in both seminal plasma and spermatozoa, methionine might be oxidized to methionine sulfoxide (=oxidized methionine) by reactive oxygen species and reduced back to methionine by methionine sulfoxide reductase. This is an important antioxidant system in many cells (Moskovitz et al., 2001; Jin et al., 2003). However, in brown trout neither reduced nor oxidized methionine decreased the spermatozoal membrane lipid peroxidation in comparison to the control. Therefore, it remains unclear whether methionine has importance as an antioxidant system in brown trout semen. Methionine is also a key intermediate metabolite in the biosynthesis of cysteine, carnitine, taurine, lecithin, phosphatidylcholine, and other phospholipids of cell metabolism (Berg et al., 2006). Therefore, it could affect brown trout spermatozoa via different metabolic pathways. In the conducted incubation experiments superoxide dismutase, ␣-tocopherol and reduced glutathione had no effect at all, while ascorbic acid, carnitine, carotene, oxidized glutathione, and ZnCl2 had negative effects at all tested concentrations. Peroxidase and glutathione reductase had no or negative effects depending on the concentration. Superoxide dismutase catalyses the breakdown of the superoxide anion into oxygen and hydrogen peroxide (Fontecave et al., 1987). Superoxide is a biologically toxic byproduct of mitochondrial respiration and is produced by phagocytes to kill invading pathogens (Fontecave et al., 1987). ␣-Tocopherol is an important lipid-soluble antioxidant, which protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction (Liebler, 1993). Diets containing ␣-tocopherol decreased the sperm lipid peroxidation in arctic char (Mansour et al., 2006) and improved the semen quality in rainbow trout (Canyurt and Akhan, 2008). Glutathione is a cysteine containing peptide whose thiol group is a reducing agent and which is maintained in the reduced form by glutathione reductase. In turn it reduces other metabolites and enzyme systems, such as ascorbate in the glutathione-ascorbate cycle, glutathione peroxidases and glutaredoxins (Berg et al., 2006). The glutathione system is an important antioxidant system in mammalian semen (Bauchè et al., 1994; Alvarez and Storey, 1989). Ascorbic acid is a monosaccharide antioxidant. In cells it is maintained in its reduced form by reaction with glutathione. Several studies indicated that ascorbic acid could play a central role as antioxidant of teleost fish semen as fish fed ascorbic acid or ascorbyl monophosphate containing diets had semen with higher motility rates or fertility rates than the control groups in rainbow trout (Ciereszko and Dabrowski, 1995, 2000) and in grass carp (Metwally and Fouad, 2009); in rainbow trout also the spermatozoal lipid peroxidation was depressed (Liu et al., 1997). The ascorbic acid concentrations measured in seminal plasma of brown trout were in a similar range to those measured in rain-

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bow trout seminal plasma by Ciereszko and Dabrowski (2000), however they were highly fluctuating as indicated by the high standard deviation of mean values. Based on the in vitro incubation experiments of the present study the role of ascorbic acid as antioxidant could not be established as supplementation of SMIS with ascorbic acid had negative effects on sperm viability in all tested concentrations. Ciereszko and Dabrowski (2000) could also not detect positive effects when salmonid spermatozoa were stored in ascorbic acid containing saline solutions. These authors suggest that the positive effect of ascorbic acid on semen quality is related to its long-term effects during spermatogenesis. In summary, the present study demonstrates that uric acid is the main antioxidant of brown trout semen. It protects spermatozoa under in vitro storage conditions. As it occurs in high quantities in seminal plasma and spermatozoa it seems to be the main antioxidant in vivo as well. Supplementation of semen storage media with uric acid is therefore beneficial to stabilize the sperm viability and can be used to improve semen storage techniques in aquaculture. Catalase also improved the viability of stored semen, however, this enzyme had importance only under in vitro conditions as it occurred in very low activities in seminal plasma and spermatozoa. Finally, brown trout semen contained also a glutathione and a methionine antioxidant system as these components were detected in seminal plasma and spermatozoa. However, as concluded from the incubation experiments these systems seem to play only a minor role for brown trout spermatozoa. Acknowledgements The investigation was funded by the Austrian ‘Fonds zur Förderung der wissenschaftlichen Forschung’ (FWF project P 20008-B11). The authors are grateful to Manfred Kletzl, the manager of the fish farm Kreuzstein for support with material. References Aitken, R.J., Buckingham, D., West, K., Wu, F.C., Zikopoulos, K., Richardson, D.W., 1992. Differential contribution of leukocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. J. Reprod. Fertil. 94, 451–462. Aitken, R.J., Harkiss, D., Buckingham, D.W., 1993. Analysis of lipid peroxidation mechanisms in human sperm. Mol. Reprod. Dev. 35, 302–315. Alvarez, J.G., Touchstone, J.C., Blasco, L., Storey, B.T., 1987. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: superoxide dismutase as major enzyme protectant against oxygen toxicity. J. Androl. 8, 338–348. Alvarez, J.G., Storey, B.T., 1989. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res. 23, 77–90. Bauchè, F., Fouchard, M.H., Jégou, B., 1994. Antioxidant systems in rat testicular cells. FEBS Lett. 349, 392–396. Berg, J.M., Tymoczko, J.L., Stryer, L., 2006. Biochemistry. W. H. Freeman Company, New York, USA. Bergmeyer, H.U., 1985. Methods of Enzymatic Analysis. VCH Verlagsgesellschaft, Weinheim, Germany.

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