Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm

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Theriogenology 77 (2012) 1129 –1136 www.theriojournal.com

Incorporation of ascorbic acid and ␣-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm S. Martínez-Páramoa,*, P. Diogoa, M.T. Dinisa, M.P. Herráezb, C. Sarasquetec, E. Cabritac a

CCMAR-Center for Marine Science, University of Algarve, Campus Gambelas, 8005-139 Faro, Portugal b Department of Molecular Biology, Area of Cell Biology, University of León, 24071 León, Spain c ICMAN-Institute of Marine Science of Andalusia, Spanish National Research Council, Av. Republica Saharaui 2, 11510 Puerto Real, Cádiz, Spain Received 19 July 2011; received in revised form 28 September 2011; accepted 8 October 2011

Abstract Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 mM ␣-tocopherol or 0.1 mM ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ⫾ 3.0 ␮m/sec, 18.3 ⫾ 1.7 ␮m/sec and 46.9 ⫾ 2.0% in extender containing 0.1 mM ␣-tocopherol or 30.6 ⫾ 3.9 ␮m/sec, 19.5 ⫾ 1.6 ␮m/sec and 47.9 ⫾ 2.2% in extender containing 1 mM ascorbic acid respect to the extender control (20.7 ⫾ 3.3 ␮m/sec, 13.8 ⫾ 1.7 ␮m/sec and 37.3 ⫾ 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of ␣-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation. © 2012 Elsevier Inc. All rights reserved. Keywords: Sea bass; Sperm cryopreservation; Antioxidants; Vitamins; Oxidative stress; Antioxidant enzymes

1. Introduction * Corresponding author. Tel: 00351 289 800 900 ext. 7374; fax: 00351 289 800 069. E-mail address: [email protected] (S. Martínez-Páramo). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.10.017

Fish sperm cryopreservation is a useful tool of undoubted interest in aquaculture, since it facilitates broodstock management in terms of synchronization of

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gamete availability from both males and females. In addition, this technique enables the genomic profile of selected breeders or selected strains to be preserved [1]. However, despite numerous advantages, this process promotes cellular damage that could compromise sperm quality in terms of motility, membrane stability or DNA integrity, impairing fertilization ability and embryo development in the last term [2– 4]. Oxidative stress because of the imbalance between the presence of reactive oxygen species (ROS) and sperm antioxidant activity is a major cause of spermatozoa damage during freezing storage [5–9]. The lipid composition of the spermatozoon membrane makes this structure very susceptible to oxidative damage due to the high content in unsaturated fatty acids [10 –11]. Because of lipid peroxidation, permeability and fluidity of membranes are modified [12] leading to spermatozoa dysfunction, reducing motility, resistance to osmotic shock and fertilization potential [11,13–14]. Moreover, protein oxidization could promote detrimental effects in sperm functionality, since plasma membrane structural proteins could be affected as well as proteins with enzymatic activity [15–17]. Fish sperm presents an antioxidant system composed by enzymatic (glutathione peroxidase and reductase, catalase, superoxide dismutase) and non-enzymatic (␣tocopherol, ascorbic acid, ␤-carotene, selenium, zinc) components that act as ROS scavengers protecting the cell structure [15,18]. In sperm, seminal plasma provides the major defense against ROS, due to the low content of cytoplasm in spermatozoa, which is removed during the final stages of spermatogenesis [11,19 –20]. However, dilution in the extender before cryopreservation reduces the concentration of those components present in seminal plasma, diminishing the antioxidant protection of sperm [21]. Several authors have demonstrated that it is possible to reduce oxidative damage by the addition of molecules with antioxidant capacity to the freezing media [9,22–26]. However, the effect of each antioxidant is species-specific, improving different parameters of sperm quality depending on the type of molecule and concentration used for each species [22]. Vitamin E (␣-tocopherol) and vitamin C (ascorbic acid) are natural antioxidants, included in the group of chain-breaking antioxidants, which scavenge radicals to terminate free radical reactions and prevent chain propagation reactions. These vitamins have different action mechanisms. Vitamin C is a water-soluble chain-breaking antioxidant which has the capacity to scavenge oxygen radicals in the aqueous phase, while vitamin E is a lipophilic antioxidant that scavenges oxygen radicals within the membrane [27]. It has been demonstrated

that the incorporation of these vitamins into the extenders improves the motility and viability of mammal sperm (boar, dog, cat, bull and human) [24 –26, 28,29]. However, the use of extenders containing antioxidants has been tested for the first time in fish by Cabrita, et al [22] in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata), although no effects in sperm motility and viability were observed after cryopreservation. Nevertheless, at DNA level, the presence of vitamins reduced DNA fragmentation in gilthead sea bream, contrary to the results obtained in European sea bass, demonstrating once again that the antioxidant effect is speciesspecific. In the present work, ascorbic acid and ␣-tocopherol were incorporated in the extenders to evaluate their capacity to improve the antioxidant system of cryopreserved European sea bass sperm (Dicentrarchus labrax) and to reduce cryodamage. For that purpose, sperm motility, viability, levels of lipid peroxidation, protein oxidation and the activity of enzymes with antioxidant capacity were evaluated to determine the antioxidant status in cryopreserved sperm. 2. Materials and methods All reagents were obtained from Sigma Aldrich (Sintra, Portugal), unless otherwise stated. The propidium iodide was purchased from Invitrogen (Barcelona, Spain). 2.1. Sperm collection For sperm collection, European sea bass (Dicentrarchus labrax) males were provided by the Aqualvor Lda fishfarm (Odiaxere, Lagos, Portugal). Sperm was collected by stripping during the natural spawning season between November and February. At the end of the period, 12 pools containing sperm from 10 to 15 males each were obtained. 2.2. Sperm dilution, cryopreservation and thawing Immediately after extraction, the sperm was diluted 1:6 (v/v) in a non-activating mineral medium (NAM) with or without vitamin supplementation. Thus, three different solutions were tested: 1) NAM, used as extender control (NAM: 59.83 mM NaCl, 1.47 mM KCL, 12.91 mM MgCl2, 3.51 mM CaCl2, 20 mM NaHCO3, 0.44 mM glucose, 1% (w/v) BSA, pH 7.7); [30] 2) NAM containing 0.1 mM ␣-tocopherol and 3) NAM containing one mM ascorbic acid. Fresh sperm was diluted in NAM without any antioxidant supplement and maintained at 4 °C for the

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determination of motility, viability, lipid peroxidation, protein oxidation and status of enzymes with antioxidant capacity in fresh samples. For cryopreservation, 10% DMSO (v/v) was added to each sperm dilution, and the mixture was loaded into 0.5 mL straws. The straws were frozen at 6.5 cm above the liquid nitrogen surface (15 min), then immersed on it and stored until used. The straws were thawed at 35 °C for 15 sec. 2.3. Evaluation of sperm quality and oxidative damage 2.3.1. Motility Sperm motility was tested in fresh and cryopreserved sperm, using the computer assisted sperm analysis (CASA). The parameters quantified were motile spermatozoa (%), curvilinear velocity (VCL; ␮m/sec), straight line velocity (VSL; ␮m/sec) and spermatozoa linearity (Lin; %). Briefly, 0.5 ␮l of diluted sperm was placed in a Makler chamber under a 10 x negative phase-contrast objective (Nikon E200, Tokyo, Japan) and motility was immediately activated with 20 ␮L of artificial seawater (513.3 mM NaCl, 10.7 mM KCl, 11.7 mM CaCl2, 54.8 mM MgSO4, 11.6 mM NaHCO3). The parameters referred to above were recorded with a Basler camera (Basler Afc, Ahrensburg, Germany) and registered with the software ISAS (Proiser, Valencia, Spain) at 10, 20, 30 and 45 sec post activation. 2.3.2. Viability The percentage of viable cells was quantified using the fluorescent dyes propidium iodide (PI) and SYBR Green. Before the addition of the fluorescent dyes, diluted sperm (1:6) was rediluted 1:1,000 in NAM, to reduce sperm concentration. Subsequently, SYBR Green (final concentration 0.25 ␮M) and PI (final concentration 18 ␮M) were added to the cell suspension. After 5 minutes of incubation in the dark at 4 °C, cell viability was quantified under an epifluorescence microscope (Nikon E200, Tokyo, Japan), equipped with triple excitation filter block DAPI-FITCTexas Red (excitation filter wavelengths: 395– 410 nm (bandpass, 403 CWL), 490 –505 nm (bandpass, 498 CWL), and 560 –580 nm (bandpass, 570 CWL)). Red (PI stained cells) and green (SYBR stained cells) cells were counted, and the percentage of viable cells (green stained) was determined. At least 100 cells per slide were counted, and two slides per pool and per treatment were observed. 2.3.3. Lipid peroxidation For lipid peroxidation determination, the concentration of malondialdehyde (MDA) was quantified using a color-

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imetric assay (kit BIOXYTECH LPO-586, OxisResearch, USA), following the protocol described by DomínguezRebolledo, et al [31] and adapted for fish sperm. Briefly, 300 ␮L of diluted sperm, including fresh and cryopreserved samples, was centrifuged at 5,000g for 5 min at 4 °C and the supernatant was discarded and substituted by NAM without BSA, to avoid any interference with the reaction. The cell suspension was incubated in a 200 ␮M sodium ascorbate solution containing 40 ␮M FeSO4 for 30 min at 37 °C in the dark to induce MDA release [32]. After that, reagents provided by the kit were added to 100 ␮L of the cell suspension (following the manufacture’s instructions) and the samples were incubated for 1 h at 45 °C in the dark. They were then centrifuged at 10,000g for 10 min at 4 °C, and 200 ␮L of supernatant were transferred to a 96-well flat-bottom transparent plate (Nunc, Roskilde, Denmark). Absorbance was read using a microplate reader at 586 nm (Synergy 4; Bio-Tek, USA) and MDA concentrations were calculated from a standard curve and presented as ␮moles of MDA per million of spermatozoa. Each sample was processed in triplicate. 2.3.4. Protein oxidation The quantification of oxidized proteins, in both fresh and cryopreserved sperm, was evaluated by the carbonyl group content, which was spectrophotometrically quantified through DNPH (2,4-dinitrophenilhidrazine) reaction at 360 nm, following the protocol described by Levine, et al[33] and adapted for fish sperm. The sperm samples were washed twice with 1 mL of NAM without BSA by centrifugation (5,000g, 5 min, 4 °C), to avoid BSA interference. Pellets were resuspended in 1 mL of 20% trichloroacetic acid (TCA) (v/v) and centrifuged (10,000g, 5 min, RT) to be incubated in 500 ␮L of 10 mM DNHP solution in the dark (20 min, RT). For all samples, a blank sample was prepared, which was incubated with 2 M HCl instead of DNPH. Afterward, 500 ␮L of 20% TCA were added to all samples which were then centrifuged (14,000g, 5 min, RT). The supernatants were discarded and 1 mL of 1:1 ethanol: ethyl acetate solution was added to wash the pellets. The samples were placed in an ultrasound water bath for 5 min and then centrifuged (14,000g, 10 min, RT). This procedure was performed trice to remove all free DNHP. Then, the samples were incubated with 1 mL of 6 M guanidine pH 6.5 at 37 °C for 10 min. After centrifugation (14,000g, 5 min, RT) the supernatants were collected and protein oxidation was estimated spectrophotometrically at 360 nm. The carbonyl content was calculated with a molar extinction coefficient of 22 mM⫺1 cm⫺1 for DNPH and was expressed as nmoles DNPH/mg protein.

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Protein was quantified using a BSA standard curve and determined spectrophotometrically at 280 nm in blank samples. Each sample was processed in triplicate. 2.4. Status of enzymes with antioxidant capacity (glutathione peroxidase, glutathione reductase and superoxide dismutase) Aliquots of 1,750 ␮L of fresh or cryopreserved sperm were centrifuged (5,000g, 5 min, 4 °C) and the pellets were stored at ⫺80 °C, for further processing. Cell extracts were prepared according to Lahnsteiner, et al [34] and stored at ⫺80 °C until used. Glutathione peroxidase (GPX), glutathione reductase (GSR) and superoxide dismutase determination (SOD) was carried out following the manufacturer’s instructions (Randox Laboratories, Ltd., UK). Absorbance was measured at 340 nm for GPX and GSR or at 505 nm for SOD using a microplate reader (Synergy 4; Bio-Tek, USA), and the activity of each enzyme was presented as units of enzyme per mg of protein. Protein content was quantified by the Lowry method [35], using the Bio-Rad DC Protein Assay kit (Bio-Rad Laboratory, Germany). Each sample was quantified in triplicate. 2.5. Statistical analysis Statistical analysis was carried out using the software package SPSS 18.0 for Windows, and results were expressed as means ⫾ SEM. To analyze the effects of antioxidants on sperm motility parameters, a general linear model with the Bonferroni correction was used (P ⬍ 0.05). Significant differences between treatments for the remaining parameters tested were determined using one-way ANOVA and identified using the SNK test (P ⬍ 0.05).

Fig. 1. Total sperm motility until 45 sec after activation, for fresh sperm, cryopreserved control (NAM) and cryopreserved with 0.1 mM ␣-tocopherol or 1 mM ascorbic acid. Bars indicate S.E.M. Different letters show differences between treatments (General linear model, Bonferroni, P ⬍ 0.05).

acid to the freezing media significantly improved total motility (31.2 ⫾ 3.0% and 30.6 ⫾ 3.9%, respectively) in comparison with the extender control (20.7 ⫾ 3.3%). In general, motility decreased linearly regardless of the treatment, until 45 sec after activation, when the last motile spermatozoa were recorded. The addition of ␣-tocopherol in cryopreserved sperm promoted similar curvilinear velocity values to those recorded in fresh sperm (around 40 ␮m/sec and 56 ␮m/sec, respectively, 10 sec after activation) (Fig. 2). In addition, both vitamins increased straight line velocity in comparison with cryopreserved sperm without antioxidant additives (Fig. 3). The presence of vitamins resulted in similar linearity percentages to those obtained in fresh sperm, significantly higher than values obtained in cryopreserved controls (Fig. 4).

3. Results 3.1. Evaluation of sperm quality and oxidative damage 3.1.1. Motility In general, the addition of vitamins to the freezing media improved parameters related with sperm motility in comparison with data obtained in samples cryopreserved with the extender control. The percentage of motile spermatozoa decreased significantly after cryopreservation, from 50.4 ⫾ 2.2% in fresh sperm to 20.7 ⫾ 3.3% in cryopreserved sperm without antioxidant additives, 10 s after activation (Fig. 1). However, the addition of ␣-tocopherol and ascorbic

3.1.2. Viability Cryopreservation promoted membrane damage since the percentage of viable cells was lower than in fresh sperm regardless of the treatment (Table 1). Therefore, the addition of vitamins did not improve these results, taking into account that the presence of ␣-tocopherol or ascorbic acid did not reduce the percentage of damaged cells after freezing. 3.1.3. Lipid peroxidation and protein oxidation Results for malondialdehyde quantification demonstrated that lipid peroxidation remained constant in all treatments (around 4 ␮mol MDA/mill spz) in fresh and cryopreserved sperm. The cryopreservation protocol

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Fig. 2. Curvilinear velocity before and after cryopreservation. Bars indicate S.E.M. Different letters show differences between treatments (General linear model, Bonferroni, P ⬍ 0.05).

did not increase lipid peroxidation, and neither did the addition of antioxidants produce any effect (Table 1). Similar to the results obtained for lipid peroxidation, the level of oxidized proteins did not increase after cryopreservation as the presence of antioxidants did not improve the results significantly, although the highest values were obtained for fresh sperm (100.7 ⫾ 21.8 nmol carbonyl/mill spz) and the lowest with the incorporation of ascorbic acid into the freezing media (66.3 ⫾ 17.5 nmol carbonyl/mill spz) (Table 1).

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Fig. 4. Percentage of linearity in fresh and cryopreserved sperm. Bars indicate S.E.M. Different letters show differences between treatments (General linear model, Bonferroni, P ⬍ 0.05).

3.2. Status of enzymes with antioxidant capacity (glutathione peroxidase, glutathione reductase and superoxide dismutase) Glutathione peroxidase quantification showed that the addition of ascorbic acid to the extender significantly increased the units of GPX per gram of protein, in comparison with the fresh sperm and cryopreserved control (36.7 ⫾ 3.3, 21.3 ⫾ 1.7 and 26.4 ⫾ 3.4 U GPX/mg prot, respectively) (Table 1). GPX activity quantified in samples cryopreserved in the presence of ␣-tocopherol did not show significant differences with the other treatments. Regarding glutathione reductase, the units of GRS per gram of protein increased in cryopreserved sperm in comparison with the fresh samples, regardless of the treatment (Table 1). Quantification of SOD was similar before and after freezing for all the extenders tested (Table 1). 4. Discussion

Fig. 3. Straight linear velocity before and after cryopreservation. Bars indicate S.E.M. Different letters show differences between treatments (General linear model, Bonferroni, P ⬍ 0.05).

Sperm cryopreservation promotes cell damage compromising fertilization success, the final result being the impairment of motility, membrane stability and spermatozoa functionality [1–2], as was previously demonstrated in sea bass sperm by Zilli, et al [4,16,17]. Oxidative stress has been hypothesized as the main cause of injuries in these functional properties. Therefore, imbalance between the ROS and the spermatozoa antioxidant system leads to metabolic and functional disorders reducing sperm motility, promoting peroxidation

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Table 1 Cell viability, lipid peroxidation, protein oxidation and enzyme activity of GPX, GSR and SOD in sperm samples, before and after cryopreservation with the different extenders. Different letters in the same line show differences between treatments for each parameter tested (ANOVA-SNK, P ⬍ 0.05). Viability (%) Lipid peroxidation (␮moles MDA/106 spz) Protein oxidation (nmoles carbonyl/mg protein) GPX (units/g protein) GRS (units/g protein) SOD (units/g protein)

Fresh

NAM

␣-tocopherol

Ascorbic acid

71.3 ⫾ 8.5a 4.0 ⫾ 0.5 100.6 ⫾ 21.8

42.4 ⫾ 5.4b 4.5 ⫾ 0.5 89.8 ⫾ 22.9

48.3 ⫾ 5.3b 3.7 ⫾ 0.6 80.7 ⫾ 21.2

40.6 ⫾ 5.6b 4.1 ⫾ 0.6 66.3 ⫾ 17.5

21.3 ⫾ 1.7b 4.4 ⫾ 0.6b 1536.2 ⫾ 260.6

26.4 ⫾ 3.4b 9.1 ⫾ 1.2a 1914.9 ⫾ 341.5

29.6 ⫾ 3.3ab 9.1 ⫾ 0.6a 1710.1 ⫾ 287.5

36.7 ⫾ 3.3a 8.2 ⫾ 0.9a 1621.1 ⫾ 298.4

of the membrane phospholipids and oxidation of proteins, besides DNA damage that compromise offspring development [2,3,5]. Although the antioxidant defense system is active in semen, its activity is limited as the amount of cytoplasm in the spermatozoa is low [14,20]. Moreover, dilution of sperm in the extender media for cryopreservation reduces the seminal plasma constituents, sperm being more vulnerable to oxidative stress. Several authors have demonstrated that it is possible to reduce the detrimental effect of ROS by the addition of antioxidants to the freezing media in several species [19,22,24 –26,29]. However, the protective effect of these compounds is species-specific, as was previously demonstrated by Cabrita, et al [22]. In the present work the addition of ␣-tocopherol and ascorbic acid to the freezing media improved sea bass sperm motility, resulting in higher percentages of motile spermatozoa with higher curvilinear velocity in comparison with the frozen control, with values of linearity similar to those obtained in fresh sperm. These results agree with those obtained in cat and canine sperm, after cryopreservation in an extender containing vitamin E (5 mM and 0.3 mM, respectively) [24,26]. Moreover, in those species, besides sperm motility and an improvement in membrane integrity was recorded immediately after thawing. However, in our study, cryopreservation reduced cell viability, regardless of the treatment, contrary to those results obtained by Zilli, et al [17], who obtained similar values of sea bass sperm viability before and after freezing. This fact could be related to the differences on the cryopreservation protocol since a different extender solution (Mounib instead of NAM) and freezing package (straws of 0.25 mL instead of 0.5 mL) were used. Recently, McCarthy, et al [36] established that osmotic stress causes oxidative stress in rhesus macaque spermatozoa, which strongly supports the hypothesis that cryopreservation-induced osmotic stress may lead to oxidative cell damage. However, according to our results, the reduction of sea bass sperm viability

after cryopreservation could be due to osmotic shock not related to oxidative stress, taking into account that the level of lipid peroxidation and protein oxidation did not increase with the cryopreservation process. During cryopreservation, osmotic stress is induced by changes in cell volume resulting from the movement of water and solutes across the sperm plasma membrane [5] as was observed in common carp by Li, et al [6]. For that reason, and taking into account that the presence of antioxidants (␣-tocopherol and ascorbic acid) did not improve membrane stability or reduce lipid and protein oxidation, in our study membrane impairment could be more associated to osmotic stress than to an oxidative process. In the presence of ROS, lipid peroxidation is one of the primary manifestations of oxidative damage, and has been linked to altered membrane structure and enzyme inactivation [6]. Thus, taking into account that it is well demonstrated that cryopreservation promotes oxidative stress [37,38], the antioxidant system of sperm in this species must be playing an important role in preventing oxidative damage, since antioxidant addition did not improve the results obtained. Recently, Lahnsteiner, et al [39] observed the same effect in salmonids presenting similar values of lipid peroxidation in fresh and cryopreserved sperm. The authors suggested that this fact might be due to the short semen handling and manipulation steps reducing ROS action time. In previous work, Lahnsteiner, et al [19] observed that the activities of oxidant defensive enzymes were low and fluctuating with the exception of SOD that plays a major role in antioxidative protection of brown trout spermatozoa under in vivo conditions. SOD scavenges both intracellular and extracellular superoxide radicals and prevents the lipid peroxidation of the plasma membrane [14]. Thus, in the present work, similar values of SOD activity in the different experimental conditions could explain why there were no significant differences in the lipid peroxidation levels before and after freezing, because a reduction in SOD

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activity might be involved in lower protection against ROS [14]. In addition, BSA present in the extender acts as a potent inhibitor of lipid peroxidation, as was previously demonstrated in human, ram and canine spermatozoa [40,41]. According to these authors, in the presence of BSA free fatty acid hydroperoxides are bound to this compound in the suspending medium and are absent from the sperm cells [40]. Reduced glutathione (GSH) is the major endogenous antioxidant produced by the cells, participating directly in the neutralization of ROS, as well as maintaining exogenous antioxidants, such as vitamins C and E in their reduced (active) forms [42]. However, Gadea, et al [42] and Stradaioli, et al [43], working with human and bovine sperm, respectively, demonstrated that GSH content in sperm decreased after cryopreservation. This molecule can be regenerated from its oxidized form (GSSG) by the GSR, whose activity is inducible upon oxidative stress, especially when lower availability of reduced glutathione reserves are present in the cells [44]. Therefore, this regulation could explain the increase in GSR activity observed in sea bass sperm after cryopreservation, activated by the imbalance of ROS promoted by cryopreservation. Consequently, higher GSR activity increased GSH content which acts as a powerful antioxidant for sea bass sperm. The role of GSR is fundamental for GPX activity, maintaining the cytosolic concentration of reduced glutathione [44], demonstrating that antioxidants act as an intricate network. In human sperm, vitamin E together with GSH and a membranebound heat labile GSH-dependent factor, presumably an enzyme, can prevent lipid peroxidation [45]. It is not clear why the presence of ␣-tocopherol and ascorbic acid increased GPX activity in sea bass sperm. However, only this fact explains the improvement in sperm motility when these vitamins were incorporated in the freezing media. Li, et al [6] suggested that GPX provided the most effective protection against cold shock and oxidative damage during cryopreservation of common carp sperm. In conclusion, results obtained in the present work demonstrated that for sea bass sperm cryopreservation, the incorporation of ␣-tocopherol and ascorbic acid at the concentrations tested did not improve sperm quality itself since only motility parameters were improved, but produced a synergic effect with sea bass sperm enzymatic antioxidant system, powerful enough to neutralize the oxidative damage promoted by cryopreservation. Moreover, the presence of these vitamins together with the antioxidant system of the spermatozoa

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improved sperm motility, which is one of the impaired parameters affected by cryopreservation.

Acknowledgments This work was supported by the CRYOSPERM project (PTDC/MAR/64,533/2006) funded by FCT national funding and AGL2011-28810 project (MICINN). S. Martínez-Páramo was supported by FCT postdoctoral fellowship (SFRH/BPD/48,520/2008) cofunded by POPHQREN, Tipologia 4.1 (FEDER and MCTES) and E. Cabrita was supported by a Ramón and Cajal research contract (RYC-2007-01650). The authors thank the Aqualvor (Alvor, Portugal) fishfarm for animals supply.

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