Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress

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Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress Eva Tvrdá a,∗ , Eva Tuˇsimová b , Anton Kováˇcik a , Duˇsan Paál c , Hana Greifová a , Abzal Abdramanov d , Norbert Lukáˇc a a Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 94976 Nitra, Slovakia b AgroBioTech Research Centre, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 94976 Nitra, Slovakia c Department of Botany and Genetics, Faculty of Natural Sciences, Constantine the Philosopher University in Nitra, Nábreˇzie mládeˇze 91, 94974 Nitra, Slovakia d Department of Veterinary Sanitary Examination and Hygiene, Faculty of Veterinary Science, Kazakh National Agrarian University, Abai street 8, 050010 Almaty city, Kazakhstan

a r t i c l e

i n f o

Article history: Received 31 March 2016 Received in revised form 19 June 2016 Accepted 20 June 2016 Available online xxx Keywords: Curcumin Oxidative stress Antioxidants Ferrous ascorbate Bulls Spermatozoa

a b s t r a c t Over the past decades, there has been an emphasis on assessment of the use of natural compounds in the prevention or repair of oxidative injury to spermatozoa. Curcumin (CUR) is a natural phenol with powerful antioxidant properties. The aim of the present study was to examine if CUR could reverse reactive oxygen species (ROS)-mediated alterations to the motility, viability and intracellular antioxidant profile of bull spermatozoa subjected to a prooxidant (i.e., ferrous ascorbate – FeAA). Spermatozoa were washed from recently collected semen samples, suspended in 2.9% sodium citrate and subjected to CUR treatment (5, 10, 25 and 50 ␮mol/L) in the presence or absence of FeAA (150 ␮mol/L FeSO4 and 750 ␮mol/L ascorbic acid) during a 6 h in vitro culture. Spermatozoa motility characteristics were assessed using the SpermVision computer-aided spermatozoa analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified using luminometry and the nitroblue-tetrazolium (NBT) test was used to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the culture to assess the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). Treatment with FeAA led to a reduced spermatozoa motility (P < 0.001), viability (P < 0.001) and decreased the antioxidant characteristics of the samples (P < 0.001) but increased the ROS generation (P < 0.001), superoxide production (P < 0.001) and lipid peroxidation (P < 0.001). The CUR treatment led to a preservation of spermatozoa motion (P < 0.001), mitochondrial activity (P < 0.001) and antioxidant characteristics (P < 0.05 with SOD and GSH; P < 0.01 with CAT and GPx), revealing the concentration range of 25–50 ␮mol/L CUR to be the most effective for sustaining spermatozoa viability. Data from the present study suggest that CUR exhibits significant protective and ROS-scavenging characteristics which may prevent oxidative insults to spermatozoa and thus preserve the functional activity of male gametes. © 2016 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. E-mail addresses: [email protected], [email protected] (E. Tvrdá).

Oxidative stress (OS) resulting from an imbalance between reactive oxygen species (ROS) generation and

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Please cite this article in press as: Tvrdá, E., et al., Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress. Anim. Reprod. Sci. (2016), http://dx.doi.org/10.1016/j.anireprosci.2016.06.008

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the available antioxidant defense mechanisms has been repeatedly associated with male reproductive dysfunction (Sikka, 1996; Hendin et al., 1999; Pasqualotto et al., 2000; Garrido et al., 2004). Spermatozoa are highly susceptible to oxidative insults as plasma membranes of these cells are rich in polyunsaturated fatty acids − the primary site of action for lipid peroxidation (LPO) (Aitken et al., 1989), while the cytoplasm is restricted to the midpiece and contains very few antioxidant systems to provide a proper protection against ROS-mediated cellular damage (Bromfield, 2014; O’Flaherty, 2014). Seminal OS may contribute to mitochondrial dysfunction (Baumber et al., 2000), DNA fragmentation (Morte et al., 2008), and oxidative breakdown of lipids and proteins (Aitken et al., 1989; Mammoto et al., 1996; Baumber et al., 2000), which are in turn associated with spermatozoa cell motility loss. A decreased capacity for sperm-oocyte fusion (Mammoto et al., 1996; Guthrie and Welch, 2012; Ahmad et al., 2015), poor fertilization rates and alterations during embryogenesis are other abnormalities that can result from oxidation events (Lewis and Aitken, 2005; Simões et al., 2013). Numerous recent studies have shown that in vitro administration of hydrophilic or lipophilic antioxidant supplements in human or veterinarian andrology practices has positive effects on the structural integrity or functional activity of male gametes (Yun et al., 2013; Petruska et al., 2014). A variety of antioxidants either scavenge ROS directly or inhibit ROS toxicity in semen of a variety of ˜ et al., mammalian or avian species (Bréque et al., 2003; Pena 2003; Zeitoun and Al-Damegh, 2015). Much attention has been devoted, particularly on use of herbal medicines or the derivatives, in the prevention and/or treatment of complications related to ROS overproduction in male reproductive cells and tissues. Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione] (CUR) is a major bioactive chemical component of turmeric powder, found in the herbal remedy and dietary spice turmeric. This vibrant yellow spice is derived from the rhizome of the plant Curcuma longa Linn., and is obtained by crushing the plant roots into powder form. The CUR is the phytochemical that gives a yellow color to turmeric and is now recognized as being responsible for most of its therapeutic effects (Aggarwal et al., 2007). CUR is a potent scavenger of an array of ROS including superoxide and hydroxyl radicals (Reddy and Lokesh, 1994) as well as nitrogen dioxide (Unnikrishnan and Rao, 1995). This molecule is an effective LPO inhibitor (Sreejayan and Rao, 1994). Inconsistent data are, however, available with respect to the effects of CUR on male fertility. Several in vivo (Salahshoor et al., 2012) as well as in vitro reports (Bucak et al., 2012; Soleimanzadeh and Saberivand, 2013) provide evidence for the involvement of CUR in energy-promoting and protective events in the testicular tissue, spermatogenesis and spermatozoa physiology. Furthermore, CUR reverses the reproductive toxicity caused by a variety of endogenous (Wei et al., 2009) or exogenous factors (ElWakf et al., 2011; Dev et al., 2013). Other studies, however, implicate a negative involvement of CUR in pathways related to human and murine spermatogenesis (Naz, 2011;

Xia et al., 2012) suggesting a cautious and thorough investigation of potentially toxic and contraceptive effects of CUR. Previous studies (Bansal and Bilaspuri, 2008a; MojicaVillegas et al., 2014; Tvrdá et al., 2015a) have emphasized that ferrous ascorbate may serve as a highly suitable ROS promoter for mammalian spermatozoa when these cells are separated from the primary antioxidant protection of the seminal plasma. Based on this inconsistent evidence, there is need to further examine the effects of CUR on spermatozoa. The present study was designed to explore the in vitro impact of CUR on bull spermatozoa exposed to oxidative stress induced by ferrous ascorbate.

2. Material and methods 2.1. Semen collection and experimental design Ejaculates (n = 50) were obtained from five adult Holstein Friesian breeding bulls (Slovak Biological Services, Nitra, Slovak Republic) that were on a regular collection schedule using an artificial vagina. Each sample had to meet the quality criteria given for the corresponding breed. Institutional and national guidelines for the care and use of animals were followed, and all experimental procedures were approved by the State Veterinary and Food Institute of Slovak Republic (no. 3398/11-221/3) and Ethics Committee. The in vitro treatment followed the protocol designed by Bansal and Bilaspuri (2008a,b, 2009). Each semen sample was centrifuged (800g) at 25 ◦ C for 5 min, seminal plasma was removed, the pellet was washed twice with 2.9% sodium citrate dissolved in distilled water (SC; pH 7.4; Centralchem, Bratislava, Slovak Republic), re-suspended in 2.9% SC using a ratio of 1:20 (for cell lysis) or 1:40 (for immediate experimental assessments) and divided into ten equal groups. To one group (Control 1; SC Control) 2.9% SC was added, and with another group (Control 2; FeAA Control) there was supplementation with an ROS inducer, i.e., ferrous ascorbate (FeAA) comprising 150 ␮mol/L FeSO4 (ferrous sulfate; FeSO4 ·7H2 O; Sigma-Aldrich, St. Louis, MO, USA) and 750 ␮mol/L ascorbic acid (Centralchem), diluted in 2.9% SC. The remaining eight (experimental) groups were supplemented with 5, 10, 25 or 50 ␮mol/L CUR (SigmaAldrich) in the presence or absence of FeAA. Specific CUR concentrations were selected based on results obtained from a previous CUR standardization study with bull semen (Tvrdá et al., 2015b). All suspensions were incubated at 37 ◦ C. After culture periods of 0, 2 and 6 h, spermatozoa motility variables, mitochondrial activity, ROS generation and intracellular superoxide production were assessed for each group. Moreover with the group where there was culturing for 6 h, centrifugation occurred at 800g at 25 ◦ C for 10 min, the media were removed and the resulting pellet was sonicated at 28 kHz for 30 s on ice using RIPA buffer (Sigma-Aldrich) with protease inhibitor cocktail suitable for mammalian cell and tissue extracts (Sigma-Aldrich). Subsequently the samples were centrifuged at 11,828g at 4 ◦ C for 15 min to purify the lysates from the residual cell

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debris. The resulting supernatants comprising the intracellular contents were stored at −80 ◦ C for further assessment. 2.2. Spermatozoa motility analysis Spermatozoa motion variables were assessed using the Computer-aided spermatozoa analysis (CASA) system comprising the SpermVision program (Minitube, Tiefenbach, Germany) and Olympus BX 51 phase contrast microscope (Olympus, Tokyo, Japan). Each sample was placed into the Makler Counting Chamber (depth 10 mm, 37 ◦ C; Sefi Medical Instruments, Haifa, Israel) and at least 1000 cells were evaluated for motility (MOT; percentage of motile spermatozoa; motility >5 ␮m/s; %) and progressive motility (PROG; percentage of progressive motile spermatozoa; motility >20 ␮m/s; %).

3

nitroblue tetrazolium chloride (2,20-bis(4-nitrophenyl)5,50-diphenyl-3,30-(3,30-dimethoxy-4,40-diphenylene) ditetrazolium chloride; Sigma-Aldrich) by the superoxide radical. The NBT salt was dissolved in PBS containing 1.5% DMSO (dimethyl sulfoxide, Sigma-Aldrich) to a final concentration of 1 mg/mL and added to the cells (100 ␮L per well). After a 1 h incubation (shaker, 37 ◦ C, 95% air atmosphere, 5% CO2 ), the cells were washed twice with PBS and centrifuged at 300g for 10 min. Lastly, the cells and formazan crystals were dissolved in 2 M KOH (potassium hydroxide; Centralchem) in DMSO. Optical density was determined at a wavelength of 620 nm against 570 nm as reference by a microplate ELISA reader (Anthos MultiRead 400). Data are expressed in percentage of the SC Control (Control 1) set to 100% (Tvrdá et al., 2015a). 2.6. Assessment of the antioxidant profile

2.3. ROS generation ROS production in each group was assessed by the chemiluminescence assay using luminol (5-amino-2, 3dihydro-1, 4-phthalazinedione; Sigma-Aldrich) as the probe (Kashou et al., 2013). The test samples consisted of luminol (10 ␮L, 5 mM) and 400 ␮L of control or experimental sample. Negative controls were prepared by replacing the spermatozoa suspension with 400 ␮L of each culture medium. Positive controls included 400 ␮L of each medium, 10 ␮L luminol and 50 ␮L hydrogen peroxide (30%; 8.8 M; Sigma-Aldrich). Chemiluminescence was measured on 48-well plates in 15 cycles of 1 min using the Glomax Multi+ Combined Spectro-Fluoro Luminometer (Promega Corporation, Madison, WI, USA). The results are expressed as relative light units (RLU)/s/106 spermatozoa cells. 2.4. Mitochondrial activity (MTT test) Spermatozoa mitochondrial activity was evaluated using the colorimetric metabolic activity (MTT) test, which is based on the conversion of a yellow tetrazolium salt (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT) to blue formazan particles by mitochondrial succinate dehydrogenase of intact mitochondria within living cells. The tetrazolium salt (Sigma-Aldrich) was dissolved in PBS (Dulbecco’s Phosphate Buffer Saline without calcium chloride and magnesium chloride; Sigma-Aldrich) at 5 mg/mL. Solution (10 ␮L) was added to each cell suspension. After a 2 h of incubation (shaker, 37 ◦ C, 95% air atmosphere, 5% CO2 ), the formazan crystals were dissolved in 80 ␮L of acidified (0.08 M HCl; Centralchem) isopropanol (Centralchem). Optical density was determined at a wavelength of 570 nm against 620 nm as reference using a microplate ELISA reader (Anthos MultiRead 400, Austria). Data are expressed as percentage of the SC Control (Control 1) set to 100% (Tvrdá et al., 2015a). 2.5. Superoxide production (NBT test) The nitroblue-tetrazolium (NBT) test was used to quantify the intracellular formation of the superoxide radical, by assessing blue NBT formazan deposits, generated by the reduction of the membrane permeable, yellow-colored,

Superoxide dismutase (SOD) activity was assessed using the Randox RANSOD commercial kit (Randox Laboratories, Crumlin, Great Britain) employing xanthine and xanthine oxidase (XO) to generate superoxide radicals, which will react with 2-(4-iodophenyl)-3-(4nitrophenol)-5-phenyltetrazolium chloride (I.N.T.) to form a red formazan dye. The SOD activity was subsequently measured by the inhibition degree of the reaction at 505 nm using the Genesys 10 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The results are expressed as U/mg protein. Catalase (CAT) activity was quantified according to Beers and Sizer (1952) by monitoring the decrease of hydrogen peroxide (H2 O2 ) at 240 nm. The calculation was based on the rate of H2 O2 decomposition, proportional to the reduction of the absorbance during 1 min measured with the Genesys 10 spectrophotometer. The values are expressed as U/mg protein. Glutathione peroxidase (GPx) activity was evaluated using the Randox RANSEL commercial kit (Randox Laboratories), applying the method of Paglia and Valentine (1967). GPx catalyzes the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase (Gr) and NADPH the oxidized glutathione is subsequently converted to the reduced form with a concomitant oxidation of NADPH to NADP+ . The decrease of absorbance was measured using the Genesys 10 spectrophotometer at 340 nm. The GPx activity is expressed as U/mg protein. Reduced glutathione (GSH) was determined by the Ellman method (1957). Each sample was treated with DTNB (5,50-dithiobis-2-nitrobenzoic acid; Ellman’s reagent; Sigma-Aldrich) which interacts with the thiol groups of GSH, cleaving the disulfide bond to give 2-nitro-5thiobenzoate (NTB− ) and creating the NTB2− dianion in water at alkaline pH. This ion has a yellow color and was quantified at 412 nm using the Genesys 10 spectrophotometer. The GSH concentration is expressed as mg/g protein. Lipid peroxidation (LPO) expressed through malondialdehyde (MDA) production was assessed with the help of the TBARS assay, modified for a 96-well plate and ELISA reader. Each sample was treated with 5% sodium dodecyl sulfate (SDS; Sigma-Aldrich), and subjected to 0.53%

Please cite this article in press as: Tvrdá, E., et al., Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress. Anim. Reprod. Sci. (2016), http://dx.doi.org/10.1016/j.anireprosci.2016.06.008

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ARTICLE IN PRESS 38.31 ± 2.48***, a 25.15 ± 1.79*, b 43.87 ± 1.86***, b 26.04 ± 1.97*, b 44.85 ± 1.99***, b 26.78 ± 1.84*, b 49.45 ± 2.23***, b 31.58 ± 2.21*, b 32.40 ± 2.47***, a 18.95 ± 2.32***, a 61.08 ± 2.78*, a 37.22 ± 1.83 MOT – spermatozoa motility, PROG – spermatozoa progressive motility. Mean ± Standard Error. * P<0.05; ** P<0.01; *** P<0.001. a Compared with Control 1 (SC Control). b Compared with Control 2 (FeAA Control).

61.26 ± 2.22*, a 38.57 ± 2.30 65.76 ± 2.74***, a 43.95 ± 2.84 51.74 ± 2.20 33.44 ± 2.88

65.10 ± 2.07***, a 42.60 ± 2.10

49.09 ± 2.40***, a 30.28 ± 2.09 56.07 ± 2.15 32.24 ± 2.60 57.65 ± 2.91*, b 33.51 ± 2.08 61.39 ± 2.11***, b 35.71 ± 2.54 47.23 ± 2.93***, a 29.74 ± 2.30 71.17 ± 2.42*, a 40.79 ± 2.07 71.58 ± 1.95*, a 40.54 ± 2.01 71.87 ± 2.33*, a 45.16 ± 2.51*, a 61.76 ± 2.35 35.04 ± 2.51

71.85 ± 2.64*, a 42.73 ± 2.27

82.83 ± 1.84 70.23 ± 2.10 83.44 ± 1.91 72.67 ± 2.12 83.54 ± 1.86 71.92 ± 2.02 75.73 ± 3.13***, a 71.46 ± 1.69 87.98 ± 0.99 73.87 ± 1.22 87.86 ± 0.99 75.16 ± 1.15 88.36 ± 0.97 76.01 ± 0.94 90.51 ± 0.83 77.77 ± 0.86 85.18 ± 2.06 74.19 ± 1.85

Time 0 h MOT [%] PROG [%] Time 2 h MOT [%] PROG [%] Time 6 h MOT [%] PROG [%]

10 ␮mol/L CUR 25 ␮mol/L CUR 50 ␮mol/L CUR Ctrl 2 (FeAA Ctrl)

Groups treated with FeAA

5 ␮mol/L CUR 10 ␮mol/L CUR 25 ␮mol/L CUR 50 ␮mol/L CUR

There were lesser (P < 0.001) spermatozoa motion characteristics in the Control 2 group as a consequence of FeAA supplementation (Table 1). For the groups in which spermatozoa were not exposed to FeAA, there were differences when comparing the groups supplemented with 25 and 50 ␮mol/L CUR with the Control 1 group (P < 0.05 at 2 h and P < 0.001 at 6 h). With all CUR supplementations, regardless of concentration, there was an abrupt decrease of spermatozoa motility as indicated by all motility variables assessed with the addition of FeAA, particularly at 6 h (P < 0.001 of culture for the PROG MOT variable; P < 0.05). Consistent with these previous findings, there was a decrease of mitochondrial activity observed after FeAA administration to the spermatozoa suspensions, with differences at all assessment times (P < 0.001; Fig. 1). All CUR

Ctrl 1 (SC Ctrl)

3. Results

Groups untreated with FeAA

a) SC Control (Control 1) group was compared to the FeAA Control (Control 2) group; b) Experimental groups not subjected FeAA treatment were compared to the SC Control exclusively (Control 1); and c) Experimental groups subjected to FeAA treatment were compared to the SC Control (Control 1) as well as to the FeAA Control (Control 2).

Groups

Statistical analyses were conducted using the GraphPad Prism program (version 3.02 for Windows; GraphPad Software, La Jolla, CA, USA, www.graphpad.com). Descriptive statistical characteristics (mean, standard error) were initially evaluated. One-way ANOVA was used for specific statistical evaluations. Dunnett’s test was applied as a follow-up test to ANOVA, based on a comparison of every mean to a control mean, and computing a confidence interval for the difference between the two means. The level of significance was set at *** (P < 0.001); ** (P < 0.01); * (P < 0.05). The comparative analysis was performed as follows, The

Table 1 Spermatozoa motility variables affected by four doses of curcumin (CUR), untreated compared with treated with ferrous ascorbate (FeAA).

2.7. Statistical analysis

5 ␮mol/L CUR

tiobarbituric acid (TBA; Sigma-Aldrich) dissolved in 20% acetic acid adjusted with NaOH (Centralchem) to pH 3.5, and subsequently boiled at 90–100 ◦ C for 1 h. Following boiling, the samples were placed on ice for 10 min and centrifuged at 1750g for 10 min. Supernatant was used to measure the end-product resulting from the reaction of MDA and TBA under high temperature and acidic conditions at 530–540 nm using the Multiskan FC microplate photometer (Thermo Fisher Scientific Inc.) (Tvrda et al., 2013). The MDA concentration is expressed as ␮mol/g protein. Protein concentration was quantified using the DiaSys Total Protein (DiaSys, Holzheim, Germany) commercial kit and the semi-automated clinical chemistry photometric analyzer Microlab 300 (Merck, Darmstadt, Germany). The measurement is based on the Biuret method, according to which copper sulfate reacts with proteins to form a violet blue color complex in alkaline solution, and the intensity of the color is directly proportional to the protein concentration when measured at 540 nm.

82.69 ± 2.52 70.49 ± 2.47

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Fig. 1. Mitochondrial activity of bull spermatozoa affected by four doses of curcumin (CUR), untreated or co-treated with ferrous ascorbate (FeAA). Each bar represents mean (±SEM) optical density as the percentage of the Control 1 (SC control), which was set to 100% and the data are expressed as a percentage of the Control 1 value. The data were obtained from five independent experiments. The level of significance was set at * P < 0.05; ** P < 0.01; *** P < 0.001. 1 Compared with Control 1; 2 Compared with Control 2.

Please cite this article in press as: Tvrdá, E., et al., Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress. Anim. Reprod. Sci. (2016), http://dx.doi.org/10.1016/j.anireprosci.2016.06.008

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ARTICLE IN PRESS 22.22 ± 1.06***, a 17.89 ± 0.98*, b ,**, a 15.12 ± 0.89***, b 13.01 ± 0.90***, b 25.05 ± 0.98***, a 9.51 ± 0.23 Mean ± Standard Error; P<0.05; P<0.01; P<0.001. a Compared with Control 1 (SC Control). b Compared with Control 2 (FeAA Control).

8.88 ± 0.36 6.92 ± 0.64**, a

*** **

6.02 ± 0.20**, a

*

8.22 ± 0.48**, a 8.01 ± 0.55*, a 5.99 ± 0.39***, b 5.18 ± 0.22***, b 9.99 ± 0.85***, a 4.22 ± 0.23 2.44 ± 0.30*, a 2.22 ± 0.25*, a

3.87 ± 0.15

3.77 ± 0.28*, a 3.44 ± 0.30*, a 2.25 ± 0.21**, b 2.05 ± 0.50**, b 4.55 ± 0.50**, a 1.66 ± 0.22 1.44 ± 0.18 1.29 ± 0.19 1.22 ± 0.20

10 ␮mol/L CUR 25 ␮mol/L CUR 50 ␮mol/L CUR Ctrl 2(FeAA Ctrl) 5 ␮mol/L CUR 10 ␮mol/L CUR 25 ␮mol/L CUR 50 ␮mol/L CUR Ctrl 1 (SC Ctrl)

Time 0 h 1.82 ± 0.21 Time 2 h 4.55 ± 0.36 Time 6 h 11.05 ± 0.43

Groups treated with FeAA Groups untreated with FeAA

Promoter systems integrating ferrous and ascorbate ions are highly suitable to induce OS in mammalian spermatozoa through the Fenton and Haber-Weiss reaction (Aitken et al., 1989; Martínez-Pastor et al., 2009). Ferrous ascorbate is a molecule that is a product of the chemistry and redox properties of iron, which as a transition metal may be able to cause oxidative depletion of important biomacromolecules including lipids, proteins and DNA (Aitken et al., 1989; Bansal and Bilaspuri, 2008b, 2009; Martínez-Pastor et al., 2009). Such alterations may in turn be associated with a disruption of the mitochondrial metabolism, ATP depletion and a subsequent decrease of spermatozoa motility (Kalla and Vasudev, 1981). Interestingly, Baumber et al. (2000) reported that although greater ROS concentrations were associated with a decreased

Groups

4. Discussion

Table 2 Reactive oxygen species (ROS) production by bull spermatozoa [RLU/s/106 sperm] affected by four doses of curcumin (CUR), untreated compared with treated with ferrous ascorbate (FeAA).

supplementations, regardless of concentration, resulted in enhanced spermatozoa viability when there was supplementation of FeAA to untreated samples (P < 0.001 with respect to 25–50 ␮mol/L CUR; P < 0.05 at 2 h; P < 0.01 in case of 5–10 ␮mol/L CUR at 6 h). Supplementation with CUR, regardles of concentration, resulted in, at least partially, prevention of the decrease of the mitochondrial activity in spermatozoa subjected to FeAA treatment, particularly with 6 h of culture (P < 0.001; Fig. 1). The decrease in bull spermatozoa motility and mitochondrial activity in the Control 2 group was accompanied by an increase of ROS and superoxide production (Table 2; Fig. 2). Compared to the FeAA-untreated Control 1 group, there was significant free radical overgeneration (P < 0.001) within a very short period after FeAA was added to the suspension (time 0 h). The addition of 25 and 50 ␮mol/L CUR was decreased the ROS as well as superoxide generation in comparison with the Control 1 group (P < 0.001 with respect to ROS at 6 h of culture). The presence of CUR in the FeAA-treated groups did not fully reverse the free radical generation, nevertheless 25 and 50 ␮mol/L CUR was very effective in decreasing both ROS (P < 0.01 at 0 h; P < 0.001 2 and 6 h of culture) and superoxide concentrations when compared to the Control 2 group (Table 2; Fig. 2). Assessment of the antioxidant profile revealed that FeAA supplementation resulted in a significant decrease of the SOD, CAT and GPx activities (P < 0.05 with respect to SOD; P < 0.001 in relation to CAT and GPx) as well as a decreased GSH concentration (P < 0.05; Table 3). There was, however, an increase of the MDA concentration (P < 0.001) after FeAA administration (Table 3). While supplementation with the 50 ␮mol/L CUR amount increased SOD activity in the FeAA-untreated samples (P < 0.05), administration of 10–50 ␮mol/L CUR increased CAT and GPx activities following FeAA treatment (P < 0.05 with 10 ␮mol/L CUR; P < 0.01 with 25 and 50 ␮mol/L CUR). Administration of 50 ␮mol/L CUR increased (P < 0.05) the GSH content when compared to the Control 2 group (Table 3). Although there were no differences in the MDA concentration among the FeAA-untreated groups, a decrease of this LPO byproduct was observed following the supplementation of all amounts of CUR to the experimental groups treated with FeAA (P < 0.001; Table 3).

5 ␮mol/L CUR

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Fig. 2. Intracellular superoxide production of bull spermatozoa affected by four doses of curcumin (CUR), untreated or co-treated with ferrous ascorbate (FeAA). Each bar represents mean (±SEM) optical density as the percentage of the Control 1 (SC control), which was set to 100% and the data are expressed as a percentage of the Control 1 value. The data were obtained from five independent experiments. The level of significance was set at * P < 0.05; ** P < 0.01; *** P < 0.001. 1 Compared with Control 1; 2 Compared with Control 2.

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2.20 ± 0.23 2.18 ± 0.28

SOD – superoxide dismutase activity, CAT – catalase activity, GPx – glutathione peroxidase activity, GSH – concentration of reduced glutathione, MDA – malondialdehyde concentration. Mean ± Standard Error. * P < 0.05; ** P < 0.01; *** P < 0.001. a Compared with Control 1 (SC Control). b Compared with Control 2 (FeAA Control).

3.51 ± 0.49***, b 3.26 ± 0.31***, b 2.67 ± 0.30***, b 2.40 ± 0.31***, b

7.58 ± 1.15 7.68 ± 1.22*, b 4.74 ± 0.68*, a 8.23 ± 1.33 8.99 ± 1.30

8.09 ± 1.04

0.035 ± 0.010 0.092 ± 0.009

0.089 ± 0.011

7.06 ± 0.21***, a

7.30 ± 1.33

0.059 ± 0.010 0.063 ± 0.013 0.065 ± 0.008

**, b ***, a

0.100 ± 0.012

2.21 ± 0.28

0.044 ± 0.010

5.89 ± 1.16

*, b **, b

6.05 ± 0.81*, b 6.73 ± 1.30**, b 6.95 ± 0.46**, b 2.65 ± 0.52***, a 7.90 ± 1.20 8.26 ± 1.14

7.05 ± 1.34

0.101 ± 0.01 0.117 ± 0.022 0.118 ± 0.02 0.122 ± 0.01 0.082 ± 0.02*, a 0.146 ± 0.02 0.161 ± 0.02 0.179 ± 0.02

5 ␮mol/L CUR 10 ␮mol/L CUR 25 ␮mol/L CUR 50 ␮mol/L CUR Ctrl 2(FeAA Ctrl) 5 ␮mol/L CUR 10 ␮mol/L CUR 25 ␮mol/L CUR 50 ␮mol/L CUR Ctrl 1 (SC Ctrl)

SOD [U/mg prot] 0.136 ± 0.02 0.202 ± 0.03*, a CAT [U/mg prot] 8.70 ± 1.13 7.49 ± 0.82 GPx [U/mg prot] 0.082 ± 0.009 0.105 ± 0.015 GSH [mg/g prot] 9.95 ± 1.22 7.97 ± 1.23 MDA [␮mol/g prot] 2.04 ± 0.25 2.39 ± 0.27

Groups treated with FeAA Groups untreated with FeAA

Groups

Table 3 Markers of oxidative balance in bull spermatozoa affected by four doses of curcumin (CUR), untreated compared with treated with ferrous ascorbate (FeAA).

6.89 ± 1.22

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spermatozoa motility in stallions, no changes were observed in the mitochondrial activity. To assess if FeAA had any impact on the intracellular ROS-scavenging capacity of spermatozoa, the present study was conducted to evaluate the activity of antioxidant enzymes, believed to be the first line of defense against oxidative insults (Agarwal et al., 2003). The SOD, CAT as well as GPx activities were less following FeAA administration, revealing a serious imbalance and inability of enzymatic antioxidants to readily detoxify the superoxide anion and peroxides generated within the cell, and to protect the cellular structures against toxic hydroxyl radicals. A similar depletion of antioxidant enzymes was previously reported by Murugan et al. (2002), Mojica-Villegas et al. (2014) and Tvrdá et al. (2015a). Unlike enzymatic antioxidantss, GSH has been reported to decrease in concentration as a response to OS in some studies (Sahoo et al., 2008; Tvrdá et al., 2015a) while in other studies there has been an increase in the GSH amount following OS induction (Zamoner et al., 2007) in male reproductive structures. The decreased GSH concentration resulting from FeAA supplementation in the present study may be a consequence of possible oxidative insults to the sulphydryl structures of GSH, responsible for the actual ROS trapping. Lipid peroxidation is known to be the primary mechanism of oxidative damage to spermatozoa (Aitken et al., 1989). During this process, male reproductive cells can have altered membrane integrity, accompanied by increased concentrations of lipid hydroperoxides, alkoxyl and/or peroxyl radicals. Escalation of LPO chain reactions may ultimately result in the production of cytotoxic aldehydes including MDA (Aitken et al., 1989; Agarwal et al., 2003), consistent with data from the present study as well as with studies where FeAA was used in studies with other mammalian spermatozoa (Baumber et al., 2000; Murugan et al., 2002; Bansal and Bilaspuri, 2008b, 2009; MojicaVillegas et al., 2014). Administration of nutritional antioxidants as a strategy to prevent or manage health conditions associated with oxidative stress has gained immense interest. Substances isolated from herbal plants such as turmeric are known to exert beneficial effects by scavenging free radicals and modulating the intricate antioxidant defense system (ElWakf et al., 2011). Data from the present study provide evidence that CUR exhibits protective effects on the spermatozoa structural integrity and functional activity under physiological in vitro conditions, as well as when there is increased oxidative damage to the germ cells. Previously collected data assessing the impact of CUR on male reproductive function are inconsistent. Salahshoor et al. (2012) reported that CUR supplementation resulted in a dose-dependent increase in important reproductive indices including spermatozoa count and motility, testis weight and testosterone concentrations in rats. Inconsitent with these findings, Naz (2011) found that the incubation of human or murine spermatozoa with CUR led to a concentration-dependent decrease in the spermatozoa forward motility, capacitation and acrosome reaction. At greater CUR concentrations, a complete inhibition of

Please cite this article in press as: Tvrdá, E., et al., Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress. Anim. Reprod. Sci. (2016), http://dx.doi.org/10.1016/j.anireprosci.2016.06.008

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spermatozoa motility and/or function was observed within 5–15 min of treatment initiation. Studies on the impact of CUR on the spermatozoa viability have indicated its involvement in the energy metabolism of male reproductive cells. Reddy and Aggarwal (1994) hypothesized that CUR in micromolar concentrations can inhibit protein kinase C (PKC), the presence, activity and localization of which has been well documented in human spermatozoa and is believed to have a role in modulating spermatozoa flagellar movement (Rotem et al., 1990a,b). The CUR-induced PKC inhibition may, therefore, be at least partially responsible for its possible spermatozoa-immobilizing activities (Rithaporn et al., 2003). The hypothesis for CUR effects on spermatozoa that has been developed from these previous studies is, however, inconsistent with findings in the present study that suggest the stimulating effects of CUR on spermatozoa motility are related to the mitochondrial activity. Furthermore, experimental outcomes of the present study are consistnet with a variety of in vivo toxicological studies where spermatozoa motility and/or activity was protected or even restored following tescticular toxicity and genotoxicity induced by cyclophosphamide (Dev et al., 2013) or aflatoxin (Mathuria and Verma, 2008). The previously collected data from which comparable results were obtained to those of the present study come from El-Wakf et al. (2011) who reported that the presence of CUR when there was a sodium nitrate-related overload increased the epididymal spermatozoa number, weight of reproductive organs, and restored the concentrations of male sex hormones and protein content in serum and reproductive tissues. The principal mechanism of action of CUR has been suggested to be related to its ability to competitively inhibit the cytochrome P450 isoenzymes responsible for the metabolic activation of a variety of carcinogens (Mathuria and Verma, 2008). Moreover, there have been several reports of this protective action to anti-inflammatory and anti-infectious activities of CUR (Srinivas et al., 1992). Such complex effects, taken together, may lead to an improved fertility and testicular performance through controlling both LPO and nitric oxide (NO) production, which may affect the resulting spermatozoa activity (Romeo et al., 2003). El-Wakf et al. (2011) speculated that the protective effect of CUR on testicular structure and function may be explained by this phenol preventing cellular damage occurring as a result of oxidative stress in the spermatogenic and Leydig cells (Aly et al., 2010). The supplementation of CUR prevented the chromium-induced decrease in weight of accessory sex organs due to normal serum testosterone concentration (Chandra et al., 2007). Moreover, CUR administration to male Wistar rats ameliorated lindane-induced reproductive toxicity in pretreatment, post treatment as well as co-treatment experimental groups (Sharma and Singh, 2010). Results for spermatozoa motility variables assessed using the CASA system in the present study are consistent with previous findings by Bucak et al. (2008, 2010) demonstrating a notable improvement in the motion characteristics of post-thawed ram spermatozoa supplemented

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with CUR supplementation. Inconsistent with this previous study based on CUR supplementation to the cryopreservation medium of bull semen a subsequent study by this group found there were not significant differences in spermatozoa motility variables with CUR supplementation (Bucak et al., 2012). The first report on the useful effects of CUR on the quality and antioxidant capacity of semen led to its use as an antioxidant supplement and cryoprotectant in spermatozoa cultures (Bucak et al., 2008). One of the possible ameliorative mechanisms of CUR on the spermatozoa motion and mitochondrial activity, hence the viability, is to scavenging of free radicals and thereby antioxidant effects that result from use of CUR. Another reason for the enhancement of spermatozoa motility observed in the present study may be the increasing activities of the internal antioxidant system, consistent with previous findings where there were changes to the total antioxidant activity that were recorded by Soleimanzadeh and Saberivand (2013) in semen supplemented with CUR. Supporting evidence for this hypothesis also comes from earlier studies of the ameliorative effects of CUR on the activity and antioxidant status of reproductive cells and tissues in metronidazole-treated mice (Karbalay-Doust and Noorafshan, 2011). The antioxidant capacity of spermatozoa is closely related to male reproductive performance, as an appropriate antioxidant balance provides a favorable environment for spermatozoa function. The decreased antioxidant capacity may be an important cause of male infertility (Agarwal et al., 2003). In vitro storage and cryopreservation of semen may cause severe alterations to the antioxidant defense capacity of spermatozoa. Based on results of the present study, the addition of CUR to isolated bull spermatozoa preserved the cellular antioxidant capacity and these findings are consistent with the previous results of Soleimanzadeh and Saberivand (2013). Furthermore, there was a significant preservation of the DNA integrity and viability in rat spermatozoa following CUR treatment that was most likely due to the stabilization of the total antioxidant equilibrium as concluded by Soleimanzadeh and Saberivand (2013). The results concerning the effect of CUR on the GSH concentration are consistent with the previous findings of Piper et al. (1998) where it was reported that the increased concentrations of GSH may be related to a decreased spermatozoa utilization of GSH as a primary consequence of the antioxidant properies of CUR or due to the increased activity of glutamyl cysteine synthase, modulated by CUR. Furthermore, Bucak et al. (2012) concluded that the addition of CUR was able to the maintain the glutathione concentrations following the freezing and thawing cycles of ram semen. The improvement of the enzymatic activity of CAT and GPx is, however, is not consistent with the findings of Salama and El-Bahr (2007) where it was observed there was a CUR-related decrease of the activity of both enzymes in cadmium-supplemented rat testicles, as a response to the decreased liberation and creation of free radicals, such as H2 O2 .

Please cite this article in press as: Tvrdá, E., et al., Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress. Anim. Reprod. Sci. (2016), http://dx.doi.org/10.1016/j.anireprosci.2016.06.008

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Inconsistent with the present results, CUR was found to exert its protective effect by modulating lipid peroxidation in a previous study (Kalpana and Menon, 2004). More specifically, CUR decreased the ROS concentrations as a result of its free radical scavenging activities and induction of detoxification enzymes (Manikandan et al., 2004) – an observation consistent with the findings in the present study. Furthermore, Rahman (2007) suggested that CUR has the ability to reverse oxidative damage, probably through its capacity to quickly and efficiently scavenge lipid peroxyl radicals before these can reach the membrane lipids. The outcomes from the present experiments suggest that CUR supplementation was able to maintain the spermatozoa membrane integrity accompanied by a significant decrease of MDA. The CUR may prevent peroxidative changes in the spermatozoa membrane, thus enhancing motility and decreasing structural or functional alterations to these cells. Such conclusions are in agreement with Ishihara et al. (2000) as well as Salama and El-Bahr (2007) where it was suggested that CUR provides protection against LPO in male reproductive structures. Data from the TBARS analysis of the present study are, however, inconsistant with findings from studies performed on ram or goat spermatozoa where there were no significant changes in the MDA concentrations with CUR supplementation. It is, therefore, speculated that CUR could protect the membrane integrity of spermatozoa against oxidative damage, even without inhibition of MDA production. It is concluded that CUR prevents the decrease of bull spermatozoa viability, functional activity and antioxidant capacity as a consequence of FeAA-induced oxidative stress. The concentration range of 25–50 ␮mol/L CUR in media was particularly effective in protecting bull spermatozoa from the damage caused by free radical generation through the prevention of lipid peroxidation, and promotion of spermatozoa viability as a consequence of protective effects of CUR on cell motility and mitochondrial metabolic activity. Furrthermore, CUR supplementation may be a suitable strategy to preserve important spermatozoa structures and functional manifestations susceptible to oxidative insults.

Conflicts of interest We declare that we have no proprietary, financial, professional or personal interest of any nature in any product, service and/or company that could have any influence on the position presented in this manuscript.

Acknowledgements This research was supported by the Research Center AgroBioTech built in accordance with the project Building Research Centre “AgroBioTech” ITMS 26220220180 and by the VEGA Project of the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and of the Slovak Academy of Sciences no. 1/0857/14.

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