Antioxidative effects of melatonin on kinetics, microscopic and oxidative parameters of cryopreserved bull spermatozoa

Animal Reproduction Science 139 (2013) 25–30

Contents lists available at SciVerse ScienceDirect

Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Antioxidative effects of melatonin on kinetics, microscopic and oxidative parameters of cryopreserved bull spermatozoa Iraj Ashrafi a,∗ , Hamid Kohram a , Farhad Farrokhi Ardabili b a b

Department of Animal Science, Faculty College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Department of Animal Science, Faculty of Agriculture, University of Urmia, Urmia, Iran

a r t i c l e

i n f o

Article history: Received 30 July 2012 Received in revised form 28 March 2013 Accepted 29 March 2013 Available online 13 April 2013 Keywords: Melatonin Antioxidant Bull semen Cryopreservation Kinetic parameters Oxidative parameters

a b s t r a c t Reactive oxygen species generated during the freeze–thawing process may reduce sperm quality. This study evaluates the effects of melatonin supplementation as an antioxidant in the semen extender on post-thaw parameters of bull spermatozoa. Melatonin was added to the citrate–egg yolk extender to yield six different final concentrations: 0, 0.1, 1, 2, 3 and 4 mM. Ejaculates were collected from six proven Holstein bulls. Semen was diluted in the extender packaged in straws, which was frozen with liquid nitrogen. The semen extender supplemented with various doses of melatonin increased (p < 0.05) total motility, progressive motility, linearity, sperm track straightness, lateral head displacement, viability, integrity of the sperm membrane and total normal morphology of sperm after the freeze–thawing process. The most effective concentration of melatonin in microscopic evaluations of the bull sperm freezing extender was 2 mM. The highest (p < 0.05) value of total antioxidant capacity (48.9 ± 2.7) and the lowest value of lipid peroxidation (2.7 ± 0.8) were achieved by inclusion of 3 mM concentration of melatonin in the semen extender and the highest activity of catalase (0.7 ± 0.1) was obtained by 2 mM melatonin. Four millimolar concentration of melatonin were reduced (p < 0.05) the progressive motility and straight linear velocity. In conclusion, supplementation of 2 or 3 mM concentration of melatonin in the semen extender improved the quality of post-thawed semen, which may associate with a reduction in lipid peroxidation as well as an increase in the total antioxidant capacity and antioxidant enzyme activity. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The viability, motility and membrane integrity of mammalian spermatozoa decrease during the cryopreservation process (Gillan and Maxwell, 1999). Cold shock during the freeze–thawing process could damage mitochondria (Pena et al., 2009) as well as plasma and acrosome membranes of spermatozoa (Meyers, 2005). It has been shown (Alvarez and Storey, 1992; Agarwal and Said, 2005) that spermatozoa that undergo the freeze–thawing process

∗ Corresponding author. Tel.: +98 9144190815; fax: +98 3262246752. E-mail addresses: i.ashrafi@ut.ac.ir, dorna [email protected] (I. Ashrafi). 0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.03.016

produce high concentrations of reactive oxygen species (ROS). Free radicals at physiological levels have beneficial effects on intracellular signalling which is involved in the regular processes of cell proliferation, differentiation and migration (Piantadosi, 2008). Physiological concentrations of ROS in terms of hydrogen peroxide (H2 O2 ), superoxide and hydroxyl radicals and likewise reactive nitrogen species in terms of nitric oxide (NO) and peroxynitrite anion (ONOO− ) may have critical roles in sperm hyperactivation, capacitation, acrosome reaction (Aitken, 1995), sperm–oocyte interaction and implantation during early embryo development (Baker and Aitken, 2004). However, excessive production of ROS may produce high levels of peroxides and free radicals that affect lipid peroxidation (LPO) and the spermatozoa membrane. The high level

26

I. Ashrafi et al. / Animal Reproduction Science 139 (2013) 25–30

of ROS may decline sperm motility (Alvarez and Storey, 1982), inactivate glycolytic enzymes, damage the acrosomal membranes (Alvarez and Storey, 1984) and oxidise DNA, which would render the sperm cell unable to fertilise the oocyte (Gil-Guzman et al., 2001). Addition of an antioxidant in the semen extender may reduce ROS production and prevent oxidative stress (Donoghue and Donoghue, 1997). Antioxidant molecules could decrease the impact of oxidative stress and therefore improve sperm quality following the freeze–thawing process (Berra and Rizzo, 2009). Melatonin (N-acetyl-5-methoxy tryptamine) is secreted by the pineal gland in the brain (Awad et al., 2006) and participates in a number of physiological functions including the control of seasonal reproduction and also affects the immune system and circadian rhythms. Many reports have substantiated the protective effects of melatonin as an antioxidant because of its high efficacy as a hydroxyl radical (• OH) scavenger (Reiter, 1998; Lee et al., 2002; El-Sokkary et al., 2003). Furthermore, melatonin has the ability to detoxify different kinds of reactive oxygen and nitrogen species involving singlet oxygen (O2 ), NO and the ONOO− anion and also its metabolite peroxynitrous acid and hydrogen peroxide (Tan et al., 2002). Melatonin also stimulates the activities of enzymes involved in metabolising ROS and preserves cell membrane fluidity. No previous study has reported the effects of melatonin in a semen extender against cryodamage to bull semen. The present study was therefore conducted to determine the effect of melatonin supplementation in the semen extender on sperm motility parameters, viability, plasma membrane integrity, morphology, total antioxidant capacity (TAC), LPO as well as antioxidant activities in terms of glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) in post-thaw bull semen. 2. Materials and methods 2.1. Semen source and preparation This trial was performed at Animal Breeding Center located in Tabriz city, northwest of Iran. Ejaculates were collected from six Holstein bulls, 3–6 years old, regularly used for breeding purpose based on their fertility estimation through in vitro and in vivo fertility tests. Semen samples were collected twice a week by an artificial vagina (45 ◦ C). The ejaculates were transferred immediately to the laboratory and submerged in a water bath (34 ◦ C), until semen evaluation. The volume of ejaculates was estimated in a conical tube graduated at 0.1-ml intervals. The sperm concentration was determined by means of an Accucell photometer (IMV Technologies, L’Aigle, France). The ejaculates were evaluated and accepted into the study if the following criteria were met: volume between 5 and 12 ml; sperm concentration of ≥1 × 109 sperm ml−1 ; percentage of motile sperms ≥70%; and ≤10% abnormal sperm. The ejaculates were then mixed in a pool, balancing the sperm contribution of each male to remove individual differences in bulls (Gil et al., 2003). The citrate–egg yolk extender (sodium citrate dihydrate (2.9 g dl−1 ); penicillin (1000 IU ml−1 ); streptomycin (1000 ␮g ml−1 ); 20% egg yolk; 7% glycerol; and

double-distilled water to make the volume up to 100 ml) was used for all the ejaculates. Melatonin (M-5250; molecular weight 232.3, Sigma–Aldrich, St. Louis, MO, USA.) was dissolved in dimethylsulphoxide (DMSO) and phosphate-buffered saline (PBS) (Ishizuka et al., 2000) and added to the extender to yield six different final concentrations: 0, 0.1, 1, 2, 3 and 4 mM. Indeed, one extender with six different concentrations of melatonin was examined. The final concentration of DMSO in all samples was 0.1%. A control group containing the same DMSO concentration was included. Each pooled ejaculate was split into six equal aliquots and diluted in the semen extender supplemented with melatonin, for a final concentration of 1 × 108 spermatozoa per ml. Then, the semen was cooled slowly at 5 ◦ C. The sample was packaged in 0.5-ml straws. The straws were sealed and the semen was frozen with liquid nitrogen vapour. The straws were cooled at approximately −15 ◦ C min−1 from +5 to −150 ◦ C. Then straws were transferred to a liquid nitrogen tank and stored. Frozen straws were thawed individually at 37 ◦ C for 30 s in a water bath for microscopic evaluation (Ashrafi et al., 2011). 2.2. Semen evaluation after the freeze–thawing process 2.2.1. Viability, motility and morphology parameters The viability of the spermatozoa in the samples was assessed by means of the nigrosin–eosin stain method. The final composition of the stain was: eosin-Y 1.67 g, nigrosin 10 g and sodium citrate 2.9 g, dissolved in 100 ml of distilled water. Sperm suspension smears were prepared by mixing a drop of sperm sample with two drops of stain on a warm slide and spreading the stain with a second slide. Viability was assessed by counting 400 sperm cells in a microscope (Olympus CX21) at 1000× magnification, using immersion oil. Sperm displaying partial or complete purple staining were considered non-viable; only sperm showing strict exclusion of stain were counted as viable (Balestri et al., 2007). Sperm motility was assessed after thawing the samples, for about 5 min at 37 ◦ C. A computer-assisted sperm motility analysis (CASA, VideoTesT-Sperm 2.1, St. Petersburg, Russia) was used to analyse sperm motion characteristics. Semen was diluted (5 ␮l semen + 45 ␮l extender) in a citrate-based extender (without egg yolk) and evaluated immediately after dilution. A 4-␮l sample of diluted semen was put onto a pre-warmed microscope slide and covered by a cover slip. Sperm motility characteristics were determined using a phase-contrast microscope (Labomed LX400; Labomed Inc., Culver City, CA, USA) with a 10× objective at 37 ◦ C. The following motility values were recorded: total motility (TM, %), progressive motility (PM, %), straight linear velocity (VSL, ␮m/s), curvilinear velocity (VCL, ␮m/s), average path velocity (VAP, ␮m/s), beat crossfrequency (BCF, Hz), lateral head displacement (ALH, ␮m), sperm track straightness (STR, %) and linearity (LIN, %). For each evaluation, five microscopic fields were analysed to include at least 400 cells. For the evaluation of sperm abnormalities, at least three drops of each sample were added to Eppendorf tubes containing 1 ml of Hancock solution (62.5 ml formalin (37%),

I. Ashrafi et al. / Animal Reproduction Science 139 (2013) 25–30

27

Table 1 Mean (±S.E.M.) percentages of sperm motion parameters, viability, HOST, total and acrosome abnormalities in freeze–thawing bull spermatozoa with different concentrations of melatonin. Parameters

Melatonin concentrations Control

TM (%) PM (%) LIN (%) STR (%) ALH (␮m VSL (␮m/s) BCF (Hz) VCL (␮m/s) VAP (␮m/s) Viability (%) HOST (%) Total abnormalities (%) Acrosome abnormality (%)

43.0 20.9 42.6 89.1 1.4 51.5 8.7 106.7 53.9 59.6 50.3 18.3 2.9

± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 mM 2.4d 2.4c 3.4bc 3.3b 0.1b 6.9a 0.1 8.5 4.4 2.3b 2.4c 2.1a 0.6ab

51.7 26.7 41.5 90.2 1.4 50.5 8.7 106.9 53.8 68.3 58.4 12.7 3.2

± ± ± ± ± ± ± ± ± ± ± ± ±

1 mM 2.0bc 2.2b 3.3c 4.8ab 0.1b 7.9ab 0.1 6.2 6.6 2.3a 2.5b 1.7c 0.9a

60.8 31.4 46.5 91.1 1.5 47.1 8.8 106.2 50.8 71.1 66.4 8.1 2.7

2 mM ± ± ± ± ± ± ± ± ± ± ± ± ±

4.0a 3.0a 3.7b 6.1ab 0.2ab 4.3ab 0.1 10.2 5.3 3.5a 1.8a 1.3d 0.4ab

62.5 30.1 51.6 95.0 1.7 49.4 8.8 109.2 50.8 69.8 68.3 6.9 2.1

3 mM ± ± ± ± ± ± ± ± ± ± ± ± ±

4.2a 2.2a 3.9a 2.5a 0.2a 3.9ab 0.2 6.3 4.4 1.1a 1.4a 1.3d 0.4b

55.4 23.9 45.5 92.4 1.7 49.6 8.8 115.5 51.4 61.8 67.8 13.2 2.5

4 mM ± ± ± ± ± ± ± ± ± ± ± ± ±

3.4b 2.7bc 2.2bc 2.1ab 0.1a 6.9ab 0.0 4.1 4.1 3.0b 1.1a 1.1c 0.4ab

47.5 15.1 45.9 92.6 1.4 41.3 8.9 99.7 46.9 61.5 57.0 15.5 3.2

± ± ± ± ± ± ± ± ± ± ± ± ±

2.8c 0.5d 2.3bc 2.7ab 0.1b 7.0b 0.0 11.0 7.5 2.5b 2.5b 1.0b 0.6a

TM: total motility (%); PM: progressive motility (%); LIN: linearity (%); STR: sperm track straightness (%); ALH: lateral head displacement (␮m); VSL: straight linear velocity (␮m/s); BCF: beat cross frequency (Hz); VCL: curvilinear velocity (␮m/s); VAP: average path velocity (␮m/s); HOST: hypo-osmotic swelling test (%). Different superscripts within rows are significantly differ, p < 0.05.

150 ml of sodium saline solution, 150 ml of buffer solution and 500 ml of double-distilled water) (Schafer and Holzmann, 2000). One drop of this mixture was put on a slide and overlaid with a cover slip. The percentages of acrosome and total abnormalities were determined by counting a total of 200 spermatozoa under phase-contrast microscopy (1000× magnification, oil immersion). 2.2.2. Assessment of membrane integrity The hypo-osmotic swelling test (HOST) was used to evaluate the functional integrity of the sperm membrane, based on curled and swollen tails. This was performed by incubating 30 ml of semen with 300 ml of a 100 mOsM hypo-osmotic solution (9 g fructose + 4.9 g sodium citrate per litre of distilled water) at 37 ◦ C for 60 min. After incubation, 0.2 ml of the mixture was spread with a cover slip on a warm slide. A total of 400 sperm were examined with 400× magnification with bright-field microscopy. Sperm with swollen or coiled tails were recorded (Buckett et al., 1997). 2.2.3. Biochemical assay The samples were thawed before the biochemical assays. An aliquot (500 ␮l) of semen from each sample was centrifuged at 800 × g for 10 min; sperm pellets were separated and washed by resuspending in PBS and recentrifuging (thrice). After the final centrifugation, 1 ml of deionised water was added to the spermatozoa and they were snap-frozen and stored at −70 ◦ C until further analysis (Roca et al., 2005). The ferric reducing/antioxidant power (FRAP) assay was used to measure TAC (Benzie and Strain, 1996). The concentrations of malondialdehyde (MDA), as indices of LPO in the sperm samples, were measured using the thiobarbituric acid reaction (Placer et al., 1966). CAT activity in the samples was measured (Goth, 1991) and total SOD activity was determined by the procedure based on the SOD ability to inhibit pyrogallolauto-oxidation (Marklund and Marklund, 1974). GPX activity was measured by the Ransel kit (Randox Laboratories Ltd., London, U.K.).

2.2.4. Statistical analysis The experiment was conducted as a completely randomised design and analysis of variance (ANOVA) was used for comparisons of means. When the ANOVA test showed statistical differences, the mean of the treatments were compared using Duncan’s multiple range tests. All data were expressed as mean ± standard error of the mean (S.E.M.). Differences were considered significant at p < 0.05. Five replicates were used in each treatment. For each replicate, three straws were thawed and pooled for evaluation of sperm parameters. 3. Results The effects of different doses of melatonin in the freezing extender on post-thaw motion parameters, viability, HOST, TM and acrosome abnormalities in bull spermatozoa are set out in Table 1. The percentages of TM and PM were higher (p < 0.05) in the freezing extender supplemented with 1 and 2 mM melatonin (60.8 ± 4.0 and 31.4 ± 3.0; 62.5 ± 4.2 and 30.1 ± 2.2, respectively). The percentage of PM did not differ (p > 0.05) between the control group (20.9 ± 2.4) and the ones frozen in extender supplemented with 3 mM melatonin (23.9 ± 2.7), while it decreased (p < 0.05) in the dose of 4 mM melatonin (15.1 ± 0.5). Addition of 2 mM melatonin in the freezing medium increased (p < 0.05) the percentage of LIN and STR (51.6 ± 3.9 and 95.0 ± 2.5, respectively) compared to other groups. ALH in 2 and 3 mM melatonin (1.7 ± 0.2, 1.7 ± 0.1 ␮m, respectively) had higher (p < 0.05) value than other treatments. Melatonin at 4 mM concentration decreased (p < 0.05) VSL (41.3 ± 7.0 ␮m/s) compared to the control group (51.5 ± 6.9 ␮m/s). No significant differences (p > 0.05) were observed in BCF, VCL and VAP in the freezing medium supplemented with melatonin following the freeze–thawing process. Immediately after thawing, the percentages of live sperm in the freezing extender with 0.1, 1 and 2 mM melatonin concentrations were higher (p < 0.05) than other groups (68.3 ± 2.3, 71.1 ± 3.5 and 69.8 ± 1.1, respectively;

28

I. Ashrafi et al. / Animal Reproduction Science 139 (2013) 25–30

Table 2 Mean (±S.E.M.) oxidative parameters in freeze–thawing bull semen with different concentrations of melatonin. Parameters

Melatonin concentrations Control

TAC (␮M Vit C) MDA (nmol/dl) CAT (U/ml) SOD (% inhibition) GPX (mU/ml)

17.7 6.1 0.0 5.5 439.2

± ± ± ± ±

0.1 mM 3.2d 0.7a 0.0e 1.7c 32.2

29.1 5.3 0.2 52.2 429.0

± ± ± ± ±

1 mM 4.3c 0.7a 0.0de 6.3b 27.4

42.5 3.6 0.5 50.4 464

± ± ± ± ±

2 mM 4.5b 1.0bc 0.1c 5.6b 25.0

40.2 3.9 1.3 53.0 453.6

3 mM ± ± ± ± ±

2.7b 0.6b 0.2a 9.3ab 33.9

48.9 2.7 0.7 60.6 466.8

4 mM ± ± ± ± ±

2.7a 0.8c 0.1b 4.9a 16.1

18.3 5.7 0.2 54 452.6

± ± ± ± ±

1.7d 0.8a 0.1d 4.6ab 30.2

TAC: total antioxidant capacity; MDA: malondialdehyde; GPX: glutathione peroxidase; CAT: catalase; SOD: levels activity of superoxide dismutase. Different superscripts within rows are significantly differ, p < 0.05.

Table 1). The HOST was improved (p < 0.05) by inclusion of all doses of melatonin in the medium. The highest values for HOST were obtained at 1, 2 and 3 mM concentrations of melatonin (66.4 ± 1.8, 68.3 ± 1.4 and 67.8 ± 1.1%, respectively). Supplementation of the medium with melatonin caused significant decrease (p < 0.05) in total abnormality rates in comparison with the control group and reached the lowest percentage of abnormal sperm with 1 and 2 mM melatonin (8.1 ± 1.3 and 6.9 ± 1.3, respectively). No treatment group affected (p > 0.05) the acrosome abnormality rates. The effects of melatonin on TAC, MDA and antioxidant activities in thawed bull semen are summarised in Table 2. Inclusion of various concentrations of melatonin in the semen extender increased (p < 0.05) TAC compared to the control (17.7 ± 3.2) group except for 4 mM melatonin (18.3 ± 1.7) and the highest values for TAC were achieved at 3 mM (48.9 ± 2.7). The level of MDA decreased (p < 0.05) with 1, 2 and 3 mM melatonin concentrations. The activity of CAT increased (p < 0.05) by inclusion of melatonin and the highest activity was at the concentration of 2 mM (1.3 ± 0.2) compared to other groups. Supplementation of the medium with various concentrations of melatonin caused a significant increase (p < 0.05) in SOD ability, which may inhibit pyrogallol auto-oxidation compared to the control group. No significant differences (p > 0.05) were detected in the activity of GPX with different concentrations of melatonin. 4. Discussion Substantial effort has been made to improve the artificial insemination (AI) techniques using frozen semen. Dilution, cooling, freezing and thawing are potential sources of stress during the semen freeze–thawing process. Cryopreservation causes an irreversible damage to enzymatic activity and sperm organelles leading to a reduction in the sperm kinetic parameters (Alvarez and Storey, 1989; Hammerstedt, 1993). Sperm fluidity, motility and ability for fertilisation, as well as structural integrity for viability, are associated with concentrations of polyunsaturated fatty acids in the plasma membrane of spermatozoa (Rao and Gangadharan, 2008). Spermatozoa are also highly susceptible to LPO due to oxidative stress, which may occur during cryopreservation (Bell et al., 1993; Aitken and Fisher, 1994; Stradaioli et al., 2007). Conversely, physiological concentrations of ROS increase membrane fluidity, preserve the fertilising ability of sperm and also have

some beneficial effects on sperm hyperactivation, capacitation and acrosome reaction (Bell et al., 1993; Aitken and Fisher, 1994; Aitken, 1995). Sperm cells and seminal plasma contain ROS scavengers, including SOD, GPX and CAT. However, an improper balance between ROS production and antioxidant enzyme activities can cause failure of sperm function (Sikka, 1996). Furthermore, the antioxidant system of seminal plasma and spermatozoa is compromised during semen cryopreservation (Alvarez and Storey, 1992). For these reasons, we used melatonin as an antioxidant in the freezing extender of bull semen to reduce the oxidative stress that may occur during the freeze–thawing process. It is suggested (Karbownik and Reiter, 2000) that melatonin stimulates the activities of antioxidant enzymes such as SOD, GPX and CAT. In consequence, melatonin reduces the number of free radicals, ROS, and also may increase the production of molecules protecting sperm cells against oxidative stress. It is reported (Reiter, 1995) that melatonin was more effective than glutathione in scavenging the highly toxic hydroxyl radical and twice as potent as vitamin E in neutralising the peroxyl radical. Further, melatonin provides the greatest molecular protection from oxidative damage than when other protective molecules are used (Yilmaz et al., 2002). SOD, CAT and GPX are important parts of antioxidant enzyme defence systems in sperm that convert superoxide (O2 − ) and peroxide (H2 O2 ) radicals into O2 and H2 O. GPX excludes peroxyl radicals from various peroxides (Irvine, 1996). CAT and SOD also eliminate O2 − produced by nicotinamide adenine dinucleotide phosphate-reduced (NADPH) oxidase (Alvarez and Storey, 1989; Jeulin et al., 1989). In our study, the activity of CAT and SOD increased by inclusion of various concentrations of melatonin in the semen extender, but no significant differences were observed in the activity of GPX. Many studies reported that melatonin has beneficial effects on preservation of mammalian sperm function and improves the microscopic parameters of spermatozoa (Sonmez et al., 2007; Fujinoki, 2008; Rao and Gangadharan, 2008; Ramadan et al., 2009; du Plessis et al., 2010; Jang et al., 2010). To our knowledge, the effect of melatonin on oxidative parameters including antioxidant enzyme activities, TAC and LPO in cryopreserved spermatozoa had not previously been assessed. In the current study, the sperm motion parameters as well as sperm viability increased with inclusion of 1 or 2 mM melatonin in the semen extender. These results are consistent with previous investigators who found higher percentages of

I. Ashrafi et al. / Animal Reproduction Science 139 (2013) 25–30

motile and viable spermatozoa in the extender supplemented with melatonin (du Plessis et al., 2010; Succu et al., 2011). Mitochondria in sperm cells encase the axosome, connect with dense fibres in the middle pieces and produce adenosine triphosphate (ATP). It has been reported (Aitken and Clarkson, 1987) that the axoneme and mitochondria in sperms may be damaged by a high level of ROS. The studies have shown that melatonin can stabilise and protect mitochondria via several mechanisms (Martin et al., 2000; Acuna et al., 2002; Leon et al., 2005; Lopez et al., 2009). Motility may increase and sperm morphology may normalise as a consequence of the protective effect of melatonin on sperm mitochondria after the freeze–thawing process (Garcia et al., 1999; Shang et al., 2004; Carretero et al., 2009; Paradies et al., 2010). Our findings also showed that an extender with 1 or 2 mM melatonin concentration decreased the percentage of sperm with abnormal morphology during cryopreservation but had no effect on the percentages of sperm with abnormal acrosome. Destruction of integrity causes a rise in the membrane permeability and a decrease in the ability of sperm to control the intracellular concentrations of ions that in turn are involved in sperm motion (Baumber et al., 2000). The results showed that inclusion of melatonin in the freezing media increased sperm plasma membrane integrity of bull semen following the freeze–thawing process. MDA production is greatly used to determine LPO in the various cell types including sperm cells (Sikka, 1996). Supplementation of the medium with 3 mM melatonin decreased the rate of MDA and LPO. These results are basically consistent with the results previously reported (Gavella and Lipovac, 2000). Moreover, inclusion of melatonin in the semen extender increased the TAC. This may be due to the stimulatory effects of melatonin on the activity of enzymes involved in antioxidant defence. The previous studies reported that melatonin has antiapoptotic effects on a wide range of cell types (Sainz et al., 2003; Jou et al., 2007), prevents capacitation and apoptoticlike changes of ram spermatozoa and also increases fertility rate (Casao et al., 2010). It is reported (Succu et al., 2011) that the addition of melatonin preserved DNA integrity in cryopreserved ram spermatozoa. Unfortunately, DNA integrity was not tested in this study, but previous reports are supported, as the levels of LPO were lower in freezing spermatozoa with melatonin. It is indicated that the protective effect of melatonin on cryopreservation injuries occurs in a dose-dependent manner (Gwayi and Bernard, 2002; Fujinoki, 2008; Succu et al., 2011). In the current study, different concentrations of melatonin (0.1–4.0 mM) in the freezing medium were assayed. The most effective concentration of melatonin in microscopic evaluations of bull sperm freezing extender was 2 mM. The highest value of TAC and the lowest value of LPO were achieved by inclusion of 3 mM concentration of melatonin in the semen extender and the highest activity of CAT was obtained by 2 mM melatonin. Extreme doses of antioxidants (4 mM of melatonin) in the freezing medium can counteract the ROS-induced oxidative stress and so impede the ROS-associated functions of spermatozoa (Roca et al., 2004).

29

In conclusion, supplementation of melatonin in the freezing medium can counteract the adverse effects of the freeze–thawing process on the motility, viability, normal morphology and plasma membrane integrity in bull spermatozoa. The results suggest that the protective effects of melatonin on spermatozoa are associated with a reduction in LPO as a consequence of increasing the TAC and antioxidant enzymes’ activity. Therefore, inclusion of 2 or 3 mM concentration of melatonin in semen extender before the freeze–thawing process may improve the efficiency of AI in cattle. Acknowledgement The authors thank Animal Breeding Center, west and northwest of country, Tabriz, Iran for supplying the semen samples and the equipment for cryopreservation. References Acuna, C.D., Escames, G., Carazo, A., Leon, J., Khaldy, H., Reiter, R.J., 2002. Melatonin, mitochondrial homeostasis and mitochondrial-related diseases. Curr. Top. Med. Chem. 2, 133–151. Agarwal, A., Said, T.M., 2005. Oxidative stress, DNA damage and apoptosis in male infertility: a clinical approach. BJU Int. 95, 503–507. Aitken, J., Fisher, H., 1994. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays 16, 259–267. Aitken, R.J., 1995. Free radicals, lipid peroxidation and sperm function. Reprod. Fertil. Dev. 7, 659–668. Aitken, R.J., Clarkson, J.S., 1987. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil. 81, 459–469. Alvarez, J.G., Storey, B.T., 1982. Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: its effect on sperm motility. Biol. Reprod. 27, 1102–1108. Alvarez, J.G., Storey, B.T., 1984. Assessment of cell damage caused by spontaneous lipid peroxidation in rabbit spermatozoa. Biol. Reprod. 30, 323–331. 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. Alvarez, J.G., Storey, B.T., 1992. Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation. J. Androl. 13, 232–241. Ashrafi, I., Kohram, H., Naijian, H., Bahreini, M., Mirzakhani, H., 2011. Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa. BMC Res. Notes 4, 547. Awad, H., Halawa, F., Mostafa, T., Atta, H., 2006. Melatonin hormone profile in infertile males. Int. J. Androl. 29, 409–413. Baker, M.A., Aitken, R.J., 2004. The importance of redox regulated pathways in sperm cell biology. Mol. Cell. Endocrinol. 216, 47–54. Balestri, F., Giannecchini, M., Sgarrella, F., Carta, M.C., Tozzi, M.G., Camici, M., 2007. Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions. Neurochem. Int. 50, 517–523. Baumber, J., Ball, B.A., Gravance, C.G., Medina, V., Davies-Morel, M.C., 2000. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J. Androl. 21, 895–902. Bell, M., Wang, R., Hellstrom, W.J., Sikka, S.C., 1993. Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm. J. Androl. 14, 472–478. Benzie, I.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76. Berra, B., Rizzo, A.M., 2009. Melatonin: circadian rhythm regulator, chronobiotic, antioxidant and beyond. Clin. Dermatol. 27, 202–209. Buckett, W.M., Luckas, M.J., Aird, I.A., Farquharson, R.G., Kingsland, C.R., Lewis-Jones, D.I., 1997. The hypo-osmotic swelling test in recurrent miscarriage. Fertil. Steril. 68, 506–509.

30

I. Ashrafi et al. / Animal Reproduction Science 139 (2013) 25–30

Carretero, M., Escames, G., Lopez, L.C., Venegas, C., Dayoub, J.C., Garcia, L., Acuna-Castroviejo, D., 2009. Long-term melatonin administration protects brain mitochondria from aging. J. Pineal Res. 47, 192–200. Casao, A., Mendoza, N., Perez-Pe, R., Grasa, P., Abecia, J.A., Forcada, F., Cebrian-Perez, J.A., Muino-Blanco, T., 2010. Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate. J. Pineal Res. 48, 39–46. Donoghue, A.M., Donoghue, D.J., 1997. Effects of water- and lipid-soluble antioxidants on turkey sperm viability, membrane integrity, and motility during liquid storage. Poult. Sci. 76, 1440–1445. du Plessis, S.S., Hagenaar, K., Lampiao, F., 2010. The in vitro effects of melatonin on human sperm function and its scavenging activities on NO and ROS. Andrologia 42, 112–116. El-Sokkary, G.H., Kamel, E.S., Reiter, R.J., 2003. Prophylactic effect of melatonin in reducing lead-induced neurotoxicity in the rat. Cell. Mol. Biol. Lett. 8, 461–470. Fujinoki, M., 2008. Melatonin-enhanced hyperactivation of hamster sperm. Reproduction 136, 533–541. Garcia, J.J., Reiter, R.J., Pie, J., Ortiz, G.G., Cabrera, J., Sainz, R.M., AcunaCastroviejo, D., 1999. Role of pinoline and melatonin in stabilizing hepatic microsomal membranes against oxidative stress. J. Bioenerg. Biomembr. 31, 609–616. Gavella, M., Lipovac, V., 2000. Antioxidative effect of melatonin on human spermatozoa. Arch. Androl. 44, 23–27. Gil-Guzman, E., Ollero, M., Lopez, M.C., Sharma, R.K., Alvarez, J.G., Thomas Jr., A.J., Agarwal, A., 2001. Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Hum. Reprod. 16, 1922–1930. Gil, J., Lundeheim, N., Soderquist, L., Rodriiuez-Martinez, H., 2003. Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen. Theriogenology 59, 1241–1255. Gillan, L., Maxwell, W.M., 1999. The functional integrity and fate of cryopreserved ram spermatozoa in the female tract. J. Reprod. Fertil. Suppl. 54, 271–283. Goth, L., 1991. A simple method for determination of serum catalase activity and revision of reference range. Clin. Chim. Acta 196, 143–151. Gwayi, N., Bernard, R.T., 2002. The effects of melatonin on sperm motility in vitro in Wistar rats. Andrologia 34, 391–396. Hammerstedt, R.H., 1993. Maintenance of bioenergetic balance in sperm and prevention of lipid peroxidation: a review of the effect on design of storage preservation systems. Reprod. Fertil. Dev. 5, 675–690. Irvine, D.S., 1996. Glutathione as a treatment for male infertility. Rev. Reprod. 1, 6–12. Ishizuka, B., Kuribayashi, Y., Murai, K., Amemiya, A., Itoh, M.T., 2000. The effect of melatonin on in vitro fertilization and embryo development in mice. J. Pineal Res. 28, 48–51. Jang, H.Y., Kim, Y.H., Kim, B.W., Park, I.C., Cheong, H.T., Kim, J.T., Park, C.K., Kong, H.S., Lee, H.K., Yang, B.K., 2010. Ameliorative effects of melatonin against hydrogen peroxide-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development. Reprod. Domest. Anim. 45, 943–950. Jeulin, C., Soufir, J.C., Weber, P., Laval-Martin, D., Calvayrac, R., 1989. Catalase activity in human spermatozoa and seminal plasma. Gamete Res. 24, 185–196. Jou, M.J., Peng, T.I., Yu, P.Z., Jou, S.B., Reiter, R.J., Chen, J.Y., Wu, H.Y., Chen, C.C., Hsu, L.F., 2007. Melatonin protects against common deletion of mitochondrial DNA-augmented mitochondrial oxidative stress and apoptosis. J. Pineal Res. 43, 389–403. Karbownik, M., Reiter, R.J., 2000. Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation. Proc. Soc. Exp. Biol. Med. 225, 9–22. Lee, Y.M., Chen, H.R., Hsiao, G., Sheu, J.R., Wang, J.J., Yen, M.H., 2002. Protective effects of melatonin on myocardial ischemia/reperfusion injury in vivo. J. Pineal Res. 33, 72–80. Leon, J., Acuna-Castroviejo, D., Escames, G., Tan, D.X., Reiter, R.J., 2005. Melatonin mitigates mitochondrial malfunction. J. Pineal Res. 38, 1–9. Lopez, A., Garcia, J.A., Escames, G., Venegas, C., Ortiz, F., Lopez, L.C., AcunaCastroviejo, D., 2009. Melatonin protects the mitochondria from oxidative damage reducing oxygen consumption, membrane potential, and superoxide anion production. J. Pineal Res. 46, 188–198.

Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474. Martin, M., Macias, M., Escames, G., Leon, J., Acuna-Castroviejo, D., 2000. Melatonin but not vitamins C and E maintains glutathione homeostasis in t-butyl hydroperoxide-induced mitochondrial oxidative stress. FASEB J. 14, 1677–1679. Meyers, S.A., 2005. Spermatozoal response to osmotic stress. Anim. Reprod. Sci. 89, 57–64. Paradies, G., Petrosillo, G., Paradies, V., Reiter, R.J., Ruggiero, F.M., 2010. Melatonin, cardiolipin and mitochondrial bioenergetics in health and disease. J. Pineal Res. 48, 297–310. Pena, F.J., Rodriguez Martinez, H., Tapia, J.A., Ortega Ferrusola, C., Gonzalez Fernandez, L., Macias Garcia, B., 2009. Mitochondria in mammalian sperm physiology and pathology: a review. Reprod. Domest. Anim. 44, 345–349. Piantadosi, C.A., 2008. Carbon monoxide, reactive oxygen signaling, and oxidative stress. Free Radic. Biol. Med. 45, 562–569. Placer, Z.A., Cushman, L.L., Johnson, B.C., 1966. Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Anal. Biochem. 16, 359–364. Ramadan, T.A., Taha, T.A., Samak, M.A., Hassan, A., 2009. Effectiveness of exposure to longday followed by melatonin treatment on semen characteristics of Damascus male goats during breeding and non-breeding seasons. Theriogenology 71, 458–468. Rao, M.V., Gangadharan, B., 2008. Antioxidative potential of melatonin against mercury induced intoxication in spermatozoa in vitro. Toxicol. In Vitro 22, 935–942. Reiter, R.J., 1995. Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J. 9, 526–533. Reiter, R.J., 1998. Oxidative damage in the central nervous system: protection by melatonin. Prog. Neurobiol. 56, 359–384. Roca, J., Gil, M.A., Hernandez, M., Parrilla, I., Vazquez, J.M., Martinez, E.A., 2004. Survival and fertility of boar spermatozoa after freeze–thawing in extender supplemented with butylated hydroxytoluene. J. Androl. 25, 397–405. Roca, J., Rodriguez, M.J., Gil, M.A., Carvajal, G., Garcia, E.M., Cuello, C., Vazquez, J.M., Martinez, E.A., 2005. Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase. J. Androl. 26, 15–24. Sainz, R.M., Mayo, J.C., Rodriguez, C., Tan, D.X., Lopez-Burillo, S., Reiter, R.J., 2003. Melatonin and cell death: differential actions on apoptosis in normal and cancer cells. Cell. Mol. Life Sci. 60, 1407–1426. Schafer, S., Holzmann, A., 2000. The use of transmigration and spermac stain to evaluate epididymal cat spermatozoa. Anim. Reprod. Sci. 59, 201–211. Shang, X., Huang, Y., Ye, Z., Yu, X., Gu, W., 2004. Protection of melatonin against damage of sperm mitochondrial function induced by reactive oxygen species. Zhonghua Nan Ke Xue 10, 604–607. Sikka, S.C., 1996. Oxidative stress and role of antioxidants in normal and abnormal sperm function. Front. Biosci. 1, e78–e86. Sonmez, M., Yuce, A., Turk, G., 2007. The protective effects of melatonin and Vitamin E on antioxidant enzyme activities and epididymal sperm characteristics of homocysteine treated male rats. Reprod. Toxicol. 23, 226–231. Stradaioli, G., Noro, T., Sylla, L., Monaci, M., 2007. Decrease in glutathione (GSH) content in bovine sperm after cryopreservation: comparison between two extenders. Theriogenology 67, 1249–1255. Succu, S., Berlinguer, F., Pasciu, V., Satta, V., Leoni, G.G., Naitana, S., 2011. Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner. J. Pineal Res. 50, 310–318. Tan, D.X., Reiter, R.J., Manchester, L.C., Yan, M.T., El-Sawi, M., Sainz, R.M., Mayo, J.C., Kohen, R., Allegra, M., Hardeland, R., 2002. Chemical and physical properties and potential mechanisms: melatonin as a broad spectrum antioxidant and free radical scavenger. Curr. Top. Med. Chem. 2, 181–197. Yilmaz, T., Celebi, S., Kukner, A.S., 2002. The protective effects of melatonin, vitamin E and octreotide on retinal edema during ischemia-reperfusion in the guinea pig retina. Eur. J. Ophthalmol. 12, 443–449.