Cell Tissue Bank DOI 10.1007/s10561-013-9412-y
ORIGINAL PAPER
Effects of in combination antioxidant supplementation on microscopic and oxidative parameters of freeze–thaw bull sperm R. Olfati Karaji • H. Daghigh Kia • I. Ashrafi
Received: 16 October 2013 / Accepted: 22 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The present study was conducted to determine the effect of reduced glutathione (GSH) and superoxide dismutase (SOD) supplementation in the bull semen freezing extender on post-thaw parameters of Holstein and Simmental bull sperm. Semen were collected from seven bulls (four Holstein and three Simmental) and cryopreserved in the Trisegg-yolk based extender as control group and supplemented with various concentrations of GSH 9 SOD (5 9 100, 7.5 9 100, 5 9 150, and 7.5 9 150 mM 9 IU mL-1) in treatment groups. Microscopic parameters were evaluated in terms of total motility parameters using computer assisted semen analysis and viability and membrane integrity were assessed using Eosin– Nigrosin stains and hypo-osmotic swelling test (HOST), respectively after thawing the semen. Malonaldialdehyde (MDA) level, SOD and glutathione peroxides (GPx) activities were assessed immediately after thawing. Results showed that supplementation of the cryopreservation medium with various concentrations of GSH 9 SOD improved total motility (TM) and progressive motility parameters for Holstein (P \ 0.05) R. Olfati Karaji H. Daghigh Kia (&) Department of Animal Science, Faculty of Agriculture, University of Tabriz, 29 Bahman Boulevard, East Azerbaijan, 5166614766 Tabriz, Iran e-mail: [email protected]; [email protected] I. Ashrafi Young Researchers and Elites Club, Science and Research Branch, Islamic Azad University, Tehran, Iran
semen, and values of TM and HOST for Simmental semen compared to the control group (P \ 0.01) after semen thawing. Addition of antioxidant to Holstein semen samples decreased the level of MDA and increased GPx activities compared to control groups (P \ 0.05). SOD activities increased in Simmental bull samples compare to the control group (P \ 0.01), but not differ in Holstein, while these activities. In conclusion, supplementation of antioxidant to the semen extender as combination (GSH 9 SOD) improved the semen post-thaw qualities which may be associated with a reduction in lipid peroxidation as well as an increase in the antioxidant enzyme activities. Keywords Bull sperm Cryopreservation Reduced glutathione Superoxide dismutase
Introduction Semen storage and cryopreservation are associated with cold shock and atmospheric oxygen (Alvarez and Storey 1982; Arav et al. 2002; Cocchia et al. 2011; Medeiros et al. 2002) that lead to higher production of reactive oxygen species (ROS) and an imbalance between free radicals and antioxidant system of extended semen. Oxygen radicals at physiological concentrations are also known to have a positive effect in intracellular signaling involved in the regular processes of cell proliferation, differentiation and
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migration (Piantadosi 2008). But, excessive production of ROS may had a toxic effect through the production of free radicals affecting functional parameters of the sperm cells such as deprivation of motility (Sikka 1996), inactivation of glycolytic enzymes and damage to the acrosomal membranes (Alvarez and Storey 1984) which would render the sperm cell unable to fertilize the oocyte (Gil-Guzman et al. 2001). During semen cryopreservation process, a large amount of the cytoplasmic component such as antioxidants will leak out to extracellular irreversibly. These events decrease enzymatic defensive system capacity of semen (Bilodeau et al. 2000; Bucak et al. 2008). Spermatozoa and seminal plasma possess an antioxidant system comprising catalase (CAT), glutathione peroxides (GPx) and superoxide dismutase (SOD) as the main antioxidant enzymatic factors (Bilodeau et al. 2000; Meister 1994) and also have glutathione (GSH), alpha-tocoferol, vitamin C, betacarotene, cysteine and hypotaurine as the non-enzymatic antioxidant to prevent oxidative damage (Agarwal et al. 2005). However, this antioxidant capacity in sperm cells, due to the small cytoplasmic component is limited for a long storage and cryopreservation. In addition, according to the several reports, cryopreservation of the semen decreased GSH content of sperm significantly as much as 64, 32 and 58–78 % in human (Gadea et al. 2011), boar (Gadea et al. 2004) and bull sperm (Bilodeau et al. 2000; Stradaioli et al. 2007), respectively, and also reduced SOD value by 50 % in bull sperm (Bilodeau et al. 2000). Therefore, mammalian sperm’s ability is not sufficient for preventing lipid peroxidation (LPO) production during the freeze–thaw process (Storey 1997; Lapointe and Bilodeau 2003). It is suggested that supplementation of antioxidant in the freezing medium may prevent excessive ROS production in semen (Donoghue and Donoghue 1997) during cryopreservation. Antioxidant molecules could decrease the impact of oxidative stress and therefore improve sperm quality after thawing. Metalloproteinase like SOD protects the sperm against O2- toxicity and also LPO by dismutation of O2 to H2O2. Previous studies reported that addition of SOD to the semen extenders improve the quality of semen (Cocchia et al. 2011; Sikka 1996) such as total motility (TM) and the percentage of viable sperm with intact acrosome following freeze–thaw process in boar semen (Roca et al. 2005a). Glutathione also is able to
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react with ROS directly (Bucak et al. 2008) and protects the sperm due to its sulfur and cysteine content. It is reported that addition of GSH to freezing extender improves the quality and fertilizing capacity of bull (Gadea et al. 2008; Perumal et al. 2011), ram (Bucak et al. 2008) and boar sperm (Gadea et al. 2005a). There are simultaneous irreversible reduction of different intracellular antioxidant contents such as SOD and GSH during semen cryopreservation process (Bilodeau et al. 2000; Gadea et al. 2011; Stradaioli et al. 2007). Further, applying single antioxidants was demonstrated to be insufficient to induce the sperm characteristics (Rossi et al. 2001; Gadea et al. 2004; Caˆmara et al. 2011). In recent years, the combination of antioxidants has been used successfully in the semen extender of human (Rossi et al. 2001), bull (Foote et al. 2002) and boar (Roca et al. 2005b). In the present study a combination of various concentrations of GSH and SOD antioxidants supplemented in semen extender of Holstein and Simmental bull semen to evaluate the quality and biochemical parameters of bull semen following freeze–thaw process. Moreover, the Holstein and Simmental breeds are major species which are commonly used in many countries as artificial insemination; conducting this research, we want to find major differences between semen samples of both Holstein and Simmental bulls after freezing– thawing process via addition of different levels of mixed antioxidants.
Materials and methods Semen source and preparation This study was performed in Animal Breeding Center of West and Northwest of Country, located in Tabriz, Iran. Ejaculates were collected from seven bulls (four Holstein and three Simmental), aged between 3 and 4 years, regularly used for breeding purpose based on their fertility estimation through in vivo fertility tests. Total of 35 ejaculates (five ejaculates per bull) were collected twice a week using an artificial vagina (45 °C). The ejaculates were transferred to the laboratory and submerged in a water bath (34 °C), until semen evaluation. The volume of ejaculate was estimated in a conical tube graduated at 0.1 mL intervals. The sperm concentration was determined by
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means of an Accucell photometer (IMV, L’Aigle, France). The ejaculates were evaluated for freezing process if the following criteria were met: volume between 5 and 12 mL; sperm concentration of C19109 sperm mL-1; motility of C70 and B10 % 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 Tris-egg-yolk based medium [60 mM citric acid, 0.25 M Tris–(hydroxymethyl)-aminomethane, 69 mM fructose, 20 % egg yolk, 7 % glycerol, 25 mg gentamycin sulfate, 5 mg tylosin tartrate, 15 mg lincomycin hydrochloride and 30 mg spectinomycin sulfate in 80 mL double distilled water] was used for the pooled ejaculates (Bilodeau et al. 2002). The extended semen samples with Tris-egg-yolk based medium were divided into the equal aliquots in order to use them in combined treatments. The treatments were prepared by applying in combination GSH and SOD (GSH 9 SOD) including 5 9 100 (I), 7.5 9 100 (II), 5 9 150 (III), 7.5 9 150 (IV) mM 9 IU mL-1 concentration, respectively. In control group (V) the Tris-egg-yolk based extender contained no additive antioxidant. Semen samples were cooled slowly to 5 °C for 2 h, and then added to the various extender treatments. The extended semen was then packaged in 0.5 mL straws. The straws were sealed via automatic filling and sealing machine (IMV Technologies, MRS 1, France) and they were frozen by a semi-automatic freezer machine (mini tube, Germany) with liquid nitrogen vapor. The straws were cooled at approximately -15 °C min-1 from ?5 to -150 °C. Then, they were transferred to a liquid nitrogen tank and stored. Frozen straws were thawed at 37 °C for 40 s in a water bath immediately before evaluation. Semen evaluation Viability and motility parameters The sperm motion parameters such as TM, PGM, VCL, VAP and VSL of frozen–thawed samples are evaluated after thawing by computer assay sperm analysis (CASA) which is equipped with a phasecontrast microscopy system (Nikon, Japan), a warm stage at 37 °C, a digital video camera (SAMSUNG, Korea) and a computer for analyzing kinetic motion
spermatozoa (Hoshmand Fanavaran, Tehran, Iran). To analyze the semen, the samples were incubated in a water bath at 37 °C for 10 min. The following motility values were recorded using CASA: Total motility (TM, %), progressive motility (PGM, %), straight linear velocity (VSL, lm s-1), curvilinear velocity (VCL, lm s-1) and average path velocity (VAP, lm s-1). For evaluation of these parameters, 5 lL drop of the sample was placed on a warmed (37 °C) slide and covered with a cover slip. Sperm motility parameters were estimated in three different microscopic fields per each semen sample. Images were obtained at 4009 magnifications using a phase contrast objective microscope (Nikon, Tokyo, Japan). The mean of the three successive estimations ([200 spermatozoa per slide) was recorded as the final data (TM and PGM percentage). The viability of spermatozoa in the samples was assessed by means of the Eosin–Nigrosin stain method after thawing. The final composition of the stain was: Eosin-Y 1.67 g, Nigrosin 10 g, and sodium citrate 2.9 g were 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. The viability of sperms was assessed by counting 400 sperm cells in a brightness microscope (Olympus DP 50) at 1,0009 magnification, using immersion oil. Sperm expression partial or complete purple staining was considered nonviable; only sperm showing strict exclusion of stain were considered as viable (Balestri et al. 2007). Assessment of functional integrity for membrane The hypoosmotic swelling test (HOST) was used to evaluate the functional integrity of the sperm membrane after thawing, based on curled and swollen tails. This was performed by incubating 30 mL of semen with 300 mL of a 100 mOsM hypoosmotic solution (9 g fructose ? 4.9 g sodium citrate per liter of distilled water) at 37 °C for 60 min. The osmotic pressure of solution was controlled and calibrated via an Osmometer (Labx, Mark III, USA). Following incubation, 0.2 mL of the mixture was spread with a cover slip on a slide. Four hundred sperm were examined with 4009 magnification with bright-field microscopy. Sperm with swollen or coiled tails were recorded (Buckett et al. 1997).
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Biochemical assay The samples (0.5 mL straw) were thawed before the beginning of biochemical assays. An aliquot (500 lL) of semen obtained from each sample was centrifuged at 8009g for 10 min, sperm pellets were separated and washed by resuspending in PBS and recentrifuging for 3 times. Following last centrifugation, 1 mL of deionized water was added to spermatozoa and they were snapfrozen and stored at -70 °C until further analysis. Lipid peroxidation Lipid peroxidation rate of sperm was estimated by measuring the level of malonaldialdehyde (MDA), using the thiobarbituric acid (TBA) based on the method described by Ohkawa et al. (1979). Malonaldialdehyde levels were measured after supplementing 100 lL of sperm cell pellet suspension added to TBA–TCA reagent (1.25 mL of 20 % w/v TCA, 1 mL of 0.2 % w/v TBA and 1.25 mL of 0.05 M H2So4). The mixture was treated in boiling water (95 °C) for 60 min. After cooling, 4 mL of butanol was added into the mixture following which it was centrifuged for 5 min at 1,500g for 5 min. The supernatant was removed and the absorbance was measured at 535 nm (biophotometer plus, Eppendorf, Germany). The MDA concentration was determined by the specific absorbance coefficient (1.56 9 105 lmol cm-3). The produced MDA is expressed as pmol mg-1 protein of sperm cell pellet. Glutathione peroxides activity Glutathione peroxides activity was measured based on the protocol of Paglia and Valentine (1967). Glutathione peroxides catalyzed the oxidation of GSH by Cumene Hyroperoxide. Owing to the presence of Glutathione reductase (GR) and nicotin amide adenine dinucleotide phosphate (NADPH), the oxidized Glutathione (GSSG) is immediately converted into the reduced form with a concomitant oxidation of NADPH to NADP?. The oxidation of NADPH to NADP? is associated with a decrease in the absorbance of 340 nm via the spectrophotometer (biophotometer plus, Eppendorf, Germany) indicating GPx activities. The rate of decrease in absorbance at 340 nm was directly proportional to the GPx activities. The reaction mixture was containing 20 lL of sample (3 mg mL-1 sperm pellet cells), 1 mL of solution (4 mM Glutathione, 5 9 10-5 u mL-1 glutathione
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reductase, 0.34 mM NADPH, 0.05 mM phosphate buffer; pH 7.2 and 4.3 mM EDTA) and 40 lL of Cumene Hyroperoxide. The absorbance of the mixture sample was measured after 20 min using a biophotometer (biophotometer plus, Eppendorf, Germany) at 340 nm wave length. Glutathione peroxides values expressed as mU mg-1 protein of sperm cell pellet. Superoxide dismutase activity Superoxide dismutase activity was determined using the method proposed by Misra and Fridovich (1972). This method was employed the Xanthine and Xanthine oxides to generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyl tetrazolium chloride (INT) to form a red Formazan dye. The SOD activity was measured by the degree of inhibition of this reaction. One unit of SOD causes a 50 % inhibition of the rate of reduction of INT under the conditions of the assay. The reaction mixture for each standard (S) consisted of 30 lL samples (sperm cells pellet) 1 mL reagent (0.05 mM Xanthine, 0.025 mM INT., 40 mM CAPS, pH, 10.2 and 0.94 mM EDTA) and 150 lL of Xanthine Oxidase. Cuveite containing mixture sample was assayed using a biophotometer (biophotometer plus, Eppendorf, Germany) at a wave length of 505 nm in two steps: (1) The initial absorbance after 30 S and (2) The second absorbance 3 min after adding Xanthine oxidase. A control was also evaluated. Superoxide dismutase value was expressed as IU g-1 protein in the sperm cell pellet. Statistical analysis Statistical analysis was performed using SAS software (version 9.1.3). Semen quality data (Total motility, progress motility, viability and membrane integrity) were subjected to arcsine transformation before being analyzed; all the data obtained from this study analyzed using the GLM procedure. Least square means were performed to determine the differences between experimental treatments throughout post-thaw evaluation.
Result The effects of different doses of GSH 9 SOD on postthaw parameters such as PGM, TM, VCL, VAP, VSL,
51.91 ± 3.80ab
II
29.36 ± 5.39
47.26 ± 2.73b 47.56 ± 3.59b
V -1
30.65 ± 3.57
49.90 ± 7.15
IV
32.93 ± 3.50
31.60 ± 3.77
33.97 ± 4.51
III
b
59.12 ± 4.98
I
a
44.83 ± 2.88
32.70 ± 4.65
b
36.95 ± 3.96
b
47.55 ± 2.44
80.63 ± 2.07
81.74 ± 8.94
85.04 ± 5.88
81.46 ± 3.98
86.14 ± 5.33
88.57 ± 2.44
87.51 ± 4.19
91.86 ± 3.56
88.65 ± 5.60
92.64 ± 3.36
VCL
-1
50.60 ± 3.35
51.62 ± 7.25
55.86 ± 3.13
53.65 ± 2.63
55.00 ± 1.74
58.90 ± 5.52
62.31 ± 3.61
65.77 ± 3.24
61.19 ± 4.77
65.21 ± 6.64
VAP
35.10 ± 4.05
37.37 ± 6.64
40.84 ± 5.78
39.18 ± 2.52
42.75 ± 5.17
41.84 ± 2.71
42.47 ± 3.65
46.61 ± 2.19
42.25 ± 4.42
45.86 ± 3.43
VSL
-1
41.32 ± 2.96b
43.39 ± 4.84b
45.19 ± 3.37
b
45.58 ± 2.75b
51.11 ± 3.69
a
46.78 ± 5.19
b
50.33 ± 1.52
ab
57.17 ± 5.48a
52.72 ± 4.44
ab
57.70 ± 2.76a
HOST (%)
55.69 ± 3.50
56.62 ± 2.09
59.06 ± 2.10
55.89 ± 3.36
61.62 ± 3.13
53.60 ± 5.19bc
54.97 ± 4.29b
60.66 ± 5.48ab
57.02 ± 4.46b
66.30 ± 4.08a
Viability (%)
I: GSH 9 SOD (5.0 9 100 mM 9 IU mL ); II: GSH 9 SOD (5.0 9 150 mM 9 IU mL ); III: GSH 9 SOD (7.5 9 100 mM 9 IU mL ); IV: GSH 9 SOD (7.5 9 150 mM 9 IU mL-1); V: Control. TM total motility, PGM progressive motility, VCL curvilinear velocity, VAP average path velocity, VSL straight linear velocity, HOST hypo-osmotic swelling test. Different superscripts (a, b, c) in the same column indicate a significant difference (P \ 0.05)
Simmental
V
IV
ab
40.83 ± 3.75a
b
48.98 ± 3.12ab
III
35.75 ± 4.33
ab
42.40 ± 4.15a
ab
53.25 ± 2.74a 48.69 ± 2.94
I
Holstein
PGM
TM (%)
Parameters
II
Treatments
Breeds
Table 1 The different concentrations of SOD 9 GSH on motion parameters, HOST and sperm viability percentage after thawing in Holstein and Simmental bull spermatozoa (Mean ± SD)
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viability and membrane integrity of sperms (positive responds to HOST) in the both breeds are represented in Table 1. Compared to the IV and control treatments, addition of GSH 9 SOD (treatment I) to the semen freezing extender of Holstein bulls increased (P \ 0.05) the percentage of TM after thawing. Regarding Simmental bulls, addition of treatment I increased (P \ 0.01) compared to the IV, II and control treatments; however no significant differences were found between other treatments in both breeds. Sperm progressive motility of Holstein samples at the levels of 5 9 100 and 7.5 9 100 (mM 9 IU mL-1) was also higher than control group after thawing (P \ 0.05). The percentage of sperm PGM did not differ (P [ 0.05) between all treatments in the Simmental samples. No significant differences (P [ 0.05) were observed in VSL, VCL and VAP in the freezing medium supplemented with doses of antioxidant in both breeds following the freeze–thawing process. Supplementation of treatment I with the medium increased viable sperm rates in comparison with the IV, II and V (control) treatments in Holstein samples evaluated after thawing (P \ 0.05). The percentage of viable sperms did not differ among all treatment in Simmental breed (P [ 0.05). Following thawing, the HOST was improved (P \ 0.05) in freezing extender supplemented with I and III treatments in Holstein (P \ 0.05) and I treatment in Simmental (P \ 0.01) samples compared to control group, however no changes were found among other treatments. Results of MDA values and enzyme (GPx and SOD) antioxidant activities in thawed both bull semen’s are summarized in Table 2. Inclusion of various concentrations of antioxidant in extended semen of Holstein caused a decrease in the level of MDA, achieving its lowest values in I and III treatments (P \ 0.05). However no significant differences were detected in the Simmental semen samples. The activity of GPx in Holstein semen samples increased by inclusion of all doses of the antioxidants (P \ 0.05) and the highest activity was detected in I treatment compared to the control group (P \ 0.05). Moreover, none of the treatment groups influenced the activity of GPx in Simmental semen samples. Superoxide dismutase activities did not differ (P [ 0.05) between control and treatment groups in Holstein extended semen while these activities increased (P \ 0.01) in the IV treatments than to the other treatments in Simmental bull samples.
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Discussion Defense systems in the sperm freezing medium may needed to overcome the oxidative stress against ROS production during freeze–thaw process which are based on the combined action of enzymatic and nonenzymatic antioxidants. According to our knowledge, the effect of combination of GSH 9 SOD antioxidants on microscopic and oxidative parameters including antioxidant enzyme activities and LPO in cryopreserved bull sperm had not previously been assessed. In the current study, we have used the combination of GSH and SOD in different concentrations, based on the data obtained from previous studies (Bucak et al. 2008; Cocchia et al. 2011; Foote et al. 2002; Gadea et al. 2004, 2011; Perumal et al. 2011; Roca et al. 2005b; Rossi et al. 2001). Previous studies have reported that the freeze–thaw process may cause significant changes in the organization of sperm membrane lipids (Gadea et al. 2005b, 2008). This happen may due to the deficiency in the endogenous antioxidant capacity and excessive production of ROS in the cells (Aitken et al. 1993; Bilodeau et al. 2000). Previous experiments in sperm of human also suggested that the freeze–thaw process enhances LPO activities (Alvarez and Storey 1992). The presence of double bonds in carbon chain polyunsaturated fatty acids in sperm cells makes them susceptible to cascade of oxidative attack and fat oxidation. There is a good evidence to indicate that the rate of lipid peroxidation correlated inversely with viability, motility and performance of the sperm (Aitken et al. 1993). Superoxide dismutase, GPx and CAT enzymes reduced the LPO cascade of membranes via scavenging of superoxide ions and hydrogen peroxide (Sikka 1996). In this investigation, the level of MDA decreased and GPx activities increased compared to the control group in Holstein samples which supplemented with 5 9 100 GSH 9 SOD levels after thawing. In this way, the reduction of MDA level may be closely related to the proper function of GPx and CAT due to converting of H2O2 into O2 and H2O. On the other hand, adding this amount of mixed antioxidant reduced lipid peroxidation positively, via improving ROS scavenger systems. An increase in SOD activities in the 7.5 9 150 concentration of Simmental semen samples may be associated with an increase in the production rate of hydrogen peroxide and in turn, it will increase the rate of sperm LPO
Cell Tissue Bank Table 2 The effect of different concentrations of SOD 9 GSH on some biochemical parameters after thawing in bull spermatozoa (Holstein and Simmental) (Mean ± SD) Breed
Treatments
MDA (pmol mg-1 protein)
GPx (mU mg-1 protein)
SOD (mU mg-1 protein)
Holstein
I
10.25 ± 0. 61b
64.16 ± 6.35a
15.76 ± 0.70
II
Simmental
ab
11.05 ± 0.76
ab
51.97 ± 7.25
15.60 ± 1.43
III
9.14 ± 0.84b
61.91 ± 4.74ab
14.99 ± 1.18
IV
11.60 ± 1.49ab
57.69 ± 4.46ab
16.14 ± 1.34
V
12.21 ± 1.07a
55.51 ± 4.55b
14.58 ± 1.30
I
14.31 ± 0.66
52.73 ± 4.63
11.12 ± 1.10b
II
14.87 ± 0.66
51.15 ± 3.92
12.42 ± 0.86b
III
14.59 ± 1.34
52.43 ± 3.00
12.23 ± 1.10b
IV
16.09 ± 1.27
48.85 ± 6.22
14.23 ± 0.71a
V
15.31 ± 0.74
46.46 ± 7.65
11.93 ± 0.95b
I: GSH 9 SOD (5.0 9 100 mM 9 IU mL-1); II: GSH 9 SOD (5.0 9 150 mM 9 IU mL-1); III: GSH 9 SOD (7.5 9 100 mM 9 IU mL-1); IV: GSH 9 SOD (7.5 9 150 mM 9 IU mL-1); V: Control. MDA malonaldialdehyde, GPx glutathione peroxidase, SOD superoxide dismutase. Different superscripts (a, b) in the same column indicate a significant difference (P \ 0.05)
simultaneously. These results are consistent with previous studies (Roca et al. 2005b) indicating that H2O2 has a more adverse impact on frozen–thawed bull spermatozoa compared to O2-. Accumulation of H2O2 may be the most common cause of increasing lipid peroxidation (0.074) due to increasing SOD activity showing no changes in GPx activity. The present study demonstrated the importance of maintaining TM (in the both breeds) and PGM (Holstein breed) parameters when their semen samples are supplemented with mixed antioxidants. According to Rossi et al. (2001) applying this way, supplementation of human freezing medium via both SOD and catalase significantly improves viability and motility of sperm cells; these results was convergence with our hypothesis considering the application of combination antioxidants supplementation on bull freezing medium. Therefore, these results could be induced from their combined and simultaneous actions (the combination of GSH and SOD or CAT and SOD) on superoxide anion and hydrogen peroxide. Previous studies have also reported a negative correlation between LPO rate and sperm motility parameters (Kasimanickam et al. 2006; Aitken et al. 1993). Our results uphold above studies since according to our study a higher values of TM or PGM are achieved in treatments that suitably reduced LPO rate via supplementation of antioxidant. These supplementations will keep the cell membranes against oxidative damage via regulation of ROS production.
In addition, it has been proved that sperm motility parameters are related to mitochondrial function which was affected in turn by the production of ATP in the inner mitochondrial membrane which is transferred into the microtubules to induce sperm motility. It is commonly believed that sperm motion reduction is primarily associated with damaged mitochondria due to freezing procedure (Ruiz-Pesini et al. 2001). Also, previous studies clarify that there is a clear correlation between mitochondrial enzymatic activities and sperm motility in human sperm (Ruiz-Pesini et al. 2001). In the other words, SOD, GPx and CAT enzyme activity disorder can be influenced on sperm motion parameters via reduced ATP production in the mitochondria. Therefore, in the present study, the improvement of sperm TM in the semen samples (I treatment) of both Holstein and Simmental breeds and PGM in Holstein is probably due to the reducing of H2O2 via GPx activity but, not increasing of SOD activity. Aitken (1995) suggested that the function of SOD may occur in the milieu with balance between SOD and glutathione, otherwise, the excess production of H2O2 via increasing SOD activities or accumulation of H2O2 without conversion into O2 and H2O by GPx may lead to the peroxidative damage. On the other hand, excessive accumulation of H2O2 in intercellular area will damage mitochondrial membrane potential decreasing motility parameters drastically. The results of this study showed that after thawing samples, PGM, VCL, VAP and VSL parameters of
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Simmental and VCL, VAP and VSL parameters of Holstein breed samples did not change significantly; it seems that the progressive of sperm cells motility, especially in Simmental samples, in Tris-egg-yolk medium have been impaired due to medium density or physical particulars, so values of these parameters discloses significantly unchanged between treatments. In Holstein semen samples, the addition of GSH 9 SOD to the freezing medium improved sperm viability compared to control group. According to previous reports, there is a negative correlation between MDA production and sperm viability (Guthrie and Welch 2012); moreover, Lipid peroxides are spontaneously generated in the sperm plasma membrane and are released by the action of phospholipase A2. Since, the peroxides are associated with impaired sperm functions and viability (Sikka 1996), the positive results obtained from these parameters in semen might be associated with reduction of phospholipase A2 and the positive changes in cell membrane, especially reduction of LPO. The higher rate of viable sperm suggested that, concentration (5 9 100) of combination antioxidants probably can protect fatty acid bands of cell membranes of spermatozoa against free radicals; which will lead to increasing viability rate. The membrane Integrity damages caused an increase in membrane permeability and a decrease in the ability of sperm to control the intracellular concentrations of ions which are involved in the sperm motility (Baumber et al. 2000). Moreover, cryodamage caused increasing permeability and transportation disorder (ions, sugars and etc.) in cell membranes via physical and/or oxidative factors during freeze–thawing process. The ultra-stretcher of membranes sperm such as proteins, lipids, phospholipids and protein/ phospholipid ratio, sorely are impaired via cold shock leading to depression of structural integrity and viability rate (Medeiros et al. 2002). Thus, it seems that in this study addition of combinative antioxidants (GSH 9 SOD) protected ultra-stretcher of membranes sperm via limiting oxidative factors. The addition of GSH 9 SOD to freezing medium increased sperm plasma membrane integrity of Holstein bull semen following the freeze–thaw process. The various responses to antioxidants’ additives between the breeds may be associated with several elements namely cholesterol/phospholipids ratio, content of lipids in the bilayer, degree of hydrocarbon chain saturation and protein/phospholipid ratio (Medeiros
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et al. 2002). Moreover, data from our study revealed different values of MDA, GPx and SOD between both breeds, while other parameters influence these values as well; therefore, the obtained variant and results disclosed functional and biochemical difference between breeds. Nichi et al. (2006) suggested that there were different resistance against ROS production between semen samples of different bull breeds. Therefore, obtained differences between results of this study from each breed is acceptable. Addition of these levels of mixed antioxidant to semen freezing extender of Simmental did not improve significantly major sperm parameters after thawing process. Hence, our result and above mentioned information suggested that, exerting lower concentrations of mixed antioxidants (lower than treatment I) may have a good effect on spermatozoa features of Simmental.
Conclusions supplementation of in combination antioxidants (GSH 9 SOD) particularly 5 9 100 and or probably 7.5 9 100 concentrations in the freezing medium can suppress the adverse effects on the motility, viability and plasma membrane integrity in bull spermatozoa during freeze–thaw process. The results indicate that the protective effects of these combination antioxidants on spermatozoa are associated with a reduction in lipid peroxidation as a consequence of increased activities of the antioxidant enzymes and synergistic effects of them to scavenge the ROS generated during the freezing process. Acknowledgments The authors thank the Animal Breeding Center of West and Northwest of the country, Sheikh Hasan, Tabriz, Iran for supplying the semen samples and the equipment for cryopreservation.
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glutathione preserves sperm function. J Androl 26(3):396–404 Gadea J, Gumbao D, Mata´s C, Romar R (2005b) Supplementation of the thawing media with reduced glutathione improves function and the in vitro fertilizing ability of boar spermatozoa after cryopreservation. J Androl 26(6):749–756 Gadea J, Gumbao D, Ca´novas S, Garcı´a-Va´zquez FA, Grullo´n LA, Gardo´n JC (2008) Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen-thawed bull spermatozoa. Int J Androl 31(1):40–49 Gadea J, Molla M, Selles E, Marco M, Garcia-Vazquez F, Gardon J (2011) Reduced glutathione content in human sperm is decreased after cryopreservation: effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology 62(1):40–46 Gil J, Lundeheim N, So¨derquist L, Rodrı´guez-Martı´nez H (2003) Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen. Theriogenology 59(5):1241–1255 Gil-Guzman E, Ollero M, Lopez M, Sharma R, Alvarez J, Thomas A, Agarwal A (2001) Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Hum Reprod 16(9):1922–1930 Guthrie H, Welch G (2012) Effects of reactive oxygen species on sperm function. Theriogenology 78(8):1700–1708 Kasimanickam R, Pelzer KD, Kasimanickam V, Swecker WS, Thatcher CD (2006) Association of classical semen parameters, sperm DNA fragmentation index, lipid peroxidation and antioxidant enzymatic activity of semen in ram-lambs. Theriogenology 65(7):1407–1421 Lapointe J, Bilodeau J-F (2003) Antioxidant defenses are modulated in the cow oviduct during the estrous cycle. Biol Reprod 68(4):1157–1164 Medeiros C, Forell F, Oliveira A, Rodrigues J (2002) Current status of sperm cryopreservation: why isn’t it better? Theriogenology 57(1):327–344 Meister A (1994) Glutathione, ascorbate, and cellular protection. Cancer Res 54:1969s–1975s Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247(10):3170–3175 Nichi M, Bols P, Zu¨ge R, Barnabe V, Goovaerts I, Barnabe R, Cortada C (2006) Seasonal variation in semen quality in Bos indicus and Bos taurus bulls raised under tropical conditions. Theriogenology 66(4):822–828 Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–358 Paglia DE, Valentine WN (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70(1):158–169 Perumal P, Selvaraju S, Selvakumar S, Barik A, Mohanty D, Das S, Das R, Mishra P (2011) Effect of pre-freeze addition of cysteine hydrochloride and reduced glutathione in semen of crossbred Jersey bulls on sperm parameters and conception rates. Reprod Domest Anim 46(4):636–641 Piantadosi CA (2008) Carbon monoxide, reactive oxygen signaling, and oxidative stress. Free Radical Biol Med 45(5):562–569
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Cell Tissue Bank Roca J, Rodriguez MJ, Gil MA, Carvajal G, Garcia EM, Cuello C, Vazquez JM, Martinez EA (2005a) Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase. J Androl 26(1):15 Roca J, Rodrı´guez MJ, Gil MA, Carvajal G, Garcia EM, Cuello C, Vazquez JM, Martinez EA (2005b) Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase. J Androl 26(1):15–24 Rossi T, Mazzilli F, Delfino M, Dondero F (2001) Improved human sperm recovery using superoxide dismutase and catalase supplementation in semen cryopreservation procedure. Cell Tissue Bank 2(1):9–13 Ruiz-Pesini E, Alvarez E, Enrı´quez JA, Lo´pez-Pe´rez MJ (2001) Association between seminal plasma carnitine and sperm
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mitochondrial enzymatic activities. Int J Androl 24(6):335–340 Sikka SC (1996) Oxidative stress and role of antioxidants in normal and abnormal sperm function. Front Biosci 1:e78– e86 Storey BT (1997) Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol Hum Reprod 3(3):203–213 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(7):1249–1255
ORIGINAL PAPER
Effects of in combination antioxidant supplementation on microscopic and oxidative parameters of freeze–thaw bull sperm R. Olfati Karaji • H. Daghigh Kia • I. Ashrafi
Received: 16 October 2013 / Accepted: 22 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The present study was conducted to determine the effect of reduced glutathione (GSH) and superoxide dismutase (SOD) supplementation in the bull semen freezing extender on post-thaw parameters of Holstein and Simmental bull sperm. Semen were collected from seven bulls (four Holstein and three Simmental) and cryopreserved in the Trisegg-yolk based extender as control group and supplemented with various concentrations of GSH 9 SOD (5 9 100, 7.5 9 100, 5 9 150, and 7.5 9 150 mM 9 IU mL-1) in treatment groups. Microscopic parameters were evaluated in terms of total motility parameters using computer assisted semen analysis and viability and membrane integrity were assessed using Eosin– Nigrosin stains and hypo-osmotic swelling test (HOST), respectively after thawing the semen. Malonaldialdehyde (MDA) level, SOD and glutathione peroxides (GPx) activities were assessed immediately after thawing. Results showed that supplementation of the cryopreservation medium with various concentrations of GSH 9 SOD improved total motility (TM) and progressive motility parameters for Holstein (P \ 0.05) R. Olfati Karaji H. Daghigh Kia (&) Department of Animal Science, Faculty of Agriculture, University of Tabriz, 29 Bahman Boulevard, East Azerbaijan, 5166614766 Tabriz, Iran e-mail: [email protected]; [email protected] I. Ashrafi Young Researchers and Elites Club, Science and Research Branch, Islamic Azad University, Tehran, Iran
semen, and values of TM and HOST for Simmental semen compared to the control group (P \ 0.01) after semen thawing. Addition of antioxidant to Holstein semen samples decreased the level of MDA and increased GPx activities compared to control groups (P \ 0.05). SOD activities increased in Simmental bull samples compare to the control group (P \ 0.01), but not differ in Holstein, while these activities. In conclusion, supplementation of antioxidant to the semen extender as combination (GSH 9 SOD) improved the semen post-thaw qualities which may be associated with a reduction in lipid peroxidation as well as an increase in the antioxidant enzyme activities. Keywords Bull sperm Cryopreservation Reduced glutathione Superoxide dismutase
Introduction Semen storage and cryopreservation are associated with cold shock and atmospheric oxygen (Alvarez and Storey 1982; Arav et al. 2002; Cocchia et al. 2011; Medeiros et al. 2002) that lead to higher production of reactive oxygen species (ROS) and an imbalance between free radicals and antioxidant system of extended semen. Oxygen radicals at physiological concentrations are also known to have a positive effect in intracellular signaling involved in the regular processes of cell proliferation, differentiation and
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migration (Piantadosi 2008). But, excessive production of ROS may had a toxic effect through the production of free radicals affecting functional parameters of the sperm cells such as deprivation of motility (Sikka 1996), inactivation of glycolytic enzymes and damage to the acrosomal membranes (Alvarez and Storey 1984) which would render the sperm cell unable to fertilize the oocyte (Gil-Guzman et al. 2001). During semen cryopreservation process, a large amount of the cytoplasmic component such as antioxidants will leak out to extracellular irreversibly. These events decrease enzymatic defensive system capacity of semen (Bilodeau et al. 2000; Bucak et al. 2008). Spermatozoa and seminal plasma possess an antioxidant system comprising catalase (CAT), glutathione peroxides (GPx) and superoxide dismutase (SOD) as the main antioxidant enzymatic factors (Bilodeau et al. 2000; Meister 1994) and also have glutathione (GSH), alpha-tocoferol, vitamin C, betacarotene, cysteine and hypotaurine as the non-enzymatic antioxidant to prevent oxidative damage (Agarwal et al. 2005). However, this antioxidant capacity in sperm cells, due to the small cytoplasmic component is limited for a long storage and cryopreservation. In addition, according to the several reports, cryopreservation of the semen decreased GSH content of sperm significantly as much as 64, 32 and 58–78 % in human (Gadea et al. 2011), boar (Gadea et al. 2004) and bull sperm (Bilodeau et al. 2000; Stradaioli et al. 2007), respectively, and also reduced SOD value by 50 % in bull sperm (Bilodeau et al. 2000). Therefore, mammalian sperm’s ability is not sufficient for preventing lipid peroxidation (LPO) production during the freeze–thaw process (Storey 1997; Lapointe and Bilodeau 2003). It is suggested that supplementation of antioxidant in the freezing medium may prevent excessive ROS production in semen (Donoghue and Donoghue 1997) during cryopreservation. Antioxidant molecules could decrease the impact of oxidative stress and therefore improve sperm quality after thawing. Metalloproteinase like SOD protects the sperm against O2- toxicity and also LPO by dismutation of O2 to H2O2. Previous studies reported that addition of SOD to the semen extenders improve the quality of semen (Cocchia et al. 2011; Sikka 1996) such as total motility (TM) and the percentage of viable sperm with intact acrosome following freeze–thaw process in boar semen (Roca et al. 2005a). Glutathione also is able to
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react with ROS directly (Bucak et al. 2008) and protects the sperm due to its sulfur and cysteine content. It is reported that addition of GSH to freezing extender improves the quality and fertilizing capacity of bull (Gadea et al. 2008; Perumal et al. 2011), ram (Bucak et al. 2008) and boar sperm (Gadea et al. 2005a). There are simultaneous irreversible reduction of different intracellular antioxidant contents such as SOD and GSH during semen cryopreservation process (Bilodeau et al. 2000; Gadea et al. 2011; Stradaioli et al. 2007). Further, applying single antioxidants was demonstrated to be insufficient to induce the sperm characteristics (Rossi et al. 2001; Gadea et al. 2004; Caˆmara et al. 2011). In recent years, the combination of antioxidants has been used successfully in the semen extender of human (Rossi et al. 2001), bull (Foote et al. 2002) and boar (Roca et al. 2005b). In the present study a combination of various concentrations of GSH and SOD antioxidants supplemented in semen extender of Holstein and Simmental bull semen to evaluate the quality and biochemical parameters of bull semen following freeze–thaw process. Moreover, the Holstein and Simmental breeds are major species which are commonly used in many countries as artificial insemination; conducting this research, we want to find major differences between semen samples of both Holstein and Simmental bulls after freezing– thawing process via addition of different levels of mixed antioxidants.
Materials and methods Semen source and preparation This study was performed in Animal Breeding Center of West and Northwest of Country, located in Tabriz, Iran. Ejaculates were collected from seven bulls (four Holstein and three Simmental), aged between 3 and 4 years, regularly used for breeding purpose based on their fertility estimation through in vivo fertility tests. Total of 35 ejaculates (five ejaculates per bull) were collected twice a week using an artificial vagina (45 °C). The ejaculates were transferred to the laboratory and submerged in a water bath (34 °C), until semen evaluation. The volume of ejaculate was estimated in a conical tube graduated at 0.1 mL intervals. The sperm concentration was determined by
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means of an Accucell photometer (IMV, L’Aigle, France). The ejaculates were evaluated for freezing process if the following criteria were met: volume between 5 and 12 mL; sperm concentration of C19109 sperm mL-1; motility of C70 and B10 % 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 Tris-egg-yolk based medium [60 mM citric acid, 0.25 M Tris–(hydroxymethyl)-aminomethane, 69 mM fructose, 20 % egg yolk, 7 % glycerol, 25 mg gentamycin sulfate, 5 mg tylosin tartrate, 15 mg lincomycin hydrochloride and 30 mg spectinomycin sulfate in 80 mL double distilled water] was used for the pooled ejaculates (Bilodeau et al. 2002). The extended semen samples with Tris-egg-yolk based medium were divided into the equal aliquots in order to use them in combined treatments. The treatments were prepared by applying in combination GSH and SOD (GSH 9 SOD) including 5 9 100 (I), 7.5 9 100 (II), 5 9 150 (III), 7.5 9 150 (IV) mM 9 IU mL-1 concentration, respectively. In control group (V) the Tris-egg-yolk based extender contained no additive antioxidant. Semen samples were cooled slowly to 5 °C for 2 h, and then added to the various extender treatments. The extended semen was then packaged in 0.5 mL straws. The straws were sealed via automatic filling and sealing machine (IMV Technologies, MRS 1, France) and they were frozen by a semi-automatic freezer machine (mini tube, Germany) with liquid nitrogen vapor. The straws were cooled at approximately -15 °C min-1 from ?5 to -150 °C. Then, they were transferred to a liquid nitrogen tank and stored. Frozen straws were thawed at 37 °C for 40 s in a water bath immediately before evaluation. Semen evaluation Viability and motility parameters The sperm motion parameters such as TM, PGM, VCL, VAP and VSL of frozen–thawed samples are evaluated after thawing by computer assay sperm analysis (CASA) which is equipped with a phasecontrast microscopy system (Nikon, Japan), a warm stage at 37 °C, a digital video camera (SAMSUNG, Korea) and a computer for analyzing kinetic motion
spermatozoa (Hoshmand Fanavaran, Tehran, Iran). To analyze the semen, the samples were incubated in a water bath at 37 °C for 10 min. The following motility values were recorded using CASA: Total motility (TM, %), progressive motility (PGM, %), straight linear velocity (VSL, lm s-1), curvilinear velocity (VCL, lm s-1) and average path velocity (VAP, lm s-1). For evaluation of these parameters, 5 lL drop of the sample was placed on a warmed (37 °C) slide and covered with a cover slip. Sperm motility parameters were estimated in three different microscopic fields per each semen sample. Images were obtained at 4009 magnifications using a phase contrast objective microscope (Nikon, Tokyo, Japan). The mean of the three successive estimations ([200 spermatozoa per slide) was recorded as the final data (TM and PGM percentage). The viability of spermatozoa in the samples was assessed by means of the Eosin–Nigrosin stain method after thawing. The final composition of the stain was: Eosin-Y 1.67 g, Nigrosin 10 g, and sodium citrate 2.9 g were 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. The viability of sperms was assessed by counting 400 sperm cells in a brightness microscope (Olympus DP 50) at 1,0009 magnification, using immersion oil. Sperm expression partial or complete purple staining was considered nonviable; only sperm showing strict exclusion of stain were considered as viable (Balestri et al. 2007). Assessment of functional integrity for membrane The hypoosmotic swelling test (HOST) was used to evaluate the functional integrity of the sperm membrane after thawing, based on curled and swollen tails. This was performed by incubating 30 mL of semen with 300 mL of a 100 mOsM hypoosmotic solution (9 g fructose ? 4.9 g sodium citrate per liter of distilled water) at 37 °C for 60 min. The osmotic pressure of solution was controlled and calibrated via an Osmometer (Labx, Mark III, USA). Following incubation, 0.2 mL of the mixture was spread with a cover slip on a slide. Four hundred sperm were examined with 4009 magnification with bright-field microscopy. Sperm with swollen or coiled tails were recorded (Buckett et al. 1997).
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Biochemical assay The samples (0.5 mL straw) were thawed before the beginning of biochemical assays. An aliquot (500 lL) of semen obtained from each sample was centrifuged at 8009g for 10 min, sperm pellets were separated and washed by resuspending in PBS and recentrifuging for 3 times. Following last centrifugation, 1 mL of deionized water was added to spermatozoa and they were snapfrozen and stored at -70 °C until further analysis. Lipid peroxidation Lipid peroxidation rate of sperm was estimated by measuring the level of malonaldialdehyde (MDA), using the thiobarbituric acid (TBA) based on the method described by Ohkawa et al. (1979). Malonaldialdehyde levels were measured after supplementing 100 lL of sperm cell pellet suspension added to TBA–TCA reagent (1.25 mL of 20 % w/v TCA, 1 mL of 0.2 % w/v TBA and 1.25 mL of 0.05 M H2So4). The mixture was treated in boiling water (95 °C) for 60 min. After cooling, 4 mL of butanol was added into the mixture following which it was centrifuged for 5 min at 1,500g for 5 min. The supernatant was removed and the absorbance was measured at 535 nm (biophotometer plus, Eppendorf, Germany). The MDA concentration was determined by the specific absorbance coefficient (1.56 9 105 lmol cm-3). The produced MDA is expressed as pmol mg-1 protein of sperm cell pellet. Glutathione peroxides activity Glutathione peroxides activity was measured based on the protocol of Paglia and Valentine (1967). Glutathione peroxides catalyzed the oxidation of GSH by Cumene Hyroperoxide. Owing to the presence of Glutathione reductase (GR) and nicotin amide adenine dinucleotide phosphate (NADPH), the oxidized Glutathione (GSSG) is immediately converted into the reduced form with a concomitant oxidation of NADPH to NADP?. The oxidation of NADPH to NADP? is associated with a decrease in the absorbance of 340 nm via the spectrophotometer (biophotometer plus, Eppendorf, Germany) indicating GPx activities. The rate of decrease in absorbance at 340 nm was directly proportional to the GPx activities. The reaction mixture was containing 20 lL of sample (3 mg mL-1 sperm pellet cells), 1 mL of solution (4 mM Glutathione, 5 9 10-5 u mL-1 glutathione
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reductase, 0.34 mM NADPH, 0.05 mM phosphate buffer; pH 7.2 and 4.3 mM EDTA) and 40 lL of Cumene Hyroperoxide. The absorbance of the mixture sample was measured after 20 min using a biophotometer (biophotometer plus, Eppendorf, Germany) at 340 nm wave length. Glutathione peroxides values expressed as mU mg-1 protein of sperm cell pellet. Superoxide dismutase activity Superoxide dismutase activity was determined using the method proposed by Misra and Fridovich (1972). This method was employed the Xanthine and Xanthine oxides to generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyl tetrazolium chloride (INT) to form a red Formazan dye. The SOD activity was measured by the degree of inhibition of this reaction. One unit of SOD causes a 50 % inhibition of the rate of reduction of INT under the conditions of the assay. The reaction mixture for each standard (S) consisted of 30 lL samples (sperm cells pellet) 1 mL reagent (0.05 mM Xanthine, 0.025 mM INT., 40 mM CAPS, pH, 10.2 and 0.94 mM EDTA) and 150 lL of Xanthine Oxidase. Cuveite containing mixture sample was assayed using a biophotometer (biophotometer plus, Eppendorf, Germany) at a wave length of 505 nm in two steps: (1) The initial absorbance after 30 S and (2) The second absorbance 3 min after adding Xanthine oxidase. A control was also evaluated. Superoxide dismutase value was expressed as IU g-1 protein in the sperm cell pellet. Statistical analysis Statistical analysis was performed using SAS software (version 9.1.3). Semen quality data (Total motility, progress motility, viability and membrane integrity) were subjected to arcsine transformation before being analyzed; all the data obtained from this study analyzed using the GLM procedure. Least square means were performed to determine the differences between experimental treatments throughout post-thaw evaluation.
Result The effects of different doses of GSH 9 SOD on postthaw parameters such as PGM, TM, VCL, VAP, VSL,
51.91 ± 3.80ab
II
29.36 ± 5.39
47.26 ± 2.73b 47.56 ± 3.59b
V -1
30.65 ± 3.57
49.90 ± 7.15
IV
32.93 ± 3.50
31.60 ± 3.77
33.97 ± 4.51
III
b
59.12 ± 4.98
I
a
44.83 ± 2.88
32.70 ± 4.65
b
36.95 ± 3.96
b
47.55 ± 2.44
80.63 ± 2.07
81.74 ± 8.94
85.04 ± 5.88
81.46 ± 3.98
86.14 ± 5.33
88.57 ± 2.44
87.51 ± 4.19
91.86 ± 3.56
88.65 ± 5.60
92.64 ± 3.36
VCL
-1
50.60 ± 3.35
51.62 ± 7.25
55.86 ± 3.13
53.65 ± 2.63
55.00 ± 1.74
58.90 ± 5.52
62.31 ± 3.61
65.77 ± 3.24
61.19 ± 4.77
65.21 ± 6.64
VAP
35.10 ± 4.05
37.37 ± 6.64
40.84 ± 5.78
39.18 ± 2.52
42.75 ± 5.17
41.84 ± 2.71
42.47 ± 3.65
46.61 ± 2.19
42.25 ± 4.42
45.86 ± 3.43
VSL
-1
41.32 ± 2.96b
43.39 ± 4.84b
45.19 ± 3.37
b
45.58 ± 2.75b
51.11 ± 3.69
a
46.78 ± 5.19
b
50.33 ± 1.52
ab
57.17 ± 5.48a
52.72 ± 4.44
ab
57.70 ± 2.76a
HOST (%)
55.69 ± 3.50
56.62 ± 2.09
59.06 ± 2.10
55.89 ± 3.36
61.62 ± 3.13
53.60 ± 5.19bc
54.97 ± 4.29b
60.66 ± 5.48ab
57.02 ± 4.46b
66.30 ± 4.08a
Viability (%)
I: GSH 9 SOD (5.0 9 100 mM 9 IU mL ); II: GSH 9 SOD (5.0 9 150 mM 9 IU mL ); III: GSH 9 SOD (7.5 9 100 mM 9 IU mL ); IV: GSH 9 SOD (7.5 9 150 mM 9 IU mL-1); V: Control. TM total motility, PGM progressive motility, VCL curvilinear velocity, VAP average path velocity, VSL straight linear velocity, HOST hypo-osmotic swelling test. Different superscripts (a, b, c) in the same column indicate a significant difference (P \ 0.05)
Simmental
V
IV
ab
40.83 ± 3.75a
b
48.98 ± 3.12ab
III
35.75 ± 4.33
ab
42.40 ± 4.15a
ab
53.25 ± 2.74a 48.69 ± 2.94
I
Holstein
PGM
TM (%)
Parameters
II
Treatments
Breeds
Table 1 The different concentrations of SOD 9 GSH on motion parameters, HOST and sperm viability percentage after thawing in Holstein and Simmental bull spermatozoa (Mean ± SD)
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viability and membrane integrity of sperms (positive responds to HOST) in the both breeds are represented in Table 1. Compared to the IV and control treatments, addition of GSH 9 SOD (treatment I) to the semen freezing extender of Holstein bulls increased (P \ 0.05) the percentage of TM after thawing. Regarding Simmental bulls, addition of treatment I increased (P \ 0.01) compared to the IV, II and control treatments; however no significant differences were found between other treatments in both breeds. Sperm progressive motility of Holstein samples at the levels of 5 9 100 and 7.5 9 100 (mM 9 IU mL-1) was also higher than control group after thawing (P \ 0.05). The percentage of sperm PGM did not differ (P [ 0.05) between all treatments in the Simmental samples. No significant differences (P [ 0.05) were observed in VSL, VCL and VAP in the freezing medium supplemented with doses of antioxidant in both breeds following the freeze–thawing process. Supplementation of treatment I with the medium increased viable sperm rates in comparison with the IV, II and V (control) treatments in Holstein samples evaluated after thawing (P \ 0.05). The percentage of viable sperms did not differ among all treatment in Simmental breed (P [ 0.05). Following thawing, the HOST was improved (P \ 0.05) in freezing extender supplemented with I and III treatments in Holstein (P \ 0.05) and I treatment in Simmental (P \ 0.01) samples compared to control group, however no changes were found among other treatments. Results of MDA values and enzyme (GPx and SOD) antioxidant activities in thawed both bull semen’s are summarized in Table 2. Inclusion of various concentrations of antioxidant in extended semen of Holstein caused a decrease in the level of MDA, achieving its lowest values in I and III treatments (P \ 0.05). However no significant differences were detected in the Simmental semen samples. The activity of GPx in Holstein semen samples increased by inclusion of all doses of the antioxidants (P \ 0.05) and the highest activity was detected in I treatment compared to the control group (P \ 0.05). Moreover, none of the treatment groups influenced the activity of GPx in Simmental semen samples. Superoxide dismutase activities did not differ (P [ 0.05) between control and treatment groups in Holstein extended semen while these activities increased (P \ 0.01) in the IV treatments than to the other treatments in Simmental bull samples.
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Discussion Defense systems in the sperm freezing medium may needed to overcome the oxidative stress against ROS production during freeze–thaw process which are based on the combined action of enzymatic and nonenzymatic antioxidants. According to our knowledge, the effect of combination of GSH 9 SOD antioxidants on microscopic and oxidative parameters including antioxidant enzyme activities and LPO in cryopreserved bull sperm had not previously been assessed. In the current study, we have used the combination of GSH and SOD in different concentrations, based on the data obtained from previous studies (Bucak et al. 2008; Cocchia et al. 2011; Foote et al. 2002; Gadea et al. 2004, 2011; Perumal et al. 2011; Roca et al. 2005b; Rossi et al. 2001). Previous studies have reported that the freeze–thaw process may cause significant changes in the organization of sperm membrane lipids (Gadea et al. 2005b, 2008). This happen may due to the deficiency in the endogenous antioxidant capacity and excessive production of ROS in the cells (Aitken et al. 1993; Bilodeau et al. 2000). Previous experiments in sperm of human also suggested that the freeze–thaw process enhances LPO activities (Alvarez and Storey 1992). The presence of double bonds in carbon chain polyunsaturated fatty acids in sperm cells makes them susceptible to cascade of oxidative attack and fat oxidation. There is a good evidence to indicate that the rate of lipid peroxidation correlated inversely with viability, motility and performance of the sperm (Aitken et al. 1993). Superoxide dismutase, GPx and CAT enzymes reduced the LPO cascade of membranes via scavenging of superoxide ions and hydrogen peroxide (Sikka 1996). In this investigation, the level of MDA decreased and GPx activities increased compared to the control group in Holstein samples which supplemented with 5 9 100 GSH 9 SOD levels after thawing. In this way, the reduction of MDA level may be closely related to the proper function of GPx and CAT due to converting of H2O2 into O2 and H2O. On the other hand, adding this amount of mixed antioxidant reduced lipid peroxidation positively, via improving ROS scavenger systems. An increase in SOD activities in the 7.5 9 150 concentration of Simmental semen samples may be associated with an increase in the production rate of hydrogen peroxide and in turn, it will increase the rate of sperm LPO
Cell Tissue Bank Table 2 The effect of different concentrations of SOD 9 GSH on some biochemical parameters after thawing in bull spermatozoa (Holstein and Simmental) (Mean ± SD) Breed
Treatments
MDA (pmol mg-1 protein)
GPx (mU mg-1 protein)
SOD (mU mg-1 protein)
Holstein
I
10.25 ± 0. 61b
64.16 ± 6.35a
15.76 ± 0.70
II
Simmental
ab
11.05 ± 0.76
ab
51.97 ± 7.25
15.60 ± 1.43
III
9.14 ± 0.84b
61.91 ± 4.74ab
14.99 ± 1.18
IV
11.60 ± 1.49ab
57.69 ± 4.46ab
16.14 ± 1.34
V
12.21 ± 1.07a
55.51 ± 4.55b
14.58 ± 1.30
I
14.31 ± 0.66
52.73 ± 4.63
11.12 ± 1.10b
II
14.87 ± 0.66
51.15 ± 3.92
12.42 ± 0.86b
III
14.59 ± 1.34
52.43 ± 3.00
12.23 ± 1.10b
IV
16.09 ± 1.27
48.85 ± 6.22
14.23 ± 0.71a
V
15.31 ± 0.74
46.46 ± 7.65
11.93 ± 0.95b
I: GSH 9 SOD (5.0 9 100 mM 9 IU mL-1); II: GSH 9 SOD (5.0 9 150 mM 9 IU mL-1); III: GSH 9 SOD (7.5 9 100 mM 9 IU mL-1); IV: GSH 9 SOD (7.5 9 150 mM 9 IU mL-1); V: Control. MDA malonaldialdehyde, GPx glutathione peroxidase, SOD superoxide dismutase. Different superscripts (a, b) in the same column indicate a significant difference (P \ 0.05)
simultaneously. These results are consistent with previous studies (Roca et al. 2005b) indicating that H2O2 has a more adverse impact on frozen–thawed bull spermatozoa compared to O2-. Accumulation of H2O2 may be the most common cause of increasing lipid peroxidation (0.074) due to increasing SOD activity showing no changes in GPx activity. The present study demonstrated the importance of maintaining TM (in the both breeds) and PGM (Holstein breed) parameters when their semen samples are supplemented with mixed antioxidants. According to Rossi et al. (2001) applying this way, supplementation of human freezing medium via both SOD and catalase significantly improves viability and motility of sperm cells; these results was convergence with our hypothesis considering the application of combination antioxidants supplementation on bull freezing medium. Therefore, these results could be induced from their combined and simultaneous actions (the combination of GSH and SOD or CAT and SOD) on superoxide anion and hydrogen peroxide. Previous studies have also reported a negative correlation between LPO rate and sperm motility parameters (Kasimanickam et al. 2006; Aitken et al. 1993). Our results uphold above studies since according to our study a higher values of TM or PGM are achieved in treatments that suitably reduced LPO rate via supplementation of antioxidant. These supplementations will keep the cell membranes against oxidative damage via regulation of ROS production.
In addition, it has been proved that sperm motility parameters are related to mitochondrial function which was affected in turn by the production of ATP in the inner mitochondrial membrane which is transferred into the microtubules to induce sperm motility. It is commonly believed that sperm motion reduction is primarily associated with damaged mitochondria due to freezing procedure (Ruiz-Pesini et al. 2001). Also, previous studies clarify that there is a clear correlation between mitochondrial enzymatic activities and sperm motility in human sperm (Ruiz-Pesini et al. 2001). In the other words, SOD, GPx and CAT enzyme activity disorder can be influenced on sperm motion parameters via reduced ATP production in the mitochondria. Therefore, in the present study, the improvement of sperm TM in the semen samples (I treatment) of both Holstein and Simmental breeds and PGM in Holstein is probably due to the reducing of H2O2 via GPx activity but, not increasing of SOD activity. Aitken (1995) suggested that the function of SOD may occur in the milieu with balance between SOD and glutathione, otherwise, the excess production of H2O2 via increasing SOD activities or accumulation of H2O2 without conversion into O2 and H2O by GPx may lead to the peroxidative damage. On the other hand, excessive accumulation of H2O2 in intercellular area will damage mitochondrial membrane potential decreasing motility parameters drastically. The results of this study showed that after thawing samples, PGM, VCL, VAP and VSL parameters of
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Cell Tissue Bank
Simmental and VCL, VAP and VSL parameters of Holstein breed samples did not change significantly; it seems that the progressive of sperm cells motility, especially in Simmental samples, in Tris-egg-yolk medium have been impaired due to medium density or physical particulars, so values of these parameters discloses significantly unchanged between treatments. In Holstein semen samples, the addition of GSH 9 SOD to the freezing medium improved sperm viability compared to control group. According to previous reports, there is a negative correlation between MDA production and sperm viability (Guthrie and Welch 2012); moreover, Lipid peroxides are spontaneously generated in the sperm plasma membrane and are released by the action of phospholipase A2. Since, the peroxides are associated with impaired sperm functions and viability (Sikka 1996), the positive results obtained from these parameters in semen might be associated with reduction of phospholipase A2 and the positive changes in cell membrane, especially reduction of LPO. The higher rate of viable sperm suggested that, concentration (5 9 100) of combination antioxidants probably can protect fatty acid bands of cell membranes of spermatozoa against free radicals; which will lead to increasing viability rate. The membrane Integrity damages caused an increase in membrane permeability and a decrease in the ability of sperm to control the intracellular concentrations of ions which are involved in the sperm motility (Baumber et al. 2000). Moreover, cryodamage caused increasing permeability and transportation disorder (ions, sugars and etc.) in cell membranes via physical and/or oxidative factors during freeze–thawing process. The ultra-stretcher of membranes sperm such as proteins, lipids, phospholipids and protein/ phospholipid ratio, sorely are impaired via cold shock leading to depression of structural integrity and viability rate (Medeiros et al. 2002). Thus, it seems that in this study addition of combinative antioxidants (GSH 9 SOD) protected ultra-stretcher of membranes sperm via limiting oxidative factors. The addition of GSH 9 SOD to freezing medium increased sperm plasma membrane integrity of Holstein bull semen following the freeze–thaw process. The various responses to antioxidants’ additives between the breeds may be associated with several elements namely cholesterol/phospholipids ratio, content of lipids in the bilayer, degree of hydrocarbon chain saturation and protein/phospholipid ratio (Medeiros
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et al. 2002). Moreover, data from our study revealed different values of MDA, GPx and SOD between both breeds, while other parameters influence these values as well; therefore, the obtained variant and results disclosed functional and biochemical difference between breeds. Nichi et al. (2006) suggested that there were different resistance against ROS production between semen samples of different bull breeds. Therefore, obtained differences between results of this study from each breed is acceptable. Addition of these levels of mixed antioxidant to semen freezing extender of Simmental did not improve significantly major sperm parameters after thawing process. Hence, our result and above mentioned information suggested that, exerting lower concentrations of mixed antioxidants (lower than treatment I) may have a good effect on spermatozoa features of Simmental.
Conclusions supplementation of in combination antioxidants (GSH 9 SOD) particularly 5 9 100 and or probably 7.5 9 100 concentrations in the freezing medium can suppress the adverse effects on the motility, viability and plasma membrane integrity in bull spermatozoa during freeze–thaw process. The results indicate that the protective effects of these combination antioxidants on spermatozoa are associated with a reduction in lipid peroxidation as a consequence of increased activities of the antioxidant enzymes and synergistic effects of them to scavenge the ROS generated during the freezing process. Acknowledgments The authors thank the Animal Breeding Center of West and Northwest of the country, Sheikh Hasan, Tabriz, Iran for supplying the semen samples and the equipment for cryopreservation.
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