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Effects of L-glutamine on boar sperm quality during liquid storage at 17°C
Accepted Manuscript Title: Effects of L-Glutamine on boar sperm quality during liquid storage at 17◦ C Authors: Shunwei Wang, Meng Sun, Na Wang, Kang Yang, Haitao Guo, Jingran Wang, Yuting Zhang, Shunli Yue, Jiabo Zhou PII: DOI: Reference:
S0378-4320(17)31045-X https://doi.org/10.1016/j.anireprosci.2018.02.011 ANIREP 5767
To appear in:
Animal Reproduction Science
Received date: Revised date: Accepted date:
19-12-2017 28-1-2018 13-2-2018
Please cite this article as: Wang S, Sun M, Wang N, Yang K, Guo H, Wang J, Zhang Y, Yue S, Zhou J, Effects of L-Glutamine on boar sperm quality during liquid storage at 17◦ C, Animal Reproduction Science (2010), https://doi.org/10.1016/j.anireprosci.2018.02.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of L-Glutamine on boar sperm quality during liquid storage at 17°C
Shunwei Wanga, Meng Suna, Na Wanga, Kang Yanga, Haitao Guoa, Jingran Wanga, Yuting Zhanga, Shunli Yueab*, Jiabo Zhouab*
College of Life Science Northeast Agricultural University, Harbin 150030, China
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Northeast Agricultural University, Harbin 150030, China
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Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province,
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ABSTRACT
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The quality of boar spermatozoa is affected by oxidative stress during preservation in vitro. It has been demonstrated that L-Glutamine (Gln) can effectively protect cells from oxidative stress-induced injury. There are, however, no reports to date evaluating the effects of Gln on boar semen liquid preservation at 17°C. The aims of the present study were to elucidate whether the addition of Gln to the extender BTS could improve the quality of boar spermatozoa when stored at 17°C and to determine the mechanism underlying Gln protection of spermatozoa against preservation-induced damage. Boar semen samples were collected and diluted with Beltsville Thawing Solution (BTS) containing different concentrations (0, 10, 20, 40 or 80 mM) of Gln. The results indicated the addition of 20 mM Gln to the BTS improved (P < 0.05) the motility, acrosome integrity and membrane integrity of boar sperm during liquid preservation. Interestingly, treatment of spermatozoa with Gln addition to the extender resulted in ROS quenching, while enhancing γ-glutamyl cysteine synthetase (γ-GCS) activity, and glutathione (GSH) content of spermatozoa. These results suggest that BTS supplemented with Gln can provide greater protective capacity to boar sperm against oxidative stress by enhancing GSH synthesis during liquid preservation. Keywords: L-Glutamine; Boar semen; Liquid preservation; Reactive oxygen species
1. Introduction
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Artificial insemination (AI) has been widely used by the modern swine production industry. Boar spermatozoa are highly susceptible to low temperatures (Yeste, 2015) because the membrane of boar spermatozoa has a low cholesterol/phospholipid ratio (Johnson et al., 2000). Liquid storage of semen has become a routine reproduction technique in pork production enterprises, as liquid stored semen is important in reducing disease risks and in increasing efficiency of semen usage (Li et al., 2017). With the emergence of long-term extenders for liquid storage, the use of cryopreserved sperm for AI in pork production enterprises has decreased (Yeste, 2015). Thus, the technical improvement of liquid storage has become more important in the swine production industry.
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The amino acid, L-Glutamine (Gln), which is the most abundant free amino acid in the body, has an important role in several cell-specific processes, including metabolism, protein synthesis and degradation, cell integrity, redox potential, and extracellular matrix (ECM) synthesis (Spodenkiewicz et al., 2016). This amino acid (Gln) is required for glutathione (GSH) synthesis, as it can be metabolized by the gamma-glutamyl cycle to produce glutathione (Roth et al., 2002). Glutathione can function as an antioxidant in semen, which provides for an intracellular defense to sperm against oxidative stress (Gadea et al., 2004). Oxidative stress is a primary factor that leads to damage of sperm functions due to greater synthesis of lipid peroxidation induced by reactive species (ROS) as a result of oxidative stress. The ROS are produced by sperm during the process of in vitro preservation, which leads to oxidative stress in sperm plasma membranes. Sperm will lose its fertilizing capacity if the polyunsaturated fatty acids in the sperm membrane are oxidized (Zhang et al., 2016). The Gln metabolism, via entry into the TCA cycle may enhance malic enzyme activity (NADP+ dependent), which will result in an increase in NADPH production (Zhang et al., 2016). This will subsequently affect the total oxidant and antioxidant status of cells. Addition of Gln to cells in vitro can lead to an increase in total glutathione concentration (Matés et al., 2002; Curi et al., 2005). The ratio of GSH to oxidized glutathione (GSSG) is the principal regulator of the cellular redox potential (Matés et al., 2002). Some studies have shown that when sperm were preserved in an extender containing glutathione, sperm quality after preservation was improved (Zhang et al., 2016; Shah et al., 2017).
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Supplementation of Gln improves the post-thaw motility and fertilizing potential of sperm in many species (Khlifaoui et al., 2005; Zhu et al., 2017). There are, however, less data available on the effect of Gln on boar sperm quality during liquid storage at 17°C. The aim of the present study was, therefore, to elucidate whether the addition of Gln to the extender could enhance boar sperm motility, acrosome integrity, and membrane integrity. The GSH content, γ-glutamyl cysteine synthetase (γ-GCS) activity, and intracellular ROS levels in spermatozoa were also measured during the processes of liquid preservation to ascertain the mechanism by which Gln protects spermatozoa during liquid storage.
2. Materials and methods
Unless noted, all chemicals and media used in this study were purchased from Sigma Chemical Co (St. Louis, MO, USA). 2.1.Semen collection
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Ejaculates were collected by gloved-hand method from five Large White male pigs, aged 1.5 to 2.5 years. The boars were allowed at least 3 days of sexual rests between collections. The semen was transported to the laboratory at 37 to 38°C within 30 min and filtered. Only semen samples with more than 70% sperm total motility (TMOT) and less than 15% morphologically abnormal spermatozoa were used (Betancourt et al., 2006; Schulze et al., 2017). Ejaculates (n = 36) were used in this experiment. The protocols for experiments using animals were subject to approval by the Animal Care and Use Committee of Northeast Agricultural University. 2.2. Semen processing
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The basic medium used for sperm extension was Beltsville Thawing Solution (BTS), which was composed of 205 mM glucose, 20.39 mM NaCl, 5.40 mM KCl, 15.01 mM NaHCO3, and 3.35 mM EDTA, penethamate and streptomycin (50 µg/mL; Kumaresan et al., 2009). The pH of the BTS extenders was adjusted to 7.2. Spermatozoa were diluted with BTS extender supplemented with different concentrations of glutamine (0, 10, 20, 40 or 80 mM).
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The diluted semen samples (3 × 107/mL) were dispersed into 100 mL plastic bottles and equilibrated for 2 h at room temperature (Day 0) prior to storage at 17°C (Thermobox, FYL-12MC-B4, China). Sperm motility and kinematic variables, membrane and acrosome integrity were analysed in the diluted semen after 2 (Day 0), 24 (Day 1), 72 (Day 3), and 120 (Day 5) h of storage.
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2.3. Assessment of semen motility and kinematic variables
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All sperm motility and kinematic variables were evaluated using the CASA system (Sperm Class Analyzer, Microptic SL, Barcelona, Spain). Semen samples were placed in a chamber and examined at 38.5°C under a phase-contrast microscopy system coupled to a video camera adapted to the Video Test Sperm system.
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2.4. Evaluation of sperm membrane integrity and acrosome integrity
Sperm membrane integrity was evaluated by the hypo-osmotic swelling test (Li et al., 2017). Briefly, 10 μL of sperm sample were added to 200 μL of pre-warmed hypotonic solution (9.0 g of fructose and 4.9 g of sodium citrate per litre of distilled water with an osmolality of 150 mOsm) and mixed thoroughly. After a 45-min incubation at 38.5°C, 200 μL 2% glutaraldehyde were added. After incubation, 15 μL of the mixture were spread with a cover slip on a warm slide. The tail coiling rate of the spermatozoa was examined using a phase-contrast microscope (Leica Wetzlar, Germany) at a magnification of 400×. Counting of
cells was conducted on individual spermatozoa in five to six different medium squares until 200 sperm had been counted. There were three technical replications for all groups.
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Acrosome integrity was analyzed using fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining as described by Zhu et al., 2017. Briefly, 200 μL semen samples were centrifuged at 2000 r/min for 4 min. The pellet was re-suspended in 1 mL of paraformaldehyde in PBS and fixed for 30 min. The spermatozoa were washed by centrifugation, the pellet was re-suspended in PBS, and smeared onto a clean glass slide for air-drying. There were 30 μL FITC-PNA solution (100 mg/mL) diluted in phosphate-buffered saline (PBS) that was spread over each slide. The slides were incubated in a dark and moist chamber at 37°C for 30 min. Subsequently, slides were rinsed with PBS, air dried in the dark and then mounted with10 µL antifade solution (Beyotime Institute of Biotechnology, Haimen, Jiangshu, China) to preserve fluorescence. The number of spermatozoa with intact acrosomes and the total number of spermatozoa were counted using an epifluorescence microscope (Leica DMIRB, Wetzlar, Germany).
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2.5. Measurement of total oxidant status (TOS), total antioxidant status (TAS), and calculation of oxidative stress index (OSI)
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Boar semen samples obtained during the different days throughout the experimental period were centrifuged at 6000 rpm for 10 minutes at room temperature to obtain supernatants. The supernatants were subsequently stored at -80°C before the analysis of TOS, TAS, and OSI. 2.5.1.TOS measurement
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Reagent 1 was composed of 150 mM xylenol orange, 140 mM NaCl and 1.35 M glycerol in a 25 mM H2SO4 solution, pH 1.75. Reagent 2 consisted of 5 mM ferrous ion and 10 mM o-dianisidine that was dissolved in a 25 mM H2SO4 solution (Qiu et al., 2016). The TOS of semen was measured by a method developed by Erel (Erel, 2004). The assay was calibrated with hydrogen peroxide and the results are expressed as μM H2O2 equivalent.
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2.5.2. TAS measurement
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For measuring total antioxidant status (TAS), Reagent 1 and 2 were prepared. Reagent 1 was composed of 0.4 M acetate buffer (pH 5.8) and Reagent 2 contained 10 mM 2.2 O-azino-di-(3-ethylbenz-thiazolinesulfonate) (ABTS) that was placed in 30mM acetate buffer (pH 3.6; Qiu et al., 2016). The TAS of semen was measured by a method developed by Erel (Erel, 2004). The reaction rate was calibrated with Trolox dissolved in phosphate buffer (30 mM, pH 7.4), and the assay results are expressed in mM Trolox equivalent (mM Trolox equiv.). 2.5.3. OSI determination
The ratio of TOS to TAS provided the oxidative stress index (OSI), an indicator of the extent of oxidative stress (Erel, 2004; Yeste, 2015). The TAS unit of mM Trolox equivalent
was converted into μM Trolox equivalent, and the OSI value was calculated as follows: OSI = [(TOS, μM H2O2 equiv.) / (TAS, μM Trolox equiv.) × 100]. 2.6. Determination of intracellular glutathione (GSH)
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The GSH content of spermatozoa was determined by using a Glutathione Quantification Kit (Beyotime Institute of Biotechnology, Haimen, Jiangshu, China) according to the manufacturer’s instructions. During the oxidation of GSH by 5,5-dithiobis-(2-nitrobenzoic acid), a yellow-colored product called 2-nitro-5 thiobenzoeic acid is generated, which can be detected photometrically by a change of absorption at 412 nm (Stradaioli et al., 2007). The contents of total glutathione (GSX), oxidized glutathione (GSSG) and reduced glutathione (GSH) were calculated according to a standard curve.
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2.7. Detection of γ-GCS activity
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The activity of γ-GCS, the key enzyme in GSH biosynthesis, was detected using a γ-GCS Assay Kit (A091, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Briefly, 1×108 spermatozoa were re-suspended in 100 mL lysis buffer, lysed by ultra-sonication and centrifuged at 12,000 g for 10 min at 4°C. The supernatants were then added to the reaction buffer, vortexed and incubated for 5 min at 37°C. Then, the mixture was inverted, and the absorbance was read at 340 nm on a Lambda-35 UV/Vis spectrometer (Perkin Elmer, Waltham, MA, USA). Analyses were performed per triplicate. Protein concentrations were determined using Bradford’s method with bovine serum albumin as the standard. The γ-GCS activity is expressed as units per mg protein.
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2.8. Measurement of intracellular ROS by flow cytometry
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Sperm intracellular ROS was assessed using 2’, 7’-dichlorofluorescein diacetate (carboxy- H2DCFDA; Molecular Probes Inc., Eugene, OR, USA) fluorescent dye. Apoptotic and dead spermatozoa were differentiated from viable spermatozoa using a counter stain dye (propidium iodide, PI). Evaluations were performed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). In brief, the sperm suspension (5×105/mL spermatozoa) was incubated with DCFDA (final concentration of 20 μM) and PI (final concentration of 1μg/mL) at 25°C for 60 min in the dark, then the labelled spermatozoa were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
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All fluorescence signals of labelled spermatozoa were analyzed with a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The FL1 (H2DCFDA green fluorescence) signals were detected between a 500 and 530 nm band-pass filter, and PI red fluorescence (580 and 630 nm) was analyzed in the FL-3 channel. The flow cytometer is capable of distinguishing 1024 channels and fluorescence intensities of both red and green fluorescence on each cell. All data were analyzed by FlowJo v10.0.7 software (Tree Star, Inc., Ashland, OR, USA).
2.9. Statistical analysis
Experiments were replicated at least three times. All percentage data were analyzed by Chi square test using SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA 2007). Differential staining data were analyzed by the Student’s t test. Data were expressed as the mean ± SEM, and a P value of less than 0.05 (P < 0.05) was considered significant. 3. Results
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3.1. Effects of different concentration of Gln on sperm quality during liquid semen storage at 17°C
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As depicted in Figure 1, the motility of boar sperm decreased during storage time (P < 0.05), however, was greater with the addition of Gln. On day 3 of storage, sperm motility was greater when the semen extender was supplemented with Gln, especially in the group supplemented with 20 mM Gln (67.63% ±1.49, P < 0.05). Similar changes in sperm motility were observed on day 5 of storage. The sperm motility in the treatment with 20 mM Gln group was greater than with other Gln concentrations groups (60.73% ± 0.69, P < 0.05, Fig. 1).
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All sperm kinematic variables were assessed by the CASA system during the storage period (Table 1). For VCL, VSL, and VAP, the values for evaluated variables decreased during storage; however, sperm storage with Gln-supplementation to the extender resulted in greater values when compared with the control group (P < 0.05; Table 1). The values of VCL, VSL, VAP were greater in the 20 mM Gln group (P < 0.05). The variables, BCF, LIN, WOB and STR, were slightly affected by inclusion of Gln in the extender and by storage time, however, there were no significant differences among treatments (P > 0.05; Table 1).
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Sperm membrane and acrosome integrity were less with longer storage time, however, were greater with Gln supplementation of extender when compared with the control (Fig. 2, 3). The proportion of spermatozoa with normal acrosomes was greater with 20 mM Gln supplementation of the extender when compared to other groups (P < 0.05).
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According to data collected in the present study, the supplementation with 20 mM Gln to extender could significantly improve boar sperm motility, acrosome integrity, and membrane integrity during liquid storage at 17°C. Thus, this concentration of Gln was chosen for the subsequent experiments.
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3.2. Effect of Gln on total oxidant and antioxidant status of liquid-stored boar sperm
On day 0 of storage, neither OSI (Fig. 4) nor GSH/GSSG ratio (Fig. 5) differed between treatment groups. The OSI value of boar sperm was greater during storage, but was less with Gln supplementation. Compared with the control group (P < 0.05), the TOS values of Gln-supplemented groups decreased from day 1 to day 5 of storage while the TAS values increased; hence, the OSI values decreased. Although OSI was less, the GSH/GSSG ratio was greater when semen was stored with Gln (P < 0.05).
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During the processes of liquid preservation, the intracellular ROS levels, GSH content, and γ-GCS activity in spermatozoa were measured to confirm whether Gln protected spermatozoa against ROS stress. Intracellular ROS levels of spermatozoa were determined by flow cytometry (Fig. 6). The percentage of spermatozoa with enhanced intracellular ROS level was not significantly different between Gln-treated and control groups on day 0 of storage when both the total sperm fraction (including nonviable spermatozoa) and viable spermatozoa (excluding nonviable spermatozoa) were evaluated. As the preservation time lengthened, the percentage of spermatozoa with greater intracellular ROS levels increased where there was no Gln extender supplementation (P < 0.05; Table 2); however, ROS levels were less in the Gln-treated group compared with the control group (P < 0.05; Fig. 6 and Table 2). On day 5 of storage, the percentage of spermatozoa with greater intracellular ROS level in the Gln-treated group was less than the control (9.45% ± 1.26 compared with 26.67% ± 1.42 in the total sperm fraction; and 6.78% ± 0.86 compared with 23.55% ± 1.64 in the viable spermatozoa fraction, Table 2).
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As depicted in Figure 7, the GSH content in spermatozoa decreased as the liquid storage time lengthened (P < 0.05). Interestingly, the extent of GSH decrease was less with the addition of Gln to the extender, resulting in a greater GSH content in the Gln-treated groups from day1 to day 5 of liquid storage compared with the control (P < 0.05; Fig. 7a).
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The effects of γ-GCS activity during boar semen liquid preservation were also investigated. The activity of γ-GCS in extender without Gln decreased over time, and was affected by Gln supplementation of extender. On day 0 of storage, sperm γ-GCS activity was similar among treatments, however, increased with Gln supplementation on day 1 of storage [(43.4 ± 1.08 compared with 22.7 ± 0.90, mean ± SEM mU/mg) (20 mM Gln compared control respectively; P < 0.05; Fig. 7b)]. The activity of sperm γ-GCS began to decrease during the subsequent days of storage; however, remained greater than values for the control [(day 3: 38.2 ± 0.55 compared with 19.6 ± 0.48; day 5: 29.70 ± 0.23 compared with 15.10 ± 0.76, mean ± SEM mU/mg) (20 mM Gln compared with control, respectively; P < 0.05)]. Glutathione synthesis in boar sperm was increased by Gln treatment during liquid preservation. In addition, treatment with 20 mM Gln improved boar sperm motility (P < 0.05; Fig. 7c). Together, the results suggest that the supplement of Gln could quench ROS, increase γ-GCS activity, enhance GSH production, and improve sperm motility during the period of storage at 17°C. 4. Discussion
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Glutamine is the most abundant free amino acid in the body, constituting more than 50% of the total intracellular free α-amino acid pool in skeletal muscle and blood of pigs (Zhu et al., 2017). The concentrations of Gln are great in seminal plasma and spermatozoa (Setchell et al., 1967; Hinton, 1990), and Gln has been used as a component of sperm-freezing extender solutions in several species (Zhu et al., 2017). These reports indicate that Gln may influence sperm quality. The aim of present study was to elucidate whether the addition of Gln to the extender could enhance boar spermatozoa quality during liquid storage at 17°C and to
determine the possible mechanism by which Gln protects spermatozoa against storage-induced damage.
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Results of the present study indicate that boar spermatozoa are very susceptible to cold shock. This occurred when freshly ejaculated boar spermatozoa were cooled from body temperature to 17°C and during the period of liquid storage, which resulted in a loss of motility of spermatozoa. The supplementation of extender with Gln enhanced the resistance of spermatozoa to cold shock (P < 0.05, Fig. 1), especially in the 20 mM Gln group, in which sperm motility was greater compared with the other groups. On day 5 of storage, sperm motility of samples supplemented with 20 mM Gln was greater than with use of the other concentrations (60.73% ± 0.69, P < 0.05, Fig. 1). The values for all sperm kinematic variables assessed by the CASA decreased during storage, but the values for these variables in Gln-treated groups were greater compared with the control group (P < 0.05; Table 1). Results of the present study are consistent with those from previous reports (Aramli et al., 2016) where motility rate and viability of post-thaw sperm of Persian Sturgeon were increased by Gln supplementation. Meanwhile, in the present study, sperm membrane and acrosome integrity was greater with Gln supplementation of extender (as shown Fig. 2 and 3) which is consistent with results reported previously in boars (De Mercado et al., 2009), bulls (Tuncer et al., 2011), mice (Guan et al., 2014), rams (Sangeeta et al., 2015) and humans (Renard et al., 1996).This indicated that the effect of Gln was biphasic and that the greater concentration of Gln may have a cytotoxic effect. Thus, in the present study, the 20 mM Gln concentration had a greater positive effect on sperm quality than the other concentrations.
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In the present study, quality of boar semen, was markedly decreased as preservation time in vitro advanced. One of the major causes of these adverse effects is oxidative stress (Zhang et al., 2016). The OSI and GSH/GSSG ratio are indicators of the total antioxidant capacity of semen (Matés et al., 2002; Zhang et al., 2015). Data from the present study indicate that the OSI values of boar sperms increased as duration of storage increased, however, OSI values were less when Gln was added to the extender. When semen was stored with Gln, OSI was less, but the GSH/GSSG ratio was greater (P < 0.05), suggesting Gln can affect the oxidant and antioxidant status of liquid-stored boar sperm. As the preservation time progressed, the percentage of spermatozoa with greater intracellular ROS levels in the Gln-treated groups were less than those of the control group (Table 2). Although the exact mechanism by which Gln protects sperm from liquid storage damage is not completely understood, the improvements observed in sperm quality in the present study may result from Gln providing protection and inhibiting excessive generation of ROS that damage the sperm plasma membrane (Zhu et al., 2017). The glutathione system is one of the major mechanisms of reducing oxidative stress. In previous studies, the freeze-thaw process reduced the GSH content of boar sperm (Giaretta et al., 2015). The addition of GSH can improve boar sperm motility, plasma membrane integrity, effective survival period and T-AOC activity, and H2O2 content during liquid preservation (Zhang et al., 2016). In the present study, the level of GSH during liquid preservation was also investigated. It was observed that sperm GSH content decreased as the duration of liquid storage time increased (P < 0.05); however, GSH content was greater with
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Gln addition to the extender during liquid preservation. Interestingly, data from the present study indicate that the addition of 20 mM Gln to the extender can not only improve sperm motility but also enhance γ-GCS activity, which is the rate limiting enzyme for GSH synthesis during boar sperm liquid preservation (P < 0.05; Fig. 7b, c). The amino acid, Gln, is required for glutathione synthesis as it provides the source of glutamate for GSH synthesis in many tissues such as the liver and skeletal muscle (Curi et al., 2005). There can be Gln transportation into cells, where Gln is metabolized to glutamate and subsequently converted to glutathione by reactions involving cysteine, γ-GCS, glycine and glutathione synthetase, which are components of the γ-glutamyl cycle (Zhu et al., 2017). Addition of Gln to cells in vitro can result in an increase in total glutathione concentration (Matés et al., 2002). These data suggest that Gln can enhance the capacity of spermatozoa to synthesize GSH during liquid preservation when these cells are subjected to oxidative stress.
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In conclusion, the use of the BTS extender containing Gln improved boar sperm motility, acrosome integrity, and membrane integrity after periods of liquid preservation. In addition, Gln can quench ROS while enhancing γ-GCS activity and GSH content of spermatozoa. The results also indicate that extender supplemented with Gln provides greater protective capacity on boar sperm during liquid preservation against oxidative stress by enhancing GSH synthesis.
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Conflict of interest
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The authors confirm that this article’s content has no conflicts of interest. Acknowledgements
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Funding
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This research was supported by the National Natural Science Foundation of China (Project No. 31372311) and Scientific Research Foundation of Northeast Agricultural University (Project No.106518001).
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Renard, P., Grizard, G., Griveau, J.F., Sion, B., Boucher, D., Le Lannou, D., 1996. Improvement of Motility and Fertilization Potential of Postthaw Human Sperm Using Glutamine. Cryobiology 33, 311–319. Roth, E., Oehler, R., Manhart, N., Exner, R., Wessner, B., Strasser, E., Spittler, A., 2002. Regulative potential of glutamine--relation to glutathione metabolism. Nutrition 18, 217–221. Sangeeta, S., Arangasamy, A., Kulkarni, S., Selvaraju, S., 2015. Role of amino acids as additives on sperm motility, plasma membrane integrity and lipid peroxidation levels at pre-freeze and post-thawed ram semen. Anim. Reprod. Sci. 161, 82–88. Schulze, M., Ammon, C., Schaefer, J., Luther, A.M., Jung, M., Waberski, D., 2017. Impact of different dilution techniques on boar sperm quality and sperm distribution of the extended ejaculate. Anim. Reprod. Sci. 182, 138–145. Setchell, B.P., Hinks, N.T., Voglmayr, J.K., Scott, T.W., 1967. Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa. Biochem. J. 105, 1061–1065. Shah, N., Singh, V., Yadav, H.P., Verma, M., Chauhan, D.S., Saxena, A., Yadav, S., Swain, D.K., 2017. Effect of reduced glutathione supplementation in semen extender on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa. Anim. Reprod. Sci. 182, 111–122. Spodenkiewicz, M., Diez-Fernandez, C., Rüfenacht, V., Gemperle-Britschgi, C., Häberle, J., 2016. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis. Biology (Basel). 5, 40. 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. Tuncer, P.B., Sarıözkan, S., Bucak, M.N., Ulutaş, P.A., Akalın, P.P., Büyükleblebici, S., Canturk, F., 2011. Effect of glutamine and sugars after bull spermatozoa cryopreservation. Theriogenology 75, 1459–1465. Yeste, M., 2015. Recent Advances in Boar Sperm Cryopreservation: State of the Art and Current Perspectives. Reprod. Domest. Anim. 50, 71–79. Zhang, X.G., Liu, Q., Wang, L.Q., Yang, G.S., Hu, J.H., 2016. Effects of glutathione on sperm quality during liquid storage in boars. Anim. Sci. J. 87, 1195–1201. Zhang, X.G., Yan, G.J., Hong, Y.J., Su, Z.Z., Yang, G.S., Li, Q.W., Hu, J.H., 2015. Effects of Bovine Serum Albumin on Boar Sperm Quality During Liquid Storage at 17°C. Reprod. Domest. Anim. 50, 263–269. Zhu, Z., Fan, X., Lv, Y., Lin, Y., Wu, Zeng, W., 2017. Glutamine protects rabbit spermatozoa against oxidative stress via glutathione synthesis during cryopreservation. Reprod. Fertil. Dev. 29, 2183–2194.
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Fig. 1. Sperm motility on different days during liquid storage of boar semen at 17°C in extenders with different concentrations of L-glutamine; Columns with different lowercase letters differ (P < 0.05), n = 6
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Fig. 2. Effect of different concentrations of Gln on boar spermatozoa membrane integrity; Columns with different lowercase letters differ (P < 0.05); n = 6
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Fig. 3. Effect of different concentrations of Gln on spermatozoa acrosome integrity; Columns with different lowercase letters differ (P < 0.05); n = 6
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Fig. 4. Total oxidant status (μM H2O2 equiv.), TAS (mM Trolox equiv.), and OSI in extenders on different days during liquid storage of boar semen with or without 20 mM Gln; Columns with different lowercase letters differ (P < 0.05); n = 6; TAS = total antioxidant status, TOS = total oxidant status, OSI = oxidative stress index
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Fig. 5. Intra-sperm GSH/GSSG ratio on different days during liquid storage of boar semen with or without 20 mM Gln; Columns with different lowercase letters differ (P < 0.05); n = 6
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Fig. 7. Effects of Gln on sperm GSH content, γ-glutamyl cysteine synthetase (γ-GCS) activity and spermatozoa motility during liquid preservation; GSH content A; γ-glutamyl cysteine synthetase (γ-GCS) activity B; and spermatozoa motility C; Data are expressed as the means ± SEM; (n = 3); Values depicted in columns with different lowercase letters (a, b) are different P < 0 .05
Table 1 Effect of different concentrations of Gln on spermatozoa kinematic variables Kinematic variables Days of Gln VCL VSL VAP BCF LIN WOB STR Storage (μm/s) (μm/s) (μm/s) (Hz) (%) (%) (%) 0 mM
94.25±4.91a
52.21±2.81a
86.21±4.32a
42.64±2.16a
55.39±0.62a
91.47±0.12a
60.56±0.60a
10 mM
92.88±0.87a
49.18±5.75a
84.36±2.51a
41.23±0.60a
52.96±4.66a
90.83±0.37a
58.29±3.38a
20 mM
93.44±2.64a
50.46±0.58a
87.13±2.90a
42.93±0.42a
54.72±0.90a
93.25±0.30a
57.91±0.40a
40 mM
92.87±3.69a
45.98±0.20a
84.05±1.52a
40.64±0.97a
49.51±0.88a
90.51±0.25a
54.71±1.19a
80 mM
91.55±0.31a
46.86.±0.54a
83.91±1.49a
40.73±1.07a
51.19±2.10a
91.65±0.57a
55.85±0.64a
0 mM
76.88±2.31a
40.14±1.65a
69.99±2.18a
37.00±1.09a
52.21±0.49a
91.04±0.14a
57.35±0.67a
10 mM
77.33±0.33a
41.95±1.76ab
71.80±0.78a
38.08±0.39a
54.25±3.53a
92.85±1.00a
58.43±4.63a
20 mM
82.81±2.41ab
45.98±1.73ab
76.27±2.27ab
38.60±0.67a
55.52±0.47a
92.10±0.13a
60.29±0.64a
40 mM
84.73±2.12b
44.07±1.82ab
76.60±1.33b
37.72±1.14a
80 mM
81.07±0.36ab
41.78±1.10b
73.21±0.68ab
37.42±0.03a
0 mM
71.96±2.03a
36.7±0.35a
65.35±2.20a
35.17±0.78a
10 mM
73.30±2.10ab
37.77±0.43a
67.08±2.50ab
36.53±0.88a
20 mM
80.99±1.73b
44.05±2.29b
75.22±1.31ab
37.61±0.46a
40 mM
80.33±2.02b
44.48±1.47b
73.73±5.53b
36.01±1.95a
80 mM
75.67±2.94ab
39.34±4.65a
68.41±3.65ab
0 mM
74.12±1.24a
38.54±0.48a
66.86±1.69a
b
b
b
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77.82±2.25
41.51±2.21
20 mM
81.27±2.03b
45.19±1.31b
40 mM
75.25±2.73ab
80 mM
50.98±4.72c
70.87±1.04
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57.53±4.82a
51.54±1.63a
90.31±0.46a
57.07±2.17a
51.05±2.21a
90.81±0.44a
56.16±2.08a
51.53±1.07a
91.51±0.21a
56.31±1.02a
54.39±4.20a
92.88±0.84a
58.56±3.89a
54.92±1.44a
91.78±0.29a
60.33±1.37a
52.00±1.26a
90.40±0.84a
57.51±3.92a
33.15±0.85a
52.00±2.17a
90.21±0.43a
57.64±2.06a
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58.57±2.09a
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52.02±0.11a
36.28±1.29 a
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10 mM
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35.72±0.52
53.35±2.16
91.07±0.43
75.51±3.84b
37.33±1.92a
55.61±1.55a
92.92±0.31a
59.85±1.47a
38.21±3.46ab
68.60±8.47a
32.34±4.24a
50.78±3.49a
91.16±0.70a
55.70±1.71a
27.46±3.16c
45.56±4.41c
33.80±2.21 a
53.87±0.52a
89.37±0.10a
60.27±0.71a
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Values are expressed as the means ± SEM (n = 6); Values associated with different lowercase letters (a, b, and c ) within the columns are different (P < 0.05); VCL = curvilinear velocity, VSL = straight-line velocity, VAP = average path velocity, BCF = beat-cross frequency, LIN = linearity (VSL/VCL), WOB = wobble (VAP/VCL), STR = straightness (VSL/VAP)
Table 2 Effects of Gln on sperm ROS production during liquid preservation Days of storage 0 1 3
5
Ctrl
8.51±1.21a
14.77±1.70a
19.58±1.63a
26.67±1.42a
Gln
8.74±0.71a
8.95±1.51b
9.24±1.37b
9.45±1.26b
Excluding nonviable cells Sperm with high ROS level (%)
Ctrl
3.89±0.91a
9.67±1.96a
14.01±1.55a
23.55±1.64a
Gln
3.06±1.42a
4.41±0.93b
5.83±1.38b
6.78±0.86b
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Including nonviable cells Sperm with high ROS level (%)
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Values are expressed as the means ± SEM (n = 6); Values associated with different lowercase letters (a and b) within a column are different (P < 0.05)
S0378-4320(17)31045-X https://doi.org/10.1016/j.anireprosci.2018.02.011 ANIREP 5767
To appear in:
Animal Reproduction Science
Received date: Revised date: Accepted date:
19-12-2017 28-1-2018 13-2-2018
Please cite this article as: Wang S, Sun M, Wang N, Yang K, Guo H, Wang J, Zhang Y, Yue S, Zhou J, Effects of L-Glutamine on boar sperm quality during liquid storage at 17◦ C, Animal Reproduction Science (2010), https://doi.org/10.1016/j.anireprosci.2018.02.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of L-Glutamine on boar sperm quality during liquid storage at 17°C
Shunwei Wanga, Meng Suna, Na Wanga, Kang Yanga, Haitao Guoa, Jingran Wanga, Yuting Zhanga, Shunli Yueab*, Jiabo Zhouab*
College of Life Science Northeast Agricultural University, Harbin 150030, China
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Northeast Agricultural University, Harbin 150030, China
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Yue);
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Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province,
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[email protected] (J Zhou)
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ABSTRACT
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The quality of boar spermatozoa is affected by oxidative stress during preservation in vitro. It has been demonstrated that L-Glutamine (Gln) can effectively protect cells from oxidative stress-induced injury. There are, however, no reports to date evaluating the effects of Gln on boar semen liquid preservation at 17°C. The aims of the present study were to elucidate whether the addition of Gln to the extender BTS could improve the quality of boar spermatozoa when stored at 17°C and to determine the mechanism underlying Gln protection of spermatozoa against preservation-induced damage. Boar semen samples were collected and diluted with Beltsville Thawing Solution (BTS) containing different concentrations (0, 10, 20, 40 or 80 mM) of Gln. The results indicated the addition of 20 mM Gln to the BTS improved (P < 0.05) the motility, acrosome integrity and membrane integrity of boar sperm during liquid preservation. Interestingly, treatment of spermatozoa with Gln addition to the extender resulted in ROS quenching, while enhancing γ-glutamyl cysteine synthetase (γ-GCS) activity, and glutathione (GSH) content of spermatozoa. These results suggest that BTS supplemented with Gln can provide greater protective capacity to boar sperm against oxidative stress by enhancing GSH synthesis during liquid preservation. Keywords: L-Glutamine; Boar semen; Liquid preservation; Reactive oxygen species
1. Introduction
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Artificial insemination (AI) has been widely used by the modern swine production industry. Boar spermatozoa are highly susceptible to low temperatures (Yeste, 2015) because the membrane of boar spermatozoa has a low cholesterol/phospholipid ratio (Johnson et al., 2000). Liquid storage of semen has become a routine reproduction technique in pork production enterprises, as liquid stored semen is important in reducing disease risks and in increasing efficiency of semen usage (Li et al., 2017). With the emergence of long-term extenders for liquid storage, the use of cryopreserved sperm for AI in pork production enterprises has decreased (Yeste, 2015). Thus, the technical improvement of liquid storage has become more important in the swine production industry.
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The amino acid, L-Glutamine (Gln), which is the most abundant free amino acid in the body, has an important role in several cell-specific processes, including metabolism, protein synthesis and degradation, cell integrity, redox potential, and extracellular matrix (ECM) synthesis (Spodenkiewicz et al., 2016). This amino acid (Gln) is required for glutathione (GSH) synthesis, as it can be metabolized by the gamma-glutamyl cycle to produce glutathione (Roth et al., 2002). Glutathione can function as an antioxidant in semen, which provides for an intracellular defense to sperm against oxidative stress (Gadea et al., 2004). Oxidative stress is a primary factor that leads to damage of sperm functions due to greater synthesis of lipid peroxidation induced by reactive species (ROS) as a result of oxidative stress. The ROS are produced by sperm during the process of in vitro preservation, which leads to oxidative stress in sperm plasma membranes. Sperm will lose its fertilizing capacity if the polyunsaturated fatty acids in the sperm membrane are oxidized (Zhang et al., 2016). The Gln metabolism, via entry into the TCA cycle may enhance malic enzyme activity (NADP+ dependent), which will result in an increase in NADPH production (Zhang et al., 2016). This will subsequently affect the total oxidant and antioxidant status of cells. Addition of Gln to cells in vitro can lead to an increase in total glutathione concentration (Matés et al., 2002; Curi et al., 2005). The ratio of GSH to oxidized glutathione (GSSG) is the principal regulator of the cellular redox potential (Matés et al., 2002). Some studies have shown that when sperm were preserved in an extender containing glutathione, sperm quality after preservation was improved (Zhang et al., 2016; Shah et al., 2017).
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Supplementation of Gln improves the post-thaw motility and fertilizing potential of sperm in many species (Khlifaoui et al., 2005; Zhu et al., 2017). There are, however, less data available on the effect of Gln on boar sperm quality during liquid storage at 17°C. The aim of the present study was, therefore, to elucidate whether the addition of Gln to the extender could enhance boar sperm motility, acrosome integrity, and membrane integrity. The GSH content, γ-glutamyl cysteine synthetase (γ-GCS) activity, and intracellular ROS levels in spermatozoa were also measured during the processes of liquid preservation to ascertain the mechanism by which Gln protects spermatozoa during liquid storage.
2. Materials and methods
Unless noted, all chemicals and media used in this study were purchased from Sigma Chemical Co (St. Louis, MO, USA). 2.1.Semen collection
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Ejaculates were collected by gloved-hand method from five Large White male pigs, aged 1.5 to 2.5 years. The boars were allowed at least 3 days of sexual rests between collections. The semen was transported to the laboratory at 37 to 38°C within 30 min and filtered. Only semen samples with more than 70% sperm total motility (TMOT) and less than 15% morphologically abnormal spermatozoa were used (Betancourt et al., 2006; Schulze et al., 2017). Ejaculates (n = 36) were used in this experiment. The protocols for experiments using animals were subject to approval by the Animal Care and Use Committee of Northeast Agricultural University. 2.2. Semen processing
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The basic medium used for sperm extension was Beltsville Thawing Solution (BTS), which was composed of 205 mM glucose, 20.39 mM NaCl, 5.40 mM KCl, 15.01 mM NaHCO3, and 3.35 mM EDTA, penethamate and streptomycin (50 µg/mL; Kumaresan et al., 2009). The pH of the BTS extenders was adjusted to 7.2. Spermatozoa were diluted with BTS extender supplemented with different concentrations of glutamine (0, 10, 20, 40 or 80 mM).
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The diluted semen samples (3 × 107/mL) were dispersed into 100 mL plastic bottles and equilibrated for 2 h at room temperature (Day 0) prior to storage at 17°C (Thermobox, FYL-12MC-B4, China). Sperm motility and kinematic variables, membrane and acrosome integrity were analysed in the diluted semen after 2 (Day 0), 24 (Day 1), 72 (Day 3), and 120 (Day 5) h of storage.
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2.3. Assessment of semen motility and kinematic variables
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All sperm motility and kinematic variables were evaluated using the CASA system (Sperm Class Analyzer, Microptic SL, Barcelona, Spain). Semen samples were placed in a chamber and examined at 38.5°C under a phase-contrast microscopy system coupled to a video camera adapted to the Video Test Sperm system.
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2.4. Evaluation of sperm membrane integrity and acrosome integrity
Sperm membrane integrity was evaluated by the hypo-osmotic swelling test (Li et al., 2017). Briefly, 10 μL of sperm sample were added to 200 μL of pre-warmed hypotonic solution (9.0 g of fructose and 4.9 g of sodium citrate per litre of distilled water with an osmolality of 150 mOsm) and mixed thoroughly. After a 45-min incubation at 38.5°C, 200 μL 2% glutaraldehyde were added. After incubation, 15 μL of the mixture were spread with a cover slip on a warm slide. The tail coiling rate of the spermatozoa was examined using a phase-contrast microscope (Leica Wetzlar, Germany) at a magnification of 400×. Counting of
cells was conducted on individual spermatozoa in five to six different medium squares until 200 sperm had been counted. There were three technical replications for all groups.
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Acrosome integrity was analyzed using fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining as described by Zhu et al., 2017. Briefly, 200 μL semen samples were centrifuged at 2000 r/min for 4 min. The pellet was re-suspended in 1 mL of paraformaldehyde in PBS and fixed for 30 min. The spermatozoa were washed by centrifugation, the pellet was re-suspended in PBS, and smeared onto a clean glass slide for air-drying. There were 30 μL FITC-PNA solution (100 mg/mL) diluted in phosphate-buffered saline (PBS) that was spread over each slide. The slides were incubated in a dark and moist chamber at 37°C for 30 min. Subsequently, slides were rinsed with PBS, air dried in the dark and then mounted with10 µL antifade solution (Beyotime Institute of Biotechnology, Haimen, Jiangshu, China) to preserve fluorescence. The number of spermatozoa with intact acrosomes and the total number of spermatozoa were counted using an epifluorescence microscope (Leica DMIRB, Wetzlar, Germany).
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2.5. Measurement of total oxidant status (TOS), total antioxidant status (TAS), and calculation of oxidative stress index (OSI)
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Boar semen samples obtained during the different days throughout the experimental period were centrifuged at 6000 rpm for 10 minutes at room temperature to obtain supernatants. The supernatants were subsequently stored at -80°C before the analysis of TOS, TAS, and OSI. 2.5.1.TOS measurement
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Reagent 1 was composed of 150 mM xylenol orange, 140 mM NaCl and 1.35 M glycerol in a 25 mM H2SO4 solution, pH 1.75. Reagent 2 consisted of 5 mM ferrous ion and 10 mM o-dianisidine that was dissolved in a 25 mM H2SO4 solution (Qiu et al., 2016). The TOS of semen was measured by a method developed by Erel (Erel, 2004). The assay was calibrated with hydrogen peroxide and the results are expressed as μM H2O2 equivalent.
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2.5.2. TAS measurement
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For measuring total antioxidant status (TAS), Reagent 1 and 2 were prepared. Reagent 1 was composed of 0.4 M acetate buffer (pH 5.8) and Reagent 2 contained 10 mM 2.2 O-azino-di-(3-ethylbenz-thiazolinesulfonate) (ABTS) that was placed in 30mM acetate buffer (pH 3.6; Qiu et al., 2016). The TAS of semen was measured by a method developed by Erel (Erel, 2004). The reaction rate was calibrated with Trolox dissolved in phosphate buffer (30 mM, pH 7.4), and the assay results are expressed in mM Trolox equivalent (mM Trolox equiv.). 2.5.3. OSI determination
The ratio of TOS to TAS provided the oxidative stress index (OSI), an indicator of the extent of oxidative stress (Erel, 2004; Yeste, 2015). The TAS unit of mM Trolox equivalent
was converted into μM Trolox equivalent, and the OSI value was calculated as follows: OSI = [(TOS, μM H2O2 equiv.) / (TAS, μM Trolox equiv.) × 100]. 2.6. Determination of intracellular glutathione (GSH)
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The GSH content of spermatozoa was determined by using a Glutathione Quantification Kit (Beyotime Institute of Biotechnology, Haimen, Jiangshu, China) according to the manufacturer’s instructions. During the oxidation of GSH by 5,5-dithiobis-(2-nitrobenzoic acid), a yellow-colored product called 2-nitro-5 thiobenzoeic acid is generated, which can be detected photometrically by a change of absorption at 412 nm (Stradaioli et al., 2007). The contents of total glutathione (GSX), oxidized glutathione (GSSG) and reduced glutathione (GSH) were calculated according to a standard curve.
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2.7. Detection of γ-GCS activity
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The activity of γ-GCS, the key enzyme in GSH biosynthesis, was detected using a γ-GCS Assay Kit (A091, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Briefly, 1×108 spermatozoa were re-suspended in 100 mL lysis buffer, lysed by ultra-sonication and centrifuged at 12,000 g for 10 min at 4°C. The supernatants were then added to the reaction buffer, vortexed and incubated for 5 min at 37°C. Then, the mixture was inverted, and the absorbance was read at 340 nm on a Lambda-35 UV/Vis spectrometer (Perkin Elmer, Waltham, MA, USA). Analyses were performed per triplicate. Protein concentrations were determined using Bradford’s method with bovine serum albumin as the standard. The γ-GCS activity is expressed as units per mg protein.
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2.8. Measurement of intracellular ROS by flow cytometry
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Sperm intracellular ROS was assessed using 2’, 7’-dichlorofluorescein diacetate (carboxy- H2DCFDA; Molecular Probes Inc., Eugene, OR, USA) fluorescent dye. Apoptotic and dead spermatozoa were differentiated from viable spermatozoa using a counter stain dye (propidium iodide, PI). Evaluations were performed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). In brief, the sperm suspension (5×105/mL spermatozoa) was incubated with DCFDA (final concentration of 20 μM) and PI (final concentration of 1μg/mL) at 25°C for 60 min in the dark, then the labelled spermatozoa were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
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All fluorescence signals of labelled spermatozoa were analyzed with a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The FL1 (H2DCFDA green fluorescence) signals were detected between a 500 and 530 nm band-pass filter, and PI red fluorescence (580 and 630 nm) was analyzed in the FL-3 channel. The flow cytometer is capable of distinguishing 1024 channels and fluorescence intensities of both red and green fluorescence on each cell. All data were analyzed by FlowJo v10.0.7 software (Tree Star, Inc., Ashland, OR, USA).
2.9. Statistical analysis
Experiments were replicated at least three times. All percentage data were analyzed by Chi square test using SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA 2007). Differential staining data were analyzed by the Student’s t test. Data were expressed as the mean ± SEM, and a P value of less than 0.05 (P < 0.05) was considered significant. 3. Results
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3.1. Effects of different concentration of Gln on sperm quality during liquid semen storage at 17°C
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As depicted in Figure 1, the motility of boar sperm decreased during storage time (P < 0.05), however, was greater with the addition of Gln. On day 3 of storage, sperm motility was greater when the semen extender was supplemented with Gln, especially in the group supplemented with 20 mM Gln (67.63% ±1.49, P < 0.05). Similar changes in sperm motility were observed on day 5 of storage. The sperm motility in the treatment with 20 mM Gln group was greater than with other Gln concentrations groups (60.73% ± 0.69, P < 0.05, Fig. 1).
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All sperm kinematic variables were assessed by the CASA system during the storage period (Table 1). For VCL, VSL, and VAP, the values for evaluated variables decreased during storage; however, sperm storage with Gln-supplementation to the extender resulted in greater values when compared with the control group (P < 0.05; Table 1). The values of VCL, VSL, VAP were greater in the 20 mM Gln group (P < 0.05). The variables, BCF, LIN, WOB and STR, were slightly affected by inclusion of Gln in the extender and by storage time, however, there were no significant differences among treatments (P > 0.05; Table 1).
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Sperm membrane and acrosome integrity were less with longer storage time, however, were greater with Gln supplementation of extender when compared with the control (Fig. 2, 3). The proportion of spermatozoa with normal acrosomes was greater with 20 mM Gln supplementation of the extender when compared to other groups (P < 0.05).
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According to data collected in the present study, the supplementation with 20 mM Gln to extender could significantly improve boar sperm motility, acrosome integrity, and membrane integrity during liquid storage at 17°C. Thus, this concentration of Gln was chosen for the subsequent experiments.
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3.2. Effect of Gln on total oxidant and antioxidant status of liquid-stored boar sperm
On day 0 of storage, neither OSI (Fig. 4) nor GSH/GSSG ratio (Fig. 5) differed between treatment groups. The OSI value of boar sperm was greater during storage, but was less with Gln supplementation. Compared with the control group (P < 0.05), the TOS values of Gln-supplemented groups decreased from day 1 to day 5 of storage while the TAS values increased; hence, the OSI values decreased. Although OSI was less, the GSH/GSSG ratio was greater when semen was stored with Gln (P < 0.05).
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During the processes of liquid preservation, the intracellular ROS levels, GSH content, and γ-GCS activity in spermatozoa were measured to confirm whether Gln protected spermatozoa against ROS stress. Intracellular ROS levels of spermatozoa were determined by flow cytometry (Fig. 6). The percentage of spermatozoa with enhanced intracellular ROS level was not significantly different between Gln-treated and control groups on day 0 of storage when both the total sperm fraction (including nonviable spermatozoa) and viable spermatozoa (excluding nonviable spermatozoa) were evaluated. As the preservation time lengthened, the percentage of spermatozoa with greater intracellular ROS levels increased where there was no Gln extender supplementation (P < 0.05; Table 2); however, ROS levels were less in the Gln-treated group compared with the control group (P < 0.05; Fig. 6 and Table 2). On day 5 of storage, the percentage of spermatozoa with greater intracellular ROS level in the Gln-treated group was less than the control (9.45% ± 1.26 compared with 26.67% ± 1.42 in the total sperm fraction; and 6.78% ± 0.86 compared with 23.55% ± 1.64 in the viable spermatozoa fraction, Table 2).
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The effects of γ-GCS activity during boar semen liquid preservation were also investigated. The activity of γ-GCS in extender without Gln decreased over time, and was affected by Gln supplementation of extender. On day 0 of storage, sperm γ-GCS activity was similar among treatments, however, increased with Gln supplementation on day 1 of storage [(43.4 ± 1.08 compared with 22.7 ± 0.90, mean ± SEM mU/mg) (20 mM Gln compared control respectively; P < 0.05; Fig. 7b)]. The activity of sperm γ-GCS began to decrease during the subsequent days of storage; however, remained greater than values for the control [(day 3: 38.2 ± 0.55 compared with 19.6 ± 0.48; day 5: 29.70 ± 0.23 compared with 15.10 ± 0.76, mean ± SEM mU/mg) (20 mM Gln compared with control, respectively; P < 0.05)]. Glutathione synthesis in boar sperm was increased by Gln treatment during liquid preservation. In addition, treatment with 20 mM Gln improved boar sperm motility (P < 0.05; Fig. 7c). Together, the results suggest that the supplement of Gln could quench ROS, increase γ-GCS activity, enhance GSH production, and improve sperm motility during the period of storage at 17°C. 4. Discussion
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Glutamine is the most abundant free amino acid in the body, constituting more than 50% of the total intracellular free α-amino acid pool in skeletal muscle and blood of pigs (Zhu et al., 2017). The concentrations of Gln are great in seminal plasma and spermatozoa (Setchell et al., 1967; Hinton, 1990), and Gln has been used as a component of sperm-freezing extender solutions in several species (Zhu et al., 2017). These reports indicate that Gln may influence sperm quality. The aim of present study was to elucidate whether the addition of Gln to the extender could enhance boar spermatozoa quality during liquid storage at 17°C and to
determine the possible mechanism by which Gln protects spermatozoa against storage-induced damage.
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Results of the present study indicate that boar spermatozoa are very susceptible to cold shock. This occurred when freshly ejaculated boar spermatozoa were cooled from body temperature to 17°C and during the period of liquid storage, which resulted in a loss of motility of spermatozoa. The supplementation of extender with Gln enhanced the resistance of spermatozoa to cold shock (P < 0.05, Fig. 1), especially in the 20 mM Gln group, in which sperm motility was greater compared with the other groups. On day 5 of storage, sperm motility of samples supplemented with 20 mM Gln was greater than with use of the other concentrations (60.73% ± 0.69, P < 0.05, Fig. 1). The values for all sperm kinematic variables assessed by the CASA decreased during storage, but the values for these variables in Gln-treated groups were greater compared with the control group (P < 0.05; Table 1). Results of the present study are consistent with those from previous reports (Aramli et al., 2016) where motility rate and viability of post-thaw sperm of Persian Sturgeon were increased by Gln supplementation. Meanwhile, in the present study, sperm membrane and acrosome integrity was greater with Gln supplementation of extender (as shown Fig. 2 and 3) which is consistent with results reported previously in boars (De Mercado et al., 2009), bulls (Tuncer et al., 2011), mice (Guan et al., 2014), rams (Sangeeta et al., 2015) and humans (Renard et al., 1996).This indicated that the effect of Gln was biphasic and that the greater concentration of Gln may have a cytotoxic effect. Thus, in the present study, the 20 mM Gln concentration had a greater positive effect on sperm quality than the other concentrations.
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In the present study, quality of boar semen, was markedly decreased as preservation time in vitro advanced. One of the major causes of these adverse effects is oxidative stress (Zhang et al., 2016). The OSI and GSH/GSSG ratio are indicators of the total antioxidant capacity of semen (Matés et al., 2002; Zhang et al., 2015). Data from the present study indicate that the OSI values of boar sperms increased as duration of storage increased, however, OSI values were less when Gln was added to the extender. When semen was stored with Gln, OSI was less, but the GSH/GSSG ratio was greater (P < 0.05), suggesting Gln can affect the oxidant and antioxidant status of liquid-stored boar sperm. As the preservation time progressed, the percentage of spermatozoa with greater intracellular ROS levels in the Gln-treated groups were less than those of the control group (Table 2). Although the exact mechanism by which Gln protects sperm from liquid storage damage is not completely understood, the improvements observed in sperm quality in the present study may result from Gln providing protection and inhibiting excessive generation of ROS that damage the sperm plasma membrane (Zhu et al., 2017). The glutathione system is one of the major mechanisms of reducing oxidative stress. In previous studies, the freeze-thaw process reduced the GSH content of boar sperm (Giaretta et al., 2015). The addition of GSH can improve boar sperm motility, plasma membrane integrity, effective survival period and T-AOC activity, and H2O2 content during liquid preservation (Zhang et al., 2016). In the present study, the level of GSH during liquid preservation was also investigated. It was observed that sperm GSH content decreased as the duration of liquid storage time increased (P < 0.05); however, GSH content was greater with
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Gln addition to the extender during liquid preservation. Interestingly, data from the present study indicate that the addition of 20 mM Gln to the extender can not only improve sperm motility but also enhance γ-GCS activity, which is the rate limiting enzyme for GSH synthesis during boar sperm liquid preservation (P < 0.05; Fig. 7b, c). The amino acid, Gln, is required for glutathione synthesis as it provides the source of glutamate for GSH synthesis in many tissues such as the liver and skeletal muscle (Curi et al., 2005). There can be Gln transportation into cells, where Gln is metabolized to glutamate and subsequently converted to glutathione by reactions involving cysteine, γ-GCS, glycine and glutathione synthetase, which are components of the γ-glutamyl cycle (Zhu et al., 2017). Addition of Gln to cells in vitro can result in an increase in total glutathione concentration (Matés et al., 2002). These data suggest that Gln can enhance the capacity of spermatozoa to synthesize GSH during liquid preservation when these cells are subjected to oxidative stress.
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In conclusion, the use of the BTS extender containing Gln improved boar sperm motility, acrosome integrity, and membrane integrity after periods of liquid preservation. In addition, Gln can quench ROS while enhancing γ-GCS activity and GSH content of spermatozoa. The results also indicate that extender supplemented with Gln provides greater protective capacity on boar sperm during liquid preservation against oxidative stress by enhancing GSH synthesis.
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Conflict of interest
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The authors confirm that this article’s content has no conflicts of interest. Acknowledgements
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Funding
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This research was supported by the National Natural Science Foundation of China (Project No. 31372311) and Scientific Research Foundation of Northeast Agricultural University (Project No.106518001).
Reference
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Fig. 1. Sperm motility on different days during liquid storage of boar semen at 17°C in extenders with different concentrations of L-glutamine; Columns with different lowercase letters differ (P < 0.05), n = 6
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Fig. 2. Effect of different concentrations of Gln on boar spermatozoa membrane integrity; Columns with different lowercase letters differ (P < 0.05); n = 6
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Fig. 3. Effect of different concentrations of Gln on spermatozoa acrosome integrity; Columns with different lowercase letters differ (P < 0.05); n = 6
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Fig. 4. Total oxidant status (μM H2O2 equiv.), TAS (mM Trolox equiv.), and OSI in extenders on different days during liquid storage of boar semen with or without 20 mM Gln; Columns with different lowercase letters differ (P < 0.05); n = 6; TAS = total antioxidant status, TOS = total oxidant status, OSI = oxidative stress index
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Fig. 5. Intra-sperm GSH/GSSG ratio on different days during liquid storage of boar semen with or without 20 mM Gln; Columns with different lowercase letters differ (P < 0.05); n = 6
I N U SC R A M ED PT CC E A Fig. 6. Flow cytometric analysis of sperm labelled with DCFDA/PI; Region Q1 (DCFDA-/PI+) represents apoptotic sperm with a relatively lesser intracellular ROS level, while Region Q2 (DCFDA+/PI+) represents an apoptotic sperm with a relatively greater intracellular ROS concentration; Region Q3 (DCFDA+/PI-) represents a viable sperm with a relatively greater intracellular ROS level; Region Q4 (DCFDA-/PI-) represents viable sperm with a relatively lesser intracellular ROS level
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Fig. 7. Effects of Gln on sperm GSH content, γ-glutamyl cysteine synthetase (γ-GCS) activity and spermatozoa motility during liquid preservation; GSH content A; γ-glutamyl cysteine synthetase (γ-GCS) activity B; and spermatozoa motility C; Data are expressed as the means ± SEM; (n = 3); Values depicted in columns with different lowercase letters (a, b) are different P < 0 .05
Table 1 Effect of different concentrations of Gln on spermatozoa kinematic variables Kinematic variables Days of Gln VCL VSL VAP BCF LIN WOB STR Storage (μm/s) (μm/s) (μm/s) (Hz) (%) (%) (%) 0 mM
94.25±4.91a
52.21±2.81a
86.21±4.32a
42.64±2.16a
55.39±0.62a
91.47±0.12a
60.56±0.60a
10 mM
92.88±0.87a
49.18±5.75a
84.36±2.51a
41.23±0.60a
52.96±4.66a
90.83±0.37a
58.29±3.38a
20 mM
93.44±2.64a
50.46±0.58a
87.13±2.90a
42.93±0.42a
54.72±0.90a
93.25±0.30a
57.91±0.40a
40 mM
92.87±3.69a
45.98±0.20a
84.05±1.52a
40.64±0.97a
49.51±0.88a
90.51±0.25a
54.71±1.19a
80 mM
91.55±0.31a
46.86.±0.54a
83.91±1.49a
40.73±1.07a
51.19±2.10a
91.65±0.57a
55.85±0.64a
0 mM
76.88±2.31a
40.14±1.65a
69.99±2.18a
37.00±1.09a
52.21±0.49a
91.04±0.14a
57.35±0.67a
10 mM
77.33±0.33a
41.95±1.76ab
71.80±0.78a
38.08±0.39a
54.25±3.53a
92.85±1.00a
58.43±4.63a
20 mM
82.81±2.41ab
45.98±1.73ab
76.27±2.27ab
38.60±0.67a
55.52±0.47a
92.10±0.13a
60.29±0.64a
40 mM
84.73±2.12b
44.07±1.82ab
76.60±1.33b
37.72±1.14a
80 mM
81.07±0.36ab
41.78±1.10b
73.21±0.68ab
37.42±0.03a
0 mM
71.96±2.03a
36.7±0.35a
65.35±2.20a
35.17±0.78a
10 mM
73.30±2.10ab
37.77±0.43a
67.08±2.50ab
36.53±0.88a
20 mM
80.99±1.73b
44.05±2.29b
75.22±1.31ab
37.61±0.46a
40 mM
80.33±2.02b
44.48±1.47b
73.73±5.53b
36.01±1.95a
80 mM
75.67±2.94ab
39.34±4.65a
68.41±3.65ab
0 mM
74.12±1.24a
38.54±0.48a
66.86±1.69a
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77.82±2.25
41.51±2.21
20 mM
81.27±2.03b
45.19±1.31b
40 mM
75.25±2.73ab
80 mM
50.98±4.72c
70.87±1.04
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57.53±4.82a
51.54±1.63a
90.31±0.46a
57.07±2.17a
51.05±2.21a
90.81±0.44a
56.16±2.08a
51.53±1.07a
91.51±0.21a
56.31±1.02a
54.39±4.20a
92.88±0.84a
58.56±3.89a
54.92±1.44a
91.78±0.29a
60.33±1.37a
52.00±1.26a
90.40±0.84a
57.51±3.92a
33.15±0.85a
52.00±2.17a
90.21±0.43a
57.64±2.06a
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58.57±2.09a
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52.02±0.11a
36.28±1.29 a
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10 mM
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35.72±0.52
53.35±2.16
91.07±0.43
75.51±3.84b
37.33±1.92a
55.61±1.55a
92.92±0.31a
59.85±1.47a
38.21±3.46ab
68.60±8.47a
32.34±4.24a
50.78±3.49a
91.16±0.70a
55.70±1.71a
27.46±3.16c
45.56±4.41c
33.80±2.21 a
53.87±0.52a
89.37±0.10a
60.27±0.71a
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Values are expressed as the means ± SEM (n = 6); Values associated with different lowercase letters (a, b, and c ) within the columns are different (P < 0.05); VCL = curvilinear velocity, VSL = straight-line velocity, VAP = average path velocity, BCF = beat-cross frequency, LIN = linearity (VSL/VCL), WOB = wobble (VAP/VCL), STR = straightness (VSL/VAP)
Table 2 Effects of Gln on sperm ROS production during liquid preservation Days of storage 0 1 3
5
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8.51±1.21a
14.77±1.70a
19.58±1.63a
26.67±1.42a
Gln
8.74±0.71a
8.95±1.51b
9.24±1.37b
9.45±1.26b
Excluding nonviable cells Sperm with high ROS level (%)
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3.89±0.91a
9.67±1.96a
14.01±1.55a
23.55±1.64a
Gln
3.06±1.42a
4.41±0.93b
5.83±1.38b
6.78±0.86b
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Values are expressed as the means ± SEM (n = 6); Values associated with different lowercase letters (a and b) within a column are different (P < 0.05)