Detection of intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) and lipid peroxidation during cryopreservation of alpaca spermatozoa
Contents
The objective of this study was to detect changes in intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) production and lipid peroxidation during cryopreservation of alpaca spermatozoa. Twelve alpaca semen samples were conventionally cryopreserved. Intracellular superoxide anion and hydrogen peroxide were evaluated by fluorescence microscopy using dihydroethidium (DHE)/YO-PRO-1 and dichlorofluorescein diacetate (H2DCFDA)/propidium iodide (PI), respectively. Evaluations were performed during cooling curve at (1) 25°C, (2) 15°C, (3) 5°C/0 min, (4) 5°C/15 min, (5) 5°C/30 min and (6) after freezing/thawing. Evaluation of lipid peroxidation by measuring malondialdehyde (MDA) was performed at 25°C, 5°C/30 min and after thawing. Maximum percentages of total spermatozoa producing superoxide anion and hydrogen peroxide were found at 5°C/30 min (62.8 ± 6.3% and 30.5 ± 5.6%, respectively), and these results were higher (p < .05) than initial (25°C: 10.8 ± 3.8% and 6.8 ± 0.7%, respectively) and after thawing (29.8 ± 9.5% and 7.5 ± 1.8%, respectively) values. However, considering only viable spermatozoa, production of superoxide anion and hydrogen peroxide during overall stabilization at 5°C (>76% and >91%, respectively) and after thawing (74.9 ± 5.0% and 78.9 ± 2.2%, respectively) was higher (p < .05) than initial values at 25°C (38.7 ± 3.1% and 53.6 ± 2.0%, respectively). Lipid peroxidation at 25°C, 5°C/30 min, and post-thawing were 346.5 ± 99.8, 401.1 ± 64.8 and 527.7 ± 142.8 ng/ml MDA, respectively. These results showed that high percentage of viable alpaca spermatozoa produces intracellular reactive species oxygen (ROS) during the cryopreservation process of alpaca semen.
1 INTRODUCTION
In alpacas, there are some detailed studies on ejaculated (Santiani, Evangelista, Valdivia, Risopatrón, & Sánchez, 2013; Santiani et al., 2005) or epididymal sperm (Banda et al., 2010; Morton, Bathgate, Evans, & Maxwell, 2007; Morton, Evans, & Maxwell, 2010) cryopreservation. However, a successful cryopreservation protocol for alpaca spermatozoa has not been developed. Acceptable pregnancy rates (60%) using artificial insemination (AI) with diluted semen are reported in alpacas (Alarcón, García, & Bravo, 2012). Nevertheless, AI with frozen semen in alpacas is not commonly used because of low sperm motility percentages (0%–20%) in alpaca spermatozoa after cryopreservation (Santiani et al., 2013).
During cryopreservation of ram semen, there is a decrease in sperm motility and viability, probably because of an increase in membrane lipid peroxidation and sperm DNA fragmentation (Kasimanickam, Pelzer, Kasimanickam, Swecker, & Thatcher, 2006; Lukanov et al., 2009). In that sense, cryopreservation process has been related to an increase in reactive oxygen species (ROS) production in ram (Santiani et al., 2014) and human (Wang, Zhang, Ikemoto, Anderson, & Loughlin, 1997) spermatozoa.
Probably, there is also oxidative stress during cryopreservation of alpaca semen, because the use of antioxidants SOD analogues during this process improves post-thaw motility and reduces DNA fragmentation (Santiani et al., 2013). Additionally, superoxide dismutase (SOD) can reduce oxidative damage during cryopreservation of ram spermatozoa (Santiani et al., 2014) and prevent sperm physiological effects mediated by ROS in human spermatozoa (De Lamirande, Tsai, Harakat, & Gagnon, 1998).
Therefore, the objective of this study was to determine whether there is oxidative stress during the process of alpaca semen cryopreservation through the detection of changes in intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) production and lipid peroxidation.
2 MATERIALS AND METHODS
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), except when otherwise indicated.
2.1 Animals and semen samples
Twelve semen samples were collected from four Huacaya-breed male alpacas during the months of October and November, in Lima, Peru, at sea level. Maintenance of alpacas as well as semen collections was carried out according to the guidelines of the Ethical Committee of Universidad Científica del Sur. Semen collections were performed by deviation of the penis into an ovine artificial vagina with internal temperature of 42°C, wrapped in an electric warming pad, while male alpacas were mounting a receptive female alpaca. Before initial evaluation, all samples were passed through a 21 ga needle several times to eliminate viscosity. Sperm motility was assessed under a coverslip (18 mm × 18 mm) on a warm glass slide using light microscopy (400× magnification), and sperm concentration was evaluated using a hemocytometer. Initial values of sperm motility and sperm concentration were 51.4 ± 6.3% and 148.3 ± 95.8 × 106 cells/ml, respectively.
2.2 Dilution, cooling curve and cryopreservation
Only ejaculates with at least 30% of motility and 50 × 106 spermatozoa/ml were diluted 1:3 on a skimmed milk-egg yolk-fructose extender (Santiani et al., 2005) warmed at 35°C. First 30 min after dilution, temperature was allowed to go down naturally (Until 25°C approximately) while samples were transported to a laboratory. Once arrived, each semen sample was divided into six aliquots for evaluation of oxidative stress in different moments of the cooling curve, stabilization period and after cryopreservation. Aliquots were slow cooled (−1°C/3 min) from 25 to 5°C during 60 min. At 5°C, dimethylacetamide as cryoprotectant agent was added (1 M final concentration). Then, samples were loaded into 0.25 ml French straws. Straws were kept at 5°C for 30 min for stabilization of cryoprotectant, then exposed to liquid nitrogen vapour for 15 min and finally plunged into liquid nitrogen (Santiani et al., 2013). Straws were thawed in water bath at 37°C for 1 min, 24 hours after cryopreservation.
2.3 Experimental design
Previously to each evaluation, samples were washed by centrifugation (600 g for 5 min) for extender removal and resuspended in PBS (250 μl). Detection of intracellular superoxide anion and hydrogen peroxide were performed using aliquots at (i) 25°C, (ii) 15°C, (iii) at the beginning of stabilization period (5°C/0 min), (iv) at 15 min of stabilization period at 5°C (5°C/15 min), (v) 30 min of stabilization period at 5°C (5°C/30 min) and (iv) after cryopreservation. In addition, lipid peroxidation and motility were assessed using samples at 25°C, 5°C/30 min and after cryopreservation.
2.4 Detection of intracellular reactive oxygen species
Dihydroethidium (DHE) and dichlorofluorescein diacetate (H2DCFDA) were used for superoxide anion and hydrogen peroxide determination, respectively, according to Mahfouz, Sharma, Lackner, Aziz, and Agarwal (2009). For superoxide anion, 10 μl of DHE working solution (125 μM, diluted in PBS; Stock solution 12.5 mM, diluted in DMSO) was added to 100 μl of washed sample and incubated for 20 min at 25°C. After incubation, 1 μl of YO-PRO-1 (Molecular Probes, Eugene, OR, USA, Stock 10 μM, diluted in DMSO) was added to each sample, just prior to evaluation. For hydrogen peroxide, 1 μl of H2DCFDA working solution (250 μM, diluted in PBS; Stock solution 25 mM, diluted in DMSO) was added to 100 μl of washed sample and incubated for 40 min at 25°C. After incubation, 1 μl of propidium iodide (PI, stock 1 mg/ml) was added to each sample, just prior to evaluation. Spermatozoa (at least 200 cells per sample) were evaluated using an i4-Epi-Lumni™ (LW Scientific, Lawrenceville, GA, USA) epifluorescence microscope. For DHE/YO-PRO-1, spermatozoa showing red/orange fluorescence were considered producing superoxide anion. For H2DCFDA/PI, spermatozoa showing green fluorescence were considered producing hydrogen peroxide. For YO-PRO-1 and PI, spermatozoa not showing green nor red, respectively, were considered viable cells. Results were expressed as percentage of total (including viable and non-viable cells) spermatozoa producing superoxide anion or hydrogen peroxide, and percentage of viable spermatozoa producing superoxide anion or hydrogen peroxide.
2.5 Lipid peroxidation assessment
Lipid peroxides were determined by the modified thiobarbituric acid assay (TBA) through measuring malondialdehyde (Aitken, Clarson, & Fishel, 1989). Briefly, each sample with a 20 × 106 spermatozoa/ml was diluted in 250 μl PBS, mixed with 125 μl of ferrous sulphate (0.2 mM) and 125 μl of sodium ascorbate (1 mM) and incubated for 60 min at 37°C. After incubation, samples placed into ice for 15 min. Then, 250 μl PBS and 250 μl 40% trichloroacetic acid were added and samples were centrifuged at 2500 g for 10 min at 4°C. Five hundred μl of supernatant was mixed with 125 μl of 2% TBA cleared with 5 μl NaOH (5 M). Reaction mixtures were heated at 90°C for 10 min and cooled at room temperature for 30 min. The absorbance was read using a Spectroquant® Pharo 300 (Merck KGaA, Darmstadt, Germany) spectrophotometer at 532 nm against a reagent blank. Results were compared with a MDA standard curve previously established in the laboratory and expressed as malondialdehyde (ng/ml).
2.6 Statistics analysis
Statistics analysis was performed using the GraphPad Prism® version 3.0 (GraphPad Software Inc, San Diego, USA) software. The effect of evaluations (At 25°C, 15°C, 5°C, 5°C/15 min, 5°C/30 min and post-thawing) on intracellular ROS production, lipid peroxidation and motility was analysed using repeated-measures ANOVA and Tukey's post hoc test. Values are means ± SEM. The level of significant was set at a p-value < .05.
3 RESULTS
Percentages of alpaca spermatozoa producing superoxide anion and hydrogen peroxide were assessed during the cryopreservation process. At the beginning of the cooling curve (25°C), spermatozoa producing superoxide anion and hydrogen peroxide were 10.8 ± 3.8% and 6.8 ± 0.7%, respectively. Then, spermatozoa producing both intracellular ROS increased gradually when reached 15°C (23.2 ± 8.1% and 10.2 ± 1.4%, respectively) and 5°C (44.0 ± 6.3% and 20.0 ± 3.4%, respectively). For superoxide anion, percentages at 5°C, 5°C/15 min and 5°C/30 min (44.0 ± 6.3%, 48.9 ± 8.4%, and 62.8 ± 6.3%, respectively) were higher (p < .05) than values at 25°C (10.8 ± 3.8%). On the same way, percentage of spermatozoa producing hydrogen peroxide at 5°C/15 min and 5°C/30 min (24.8 ± 4.6% and 30.5 ± 5.6%, respectively) were higher (p < .05) than values at 25°C (6.8 ± 0.6%). After freezing/thawing, percentages returned to basal values for superoxide anion (29.8 ± 9.5%) and hydrogen peroxide (7.5 ± 1.8%) and were lower (p < .05) than 5°C/30 min (Figure 1).
In relation to viable spermatozoa, at 5°C, 5°C/15 min, 5°C/30 min and after thawing, production of superoxide anion (77.0 ± 4.7%, 79.8 ± 5.6%, 82.8 ± 3.8% and 74.9 ± 5.0%, respectively) and hydrogen peroxide (93.5 ± 2.0%, 92.0 ± 2.2%, 93.4 ± 1.6% and 78.9 ± 2.2%, respectively) were higher (p < .05) than initial values at 25°C (38.7 ± 3.1% and 53.6 ± 2.0%, respectively) (Figure 2).
Lipid peroxidation was similar (p > .05) when evaluated MDA at 25°C (346.5 ± 99.8 ng/ml), 5°C (401.1 ± 64.8. ng/ml) and after thawing (527.6 ± 142.8 ng/ml). However, sperm motility was higher (p < .05) at 25°C (51.4 ± 6.3%) and 5°C/30 min (38.6 ± 6.2%) than after thawing (11.3 ± 4.4%) (Figure 3).
4 DISCUSSION
The present study demonstrates that there is an increase in ROS production by alpaca spermatozoa during the process of cryopreservation, similar to previous studies in human (Wang et al., 1997), bull (Chatterjee & Gagnon, 2001), canine (Kim, Yu, & Kim, 2010) and ram (Santiani et al., 2014) spermatozoa. Percentages of spermatozoa producing superoxide anion and hydrogen peroxide were increasing gradually while temperature was decreasing, reaching maximum values during stabilization period at 5°C. Nevertheless, production of ROS after freezing/thawing apparently was significantly lower, similar than initial values. In the same way, Wang et al. (1997) and Santiani et al. (2014) reported that despite a significant increase in ROS at 5°C, ROS levels returned to baseline values after freezing/thawing. It was explained because most ROS are highly unstable and their lifetime under cellular conditions is relatively short, being in the nanosecond range (Newton & Milligan, 2006). In this way, it was believed that the oxidative stress is limited mainly to the end of stabilization period of spermatozoa, because it is not detectable after cryopreservation. In that sense, Kim et al. (2010) reported that percentages of spermatozoa producing hydrogen peroxide are similar between raw and frozen-thawed canine semen.
Plasma membrane in spermatozoa is the primary site of cold-induced damage (Bailey et al., 2008) and changes in the structure and/or function of the sperm plasma membrane has been associated with ROS (Wang et al., 1997). Increasing of ROS at 5°C could be related to the activation of some mechanisms of intracellular ROS production as NADPH oxidase activity (Medeiros, Forell, Oliveira, & Rodrigues, 2002) caused by altered distribution of sulphhydryl groups (Chatterjee, de Lamirande, & Gagnon, 2001) or changes in the location of lipids and phospholipids in the plasma membrane (Watson, 2000). However, our results indicate that although peaks of ROS production were found when analysing total (viable and non-viable) spermatozoa, a high percentage of viable alpaca spermatozoa continue producing superoxide anion and hydrogen peroxide after freezing/thawing. Similarly, after cryopreservation, canine (Kim et al., 2010) and boar (Kim, Lee, & Kim, 2011) spermatozoa producing hydrogen peroxide increased significantly in viable spermatozoa, excluding non-viable cells in analysis. In this regard, the analysis of ROS production considering only viable cells shows that there is not a significant decrease in ROS production after freezing/thawing and that the reduced levels of ROS found when analysing total spermatozoa would be mainly caused by the diminution of viable spermatozoa and not for the impairment in mechanisms of ROS production in viable spermatozoa after thawing. In addition, the decrease in superoxide dismutase activity and levels of reduced glutathione reported after cryopreservation of bull spermatozoa (Bilodeau, Chatterjee, Sirard, & Gagnon, 2000) could be also occurring in alpaca spermatozoa and explain high ROS production observed after thawing.
In the present study, we did not found significant increase in MDA as indicator of lipid peroxidation during cooling nor after thawing. However, highest levels of MDA were found in thawed samples, similar to reported in ram semen (Santiani et al., 2014). Nevertheless, significant increase in lipid peroxidation has been reported after cryopreservation of human (Alvarez & Storey, 1992) and bull (Chatterjee & Gagnon, 2001) semen. Recently, using BODIPY 581/591 C11, percentage of lipid peroxidation have been described as less than 1% (Santiani, Ugarelli, & Evangelista-Vargas, 2016) and around 10% (Santiani et al., 2015) in raw and cryopreserved alpaca spermatozoa, respectively. These results could indicate that there is a slight increase in lipid peroxidation during cryopreservation of alpaca spermatozoa.
Reduced alpaca sperm motility after cryopreservation found in this study is similar to previous reports freeze/thawed epididymal (Morton et al., 2007, 2010) and ejaculated spermatozoa (Santiani et al., 2005, 2013). Furthermore, addition of antioxidants analogues of superoxide dismutase in alpaca semen extender prevents loss of motility and DNA fragmentation (Santiani et al., 2013). In this way, it is probably that high levels of viable spermatozoa producing superoxide anion and hydrogen peroxide could be related to low sperm motility, as reported in ram semen (Peris, Bilodeau, Dufour, & Bailey, 2007; Stefanov et al., 2004).
In conclusion, intracellular production of superoxide anion and hydrogen peroxide during cryopreservation of alpaca semen is demonstrated, and this effect could be related to decrease in sperm motility.
ACKNOWLEDGEMENTS
We wish to thank Ximena Trelles, Daniel Muchotrigo and Katherine Choez for their assistance in the project and Universidad Nacional Daniel Alcides Carrión (UNDAC), Cerro de Pasco, Perú for providing alpaca males. This research was supported by the National Science and Technology Council (CONCYTEC) of Peru, under the grant PROCYT N°366-2012- CONCYTEC-OAJ.
CONFLICT OF INTEREST
None of the authors have any conflict of interest to declare.
AUTHOR CONTRIBUTIONS
Shirley Evangelista-Vargas was responsible for the design of the study and analysis of semen, ROS and MDA. Alexei Santiani was responsible for alpaca semen collection, data analysis and redaction of the manuscript.
