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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

Reactive oxygen species (ROS) are one of several crucial factors that cause mammalian sperm damage during handling and preservation. Their deleterious effects can be restricted by the action of antioxidants. The present study aimed at investigating: (i) effects of cold storage prior to freezing on activities of enzymatic antioxidants (superoxide dismutase; SOD and glutathione peroxidase; GPx) and level of total reactive oxygen species (tROS); and (ii) effects of SOD or SOD plus GPx supplementation to chilled (3 and 96 h) and frozen-thawed semen. Six privately owned dogs were included. Experiment I: Each pooled semen was divided into three equal aliquots, which were subjected to chilled storage for 3, 24 or 96 h prior to freezing (n = 7). The activities of SOD and GPx in sperm cells and tROS level in chilled and frozen-thawed semen were measured. Experiment II: Pooled semen was divided to be cold stored for 3 or 96 h in three different extenders; (i) Uppsala Equex extender (control), (ii) Uppsala Equex extender supplemented SOD, or (iii) Uppsala Equex extender supplemented with SOD plus GPx. Sperm motility, viability and integrity of acrosome and DNA was evaluated after cold storage and frozen-thawed. The cold storage from 24 h prior to freezing resulted in a decrease in the SOD activity in the frozen-thawed sperm cells whereas the GPx activity and tROS levels were not affected. In addition, the supplementation of SOD plus GPx enhanced the percentage of sperm viability and DNA integrity after cold stored and frozen-thawed. In sum, the SOD activity is compromised by cold storage prior to freezing of dog semen. Addition of GPx is suggested to assist SOD to complete the enzymatic ROS scavenging system in the dog sperm.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

Technology for semen cryopreservation draws a striking attraction since artificial insemination (AI) has been introduced as a powerful tool to manage dog breeding programs. Although cold storage of dog semen at 4–5°C could maintain fertility and quality up to 7 days (Ponglowhapan et al. 2004), freezing of sperm in liquid nitrogen facilitates long-term storage. In dog breeding, the process of freezing is usually not possible at kennels. Semen is generally collected on site, cold stored and transported to perform cryopreservation at a well-equipped laboratory (Hermansson and Linde Forsberg 2006). During sperm preparation and cryopreservation processes, oxidative stress, an imbalance condition between naive antioxidant and reactive oxygen species (ROS) level, has been considered one of the crucial factors causing sperm damage i.e. decrease of sperm motility, membrane fluidity, increase of membrane disruption, impairment of DNA and mitochondrial function (Agarwal et al. 2003). The ROS, superoxide radicals (O2), hydroxyl radicals (OH˙), hydrogen peroxide (H2O2), are free radicals which freely attack polyunsaturated fatty acids on mammalian sperm membrane lipid peroxidation, with subsequent failure of fertility (Agarwal et al. 2003). The ROS are generated by an oxygen consumption of sperm themselves during storage under aerobic condition, and their levels increase after sperm cryopreservation and post-thaw incubation process (Chatterjee and Gagnon 2001; Agarwal et al. 2003). The deleterious effect of ROS can be minimized by the action of enzymatic and non-enzymatic antioxidants (Agarwal et al. 2003). The enzymatic antioxidants comprise of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD). They are abundant and constituent in sperm and normal morphology, male accessory glands, epididymis and seminal plasma (Chabory et al. 2010). However, when dog semen is frozen in the laboratory seminal plasma is routinely discarded which may result in an insufficient enzymatic antioxidant level. The SOD is the first line defense mechanism against the harmful effects of ROS by catalyzing O2 from the cytosol of living cells; however, H2O2 is generated (Silva 2006). The CAT and GPx can potentially act as H2O2 detoxifier to water (Silva 2006). Supplementation of enzymatic and non-enzymatic antioxidants (i.e. vitamin C, N-acetyl cysteine) to the semen extender has been shown to improve dog semen quality after short and long-term cold storage (Michael et al. 2009). Among three different enzymatic antioxidants, only the action of CAT supplemented to dog semen extender has been investigated (Michael et al. 2009).

The aim of the present study was to investigate; (i) the activities of natural enzymatic antioxidants (SOD and GPx) and the level of total reactive oxygen species (tROS) in chilled (3, 24 and 96 h) and frozen-thawed dog semen, and (ii) the effects of enzymatic antioxidants (SOD or SOD plus GPx) addition to chilled (3 and 96 h) and frozen-thawed dog semen.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

Animals

Six privately owned Siberian husky (n = 3) and beagle (n = 3) dogs of proven fertility, aged between 2 and 5 years old, with semen quality of ≥70% sperm motility and ≥70% normal sperm morphology were included in this study.

Experimental design

Experiment I was to determine activities of SOD and GPx, level of tROS in chilled and frozen-thawed semen. Three ejaculates were pooled to obtain the total sperm number of 1200 × 106. Three aliquots of each pooled sample (n = 7) were subjected to chilled storage for 3, 24 or 96 h prior to freezing. The activities of SOD, GPx and tROS level were assessed after cooling (3, 24 or 96 h) and after freezing/thawing. Experiment II was performed to study protective effects of SOD and GPx supplementation to chilled and frozen-thawed semen. Two or three ejaculates were pooled to constitute a total of 600 × 106 sperm. The pooled semen (n = 9) was centrifuged, resuspended and cold stored for 3 and 96 h in three different semen extenders; (i) Uppsala Equex extender (control) (Rota et al. 1997), (ii) Uppsala Equex extender supplemented with 100 IU SOD (SOD), or (iii) Uppsala Equex extender supplemented with 100 IU SOD plus 5 IU GPx (SOD plus GPx). Furthermore, the 3 h cold stored sperm sample was continuously processed for cryopreservation. The Sperm quality (motility, viability and integrity of acrosome and DNA) was assessed after cold storage (3 and 96 h) and at 0 and 6 h post-thaw.

Semen processing

Semen extender and freezing was performed according to the Uppsala system (Rota et al. 1997).

Extraction of antioxidative enzyme from the sperm cells

Antioxidative enzyme extraction was performed using a hyposmotical-thermical shock method (Cassani et al. 2005). Briefly, sperm pellet was resuspended with 1 ml normal saline and centrifuged at 600 g for 5 min twice. The supernatant was discarded. The sperm pellet was resuspended with 1 ml double distilled water, cryopreserved at −20°C for 2 h and thawed at room temperature (repeated twice). The sample was then centrifuged at 20 000× g 4°C for 20 min. A 0.5 ml aliquot of supernatant was stored at −20°C until analysed.

Antioxidative enzymes and total reactive oxygen species measurement

Superoxide dismutase (SOD) assay

Superoxide dismutase activity was measured by a Ransod Kit (Randox Laboratories Ltd., Antrim, UK) (Tavilani et al. 2008). The principle of the assay is as followed; superoxide radicals are generated by xanthine-xanthine oxidase system. The superoxide radicals react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (I.N.T.) to form a red formazan dye while SOD inhibits superoxide radical activity. Thus in this assay, one unit of SOD activity was the amount of SOD that caused a 50% inhibition of the I.N.T. rate reduction under the assay condition. Briefly, 0.05 ml of 10-fold diluted extracted antioxidative enzyme sample and Ransod sample diluents were thoroughly mixed with commercial test kit-mixed substrate (0.05 mm/l xanthine and 0.025 mm/l I.N.T.). A 0.25-ml xanthine oxidase was added to the sample. The level of SOD activity was measured by a spectrophotometer at 505 nm wavelength at 37°C against air.

Glutathione peroxidase (GPx) assay

Glutathione peroxidase activity was measured by a Ransel Kit (Randox Laboratories) (Tavilani et al. 2008). The principle of the assay is as follows; GPx catalyze glutathione (GSH)-cumene hydroperoxide reaction to oxidized GSH (GSSG). With the reaction between GSSG, reduced nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione reductase (GR), nicotinamide adenine dinucleotide phosphate (NADP+) is generated. The GPx activity level was indirectly measured by the increase of NADP+ level. Briefly, 0.02 ml of 10-fold diluted extracted antioxidative enzyme sample and Ransel sample diluents was thoroughly mixed with reagent I (4 mm/l GSH, 0.5 U/l GR and 0.34 nm/l NADPH) and 0.18 mm/l cumene hydroperoxide. The level of GPx activity was measured by a spectrophotometer at 340 nm wavelength at 37°C against air.

Total reactive oxygen species

Level of tROS was measured using the method described by Michael et al. (2008). Briefly, 0.1 ml of sample diluted with 0.89 ml double distilled water, was mixed with 1 mm luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma Chemical Co, St. Louis, MO, USA) which has been considered as the sensitive chemiluminescent probe that could react with several types of ROS. The absorbance was spectrophotometrically measured at 380 nm wavelength. The level of tROS (Ɛ) was calculated and reported followed the equitation of Lambert Beer's Law; A = ƐdC. (A, absorbance; Ɛ, molar extinction coefficient; d, path length in cm and C, molar concentration).

Evaluation of sperm quality

Sperm concentration was determined using a haemocytometer chamber (Boeco, Hamburg, Germany). William's staining method was used for sperm head morphology assessment, whereas sperm fixed in formal saline were evaluated for midpiece and tail morphology. Percentage of motile sperm was assessed before and after cryopreservation under a phase contrast microscope at 200 × magnification. Sperm membrane integrity was determined by a double-fluorescent labeling technique; SYBR-14 and propidium iodide (Dead/Alive Kit; Molecular Probe Inc., Eugene, OR, USA) and acrosome integrity was assessed by staining with FITC-PNA (Sigma) and propidium iodide (Molecular Probe) as described by Axnér et al. (2004). Sperm DNA integrity was evaluated using acridine orange (AO) (Sigma) as described by Thuwanut et al. (2008).

Statistical analysis

Data were analyzed using a mixed procedure (version 9.0; SAS Institute Inc., 2002, Cary, NC, USA). Normal distribution of residuals from the statistical models was tested using the univariate procedure option normal. The statistical models included the fixed effects of individual dogs, cold storage and post-thaw incubation time as well as types of semen extenders. The dependent variables (sperm quality, SOD and GPx activities) were evaluated using anova (glm procedure). The levels of tROS were compared using npar1way procedure and a Kruskall-Wallis test. Values were presented as mean ± SD. The level of significance was set at p ≤ 0.05.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

Experiment I: the activities of SOD and GPx in the sperm cell and the tROS level in extended semen

The SOD and GPx activities in the dog semen did not differ throughout the cold storage (p > 0.05). However, the SOD activity decreased in frozen-thawed samples after cold storage for 24 and 96 h (p < 0.05). There were no significant differences in the tROS levels among periods of cold storage time or between chilled and frozen-thawed samples (p > 0.05) (Table 1).

Table 1. Activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in dog sperm cell and levels of total reactive oxygen species (tROS) in extended semen (mean ± SD)
Parameter3 h24 h96 h
ChilledFrozenChilledFrozenChilledFrozen
  1. Different letters (a, b) within the same row indicate significant differences (p < 0.05).

SOD activity (Unit/50 × 106 sperm)110.7 ± 2.8109.9 ± 2.3a106.7 ± 2.2103.1 ± 2.4b106.8 ± 2.0103.5 ± 5.5b
GPx activity (Unit/50 × 106 sperm)215.3 ± 11.1237.6 ± 18.4218.6 ± 7.0216.8 ± 4.3221.8 ± 5.4219.3 ± 4.9
tROS (10−7/molar)237.2 ± 149.1326.4 ± 316.6181.7 ± 70.6244.9 ± 176.9259.9 ± 210.0370.7 ± 297.5

Experiment II: the effects of SOD or SOD plus GPx addition to chilled and frozen-thawed semen

Supplementation of the SOD plus GPx enhanced percentage of sperm viability after 96 h cold-storage compared to the control and the SOD extender (p < 0.05) (Table 2). Immediately after thawing, the SOD plus GPx extender had higher number of viable sperm than the control (p < 0.05) but no difference was found compared to SOD extender (p > 0.05). At 0 and 6 h post-thaw, the sperm number with intact DNA was higher in the SOD plus GPx extender compared to the control (p < 0.05) but did not differ from the SOD extender (Table 2).

Table 2. Dog sperm quality (mean ± SD) after cold storage and subsequent freezing and thawing in an Uppsala Equex extender without antioxidative enzyme supplementation (control), with 100 IU superoxide dismutase (SOD) plus 5 IU glutathione peroxidase (GPx) (SOD + GPx Ext), or with 100 IU SOD (SOD Ext)
Sperm quality (%)ControlSOD + GPx ExtSOD Ext
  1. Different letters (a, b) within the same row indicate significant differences (p < 0.05).

Chilled 3 h
Motility78.9 ± 6.0a,b82.8 ± 6.7a73.3 ± 8.7b
Viability84.1 ± 6.7a86.7 ± 4.8a76.5 ± 6.7b
Intact DNA95.1 ± 5.1a,b96.2 ± 2.8a88.8 ± 10.3b
Intact acrosome76.2 ± 13.182.6 ± 12.371.3 ± 16.4
Chilled 96 h
Motility60.6 ± 23.566.1 ± 20.055.0 ± 27.2
Viability70.1 ± 6.2a79.3 ± 8.3b66.2 ± 6.7a
Intact DNA91.6 ± 9.894.8 ± 6.386.1 ± 14.2
Intact acrosome60.6 ± 16.859.2 ± 24.058.4 ± 24.1
At 0 h post thaw   
Motility40.0 ± 18.052.2 ± 9.744.0 ± 21.8
Viability34.1 ± 14.0a47.6 ± 12.0b37.9 ± 14.8a,b
Intact DNA80.3 ± 6.3a86.2 ± 6.5b80.9 ± 5.1a,b
Intact acrosome64.1 ± 20.865.2 ± 17.764.2 ± 15.1
At 6 h post-thaw
Motility28.3 ± 19.831.7 ± 17.020.0 ± 16.6
Viability24.5 ± 13.136.1 ± 13.330.3 ± 15.5
Intact DNA72.8 ± 12.0a84.5 ± 9.9b74.6 ± 12.7a,b
Intact acrosome32.5 ± 15.436.2 ± 14.029.2 ± 16.2

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

In this study, we demonstrated the effect of cold storage prior to freezing on enzymatic antioxidant system as well as the combined effect of SOD and GPx on improving the quality of chilled and frozen-thawed dog semen. In the present study, the cold storage up to 96 h did not affect the SOD activity of the dog sperm. Our results were supported by the unchanged production of superoxide anion (O2), the SOD substrate, during the dog semen cold stored at 4°C for 72 h in the previous study (Michael et al. 2009), suggesting that during cold storage for up to 96 h the SOD antioxidant system efficiently neutralized O2 production. However, the SOD activity was deteriorated by freezing and thawing to the extent with increasing time of cold storage prior to freezing. Accordingly, in this study, the GPx activity did not decline throughout the cold storage time. The result was in accordance with the steady level of GPx in the bull sperm cold stored for 72 h (Nair et al. 2006). Moreover, the unchanged tROS in this study suggested that the antioxidant system likely defended efficiently during the cold storage of the dog sperm.

In this study, the semen extender supplemented with SOD plus GPx improved the sperm quality of both chilled and frozen-thawed semen particularly sperm viability and DNA integrity. It is likely that the presence of GPx was necessary to transform H2O2 generated by the SOD scavenging system into a harmless product, H2O (Chabory et al. 2010). Without GPx, H2O2 accumulation leads to hydroxyl radical (OH2) production, which attacks cell membrane and nucleic acids, ultimately contributing to cell death (Chabory et al. 2010). Moreover, the H2O2 itself can cause damage to the sperm membrane, mitochondrial damage, DNA fragmentation and induce a caspase-3 activity level leading to sperm apoptosis (Agarwal et al. 2003).

In conclusion, the cold storage prior to freezing compromised the SOD activity in the dog sperm cells. Moreover, GPx seemed to work efficiently in corporate with SOD to scavenge the ROS in chilled and frozen-thawed dog semen Thus, addition of GPx is suggested when dog semen extender is supplemented with SOD to protect sperm viability and DNA integrity.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

This study was funded by Thailand Research Fund (MRG-WI515S017). The authors are grateful for Dr. Nuthee Am-in for his assistance on statistical analysis.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References

KC was responsible for the study design, discussion and manuscript revision. AC was responsible for sample collection, SOD, GPx and tROS analyses. PT was responsible for sperm analyses and manuscript preparation. SP was responsible for manuscript revision.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References
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