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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgement
  9. Conflicts of interest
  10. Funding
  11. References

Oxidative stress (OS) has been recognized as one of the most important causes of male infertility. The antioxidant activities of seminal plasma and epididymal fluid are not enough to prevent OS, which can damage sperm membranes and DNA, so antioxidant supplementation has been used as a treatment of male infertility. The aim of this experiment was to evaluate the DNA peroxidation before and after antioxidant supplementation with vitamin C and E in dogs with and without fertility problems. A total of eleven dogs were used and were divided in two groups: fertile group (G1), dogs with normal spermiogram (n = 5); subfertile group (G2): dogs with low sperm count (<20 × 106 sptz/ml) and/or more than 30% of total sperm pathology (n = 6). Both groups received 500 mg/day of vitamin C and 500 mg/day of vitamin E for 60 days. A semen sample was collected before (M1) and after (M2) oral supplementation. Samples were analysed for DNA peroxidation by measuring the 8-hydroxy-2′-deoxyguanosine concentration. No significant difference was observed between groups at either time. Oral supplementation with 500 mg/day of vitamin C and 500 mg/day of vitamin E did not change the DNA peroxidation in fertile and subfertile dogs.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgement
  9. Conflicts of interest
  10. Funding
  11. References

Although the traditional semen parameters like concentration, motility and morphology are often used to classify male infertility, it has become apparent that none of these are sufficient for the prediction of male fertility. During the last decade, the search for better predictors of male fertility has resulted in an increased focus on sperm DNA integrity (Bungum 2012). Oxidative stress (OS) has been associated with DNA damage to spermatozoa (Twigg et al. 1998). Spermatozoa are sensitive to OS because they possess limited endogenous antioxidant protection while presenting abundant substrates for free radical attack in the form of unsaturated fatty acids and DNA (Koppers et al. 2010). Peroxidation of DNA can lead to chromatin cross-linking, base changes and DNA strand breaks (Twigg et al. 1998); therefore, embryonic development and pregnancy rate can be influenced by the rate of peroxidation found in DNA (Fraga et al. 1991). The expanding research interest in the last two decades on reactive oxygen species (ROS), OS and male infertility has led to the development of various techniques for evaluating oxidative DNA damage in spermatozoa. Among various oxidative DNA adducts, 8-hydroxydeoxyguanosine (8-OHdG) has been selected as a representative of oxidative DNA damage owing to its high specificity, potent mutagenicity and relative abundance in DNA. It offers a specific and quantitative biomarker on the extent of oxidative DNA damage caused by ROS in sperm (Shen and Ong 2000). For example, OS occurs when there is an imbalance between the concentrations of ROS and antioxidants and the freeze-thawing of spermatozoa is associated with an increase in ROS generation (Ball et al. 2001). It is believed that a dietary supplementation with antioxidants may reduce the sperm DNA damage even if sperm cells were submitted to cryopreservation (Fraga et al. 1991).

The aim of this work was to compare peroxidation damage of DNA of frozen-thawed sperm from fertile and subfertile dogs after oral supplementation with vitamin C and E.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgement
  9. Conflicts of interest
  10. Funding
  11. References

Eleven healthy and sexually mature dogs were used in this study. They were from different breeds (4 Labrador Retriever, 3 German Sheperd, 1 Rottweiler, 1 Boxer, 1 Beagle, 1 Mongrel) and age ranging between 2 and 8 years. The dogs were from Military Police from Bauru, state of São Paulo, Brazil. All dogs were fed with a commercial dog food (Maxi adulto; Royal Canin) and water ad libitum. Based on the results of five previous semen analyses, dogs were divided in two groups – fertile dogs (G1): five dogs that had normal spermiogram; subfertile dogs (G2): six dogs that had low sperm count <20 × 106 spermatozoa/ml and/or more than 30% of total sperm pathology. After the first semen collection (M1), all dogs received an oral supplementation of 500 mg/day of vitamin C and 500 mg/day of vitamin E for 60 days, when semen was again collected (M2). After semen collection, an aliquot was removed to analyse sperm motility, concentration and pathology, and semen was centrifuged at 800 × g for 10 min. The seminal plasma was removed, and pellets were resuspended with TRIS/egg yolk/glycerol extender [composition: 3 g TRIS; 1.7 g citric acid; 1.25 g Fructose; 0.020 g amikacin sulphate; 20 ml egg yolk; 1 ml Orvus es Paste (OEP), 8 ml glycerol and distilled water to 100 ml, pH 6.8] to obtain a concentration of 30 × 106 spermatozoa/ml. The extended semen was loaded into 0.5-ml straws; then they were cooled to 4°C for 1 h, and then placed in liquid nitrogen vapour for 20 min. Immediately after, the straws were immersed in liquid nitrogen. The straws were thawed at 70°C for 8 s. To quantify the degree of DNA peroxidation after thawing, a commercial rapid sensitive competitive immunoassay for the detection and quantification of 8-OHdG was used (DNA Damage Elisa Kit; Assay designs Ann Arbor, MI, USA). Values were expressed in nanograms per millilitre of semen.

Data were analysed by anova using the General Linear Models procedure of sas (SAS Institute, Inc., Cary, NC, USA). For all analyses, p < 0.05 was considered significant.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgement
  9. Conflicts of interest
  10. Funding
  11. References

No significant difference was observed between either groups at any time period (p < 0.05). (Table 1).

Table 1. Mean values of sperm DNA peroxidation obtained after thawing the semen of dogs from both groups: fertile group (G1) and subfertile group (G2), before (M1) and after 60 days (M2) of oral supplementation with vitamin C and E. Values are expressed in ng/ml of 8-hydroxy-2′-deoxyguanosine
GroupMoment
M1M2
  1. Means with different letters indicate significant differences (p < 0.05).

G163.66 ± 4.73A64.88 ± 7.27A
G259.69 ± 6.68A67.44 ± 5.94A

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgement
  9. Conflicts of interest
  10. Funding
  11. References

Although necessary for survival, oxygen also leads to production of ROS, which, in a process named OS, can damage sperm DNA. Oxidative stress is caused by an imbalance between the antioxidant ability in seminal plasma and the production of ROS leading to the formation of oxidative products such as 8-OHdG (Bungum 2012). Normally, sperm cells are protected by seminal plasma, which contains a variety of antioxidants that counteract the damaging effects of ROS, but sperm preparation for cryopreservation often involves the removal of seminal plasma; consequently, cryopreservation of spermatozoa may subject sperm cells to OS and potential DNA damage (Ball et al. 2001). Oral supplementation with antioxidants may feasibly reduce the negative ROS impact on spermatozoa. In the present study, samples were frozen/thawed to increase ROS level, and the aim was to observe whether oral supplementation with antioxidants for 60 days could prevent or decrease DNA peroxidation. It is important to recognize that there may have been adequate antioxidant in the commercial diets fed to these dogs, and the study was examining the effect of extra supplementation.

No difference was observed between moments and groups, which also suggests that probably DNA peroxidation was not one of the factors that caused subfertility of our experiment group (G2). These results are in disagreement with others previous reported, where the levels of 8-OHdG in sperm DNA from infertile men were significantly higher than controls, and where a combination of antioxidant supplements given to infertile men resulted in a significant reduction of 8-OHdG concentrations in sperm DNA (Kodama et al. 1997). Using only vitamin C for 28 days, Fraga et al. (1991) also observed a decrease of sperm DNA damage. Dogs, unlike humans can synthesize vitamin C; this could be at least one reason why the oral supplementation with this vitamin did not have the same effect as seen in men.

Conclusion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgement
  9. Conflicts of interest
  10. Funding
  11. References

Oral supplementation with 500 mg of vitamins C and E did not decrease DNA peroxidation in frozen-thawed semen from fertile and subfertile dogs.

Funding

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgement
  9. Conflicts of interest
  10. Funding
  11. References

The authors acknowledge financial support from FAPESP (process number 08/53497-1).

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgement
  9. Conflicts of interest
  10. Funding
  11. References