Advertisement

Effects of anti-lipid peroxidases on frozen-thawed boar spermatozoa

  • Sarah J. Casey
  • Rachel Taupier
  • Brian D. Whitaker
Report

Abstract

This study evaluated the effects of anti-lipid peroxidases when supplemented to the thawing and incubation media of frozen-thawed boar spermatozoa. Semen pellets were thawed and incubated in media with 1.0 mM α-tocopherol or diethylenetriamine. After 1 h, the acrosome reaction was induced using calcium ionophore A23187, and acrosomes were evaluated using Wells–Awa staining. The number of spermatozoa with fragmented DNA was evaluated using silver staining after single-cell gel electrophoresis. Membrane lipid peroxidation was measured by the end point generation of malondialdehyde. The diethylenetriamine-supplemented media had a higher (P < 0.05) percentage of acrosome-reacted spermatozoa (84.4 ± 4.1%) compared to the control (78.3 ± 4.2%) and α-tocopherol-supplemented (78.0 ± 3.9%). The control had a higher (P < 0.05) percentage of spermatozoa with fragmented DNA (59.3 ± 4.3%) compared to the DETA (28.7 ± 4.1%) and α-tocopherol supplementation (28.0 ± 3.8%). Spermatozoa supplemented with diethylenetriamine had higher amounts (P < 0.05) of malondialdehyde generated (3.60 ± 0.05 μM/107 cells) compared to the α-tocopherol supplementation (0.14 ± 0.05 μM/107 cells) and the control (0.12 ± 0.05 μM/107 cells). These results indicate that supplementing with either 1.0 mM diethylenetriamine or α-tocopherol during semen thawing and incubation protects against DNA fragmentation, and diethylenetriamine increases the percent of spermatozoa capable of completing the acrosome reaction that could induce membrane lipid peroxidation.

Keywords

Sperm Anti-oxidants α-Tocopherol Lipid peroxidation 

References

  1. Abeydeera L. R. In vitro production of embryos in swine. Theriogenology 57: 256–273; 2002.Google Scholar
  2. Abeydeera L. R.; Day B. N. In vitro penetration of pig oocytes in a modified tris-buffered medium: effect of BSA, caffeine, and calcium. Theriogenology 48: 537–544; 1997.PubMedCrossRefGoogle Scholar
  3. Armstrong J. S.; Rajasekaran M.; Chamulitrat W.; Gatti P.; Hellstrom W. J.; Sikka S. C. Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radic. Bio. Med. 26: 1377–1390; 1999.CrossRefGoogle Scholar
  4. Berger T.; Turner K.; Meizel S.; Hendrick J. Zona pellucida-induced acrosome reaction in boar sperm. Biol. Reprod. 40: 525–530; 1989.PubMedCrossRefGoogle Scholar
  5. Bilodeau J. F.; Chatterjee S.; Sirard M. A.; Gagnon C. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol. Reprod. Dev. 55: 282–288; 2000.PubMedCrossRefGoogle Scholar
  6. Bocca L.; Valenti S.; Cuttica C. M.; Spaziante R.; Giordano G.; Giusti M. Nitric oxide biphasically modulates GH secretion in cultured cells of GH-secreting human pituitary adenomas. Minerva. Endocrinol. 25: 55–59; 2000.PubMedGoogle Scholar
  7. Chatterjee S.; Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol. Reprod. Dev. 59: 451–458; 2001.PubMedCrossRefGoogle Scholar
  8. Chow C. K. Vitamin E and oxidative stress. Free Radical Biol. Med. 11: 215–232; 1991.CrossRefGoogle Scholar
  9. Coyan K.; Başpınar N.; Bucak M. N.; Akalın P. P.; Ataman M. B.; Omür D.; Güngör S.; Küçükgünay S.; Ozkalp B.; Sarıözkan S. Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen. Res. Vet. Sci. 89: 426–431; 2010.PubMedCrossRefGoogle Scholar
  10. Dapino D. G.; Marini P. E.; Cabada M. O. Effect of heparin on in vitro capacitation of boar sperm. Biol. Res. 39: 631–639; 2006.PubMedCrossRefGoogle Scholar
  11. de Lamirande E.; Lamothe G. Reactive oxygen-induced reactive oxygen formation during human sperm capacitation. Free Radic. Bio. Med. 46: 502–510; 2009.CrossRefGoogle Scholar
  12. du Plessis S. S.; Hagenaar K.; Lampiao F. The in vitro effects of melatonin on human sperm function and its scavenging activities on NO and ROS. Andrologia 42: 112–116; 2010.PubMedCrossRefGoogle Scholar
  13. Eddy E. M.; O’Brien D. The spermatozoon. In: Knobil E.; Neill J. D. (eds) The physiology of reproduction. Raven, New York, pp 29–77; 1994.Google Scholar
  14. Fraser L.; Strzeżek J. The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5°C and 16°C. Folia Histochem. Cytobiol. 40: 530–536; 2004.Google Scholar
  15. Fraser L.; Strzeżek J. Effects of freezing-thawing on DNA integrity of boar spermatozoa assessed by neutral comet assay. Reprod. Domest. Anim. 40: 530–536; 2005.PubMedCrossRefGoogle Scholar
  16. Fraser L.; Strzeżek J. Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezing-thawing? Theriogenology 68: 248–257; 2007.PubMedCrossRefGoogle Scholar
  17. Hernandez M.; Roca J.; Calvete J.; Sanz L.; Muino-Blanco T.; Cebrian-Perez J. A.; Valazquez J. M.; Martinez E. A. Cryosurvival and in vitro fertilizing capacity postthaw is improved when boar spermatozoa are frozen in the presence of seminal plasma from good freezer boars. J. Androl. 28: 689–697; 2007.PubMedCrossRefGoogle Scholar
  18. Hsu P. C.; Liu M. Y.; Hsu C. C.; Chen L. Y.; Guo Y. L. Effects of vitamin E and/or C on reactive oxygen species-related lead toxicity in the rat sperm. Toxicology 128: 169–179; 1998.PubMedCrossRefGoogle Scholar
  19. Jeong Y. J.; Kim M. K.; Song H. J.; Kang E. J.; Ock S. A.; Kumar B. M.; Balasubramanian S.; Rho G. J. Effect of alpha-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes. Cryobiology 58: 181–189; 2009.PubMedCrossRefGoogle Scholar
  20. Miller J. K.; Brzezinska-Slebodzinska E.; Madsen F. C. Oxidative stress, antioxidants and animal function. J. Dairy Sci. 76: 2812–2823; 1993.PubMedCrossRefGoogle Scholar
  21. Moran J. M.; Madejón L.; Ortega-Ferrusola C.; Peña F. J. Nitric oxide induces caspase activity in boar spermatozoa. Theriogenology 70: 91–96; 2008.PubMedCrossRefGoogle Scholar
  22. Nadin S. B.; Vargas-Roig L. M.; Ciocca D. R. A silver staining method for single-cell gel assay. J. Histochem. Cytochem. 49: 1183–1186; 2001.PubMedCrossRefGoogle Scholar
  23. Peña F. J.; Johannisson A.; Wallgren M.; Rodrıguez-Martınez H. Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim. Reprod. Sci. 78: 85–98; 2003.PubMedCrossRefGoogle Scholar
  24. Satorre M. M.; Breininger E.; Beconi M. T.; Beorlegui N. B. Protein tyrosine phosphorylation under capacitating conditions in porcine fresh spermatozoa and sperm cryopreserved with and without alpha tocopherol. Andrologia 41: 184–192; 2009.PubMedCrossRefGoogle Scholar
  25. Tardiff S.; Sirard M. A.; Sullivan R.; Bailey J. Identification of capacitation-associated phosphoproteins in porcine sperm electroportated with ATP-γ-32 P. Mol. Reprod. Dev. 54: 292–302; 1999.CrossRefGoogle Scholar
  26. Tao Y.; Chen H.; Tian N. N.; Huo D. T.; Li G.; Zhang Y. H.; Liu Y.; Fang F. G.; Ding J. P.; Zhang X. R. Effects of L-ascorbic acid, alpha-tocopherol and co-culture on in vitro developmental potential of porcine cumulus cells free oocytes. Reprod. Domest. Anim. 45: 19–25; 2010.Google Scholar
  27. Tao Y.; Zhou B.; Xia G.; Wang F.; Wu Z.; Fu M. Exposure to L-ascorbic acid or α-tocopherol facilitates the development of porcine denuded oocytes from metaphase I to metaphase II and prevents cumulus cells from fragmentation. Reprod. Domest. Anim. 39: 52–57; 2004.PubMedCrossRefGoogle Scholar
  28. Warren S.; Patel S.; Kapron C. M. The effect of vitamin E exposure on cadmium toxicity in mouse embryo cells in vitro. Toxicology 142: 119–126; 2000.PubMedCrossRefGoogle Scholar
  29. Whitaker B. D.; Carle B.; Mukai T.; Simpson A.; Vu L.; Knight J. W. Effect of exogenous glutathione supplementation on motility, viability, and DNA integrity of frozen-thawed boar semen. Anim. Reprod. 5: 127–131; 2008.Google Scholar
  30. Whitaker B. D.; Knight J. W. Exogenous γ-glutamyl cycle compounds supplemented to in vitro maturation medium influence in vitro fertilization, culture, and viability of porcine oocytes and embryos. Theriogenology 62: 311–322; 2004.PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2011

Authors and Affiliations

  • Sarah J. Casey
    • 1
  • Rachel Taupier
    • 1
  • Brian D. Whitaker
    • 2
  1. 1.Department of BiologyFerrum CollegeFerrumUSA
  2. 2.Department of Animal SciencesUniversity of FindlayFindlayUSA

Personalised recommendations