Combined Effect of Trolox and EDTA on Frozen-Thawed Sperm Quality

Sara Keshtgar, Farideh Iravanpour, Behrooz Gharesi-Fard, Marjaneh Kazerooni

Abstract


The freezing and thawing process not only is associated with serious damage to sperm such as damage to the plasma membrane and the acrosomal membrane but also changes the membrane permeability to some ions including calcium. Also, the generation of oxygen free radicals is increased during the freezing-thawing process. The purpose of this study was to evaluate of the effects of Trolox as an antioxidant and edetic acid (EDTA) as a calcium chelator on frozen-thawed (FT) sperm and compare these effects with those on fresh sperm. This study was done on these men of 25 healthy men, who referred to Shiraz Infertility Centerbetween2012 and2013. Normal samples were transferred to the ReproductivePhysiology Laboratory, Department of Physiology,Shiraz University of Medical Sciences, Shiraz. The samples were divided into two groups randomly: fresh and FT sperm groups. Each group was divided into five subgroups:control group, the solvent group (0.1%dimethyl sulfoxide [DMSO]), Trolox group (200μM), EDTA group (1.1mM), and Trolox+EDTA group. The percentages of motility, viability, and acrosome-reacted sperm were tested. The percentages of motility and viability in the FT sperm were lower than those in the fresh sperm. The progressive motility of the FT sperm was improved nonsignificantly with Trolox+EDTA. However, the effect of Trolox+EDTA on the progressive motility of the FT sperm was much more than that on the fresh sperm. The fewest acrosome-reacted sperm were observed in the EDTA-containingFT sperm. Antioxidant supplementation or omission of extracellular calcium may partly improve motility and also reduce acrosomal damage in FT sperm.


Keywords


Freezing; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Edetic Acid; Sperm Motility; Acrosome reaction

Full Text:

PDF

References


Day JG, Stacey G. Cryopreservation and freeze-drying protocols. 2nd ed. New Jersey: Humana Press; 2007. 347 p.

World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: World Health Organization; 2010. 271 p.

AbdelHafez F, Bedaiwy M, El-Nashar SA, Sabanegh E, Desai N. Techniques for cryopreservation of individual or small numbers of human spermatozoa: a systematic review. Hum Reprod Update. 2009;15:153-64. doi: 10.1093/humupd/dmn061. PubMed PMID: 19109313.

Di Santo M, Tarozzi N, Nadalini M, Borini A. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART. Adv Urol. 2012;2012:854837. doi: 10.1155/2012/854837. PubMed PMID: 22194740; PubMed Central PMCID: PMC3238352.

Martinez-Soto JC, Garcia-Vazquez FA, Gumbao D, Landeras J, Gadea J. Assessment of two thawing processes of cryopreserved human sperm in pellets. Cryobiology. 2011;63:131-6. doi: 10.1016/j.cryobiol.2011.08.001. PubMed PMID: 21884688.

Kadirvel G, Kumar S, Kumaresan A, Kathiravan P. Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium. Anim Reprod Sci. 2009;116:244-53. doi: 10.1016/j.anireprosci.2009.02.003. PubMed PMID: 19261396.

Silva SV, Soares AT, Batista AM, Almeida FC, Nunes JF, Peixoto CA, et al. Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation. Anim Reprod Sci. 2013;137:37-44. doi: 10.1016/j.anireprosci.2012.12.002. PubMed PMID: 23280096.

Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J Assist Reprod Genet. 2008;25:403-11. doi: 10.1007/s10815-008-9232-3. PubMed PMID: 18704674; PubMed Central PMCID: PMC2582121.

Peris SI, Bilodeau JF, Dufour M, Bailey JL. Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Mol Reprod Dev. 2007;74:878-92. doi: 10.1002/mrd.20686. PubMed PMID: 17186553.

Chatterjee S, Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev. 2001;59:451-8. doi: 10.1002/mrd.1052. PubMed PMID: 11468782.

Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez FA, Gardon JC. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology. 2011;62:40-6. doi: 10.1016/j.cryobiol.2010.12.001. PubMed PMID: 21156167.

Bath ML. Inhibition of in vitro fertilizing capacity of cryopreserved mouse sperm by factors released by damaged sperm, and stimulation by glutathione. PLoS One. 2010;5:e9387. doi: 10.1371/journal.pone.0009387. PubMed PMID: 20195370; PubMed Central PMCID: PMC2827551.

Bilodeau JF, Chatterjee S, Sirard MA, Gagnon C. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol Reprod Dev. 2000;55:282-8. doi: 10.1002/(SICI)1098-2795(200003)55:3<282::AID-MRD6>3.0.CO;2-7. PubMed PMID: 10657047.

Maia Mda S, Bicudo SD, Sicherle CC, Rodello L, Gallego IC. Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with antioxidants. Anim Reprod Sci. 2010;122:118-23. doi: 10.1016/j.anireprosci.2010.08.004. PubMed PMID: 20813469.

da Silva Maia M, Bicudo SD, Azevedo HC, Sicherle CC, de Sousa DB, Rodello L. Motility and viability of ram sperm cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants. Small Ruminant Research. 2009;85:85-90. doi: 10.1016/j.smallrumres.2009.07.001.

Martinez-Pastor F, Aisen E, Fernandez-Santos MR, Esteso MC, Maroto-Morales A, Garcia-Alvarez O, et al. Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa. Reproduction. 2009;137:225-35. doi: 10.1530/REP-08-0357. PubMed PMID: 19028926.

Minaei MB, Barbarestani M, Nekoonam S, Abdolvahabi MA, Takzare N, Asadi MH, et al. Effect of Trolox addition to cryopreservation media on human sperm motility. Iran J Reprod Med. 2012;10:99-104. PubMed PMID: 25242981; PubMed Central PMCID: PMC4163270.

Breininger E, Beorlegui NB, O'Flaherty CM, Beconi MT. Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen. Theriogenology. 2005;63:2126-35. doi: 10.1016/j.theriogenology.2004.08.016. PubMed PMID: 15826678.

Kalthur G, Raj S, Thiyagarajan A, Kumar S, Kumar P, Adiga SK. Vitamin E supplementation in semen-freezing medium improves the motility and protects sperm from freeze-thaw-induced DNA damage. Fertil Steril. 2011;95:1149-51. doi: 10.1016/j.fertnstert.2010.10.005. PubMed PMID: 21067726.

Pena FJ, Johannisson A, Wallgren M, Rodriguez Martinez H. Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim Reprod Sci. 2003;78:85-98. PubMed PMID: 12753785.

Yoshimoto T, Nakamura S, Yamauchi S, Muto N, Nakada T, Ashizawa K, et al. Improvement of the post-thaw qualities of Okinawan native pig spermatozoa frozen in an extender supplemented with ascorbic acid 2-O-alpha-glucoside. Cryobiology. 2008;57:30-6. doi: 10.1016/j.cryobiol.2008.05.002. PubMed PMID: 18589410.

Okazaki T, Yoshida S, Teshima H, Shimada M. The addition of calcium ion chelator, EGTA to thawing solution improves fertilizing ability in frozen-thawed boar sperm. Anim Sci J. 2011;82:412-9. doi: 10.1111/j.1740-0929.2010.00856.x. PubMed PMID: 21615834.

Patrat C, Serres C, Jouannet P. The acrosome reaction in human spermatozoa. Biol Cell. 2000;92:255-66. doi: 10.1016/S0248-4900(00)01072-8. PubMed PMID: 11043413.

O'Toole CM, Arnoult C, Darszon A, Steinhardt RA, Florman HM. Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction. Mol Biol Cell. 2000;11:1571-84. PubMed PMID: 10793136; PubMed Central PMCID: PMC14868.

Kalive M, Faust JJ, Koeneman BA, Capco DG. Involvement of the PKC family in regulation of early development. Mol Reprod Dev. 2010;77:95-104. doi: 10.1002/mrd.21112. PubMed PMID: 19777543.

Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil. 1987;81:459-69. PubMed PMID: 2828610.

Baker MA, Aitken RJ. The importance of redox regulated pathways in sperm cell biology. Mol Cell Endocrinol. 2004;216:47-54. doi: 10.1016/j.mce.2003.10.068. PubMed PMID: 15109744.

Burnaugh L, Sabeur K, Ball BA. Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium. Theriogenology. 2007;67:580-9. doi: 10.1016/j.theriogenology.2006.07.021. PubMed PMID: 17045638.

Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod. 1989;41:183-97. doi: 10.1095/biolreprod41.1.183. PubMed PMID: 2553141.

Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl. 1989;10:214-20. doi: 10.1002/j.1939-4640.1989.tb00091.x. PubMed PMID: 2501260.

Anel L, Gomes-Alves S, Alvarez M, Borragan S, Anel E, Nicolas M, et al. Effect of basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa. Theriogenology. 2010;74:643-51. doi: 10.1016/j.theriogenology.2010.03.004. PubMed PMID: 20494423.

Hossain MS, Johannisson A, Siqueira AP, Wallgren M, Rodriguez-Martinez H. Spermatozoa in the sperm-peak-fraction of the boar ejaculate show a lower flow of Ca(2+) under capacitation conditions post-thaw which might account for their higher membrane stability after cryopreservation. Anim Reprod Sci. 2011;128:37-44. doi: 10.1016/j.anireprosci.2011.08.006. PubMed PMID: 21962836.

Donnelly ET, McClure N, Lewis SE. Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertil Steril. 2001;76:892-900. doi: 10.1016/S0015-0282(01)02834-5. PubMed PMID: 11704107.

Mendoza C, Carreras A, Moos J, Tesarik J. Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil. 1992;95:755-63. doi: 10.1530/jrf.0.0950755. PubMed PMID: 1383539.

Chi HJ, Kim JH, Ryu CS, Lee JY, Park JS, Chung DY, et al. Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Hum Reprod. 2008;23:1023-8. doi: 10.1093/humrep/den060. PubMed PMID: 18325884.

Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl. 2000;21:1-7. PubMed PMID: 10670514.

Aisen EG, Alvarez HL, Venturino A, Garde JJ. Effect of trehalose and EDTA on cryoprotective action of ram semen diluents. Theriogenology. 2000;53:1053-61. doi: 10.1016/S0093-691X(00)00251-X. PubMed PMID: 10798483.


View Counter: Abstract | 688 | and PDF | 0 |

Refbacks

  • There are currently no refbacks.


pISSN: 0253-0716         eISSN: 1735-3688