SpringerLink
Log in

Evaluating Damages to Sperm DNA

  • Chapter
  • First Online:
Protocols in Semen Biology (Comparing Assays)

  • 818 Accesses

Abstract

It is an accepted fact that reproduction of the species is the primary goal for all the species from an evolutionary standpoint. The integrity of the DNA plays a crucial part towards achieving this goal. The routine semen quality evaluation parameters do not reflect the integrity of the nuclear material. This chapter dwells into protocols which are commonly employed to assess integrity of the DNA. The chapter has outlined DNA integrity into three categories, viz. DNA condensation, nicks and breaks and fragmentation, and various assays to measure them. These assays are further classified into four groups as cytochemical, fluorescent probes, flow cytometry and transmission electron microscopy. A comparison of these assays vis-à-vis their merit has been provided. Because of ease of performing cytochemical assays, we have outlined procedures of spermatozoa staining with acridine orange, aniline blue, toluidine blue, chromomycin A3 and sperm chromatin structure assay (SCSA) using flow cytometry.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

Literature Cited

  1. Agarwal A, Erenpreiss J, Sharma R (2009) Sperm chromatin assessment. In: Gardner DK, Weissman A, Howles CM, Shoham Z (eds) Textbook of assisted reproductive technologies, 3rd edn. Informa Healthcare, London, pp 67–84

    Google Scholar 

  2. Auger J, Mesbah M, Huber C, Dadoune JP (1990) Aniline blue staining as a marker of sperm chromatin defects associated with different semen characteristics discriminates between proven fertile and suspected infertile men. Int J Androl 13:452–462

    Article  CAS  PubMed  Google Scholar 

  3. Bochenek M, Smorag Z, Pilch J (2001) Sperm chromatin structure assay of bulls qualified for artificial insemination. Theriogenology 56:557–567

    Article  CAS  PubMed  Google Scholar 

  4. Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT (2006) Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl 27:53–59

    Article  CAS  PubMed  Google Scholar 

  5. Dadoune JP, Mayaux MJ, Guihard-Moscato ML (1988) Correlation between defects in chromatin condensation of human spermatozoa stained by aniline blue and semen characteristics. Andrologia 20:211–217

    Article  CAS  PubMed  Google Scholar 

  6. Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR (1975) Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 90:411–428

    Article  CAS  PubMed  Google Scholar 

  7. Erenpreiss J, Jepson K, Giwercman A, Tsarev I, Erenpreisa J, Spano M (2004) Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod 19:2277–2282

    Article  CAS  PubMed  Google Scholar 

  8. Esterhuizen A, Franken D, Lourens JGH, Prinsloo E, Rooyen LHV (2000) Sperm chromatin packaging as an indicator of in-vitro fertilization rates. Hum Reprod 15:657–661

    Article  CAS  PubMed  Google Scholar 

  9. Evenson DP (1999) Loss in livestock breeding efficiency due to uncompensable sperm nuclear defects. Reprod Fertil Dev 11:1–15

    Article  CAS  PubMed  Google Scholar 

  10. Evenson DP, Darzynkiewicz Z, Melamed MR (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131–1133

    Article  CAS  PubMed  Google Scholar 

  11. Fraser L (2004) Structural damage to nuclearDNAin mammalian spermatozoa: its evaluation techniques and relationship with male infertility. Pol J Vet Sci 7:311–321

    CAS  PubMed  Google Scholar 

  12. Graham JK (2001) Assessment of sperm quality: a flow cytometric approach. Anim Reprod Sci 68:239–247

    Article  CAS  PubMed  Google Scholar 

  13. Hallap T, Nagy S, Haard M, Jaakma U, Johannisson A, Rodriguez-Martinez H (2005) Sperm chromatin stability in frozen-thawed semen is maintained over age in AI bulls. Theriogenology 63:1752–1763

    Article  CAS  PubMed  Google Scholar 

  14. Hunter RHF, Rodriguez-Martinez H (2002) Analysing mammalian fertilisation: reservations and potential pitfalls with an in vitro approach. Zygote 10:11–15

    Article  CAS  PubMed  Google Scholar 

  15. Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ (2000) DNA integrity in human spermatozoa: relationships with semen quality. J Androl 21:33–44

    CAS  PubMed  Google Scholar 

  16. Kajstura M, Halicka HD, Pryjma J, Darzynkiewicz Z (2007) Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete “sub-G1” peaks on DNA content histograms. Cytometry 71A:125–131

    Article  CAS  Google Scholar 

  17. Kim H, Kang MJ, Kim SA, Oh SK, Kim H, Ku SY, Kim SY, Moon SY, Choi YM (2013) The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis. Clin Exp Reprod med 40(1):23–28

    Article  PubMed  PubMed Central  Google Scholar 

  18. Liu DY, Baker HWG (1992) Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding, and fertilization rates in vitro. Fertil Steril 58:1178–1184

    Article  CAS  PubMed  Google Scholar 

  19. Manicardi G, Bianchi P, Pantano S, Azzoni S, Bizzaro D, Bianchi U, Sakka D (1995) Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biol Reprod 52:864–867

    Article  CAS  PubMed  Google Scholar 

  20. Marcon L, Boissonneault G (2004) Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling. Biol Reprod 70:910–918

    Article  CAS  PubMed  Google Scholar 

  21. Mello ML (1982) Induced metachromasia in bull spermatozoa. Histochemistry 74:387–392

    Article  CAS  PubMed  Google Scholar 

  22. Muriel L, Garrido N, Fernandez JL, Remohi J, Pellicer A, de los Santos MJ (2006) Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 85:371–383

    Article  CAS  PubMed  Google Scholar 

  23. Nagata S (2000) Apoptotic DNA fragmentation. Exp Cell res 256:12–18

    Article  CAS  PubMed  Google Scholar 

  24. Nanassy L, Carrell D (2008) Paternal effects on early embryogenesis. J Exp Clin Assist Reprod 5:1–9

    Article  Google Scholar 

  25. Nijs M, Creemers E, Cox A, Franssen K, Janssen M, Vanheusden E, Jonge CD, Ombelet W (2009) Chromomycin A3 staining, sperm chromatin structure assay and hyaluronic acid binding assay as predictors for assisted reproductive outcome. Reprod Biomed Online 19(5):671–684

    Article  CAS  PubMed  Google Scholar 

  26. Park YS, Kim MK, Lee SH, Cho JW, Song IO, Seo JT (2011) Efficacy of testicular sperm chromatin condensation assay using aniline blue eosin staining in the IVF-ET cycle. Clin Exp Reprod med 38:142–147

    Article  PubMed  PubMed Central  Google Scholar 

  27. Schulte RT, Ohl DA, Sigman M, Smith GD (2010) Sperm DNA damage in male infertility: etiologies, assays, and outcomes. Assist Reprod Genet 27:3–12

    Article  Google Scholar 

  28. Sergerie M, Laforest G, Bujan L, Bissonnette F, Bleau G (2005) Sperm DNA fragmentation: threshold value in male fertility. Hum Reprod 20:3446–3451

    Article  CAS  PubMed  Google Scholar 

  29. Silva PF, Gadella BM (2006) Detection of damage in mammalian sperm cells. Theriogenology 65:958–978

    Article  CAS  PubMed  Google Scholar 

  30. Srivastava SK, Shinde S, Sushobhit Kumar Singh, Mehrotra S, Med Ram Verma, Singh AK, Nandi S, Srivastava N, Singh SK, Kumar H, Ghosh SK, 2016. Anti-sperm antibodies in repeaters: frequency, detection and validation of threshold levels employing sperm-immobilization, agglutination and immunoperoxidase assay in cattle. Reprod Domest Anim. DOI: 10.1111/rda.12877.

  31. Talebi AR, Vahidi S, Aflatoonian A, Ghasemi N, Ghasemzadeh J, Firoozabadi RD (2012) Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions. Andrologia 44(Suppl 1):462–470

    Article  PubMed  Google Scholar 

  32. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S (1984) A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 42:87–91

    Article  CAS  PubMed  Google Scholar 

  33. Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ (1998) Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol hum Reprod 4:439–445

    Article  CAS  PubMed  Google Scholar 

  34. Wong A, Chuan SS, Patton WC, Jacobson JD, Corselli J, Chan PJ (2008) Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones. Fertil Steril 90:1999–2002

    Article  PubMed  Google Scholar 

  35. Zini A, Kamal K, Phang D, Willis J, Jarvi K (2001) Biologic variability of sperm DNA denaturation in infertile men. Urology 58:258–261

    Article  CAS  PubMed  Google Scholar 

Key References

  1. Manicardi and co-workers (1995) See above [23] Excellent explanation of DNA nicks and breaks and staining procedure using Chromomycin A3.

    Google Scholar 

  2. Ryan and co-workers (2010) See above [32] Detailed explanation of several assays employed to assess DNA integrity and their comparative merits.

    Google Scholar 

  3. Silva and Gadella (2006) See above [14] In-depth discussion and excellent background information related to merits of different staining techniques and principle behind them.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Quality Control, Semen Freezing Laboratory, ICAR-Central Institute for Research on Cattle, Meerut, 250001, Uttar Pradesh, India

    N. Srivastava

  2. Animal Physiology and Embryo Transfer Laboratory, ICAR-Central Institute for Research on Cattle, Meerut, 250001, Uttar Pradesh, India

    Megha Pande

Authors
  1. N. Srivastava
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Megha Pande
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to N. Srivastava .

Editor information

Editors and Affiliations

  1. Quality Control, Semen Freezing Laboratory, ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India

    N. Srivastava

  2. Animal Physiology and Embryo Transfer Laboratory, ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India

    Megha Pande

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Nature Singapore Pte Ltd.

About this chapter

Cite this chapter

Srivastava, N., Pande, M. (2017). Evaluating Damages to Sperm DNA. In: Srivastava, N., Pande, M. (eds) Protocols in Semen Biology (Comparing Assays). Springer, Singapore. https://doi.org/10.1007/978-981-10-5200-2_15

Download citation

Publish with us

Policies and ethics

Search

Navigation