Sperm Cleanup and Centrifugation Processing for Cryopreservation

  • Harald SiemeEmail author
  • Harriëtte Oldenhof
Part of the Methods in Molecular Biology book series (MIMB, volume 1257)


Fertility rates with artificial insemination are highest with good-quality sperm samples. Therefore, nonviable sperm, cellular debris, and seminal plasma are preferably removed from semen samples prior to use or for preservation. Such compounds are sources where reactive oxygen species are generated during storage or upon cryopreservation, impairing sperm function. In this chapter we describe methods to remove seminal plasma and cellular debris from sperm samples, and for selecting morphologically normal motile sperm. The methods that are described here include: ordinary centrifugation, sperm swim-up, glass wool and Sephadex filtration/adherence, and single-layer as well as discontinuous two-layer iodixanol density gradient centrifugation.

Key words

Semen Spermatozoa Sperm selection Subpopulations Cleanup 


  1. 1.
    Henkel R (2012) Sperm preparation: state-of-the-art–physiological aspects and application of advanced sperm preparation methods. Asian J Androl 14:260–269CrossRefGoogle Scholar
  2. 2.
    Mahadevan M, Baker G (1984) Assessment and preparation of semen for in vitro fertilization. In: Wood C, Trounson AO (eds) Clinical in vitro fertilization. Springer, Berlin, p 83CrossRefGoogle Scholar
  3. 3.
    Jeyendran RS, Perez-Palaez M, Crabo BG (1986) Concentration of viable spermatozoa for artificial insemination. Fertil Steril 45:132–134Google Scholar
  4. 4.
    Graham EF, Graham JK (1978) The effect of whole ejaculate filtration on the morphology and the fertility of bovine semen. J Dairy Sci 73:91–97CrossRefGoogle Scholar
  5. 5.
    Sieme H, Martinsson G, Rauterberg H, Walter K, Aurich C, Petzoldt R, Klug E (2003) Application of techniques for sperm selection in fresh and frozen-thawed stallion semen. Reprod Dom Anim 38:134–140CrossRefGoogle Scholar
  6. 6.
    Henkel RR, Schill WB (2003) Sperm preparation for ART. Reprod Biol Endocrinol 1:108CrossRefGoogle Scholar
  7. 7.
    Sakkas D (2013) Novel technologies for selecting the best sperm for in vitro fertilization and intracytoplasmic sperm injection. Fert Steril 99:1023–1029CrossRefGoogle Scholar
  8. 8.
    Mehta A, Sigman M (2014) Identification and preparation of sperm for ART. Urol Clin North Am 41:169–180CrossRefGoogle Scholar
  9. 9.
    Aitken RJ, Clarkson JS (1988) Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 9:367–376Google Scholar
  10. 10.
    Mortimer D (1994) Sperm recovery techniques to maximize fertilizing capacity. Reprod Fertil Dev 6:25–31CrossRefGoogle Scholar
  11. 11.
    Katkov I, Mazur P (1999) Factors affecting yield and survival of cells when suspensions are subjected to centrifugation. Cell Biochem Biophys 31:231–245CrossRefGoogle Scholar
  12. 12.
    Knop K, Hoffmann N, Rath D, Sieme H (2005) Effects of cushioned centrifugation technique on sperm recovery and sperm quality in stallions with good and poor semen freezability. Anim Reprod Sci 89:294–297Google Scholar
  13. 13.
    Morrell JM, Rodriguez-Martinez H, Johannisson A (2010) Single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size: data from 3 breeding seasons. Equine Vet J 42:579–585CrossRefGoogle Scholar
  14. 14.
    Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H (2009) Morphology and chromatin integrity of stallion spermatozoa prepared by density gradient and single layer centrifugation through silica colloids. Reprod Domest Anim 44:512–517CrossRefGoogle Scholar
  15. 15.
    Mortimer D, Mortimer ST (2013) Density gradient separation of sperm for artificial insemination. In: Carrell DT, Aston KI (eds) Spermatogenesis: methods and protocols, vol 927, Methods in molecular biology. Humana Press, New York, NY, pp 217–226CrossRefGoogle Scholar
  16. 16.
    Stuhtmann G, Oldenhof H, Peters P, Klewitz J, Martinsson G, Sieme H (2012) Iodixanol density centrifugation for selecting stallion sperm for cold storage and cryopreservation. Anim Reprod Sci 133:184–190CrossRefGoogle Scholar
  17. 17.
    Sieme H (2011) Freezing semen. In: McKinnon AO, Squires EL, Vaala WE, Varner DD (eds) Equine reproduction, 2nd edn. Blackwell Publishing Ltd., Chichester, pp 2972–2982Google Scholar
  18. 18.
    Smith TT, Byers M, Kaftani D, Whitford W (1997) The use of iodixanol as a density gradient material for separating human sperm from semen. Arch Androl 38:223–230CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Unit for Reproductive Medicine, Clinic for HorsesUniversity of Veterinary Medicine HannoverHannoverGermany

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