Skip to main content

Cryopreservation of Semen from Domestic Livestock: Bovine, Equine, and Porcine Sperm

  • 1681 Accesses

Part of the Methods in Molecular Biology book series (MIMB,volume 2180)


In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemination doses in straws, freezing at a defined cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk (INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore, simple methods are presented for cooling and freezing of sperm at defined cooling rates.

Key words

  • Bull
  • Cryopreservation
  • Lactose-egg yolk extender
  • Skim milk extender
  • Pig
  • Semen
  • Spermatozoa
  • Stallion
  • TRIS-egg yolk extender

This is a preview of subscription content, access via your institution.

Buying options

USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-0783-1_15
  • Chapter length: 13 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
USD   119.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-0783-1
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   159.99
Price excludes VAT (USA)
Hardcover Book
USD   249.99
Price excludes VAT (USA)
Fig. 1

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. Curry MR (2000) Cryopreservation of semen from domestic livestock. Rev Reprod 5:46–52

    CAS  CrossRef  Google Scholar 

  2. Samper JC, Morris CA (1998) Current methods for stallion semen cryopreservation: a survey. Theriogenology 49:895–903

    CAS  CrossRef  Google Scholar 

  3. Yeste M (2017) State-of-the-art of boar sperm preservation in liquid and frozen state. Anim Reprod 14:69–81

    CrossRef  Google Scholar 

  4. Barbas JP, Mascarenhas RD (2009) Cryopreservation of domestic animal sperm cells. Cell Tissue Bank 10:49–62

    CAS  CrossRef  Google Scholar 

  5. Benson JD, Woods EJ, Walters EM, Critser JK (2012) The cryobiology of spermatozoa. Theriogenology 78:1682–1699

    CAS  CrossRef  Google Scholar 

  6. Woelders H, Matthijs A, Zuidberg CA, Chaveiro AEN (2005) Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws. Theriogenology 63:383–395

    CAS  CrossRef  Google Scholar 

  7. Morris GJ, Faszer K, Green JE, Draper D, Grout BW, Fonseca F (2007) Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation. Theriogenology 68:804–812

    CAS  CrossRef  Google Scholar 

  8. Oldenhof H, Bigalk J, Hettel C, de Oliveira BL, Sydykov B, Bajcsy ÁC, Sieme H, Wolkers WF (2017) Stallion sperm cryopreservation using various permeating agents: interplay between concentration and cooling rate. Biopreserv Biobank 15:422–431

    CAS  CrossRef  Google Scholar 

  9. Vidament M, Ecot P, Noue P, Bourgeois C, Magistrini M, Palmer E (2000) Centrifugation and addition of glycerol at 22°C instead of 4°C improve post-thaw motility and fertility of stallion spermatozoa. Theriogenology 54:907–919

    CAS  CrossRef  Google Scholar 

  10. Wall RJ, Foote RH (1999) Fertility of bull sperm frozen and stored in clarified egg yolk-tris-glycerol extender. J Dairy Sci 82:817–821

    CAS  CrossRef  Google Scholar 

  11. Pursel VG, Johnson LA (1975) Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure. J Anim Sci 40:99–102

    CAS  CrossRef  Google Scholar 

  12. Westendorf P, Richter L, Treu H (1975) Zur Tiefgefrierung von Ebersperma. Labor-und Besamungsergebnisse mit dem Hülsenberger Pailetten-Verfahren. Dtsch Tierärztl Wschr 82:261–267

    CAS  Google Scholar 

  13. Carvajal G, Cuello C, Ruiz M, Vázquez JM, Martínez EA, Roca J (2004) Effects of centrifugation before freezing on boar sperm cryosurvival. J Androl 25:389–396

    CrossRef  Google Scholar 

Download references


Employees of the Unit for Reproductive Medicine of the University of Veterinary Medicine Hannover (Hannover, Germany), National Stud of Lower Saxony (Celle, Germany), and Masterrind GmbH (Verden, Germany) are acknowledged for sharing protocols.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Harriëtte Oldenhof .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Oldenhof, H., Wolkers, W.F., Sieme, H. (2021). Cryopreservation of Semen from Domestic Livestock: Bovine, Equine, and Porcine Sperm. In: Wolkers, W.F., Oldenhof, H. (eds) Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology, vol 2180. Humana, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0782-4

  • Online ISBN: 978-1-0716-0783-1

  • eBook Packages: Springer Protocols