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Cryopreservation of Bovine, Equine, and Porcine Sperm.
Abstract: In livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation enables long-term storage and transport of insemination doses, thereby securing genetic material for later use. Although specifics may vary, commonly used sperm cryopreservation protocols for domestic species feature similar processing steps as outlined in this chapter. We here describe common procedures for cryopreservation of bull, stallion and boar sperm. This includes preparation and use of the following diluents: TRIS egg yolk extenders (TEY) for bull sperm, ordinary and freezing skim milk extenders (INRA-82) for stallion sperm, and Beltsville thawing solution (BTS) and lactose egg yolk extenders (LEY/GO) for use with boar sperm. Details and schematic presentations are given for dilution with cryoprotective agents, using a one-step versus a two-step approach. Furthermore, simple methods are presented for packaging of insemination doses in straws, slow cooling of sperm samples to 5°C, controlled freezing in a polystyrene box with liquid nitrogen vapor, and rapid warming at 37°C.
© 2026. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Publication Date: 2026-09-13 PubMed ID: 42732501DOI: 10.1007/978-1-0716-5416-3_11Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Cryopreservation techniques for bull, stallion, and boar sperm are crucial for livestock breeding, enabling long-term storage and use of genetic material through artificial insemination. This research outlines standardized procedures and extenders used for freezing and thawing sperm from these species.
Introduction to Sperm Cryopreservation in Livestock
- Cryopreservation allows semen to be stored long-term for artificial insemination, supporting genetic diversity and breeding programs.
- Common steps exist across species despite some protocol variation.
- The focus is on bovine (bull), equine (stallion), and porcine (boar) sperm due to their agricultural importance.
Extenders and Preparation for Cryopreservation
- Extenders are solutions that protect sperm during freezing and thawing, providing nutrients and stabilizing membranes.
- Different extenders are optimized for each species:
- Bull sperm: TRIS egg yolk extenders (TEY) are used, which contain buffering agents and egg yolk for membrane protection.
- Stallion sperm: Skim milk-based extenders (INRA-82) are employed, with both ordinary and freezing variants tailored for cold tolerance.
- Boar sperm: Beltsville Thawing Solution (BTS) is used as a dilution medium, while lactose egg yolk extenders (LEY or GO) are applied for freezing.
- Each extender is designed to support species-specific sperm physiology and enhance survival after thawing.
Cryoprotective Agents and Freezing Protocols
- Cryoprotective agents (CPAs) are added to extenders to prevent ice crystal formation and cellular damage during freezing.
- Two general approaches to CPA addition:
- One-step method: CPA is added in a single dilution step before freezing.
- Two-step method: CPA is added gradually across two dilution steps to reduce osmotic shock.
- Choice of method depends on sperm species and laboratory protocols.
Packaging and Cooling Procedures
- Sperm samples are packaged into straws, which are standardized containers facilitating easy handling and storage.
- Cooling involves gradually lowering the temperature to 5°C to prepare sperm for freezing:
- Slow cooling reduces thermal shock that can damage sperm membranes.
- Freezing is controlled by placing straws in a polystyrene box suspended above liquid nitrogen to cool by vapor:
- This method allows consistent cooling rates critical for optimal freezing.
Thawing Techniques
- Thawed rapidly at 37°C to avoid recrystallization and minimize damage.
- Rapid warming helps restore sperm function and viability, maximizing fertility outcomes.
Summary
- The chapter provides detailed protocols for semen cryopreservation in cow, horse, and pig species.
- Use of specific extenders and controlled temperature management optimize sperm survival and post-thaw fertility.
- Overall, these methods support effective artificial insemination by preserving high-quality genetic material.
Cite This Article
APA
Oldenhof H, Wolkers WF, Tönißen A, Sieme H.
(2026).
Cryopreservation of Bovine, Equine, and Porcine Sperm.
Methods Mol Biol, 3060, 319-337.
https://doi.org/10.1007/978-1-0716-5416-3_11 Publication
Researcher Affiliations
- Unit for Reproductive Medicine - Clinic for Horses, University of Veterinary Medicine Hannover, Bünteweg 15, 30559, Hannover, Germany.
- Biostabilization Laboratory - Lower Saxony Center for Biomedical Engineering, Implant Research and Development, University of Veterinary Medicine Hannover, Hannover, Germany.
- Unit for Reproductive Medicine - Clinic for Horses, University of Veterinary Medicine Hannover, Bünteweg 15, 30559, Hannover, Germany.
- Biostabilization Laboratory - Lower Saxony Center for Biomedical Engineering, Implant Research and Development, University of Veterinary Medicine Hannover, Hannover, Germany.
- Unit for Reproductive Medicine - Clinic for Horses, University of Veterinary Medicine Hannover, Bünteweg 15, 30559, Hannover, Germany.
- Unit for Reproductive Medicine - Clinic for Horses, University of Veterinary Medicine Hannover, Bünteweg 15, 30559, Hannover, Germany.
MeSH Terms
- Animals
- Cryopreservation / methods
- Cryopreservation / veterinary
- Male
- Semen Preservation / methods
- Semen Preservation / veterinary
- Horses
- Spermatozoa / cytology
- Spermatozoa / drug effects
- Cryoprotective Agents / pharmacology
- Swine
- Cattle
- Insemination, Artificial / methods
References
This article includes 11 references
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