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Home / Equine Research Bank / Methods Mol Biol / Cryopreservation of Horse Sperm.
Methods in molecular biology (Clifton, N.J.)2025; 2897; 193-206; doi: 10.1007/978-1-0716-4406-5_14

Cryopreservation of Horse Sperm.

Abstract: Cryopreservation is currently the only strategy for long-term conservation of equine sperm. To get optimal post-thaw sperm survival, carefully following each step of the freezing protocol is crucial. First, one needs to obtain and exhaustively analyze an ejaculate of good sperm quality. Then, the seminal plasma is removed by centrifugation, and the resulting pellet is resuspended in a certain volume of the freezing medium to reach the right sperm concentration. Finally, sperm samples are packaged into 0.5-mL straws, cooled, and frozen using an automatic, controlled-rate freezer. Once the temperature reaches -120 °C, straws are plunged into liquid nitrogen and stored in tanks until their further use.
© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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Publication Date: 2025-04-09 PubMed ID: 40202637PubMed Central: 7278951DOI: 10.1007/978-1-0716-4406-5_14Google Scholar: Lookup
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Summary

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This research paper examines the process of preserving horse sperm through cryopreservation. It details the essential steps of the procedure, from obtaining the sperm to final storage.

Acquiring and Analyzing Quality Sperm

  • The first stage of the cryopreservation process involves obtaining an ejaculate of good sperm quality. The quality of the sperm is then thoroughly analyzed, as certain aspects of its characteristics, such as motility and morphology, play an essential role in successful cryopreservation.

Removing Seminal Plasma

  • After acquiring sperm of satisfactory quality, the subsequent step consists of removing the seminal plasma. This is done through a process known as centrifugation, which separates denser substances like sperm from less dense materials like plasma. Saparation is significant to ensure the sperm remains unadulterated and undamaged.

Freezing Medium

  • Once the seminal plasma has been removed, the sperm sample is then resuspended in a special solution known as the freezing medium. This medium helps to preserve the sperm during freezing and thawing. The volume of the medium is carefully adjusted to ensure the proper sperm concentration is achieved for optimal cryopreservation.

Sample Packaging, Cooling, and Freezing

  • After the sperm has been suspended in the freezing medium, the sample is packaged into 0.5-mL straws. This packaging process involves precise measurement and careful handling to maintain the integrity of the sperm cells.
  • These packaged samples are then subjected to cooling and freezing. The cooling and freezing steps are carried out using an automatic, controlled-rate freezer to ensure a gradual decrease in temperature, which is crucial to prevent the formation of ice crystals that could damage cell integrity.
  • Once the temperature reaches -120 °C, the samples are plunged into liquid nitrogen for final freezing and preservation. This step completes the cryopreservation process and the straws are now ready to be stored in liquid nitrogen tanks for long-term storage until required for use.

Cite This Article

APA
Miró J, Martínez-Rodero I, Yeste M, Catalán J. (2025). Cryopreservation of Horse Sperm. Methods Mol Biol, 2897, 193-206. https://doi.org/10.1007/978-1-0716-4406-5_14

Publication

Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
NlmUniqueID: 9214969
Country: United States
Language: English
Volume: 2897
Pages: 193-206

Researcher Affiliations

Miró, Jordi
  • Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain. jordi.miro@uab.cat.
Martínez-Rodero, Iris
  • Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain.
Yeste, Marc
  • Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, Girona, Spain.
  • Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, Girona, Spain.
  • Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain.
Catalán, Jaime
  • Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain.
  • Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, Girona, Spain.
  • Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, Girona, Spain.

MeSH Terms

  • Animals
  • Cryopreservation / methods
  • Cryopreservation / veterinary
  • Horses
  • Male
  • Semen Preservation / methods
  • Semen Preservation / veterinary
  • Spermatozoa / cytology
  • Cryoprotective Agents / pharmacology
  • Semen Analysis

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