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Animals : an open access journal from MDPI2025; 15(13); 1881; doi: 10.3390/ani15131881

Effect of Centrifugation of Stallion Semen Through a Low Density Colloid Prior to Freezing on Sperm Cryosurvival.

Abstract: Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation) were evaluated before and after freezing using kinematic and flow cytometric analysis. The parameters for fresh samples were within the normal range for stallion semen but were lower after thawing. There were few differences between the three preparation methods. Interestingly, DNA fragmentation was affected most by the sperm preparation method, being lowest for SLC through high density Equicoll, although SLC through low density Equicoll was effective for some stallions. Some differences were observed in the proportions of live or dead spermatozoa positive for hydrogen peroxide. In conclusion, all of these methods would be suitable for the preparation of semen prior to cryopreservation, but Single Layer Centrifugation through high density Equicoll was the most effective in removing spermatozoa with damaged DNA.
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Publication Date: 2025-06-25 PubMed ID: 40646780PubMed Central: PMC12249079DOI: 10.3390/ani15131881Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research investigates the impact of a particular method of centrifugation on the survival rate of horse sperm post-freezing. The study reveals that all three tested techniques for seminal plasma removal are viable for preparing sperm before cryopreservation, but centrifugation using a high-density colloid yielded the best results in removing sperm with damaged DNA.

Objectives and Methodology

  • The primary objective of the study was to observe the effects of sperm cryopreservation, a process known to adversely affect sperm quality due to increased production of reactive oxygen species.
  • The researchers investigated three different methods to remove seminal plasma: washing and Single Layer Centrifugation (SLC) through high or low-density Equicoll.
  • A total of 29 ejaculates from ten stallions were prepared for freezing.
  • The researchers evaluated sperm quality parameters including kinematics, plasma membrane integrity, production of superoxide and hydrogen peroxide, mitochondrial membrane potential, and DNA fragmentation before and after freezing.
  • Flow cytometric analysis was used to assess these quality parameters.

Findings

  • The parameters for fresh semen samples were within the normal range for stallion sperm, but were comparatively lower after the thawing process.
  • The researchers found only minor differences between the three seminal plasma removal methods they tested.
  • DNA fragmentation appeared to be most affected by the sperm preparation method. The lowest fragmentation rates occurred with SLC through high-density Equicoll, while SLC through low-density Equicoll was effective for some horses.
  • Differences were also identified in the proportions of live or dead spermatozoa positive for hydrogen peroxide.

Conclusion

  • The results suggest that all the methods tested are suitable for preparing sperm prior to cryopreservation. However, SLC through high-density Equicoll emerged as the most effective solution for excluding spermatozoa with damaged DNA.

Cite This Article

APA
Al-Kass Z, Morrell JM, Ntallaris T. (2025). Effect of Centrifugation of Stallion Semen Through a Low Density Colloid Prior to Freezing on Sperm Cryosurvival. Animals (Basel), 15(13), 1881. https://doi.org/10.3390/ani15131881

Publication

Animals : an open access journal from MDPI
ISSN: 2076-2615
NlmUniqueID: 101635614
Country: Switzerland
Language: English
Volume: 15
Issue: 13
PII: 1881

Researcher Affiliations

Al-Kass, Ziyad
  • Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul 41002, Iraq.
  • Department of Clinical Sciences, Swedish University of Agricultural Sciences, P.O. Box 7054, SE-75007 Uppsala, Sweden.
Morrell, Jane M
  • Department of Clinical Sciences, Swedish University of Agricultural Sciences, P.O. Box 7054, SE-75007 Uppsala, Sweden.
Ntallaris, Theodoros
  • Department of Clinical Sciences, Swedish University of Agricultural Sciences, P.O. Box 7054, SE-75007 Uppsala, Sweden.

Grant Funding

  • NA / Linnea and Axel Ericsson Foundation, Sweden
  • NA / Seydlitz Foundation, Sweden

Conflict of Interest Statement

J.M.M. is the inventor and one of the patent holders of the colloids used in this study. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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