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Reproduction in domestic animals = Zuchthygiene2025; 60(10); e70135; doi: 10.1111/rda.70135

Optimising Stallion Semen Cryopreservation: Preliminary Insights Into Pre-Centrifugation Extender Effects.

Abstract: This study evaluated the effects of cholesterol, pentoxifylline and casein, with or without skim milk, added to extenders during pre-centrifugation on equine sperm cryosurvival. Seven ejaculates from four stallions (n = 28) were divided into four groups: SM (skim milk), SMP (SM + pentoxifylline), SMCho (SM + cholesterol) and ChoCa (cholesterol + casein). After centrifugation and freezing, sperm kinematics and plasma membrane integrity were assessed immediately and 30 min post-thaw. SMCho and ChoCa showed superior results compared with SM and SMP. These findings indicate that cholesterol-based extenders improve post-thaw sperm quality when added before cryopreservation.
© 2025 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.
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Publication Date: 2025-10-22 PubMed ID: 41121986PubMed Central: PMC12541287DOI: 10.1111/rda.70135Google 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.

Research Overview

  • This study investigated how adding certain substances to extenders before centrifugation influences the survival and quality of frozen-thawed stallion sperm.
  • The goal was to improve cryopreservation outcomes by testing different extender formulations.

Background and Objective

  • Cryopreservation of stallion semen is important for breeding management, but freezing and thawing processes can damage sperm cells.
  • Extenders are solutions used to preserve sperm, providing nutrients and protection during freezing.
  • The study focused on evaluating the effects of adding cholesterol, pentoxifylline, and casein—known to influence sperm membrane stability and motility—to extenders applied before centrifugation and freezing.
  • The aim was to determine which extender formulations best improve post-thaw sperm function and integrity.

Methodology

  • Sample Collection:
    • Seven ejaculates collected from four stallions, totaling 28 samples.
  • Experimental Groups:
    • SM: Skim milk extender alone (control group)
    • SMP: Skim milk + pentoxifylline
    • SMCho: Skim milk + cholesterol
    • ChoCa: Cholesterol + casein (without skim milk)
  • Procedure:
    • Sperm were diluted with respective extenders before centrifugation.
    • Samples were centrifuged, frozen, and later thawed for analysis.
  • Assessment:
    • Sperm kinematics (motility parameters) and plasma membrane integrity were evaluated immediately after thawing and 30 minutes post-thaw.

Key Findings

  • Extenders containing cholesterol (SMCho and ChoCa) showed improved sperm motility and membrane integrity compared to those containing skim milk alone or skim milk plus pentoxifylline.
  • The presence of cholesterol in the extender before centrifugation enhanced sperm cryosurvival.
  • Casein combined with cholesterol (ChoCa) performed similarly well to cholesterol plus skim milk, suggesting casein is an effective protective agent in the extender.
  • Pentoxifylline addition did not significantly improve post-thaw sperm quality relative to the control with skim milk.

Implications and Conclusions

  • The study suggests that including cholesterol-based components in extenders before centrifugation positively influences the survival and functional quality of frozen-thawed stallion sperm.
  • This enhancement likely results from cholesterol stabilizing sperm plasma membranes, which are vulnerable to damage during freezing and thawing.
  • Choosing appropriate extender formulations can optimize cryopreservation protocols, potentially improving fertility outcomes for stallions.
  • The findings encourage further detailed studies to confirm and expand on these preliminary insights and to explore the mechanisms involved.

Cite This Article

APA
de Zutter BM, de Paula Freitas-Dell'Aqua C, Dell'Aqua-Junior JA, Monteiro GA, Troncarelli T, Papa FO. (2025). Optimising Stallion Semen Cryopreservation: Preliminary Insights Into Pre-Centrifugation Extender Effects. Reprod Domest Anim, 60(10), e70135. https://doi.org/10.1111/rda.70135

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 60
Issue: 10
Pages: e70135
PII: e70135

Researcher Affiliations

de Zutter, Bruna Merci
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
de Paula Freitas-Dell'Aqua, Camila
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Dell'Aqua-Junior, José Antônio
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Monteiro, Gabriel Augusto
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Troncarelli, Tiago
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Papa, Frederico Ozanam
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

MeSH Terms

  • Animals
  • Male
  • Horses
  • Cryopreservation / veterinary
  • Cryopreservation / methods
  • Semen Preservation / veterinary
  • Semen Preservation / methods
  • Cryoprotective Agents / pharmacology
  • Spermatozoa / physiology
  • Spermatozoa / drug effects
  • Cholesterol / pharmacology
  • Pentoxifylline / pharmacology
  • Sperm Motility / drug effects
  • Semen Analysis / veterinary
  • Centrifugation / veterinary
  • Caseins / pharmacology
  • Milk

Conflict of Interest Statement

The authors declare no conflicts of interest.

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