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Reproduction in domestic animals = Zuchthygiene2025; 60(9); e70126; doi: 10.1111/rda.70126

Evaluation of a Chemically Defined, Long-Term Extender for Liquid Storage of Stallion Semen.

Abstract: Efficient use of stallion semen in liquid state is limited by its relatively short shelf-life. A chemically defined extender (Beyond) is now available for long-term liquid semen preservation. The objectives of the present study were to compare Beyond with milk extenders for the preservation of semen at two temperatures, and to evaluate fertility of semen cooled for 4-8 days before artificial insemination. Semen was processed using different extenders: milk, cholesterol (BotuSemen Special); milk-based (INRA 96); and Beyond. Sperm motility, membrane and acrosome integrity, and chromatin structure were evaluated in semen stored at 17°C for 7 days or at 5°C for 14 days. Sperm motility decreased in the first few days of storage regardless of extender or storage temperature. Sperm motility continued to decline at relatively constant rates in semen extended in milk extenders, but the rate of decline was substantially reduced with Beyond. Sperm motility in semen extended with Beyond was greater than in semen extended with milk extenders after 4 days of storage at 17°C, or after 7 days of storage at 5°C. Extender did not affect sperm DNA damage during storage, but sperm with intact membrane and intact acrosome were lower with Beyond. Inseminations with semen stored with Beyond at 5°C for an average of 5.5 days resulted in embryos in 61% of cycles (11/18). In conclusion, Beyond extender resulted in greater sperm motility longevity when compared to milk extenders, especially when semen was stored at 5°C. Satisfactory fertility was obtained with semen cooled for 4-8 days before artificial insemination.
© 2025 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.
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Publication Date: 2025-09-26 PubMed ID: 41002042PubMed Central: PMC12465434DOI: 10.1111/rda.70126Google Scholar: Lookup
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  • Journal Article

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.

Overview

  • This study evaluated the effectiveness of a new chemically defined extender called Beyond for preserving stallion semen in liquid form over extended periods, comparing it to traditional milk-based extenders and assessing fertility outcomes after storage.

Background

  • Stallion semen in liquid form typically has a short shelf-life, limiting its use in artificial insemination.
  • Extenders are solutions used to preserve semen by maintaining sperm viability during storage.
  • Milk-based extenders have been the standard but have limitations in maintaining sperm motility and integrity over time.
  • Beyond is a newly available chemically defined extender designed for long-term preservation of stallion semen.

Objectives

  • Compare the performance of Beyond versus milk-based extenders (including BotuSemen Special and INRA 96) in preserving stallion semen at two temperatures: 17°C and 5°C.
  • Evaluate sperm parameters such as motility, membrane integrity, acrosome integrity, and chromatin structure over storage periods of up to 14 days.
  • Assess fertility outcomes after artificial insemination using semen cooled with Beyond extender for 4-8 days.

Methodology

  • Semen samples were divided into groups and extended using different extenders: milk, cholesterol-enriched BotuSemen Special, milk-based INRA 96, and the chemically defined Beyond extender.
  • Samples were stored at either 17°C for 7 days or 5°C for 14 days.
  • Sperm evaluations included measuring motility, membrane and acrosome integrity (both critical for sperm function), and chromatin structure (to assess DNA integrity).
  • Fertility trials were conducted by artificial insemination of mares using semen stored with Beyond at 5°C for an average of approximately 5.5 days.

Key Findings

  • Sperm motility declined in the first few days in all groups regardless of the extender or storage temperature.
  • After the initial decline, sperm motility continued to decrease at a relatively constant rate in milk-based extenders.
  • In contrast, the rate of motility decline was substantially slower when using Beyond extender.
  • After 4 days at 17°C or 7 days at 5°C, sperm motility was significantly higher in samples stored with Beyond compared to those with milk-based extenders.
  • There was no noticeable impact of extender type on sperm DNA damage during storage, indicating chromatin integrity was maintained similarly across extenders.
  • Interestingly, the percentage of sperm with intact membranes and acrosomes was lower in samples stored with Beyond, suggesting some effects on certain sperm cell structures.
  • Artificial insemination using semen stored with Beyond at 5°C for 4-8 days achieved a pregnancy (embryo) rate of 61% (11 pregnancies out of 18 cycles), demonstrating satisfactory fertility.

Conclusions

  • The chemically defined Beyond extender provided better preservation of sperm motility over time compared to conventional milk-based extenders, particularly at lower storage temperatures (5°C).
  • Despite some reduction in sperm membrane and acrosome integrity, the overall fertility using semen stored longer term with Beyond remained acceptable.
  • This suggests that Beyond extender may improve the practical storage duration for stallion semen in liquid form, potentially allowing for more flexible and efficient use in artificial insemination.

Implications for Practice

  • Beyond extender could be advantageous for breeding programs requiring extended storage of stallion semen without freezing.
  • Improved motility longevity can enhance insemination timing and logistics, especially when shipping or coordinating breeding across distances.
  • Further research may be warranted to optimize extender formulations to maintain membrane and acrosome integrity alongside motility.

Cite This Article

APA
Brito LFC, Linardi RL, Rosales LAS, Balamurugan NS, Hernández-Avilés C, Ramírez-Agámez L. (2025). Evaluation of a Chemically Defined, Long-Term Extender for Liquid Storage of Stallion Semen. Reprod Domest Anim, 60(9), e70126. https://doi.org/10.1111/rda.70126

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 60
Issue: 9
Pages: e70126
PII: e70126

Researcher Affiliations

Brito, Leonardo F C
  • Department of Clinical Studies - New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA.
Linardi, Renata L
  • Department of Clinical Studies - New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA.
Rosales, Leslie A S
  • Department of Clinical Studies - New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA.
Balamurugan, Nithiya Sri
  • Department of Clinical Studies - New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA.
Hernández-Avilés, Camilo
  • Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA.
Ramírez-Agámez, Luisa
  • Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA.

MeSH Terms

  • Animals
  • Semen Preservation / veterinary
  • Semen Preservation / methods
  • Male
  • Horses / physiology
  • Sperm Motility / drug effects
  • Insemination, Artificial / veterinary
  • Spermatozoa / physiology
  • Spermatozoa / drug effects
  • Semen / physiology
  • Acrosome / physiology
  • Female
  • Milk
  • Cholesterol / pharmacology
  • Semen Analysis / veterinary
  • DNA Damage

Grant Funding

  • Georgia and Philip Hofmann Endowment
  • Legends Premier Stallion Season Auction

Conflict of Interest Statement

The authors declare no conflicts of interest.

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