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Andrology2025; 14(2); 583-593; doi: 10.1111/andr.70139

Incubation of Frozen-Thawed Semen Under Capacitating Conditions Supports Successful In Vitro Fertilization and Improves Intracytoplasmic Sperm Injection-Results in Horses.

Abstract: In 2022, a repeatable protocol for in vitro fertilization (IVF) using fresh semen was established in horses. This facilitated successful capacitation of equine semen allowing to explore novel applications. Objective: We aimed to extend this technique to IVF with frozen-thawed semen and intracytoplasmic sperm injection (ICSI), and determine the outcome parameters such as blastocyst production and euploidy rates. Methods: A total of 221 oocytes were subjected to either IVF with frozen-thawed semen, ICSI with frozen-thawed semen incubated under capacitating conditions (ICSI cap) or control ICSI with washed frozen-thawed semen. Cleavage and blastocyst rates were assessed and compared across the three groups using one-way ANOVA. Shallow whole genome sequencing was performed on embryos obtained from IVF and ICSI cap. Results: We established a repeatable protocol for IVF with frozen-thawed semen resulting in higher blastocyst rates per collected oocyte (22.4%) when compared to control ICSI (16.4%) (p = 0.048). Furthermore, the use of semen incubated under capacitating conditions for ICSI resulted in higher blastocyst rates than washed sperm, with 69.0% versus 50.0% blastocysts per cleaved embryo (p = 0.03) and 27.8% versus 16.4% blastocysts per collected oocyte (p = 0.04), respectively. It also yielded higher blastocyst rates per cleaved embryo than IVF, with 69.0% versus 45.9% (p = 0.04). The average day of blastocyst formation was not different between the three groups (p = 0.73). Shallow whole genome sequencing revealed no differences in aneuploidy rates between IVF (1/17) and ICSI cap (0/18) (p = 0.49). Conclusions: The incubation of sperm under capacitating conditions for use in ICSI or IVF with frozen-thawed semen may represent a novel method to improve the clinical efficiency of equine IVP embryos, without affecting aneuploidy rates.
© 2025 American Society of Andrology and European Academy of Andrology.
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Publication Date: 2025-10-29 PubMed ID: 41159952DOI: 10.1111/andr.70139Google 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.

Overview

  • This study evaluates a new method for improving fertilization and embryo development in horses using frozen-thawed semen by incubating sperm under capacitating conditions for both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).
  • The research compares fertilization outcomes and embryo quality, including blastocyst formation rates and chromosomal normality, using this approach versus conventional techniques.

Background

  • In 2022, a reproducible IVF protocol using fresh horse semen was established, enabling successful capacitation—a process that sperm undergo to become capable of fertilizing an egg—in equine semen.
  • This advancement opened possibilities to apply capacitation methods to frozen-thawed semen, which is commonly used in equine reproduction but often associated with lower fertilization success.
  • ICSI, where a single sperm is injected directly into an egg, is another technique used to assist fertilization, especially with compromised semen quality.
  • The goal was to test whether incubating frozen-thawed semen under capacitating conditions would enhance embryo development outcomes in both IVF and ICSI procedures.

Objectives

  • Extend the previously developed capacitation-based IVF protocol to frozen-thawed equine semen.
  • Evaluate effects of capacitation incubation on ICSI outcomes using frozen-thawed semen.
  • Measure important parameters: cleavage rates (early embryo cell division), blastocyst production rates (advanced embryo stage), and euploidy rates (chromosomal normality) in resulting embryos.

Methods

  • Collected 221 equine oocytes (egg cells) and divided them into three experimental groups:
    • IVF group with frozen-thawed semen incubated under capacitating conditions.
    • ICSI with frozen-thawed semen incubated under capacitating conditions (“ICSI cap”).
    • Control ICSI group with washed, frozen-thawed semen without capacitation incubation.
  • Compared cleavage rates, blastocyst formation rates, and timing of blastocyst development across groups using statistical analysis (one-way ANOVA).
  • Performed shallow whole genome sequencing on embryos from the IVF and ICSI cap groups to detect any differences in chromosomal abnormalities (aneuploidy rates).

Key Findings

  • Using capacitated frozen-thawed semen in IVF resulted in a significantly higher blastocyst formation rate (22.4% per collected oocyte) compared to control ICSI (16.4%).
  • In ICSI, capacitation incubation markedly improved outcomes:
    • Blastocyst rates per cleaved embryo were 69.0% for ICSI cap versus 50.0% for control ICSI.
    • Blastocyst rates per collected oocyte were 27.8% for ICSI cap versus 16.4% for control ICSI.
  • Blastocyst rates per cleaved embryo were also higher in ICSI cap (69.0%) compared to IVF (45.9%), suggesting capacitation incubation may be particularly beneficial in ICSI procedures.
  • The timing of blastocyst formation (average day) showed no significant differences among the three groups, indicating the technique does not alter developmental speed.
  • Genomic analysis showed no significant differences in chromosomal abnormality rates between embryos derived via IVF (1 out of 17 aneuploid) and ICSI cap (0 out of 18), supporting the genomic safety of the capacitation incubation approach.

Conclusions and Implications

  • Incubating frozen-thawed equine semen under capacitating conditions improves embryo development success in both IVF and ICSI without increasing chromosomal abnormalities.
  • This new method potentially enhances the clinical efficiency and outcomes of assisted reproductive technologies in horses, which can be valuable for breeding programs, especially when relying on frozen semen.
  • The approach allows better use of compromised or frozen semen by improving sperm fertilizing capability prior to fertilization procedures.
  • Further research may focus on optimizing capacitation conditions and exploring long-term viability and health of offspring produced using these techniques.

Cite This Article

APA
Broothaers K, Angel-Velez D, Molto FLG, Hedia M, Coster T, Govaere J, Soom AV, Menten B, Smits K. (2025). Incubation of Frozen-Thawed Semen Under Capacitating Conditions Supports Successful In Vitro Fertilization and Improves Intracytoplasmic Sperm Injection-Results in Horses. Andrology, 14(2), 583-593. https://doi.org/10.1111/andr.70139

Publication

Andrology
ISSN: 2047-2927
NlmUniqueID: 101585129
Country: England
Language: English
Volume: 14
Issue: 2
Pages: 583-593

Researcher Affiliations

Broothaers, Klaartje
  • Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium.
Angel-Velez, Daniel
  • Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium.
  • School of Veterinary Medicine, Universidad CES, Medellin, Colombia.
Molto, Fabiola Le Gaffric
  • Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium.
Hedia, Mohamed
  • Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium.
  • Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.
  • Gamete Research Centre, Laboratory for Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
Coster, Tine De
  • Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium.
Govaere, Jan
  • Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium.
Soom, Ann Van
  • Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium.
Menten, Björn
  • Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
Smits, Katrien
  • Department of Internal Medicine, Reproduction and Population Medicine, Ghent University, Merelbeke, Belgium.

MeSH Terms

  • Animals
  • Horses
  • Sperm Injections, Intracytoplasmic / veterinary
  • Sperm Injections, Intracytoplasmic / methods
  • Male
  • Fertilization in Vitro / veterinary
  • Fertilization in Vitro / methods
  • Female
  • Cryopreservation / veterinary
  • Semen Preservation / veterinary
  • Semen Preservation / methods
  • Semen / physiology
  • Blastocyst
  • Pregnancy

Grant Funding

  • BOF/STA/202202/008 / by Ghent University

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