Reproduction in domestic animals = Zuchthygiene2026; 61(10); e70327; doi: 10.1111/rda.70327

Effect of Different Equilibration Times in the Freezing Process on Post-Thaw Motility Parameters in Equine Spermatozoa.

Farnia, H
·
Sautejeau-Rizzoni, P
·
Chekchouk, M
·
Belala, R
·
Mimoune, N
·
Teixeira Fangueiro, L
·
Thorin, C
·
Leborgne, I
·
Le-Guiriec, M
·
Bencharif, D

Abstract: Cryopreservation of stallion semen remains suboptimal, with approximately one-third of stallions producing semen that is poorly tolerant to freezing. As optimization of pre-freezing procedures may improve post-thaw sperm quality, this study evaluated the effect of different equilibration times before freezing on the post-thaw quality of stallion semen. For this purpose, 21 ejaculates were initially collected from 5 stallions and 15 were included in the final analysis after predefined exclusions. These ejaculates were separated from the seminal plasma via centrifugation in INRA 96 medium. After removal of the supernatant, each ejaculate was divided into three aliquots and equilibrated at 4°C for 2, 4, or 6 h before freezing. The pellets were mixed with INRA FREEZE solution at a concentration of 100 million spermatozoa per millilitre. Subsequently, the semen was frozen in 0.5 mL straws after being kept at the designated temperature at +4°C for either 2, 4 or 6 h post-cooling. Post-thaw sperm total motility, progressive motility and kinematic parameters were assessed by computer-assisted sperm analysis (CASA). Total motility increased with equilibration time, from 27% ± 7% after 2 h to 34% ± 10% after 4 h and 40% ± 7% after 6 h (p < 0.05). Progressive motility was significantly lower after 2 h (15% ± 6%) than after 4 h (20% ± 7%) and 6 h (22% ± 7%) of equilibration (p < 0.05), whereas no significant difference was observed between 4 and 6 h. In contrast, equilibration time did not significantly affect sperm kinematic parameters, including VAP, VSL, VCL, ALH and LIN. These findings indicate that extending the equilibration period to 6 h at 4°C before freezing improves post-thaw total and progressive sperm motility in stallions without altering the movement characteristics of surviving spermatozoa.

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Publication Date: 2026-10-12 PubMed ID: 42862011DOI: 10.1111/rda.70327Google 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 investigated how varying the time stallion semen is kept cold before freezing (called equilibration time) affects sperm movement and quality after thawing.
The research found that longer equilibration times (up to 6 hours at 4°C) improved sperm motility after thawing, which is crucial for fertility, without changing the detailed movement patterns of the sperm.

Background and Objective

Cryopreservation (freezing) of stallion semen is important for breeding but currently not ideal, with about one-third of stallions producing semen that does not tolerate freezing well.
Post-thaw sperm quality can be influenced by how the semen is handled before freezing, particularly the equilibration time at low temperature, which may impact sperm survival and motility.
The objective was to determine how different equilibration times (2, 4, and 6 hours at 4°C) before freezing affect post-thaw sperm motility and movement parameters in stallion semen.

Methods

Semen collection:
- 21 ejaculates collected from 5 stallions initially.
- After applying exclusion criteria, 15 ejaculates were included in the analysis.
Preparation:
- Seminal plasma was removed by centrifugation using INRA 96 medium.
- The sperm pellet was divided into three aliquots for different treatments.
Equilibration:
- Each aliquot equilibrated at 4°C for 2, 4, or 6 hours.
- Equilibration is the period sperm samples remain at low temperature before freezing, allowing cells to adapt and reduce damage during freezing.
Freezing:
- Samples mixed with INRA FREEZE solution to reach a concentration of 100 million sperm/mL.
- Frozen in 0.5 mL straws after the specified equilibration period.
Post-thaw analysis:
- Semen thawed and assessed for total motility, progressive motility, and kinematic parameters using computer-assisted sperm analysis (CASA).
- Kinematic parameters measured include average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and linearity (LIN).

Results

Total motility (percentage of moving sperm):
- Increased with longer equilibration time.
- 27% ± 7% after 2 hours.
- 34% ± 10% after 4 hours.
- 40% ± 7% after 6 hours.
- Differences were statistically significant (p < 0.05).
Progressive motility (percentage of sperm moving actively forward):
- Lower after 2 hours (15% ± 6%) compared to 4 hours (20% ± 7%) and 6 hours (22% ± 7%).
- No significant difference between 4 and 6 hours.
- Indicates improvement with longer equilibration but plateaus after 4 hours.
Kinematic parameters (VAP, VSL, VCL, ALH, LIN):
- No significant changes between different equilibration times.
- Suggests that while more sperm survive and move, their quality of movement remains consistent regardless of equilibration time.

Conclusions and Implications

Lengthening the equilibration time to 6 hours at 4°C before freezing improves overall motility and progressive motility of post-thaw stallion sperm.
This improvement occurs without altering the detailed movement characteristics of the surviving sperm cells.
These findings are valuable for optimizing semen cryopreservation protocols, potentially enhancing fertility outcomes in equine breeding programs.
Longer equilibration times may help stallions with initially poor semen freezability by increasing sperm survival and motility post-thaw.

Cite This Article

APA

Farnia H, Sautejeau-Rizzoni P, Chekchouk M, Belala R, Mimoune N, Teixeira Fangueiro L, Thorin C, Leborgne I, Le-Guiriec M, Bencharif D. (2026). Effect of Different Equilibration Times in the Freezing Process on Post-Thaw Motility Parameters in Equine Spermatozoa. Reprod Domest Anim, 61(10), e70327. https://doi.org/10.1111/rda.70327

Publication

Reproduction in domestic animals = Zuchthygiene

ISSN: 1439-0531

NlmUniqueID: 9015668

Country: Germany

Language: English

Volume: 61

Issue: 10

Pages: e70327

Researcher Affiliations

Farnia, Hamed

Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: College of Veterinary Medicine, Food Science and Engineering, Nantes, France.

Sautejeau-Rizzoni, Paola

Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: College of Veterinary Medicine, Food Science and Engineering, Nantes, France.

Chekchouk, Mouna

Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: College of Veterinary Medicine, Food Science and Engineering, Nantes, France.

Belala, Rédha

Biotechnologies Laboratory Related to Animal Reproduction (LBRA), Institute of Veterinary Medicine, Saad Dahleb Blida University 1, Blida, Algeria.

Mimoune, Nora

Higher National Veterinary School, Algiers, Algeria.

Teixeira Fangueiro, Lucas

Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: College of Veterinary Medicine, Food Science and Engineering, Nantes, France.

Thorin, Chantal

Department of Statistics, ONIRIS: College of Veterinary Medicine, Food Science and Engineering, Nantes, France.

Leborgne, Ingrid

Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: College of Veterinary Medicine, Food Science and Engineering, Nantes, France.

Le-Guiriec, Morgane

Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: College of Veterinary Medicine, Food Science and Engineering, Nantes, France.
Center for Research in Transplantation and Translational Immunology, UMR 1064, INSERM, Nantes Université, CHU Nantes, Nantes, France.

Bencharif, Djemil

Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: College of Veterinary Medicine, Food Science and Engineering, Nantes, France.
Center for Research in Transplantation and Translational Immunology, UMR 1064, INSERM, Nantes Université, CHU Nantes, Nantes, France.

MeSH Terms

Animals
Male
Horses / physiology
Sperm Motility
Cryopreservation / veterinary
Cryopreservation / methods
Semen Preservation / veterinary
Semen Preservation / methods
Spermatozoa / physiology
Time Factors
Freezing
Semen Analysis / veterinary

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