Effect of pre-freeze sperm concentration, freezing extender, and epididymal flushing technique on post-thaw quality of cryopreserved epididymal stallion sperm.
Abstract: Cryopreservation of stallion epididymal sperm has become a common clinical procedure after routine castration, euthanasia, or acute death. Unique features of epididymal sperm compared to ejaculated sperm include the requirement to remove sperm from the cauda epididymis, lack of exposure to seminal plasma, and potential recovery of large sperm numbers. In this study, the effect of the flushing technique (Extender (INRA) vs. AIR), freezing extender (LE, CMLE, MFR5, CFR5, or BOTU), and the concentration at which sperm are cryopreserved (200, 400, or 800 × 10 sperm/mL) on post-thaw epididymal sperm quality were studied. More total sperm were recovered when the cauda epididymides were flushed with INRA (14 × 10) compared to AIR (10 × 10). Epididymal sperm cryopreserved in BOTU and CMLE yielded higher post-thaw total and progressive motility than LE, MFR5, and CFR5 (P < 0.05); BOTU, MFR5, and CFR5 yielded higher post-thaw viable/acrosome-intact sperm than CMLE or LE (P < 0.05); CMLE, CFR5, and BOTU yielded higher post-thaw curvilinear velocity than LE or MFR5 (P < 0.05); an effect of extender on sperm DNA damage (COMP) was not observed (P > 0.05). Overall, similar post-thaw epididymal sperm quality parameters were observed among different cryopreserved sperm concentrations (P < 0.05). This study provides evidence of some factors that can affect the post-thaw quality of epididymal sperm from stallions.
Published by Elsevier Inc.
Publication Date: 2025-03-31 PubMed ID: 40203729DOI: 10.1016/j.theriogenology.2025.117418Google Scholar: Lookup
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Summary
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Effect of pre-freeze sperm concentration, freezing extender, and epididymal flushing technique on post-thaw quality of cryopreserved epididymal stallion sperm.
Overview
- This study investigated how different methods of collecting and freezing stallion epididymal sperm affect the quality of sperm after thawing.
- The researchers evaluated the impact of flushing technique, freezing extender used, and sperm concentration before freezing on key sperm quality measures following cryopreservation.
Background
- Stallion epididymal sperm cryopreservation is increasingly applied after castration, euthanasia, or sudden death to preserve fertility.
- Epididymal sperm are distinct from ejaculated sperm because they are collected from the cauda epididymis, have not been exposed to seminal plasma, and may be harvested in large quantities.
Experimental Variables
- Flushing technique: Cauda epididymides were flushed with two different extenders:
- INRA extender
- AIR extender
- Freezing extenders tested: Five extenders were evaluated for freezing sperm:
- LE (Lactose-Egg yolk)
- CMLE (Complex medium Lactose-Egg yolk)
- MFR5
- CFR5
- BOTU
- Sperm concentration before freezing: Sperm were cryopreserved at three concentrations:
- 200 × 10^6 sperm/mL
- 400 × 10^6 sperm/mL
- 800 × 10^6 sperm/mL
Key Findings
- Sperm recovery: More total sperm were recovered when using INRA extender (14 × 10^6) compared to AIR (10 × 10^6) for flushing the cauda epididymis.
- Post-thaw motility:
- Extenders BOTU and CMLE resulted in higher total and progressive motility after thawing than LE, MFR5, and CFR5 (P < 0.05).
- Sperm viability and acrosome integrity:
- BOTU, MFR5, and CFR5 extenders yielded higher percentages of viable and acrosome-intact sperm post-thaw than CMLE or LE (P < 0.05).
- Sperm velocity:
- CMLE, CFR5, and BOTU extenders produced sperm with higher curvilinear velocity post-thaw than LE or MFR5 (P < 0.05).
- Sperm DNA damage: No significant effect of the extender on DNA damage was observed (P > 0.05).
- Effect of sperm concentration: There were no significant differences in post-thaw sperm quality parameters among the three tested pre-freeze sperm concentrations (P < 0.05).
Implications and Conclusions
- The choice of flushing technique and freezing extender has a significant influence on the quality of frozen-thawed stallion epididymal sperm.
- INRA extender is more effective for flushing to maximize sperm recovery from cauda epididymis.
- Extenders BOTU and CMLE enhance motility, while BOTU, MFR5, and CFR5 are better for preserving sperm viability and acrosome integrity.
- Sperm concentration before freezing within the tested range does not greatly affect post-thaw quality.
- These findings can guide best practices in cryopreservation protocols for epididymal stallion sperm to improve fertility potential after thawing.
Cite This Article
APA
Spacek SG, Hernández-Avilés C, Ramírez-Agámez L, Pearson M, Love CC.
(2025).
Effect of pre-freeze sperm concentration, freezing extender, and epididymal flushing technique on post-thaw quality of cryopreserved epididymal stallion sperm.
Theriogenology, 241, 117418.
https://doi.org/10.1016/j.theriogenology.2025.117418 Publication
Researcher Affiliations
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA. Electronic address: clove@cvm.tamu.edu.
MeSH Terms
- Animals
- Male
- Cryopreservation / veterinary
- Cryopreservation / methods
- Horses / physiology
- Semen Preservation / veterinary
- Semen Preservation / methods
- Epididymis / cytology
- Spermatozoa / physiology
- Cryoprotective Agents / pharmacology
- Semen Analysis / veterinary
- Sperm Count / veterinary
- Freezing
- Sperm Motility
Conflict of Interest Statement
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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