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A New Method for Evaluating Stallion Sperm Viability and Mitochondrial Membrane Potential in Fixed Semen Samples
F J Peña1,2, B A Ball2, E L Squires2
1Department of Animal Medicine, Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Cytometry. Part B, Clinical Cytometry
|December 30, 2016
Summary
This study validates a new flow cytometry method for stallion sperm quality assessment using fixable dyes. Preserving and fixing sperm samples for up to 72 hours yields reliable results, improving accessibility for veterinary practices.
Area of Science:
- Veterinary Science
- Reproductive Biology
- Cell Biology
Background:
- Flow cytometry offers multiparametric stallion sperm quality assessment but is limited by instrument availability in veterinary practice.
- Preserving sperm samples with fixable probes could enable later analysis, broadening flow cytometry's application in andrology.
Purpose of the Study:
- To validate a novel flow cytometry assay for stallion sperm viability and mitochondrial membrane potential using fixable probes.
- To assess the stability of stained and fixed sperm samples over a 72-hour storage period.
Main Methods:
- Validated a combination of Zombie Green® (live/dead) and MitoTracker Deep Red® (mitochondrial membrane potential) fixable probes.
- Compared results with traditional non-fixable membrane assays (SYBR-14/PI).
- Assessed sperm samples after 72 hours of storage post-staining and fixation.
Main Results:
- The validated assay demonstrated feasibility for assessing stallion sperm quality.
- Fixable probes provided reliable data on sperm viability and mitochondrial function.
- Stained and fixed samples stored for 72 hours yielded results equivalent to same-day analysis.
Conclusions:
- A new protocol using fixable probes for flow cytometry analysis of stallion sperm is feasible.
- This method allows for storage and transport of samples, overcoming limitations of flow cytometer availability.
- The protocol is expected to facilitate wider adoption of flow cytometry in equine andrology.
