A New Method for Evaluating Stallion Sperm Viability and Mitochondrial Membrane Potential in Fixed Semen Samples

F. J. Peña, B. A. Ball, E. L. Squires

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


Multiparametric assessment of stallion sperm quality using flow cytometry can be a useful adjunct in semen evaluation; however, the availability of flow cytometers in veterinary practice is limited. The ability to preserve and transport sperm samples for later flow cytometric analysis using fixable probes would potentially facilitate this process. In the current study, we validated the combination of live/dead Zombie Green ® (a fixable dye used to assess live and dead sperm) and MitoTracker Deep Red ® (used to assess mitochondrial membrane potential). The assay was validated against classic, non-fixable, membrane assays (SYBR-14/PI). Our results demonstrated the feasibility of the assay. In conclusion, stained and fixed semen samples stored for 72 h obtained equivalent results to the exam on the same day; this new protocol shall facilitate the wider use of flow cytometry in stallion andrology in the future.

Original languageEnglish
Pages (from-to)302-311
Number of pages10
JournalCytometry Part B - Clinical Cytometry
Issue number2
StatePublished - Mar 2018

Bibliographical note

Funding Information:
*Correspondence to: Dr. F.J. Peña, Laboratory of Equine Spermatol-ogy and Reproduction, Faculty of Veterinary Medicine University of Extremadura Adv. de la Universidad s/n 10003 Cáceres Spain E-mail: fjuanpvega@unex.es Grant sponsor: Ministerio de Economía y Competitividad-FEDER in Madrid, Spain; Grant number: AGL2013-43211-R. Grant sponsor: Ministerio de Educación; Grant number: PRX14000/95. Grant sponsor: Junta de Extremadura-FEDER; Grant number: GR 10010, PCE1002. Grant sponsor: Clay Fellowship of the University of Kentucky.

Publisher Copyright:
© 2016 International Clinical Cytometry Society


  • fixable dyes
  • flow cytometry
  • membranes
  • mitochondria
  • sperm
  • stallion

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology


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