FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Cytometry. Part B, Clinical cytometry2017; 94(2); 302-311; doi: 10.1002/cyto.b.21506

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

Abstract: 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. © 2017 International Clinical Cytometry Society.
© 2016 International Clinical Cytometry Society.
Get Full Text
Publication Date: 2017-02-08 PubMed ID: 28033647DOI: 10.1002/cyto.b.21506Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

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.

The research article provides insights into a new method for evaluating the viability of stallion sperm and mitochondrial membrane potential in fixed semen samples. It introduces the innovative use of live/dead Zombie Green and MitoTracker Deep Red dyes as flow cytometry methods and compares this new method to the classic membrane assays.

Objective of the Study

  • The study’s main objective was to validate a new technique for stallion sperm’s multiparametric assessment using flow cytometry with the help of Zombie Green fixable dye and MitoTracker Deep Red.

Limitations in Current Practice

  • The researchers noted that in-veterinary practice, the use of flow cytometers is limited due to the unavailability of instrument.
  • There was also a challenge with the preservation and transport of sperm samples for later analysis using fixable probes.

Proposed Solution and Methodology

  • The researchers validated the use of a combination of live/dead Zombie Green, a fixable dye used to assess live and dead sperm, and MitoTracker Deep Red, a dye used to assess mitochondrial membrane potential.
  • This method was tested against classical, non-fixable, membrane assays (SYBR-14/PI).
  • The primary intention was to establish a durable, transportable method that still provided precise data on sperm viability and mitochondrial membrane potential.

Results and Conclusion

  • Results from the new assay method demonstrated that staining and fixing semen samples then storing them for 72 hours produced equivalent results to tests run on the same day.
  • Through the implementation of this new methodology, the researchers concluded that there would be wider use of flow cytometry for stallion andrology in the future because of its improved accessibility and usability.

Implications of the Study

  • This research paves the way for more accessible and effective testing of stallion sperm potency and quality, contributing notably to the fields of animal husbandry and veterinary science.
  • The validated method could improve efficiency and reduce the technical limitations currently experienced in stallion andrology.

Cite This Article

APA
Peña FJ, Ball BA, Squires EL. (2017). A New Method for Evaluating Stallion Sperm Viability and Mitochondrial Membrane Potential in Fixed Semen Samples. Cytometry B Clin Cytom, 94(2), 302-311. https://doi.org/10.1002/cyto.b.21506

Publication

Cytometry. Part B, Clinical cytometry
ISSN: 1552-4957
NlmUniqueID: 101235690
Country: United States
Language: English
Volume: 94
Issue: 2
Pages: 302-311

Researcher Affiliations

Peña, F J
  • Department of Animal Medicine, Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Kentucky.
Ball, B A
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Kentucky.
Squires, E L
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Kentucky.

MeSH Terms

  • Animals
  • Cell Survival / physiology
  • Flow Cytometry / methods
  • Fluorescent Dyes / pharmacology
  • Horses / physiology
  • Male
  • Membrane Potential, Mitochondrial / physiology
  • Semen / physiology
  • Semen Analysis / methods
  • Spermatozoa / physiology
  • Staining and Labeling / methods

Citations

This article has been cited 16 times.
  1. Podico G, Spencer KM, Magalhaes HB, Canisso IF. Semen Quality of the First and Second Ejaculates Collected from Breeding Inactive Stallions after Cooling and Freezing. Vet Sci 2023 Feb 21;10(3).
    doi: 10.3390/vetsci10030173pubmed: 36977212google scholar: lookup
  2. Kalwar Q, Chu M, Korejo RA, Soomro H, Yan P. Cryopreservation of Yak Semen: A Comprehensive Review. Animals (Basel) 2022 Dec 7;12(24).
    doi: 10.3390/ani12243451pubmed: 36552371google scholar: lookup
  3. Podico G, Canisso IF. Retrograde Flushing Followed by Slicing Float-Up as an Approach to Optimize Epididymal Sperm Recovery for the Purpose of Cryopreservation in Equids. Animals (Basel) 2022 Jul 14;12(14).
    doi: 10.3390/ani12141802pubmed: 35883349google scholar: lookup
  4. Mocé ML, Esteve IC, Pérez-Fuentes S, Gómez EA, Mocé E. Microbiota in Goat Buck Ejaculates Differs Between Breeding and Non-breeding Seasons. Front Vet Sci 2022;9:867671.
    doi: 10.3389/fvets.2022.867671pubmed: 35647092google scholar: lookup
  5. Lago-Alvarez Y, Podico G, Segabinazzi LG, Cunha LL, Barbosa L, Arnold CE, Lima FS, King LT, McLean AK, Canisso IF. Donkey Epididymal Transport for Semen Cooling and Freezing. Animals (Basel) 2020 Nov 25;10(12).
    doi: 10.3390/ani10122209pubmed: 33255737google scholar: lookup
  6. F Riesco M, Anel-Lopez L, Neila-Montero M, Palacin-Martinez C, Montes-Garrido R, Alvarez M, de Paz P, Anel L. ProAKAP4 as Novel Molecular Marker of Sperm Quality in Ram: An Integrative Study in Fresh, Cooled and Cryopreserved Sperm. Biomolecules 2020 Jul 14;10(7).
    doi: 10.3390/biom10071046pubmed: 32674525google scholar: lookup
  7. Marzano G, Moscatelli N, Di Giacomo M, Martino NA, Lacalandra GM, Dell'Aquila ME, Maruccio G, Primiceri E, Chiriacò MS, Zara V, Ferramosca A. Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm. Biology (Basel) 2020 Jan 27;9(2).
    doi: 10.3390/biology9020022pubmed: 32012799google scholar: lookup
  8. Peña FJ, O'Flaherty C, Ortiz Rodríguez JM, Martín Cano FE, Gaitskell-Phillips GL, Gil MC, Ortega Ferrusola C. Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa. Antioxidants (Basel) 2019 Nov 19;8(11).
    doi: 10.3390/antiox8110567pubmed: 31752408google scholar: lookup
  9. Ugur MR, Saber Abdelrahman A, Evans HC, Gilmore AA, Hitit M, Arifiantini RI, Purwantara B, Kaya A, Memili E. Advances in Cryopreservation of Bull Sperm. Front Vet Sci 2019;6:268.
    doi: 10.3389/fvets.2019.00268pubmed: 31552277google scholar: lookup
  10. Eser A, Alakuş A, Bağcı K, Cihangiroğlu AÇ, Yağcıoğlu S, Arıcı R, Demir K. Precursor A-Kinase Anchor Protein 4 as a Predictive Biomarker of Post-Thaw Semen Quality in Goats. Vet Sci 2025 Oct 16;12(10).
    doi: 10.3390/vetsci12101003pubmed: 41150143google scholar: lookup
  11. Martínez-Rodrigo L, Gacem S, Brüggemann M, Pinto S, Mocé E, Serrano-Jara D, García ML, Yániz JL, Silvestre MA. Evaluating DAPI stain to assess sperm membrane integrity by flow cytometry in livestock species. Vet Res Commun 2025 Oct 20;49(6):357.
    doi: 10.1007/s11259-025-10948-wpubmed: 41114779google scholar: lookup
  12. Santiani A, Cucho M, Delgado J, Juárez J, Ruiz L, Evangelista-Vargas S. Field-Compatible Cytometric Assessment of Epididymal Alpaca Sperm Viability and Acrosomal Integrity Using Fluorochrome. Animals (Basel) 2025 Aug 5;15(15).
    doi: 10.3390/ani15152282pubmed: 40805072google scholar: lookup
  13. Ni J, Li X, Wu Y, Tu X, Zhang X, Wang L, Xie H, Hou Y, Dou H, Zhao S. CD81 Aggravates Ovarian Cancer Progression via p-Cresyl Sulfate-Mediated Mitophagy in Tim4(+) Tumour-Associated Macrophages. J Cell Mol Med 2025 Jul;29(13):e70701.
    doi: 10.1111/jcmm.70701pubmed: 40604335google scholar: lookup
  14. Hai E, Li B, Song Y, Zhang J, Zhang J. Ferroptosis emerges as the predominant form of regulated cell death in goat sperm cryopreservation. J Anim Sci Biotechnol 2025 Feb 18;16(1):26.
    doi: 10.1186/s40104-025-01158-0pubmed: 39966967google scholar: lookup
  15. Egyptien S, Deleuze S, Ledeck J, Ponthier J. Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions. Animals (Basel) 2023 Oct 6;13(19).
    doi: 10.3390/ani13193123pubmed: 37835729google scholar: lookup
  16. Paul P, Iyer S, Nadella RK, Nayak R, Chellappa AS, Ambardar S, Sud R, Sukumaran SK, Purushottam M, Jain S, Viswanath B. Lithium response in bipolar disorder correlates with improved cell viability of patient derived cell lines. Sci Rep 2020 May 4;10(1):7428.
    doi: 10.1038/s41598-020-64202-1pubmed: 32366893google scholar: lookup
Subscribe to our newsletter
Join 99,727 horse owners like you who receive our email updates on horse health and nutrition.