Multiparametric Flow Cytometry for Determination of Viability, Caspase 3 and 7 Activity, and Lipid Peroxidation Adduct (4-Hydroxynonenal) in Equine Spermatozoa.
Abstract: Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flow cytometry analysis in equine spermatozoa using three sources of excitation and polychromatic flow cytometry for the detection of 4HNE, a lipid peroxidation adduct (by anti-4HNE antibody), apoptotic markers (by caspases 3 and 7 activity), and live/dead spermatozoa (by ethidium-homodimer) excluding the debris with Hoechst 33342 staining and gating. This multiparametric analysis allows the simultaneous detection of different spermatic parameters, providing useful information for the characterization of a seminal sample and fertility estimation. © 2023 Wiley Periodicals LLC. Basic Protocol: Determination of viability, caspase 3 and 7 activity, and 4-hydroxynonenal in equine spermatozoa by flow cytometry.
© 2023 Wiley Periodicals LLC.
Publication Date: 2023-09-06 PubMed ID: 37672491DOI: 10.1002/cpz1.885Google Scholar: Lookup
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Summary
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This scientific research paper presents the application of multiparametric flow cytometry for analyzing equine spermatozoa. It examines the viability and determination of activities of caspase 3 and 7 and the detection of lipid peroxidation, which contribute to the understanding of spermatozoa conditions and fertility estimation.
Introduction to Flow Cytometry
In this study, flow cytometry, a sophistical technique used for analyzing cell samples at a single-cell level, is the main tool employed. It’s particularly useful in studying multipopulations like spermatozoa. The advent of multiparametric cytometers has greatly enhanced this analysis, enabling the examination of various cellular features that include:
- Protein expression
- DNA analysis
- Mitochondrial activity
These cytometers employ either traditional fluorescent dyes or fluorophore-conjugated antibodies to individually stain each cell or cellular component. Following high-speed analysis, the resulting data is displayed and interpreted using a dot-plot system.
Study Methodology
In this research, the team used multiparametric flow cytometry to analyze equine spermatoza. The research utilized three sources of excitation and polychromatic flow cytometry. The parameters analyzed include:
- 4HNE detection – a lipid peroxidation adduct assessed using an anti-4HNE antibody
- Assessing apoptotic markers – measured by caspase 3 and 7 activity
- Examining live and nonviable spermatozoa- determined by using ethidium-homodimer
To exclude debris during the analysis, the team used Hoechst 33342 staining and gating.
Implication of the Multiparametric Analysis
The multiparametric analysis performed in this study allows for simultaneous detection of diverse spermatic parameters. This aligned detection offers valuable information that can be used in characterizing seminal samples as well as estimating fertility. It essentially gives a comprehensive understanding of the sperm quality, important for both natural conception and assisted reproduction techniques. This application thus provides a valuable contribution to the field of veterinary reproductive biology and medicine.
Cite This Article
APA
da Silva CMB, Cano FEM, Gaitskell-Phillips G, Vega FJP.
(2023).
Multiparametric Flow Cytometry for Determination of Viability, Caspase 3 and 7 Activity, and Lipid Peroxidation Adduct (4-Hydroxynonenal) in Equine Spermatozoa.
Curr Protoc, 3(9), e885.
https://doi.org/10.1002/cpz1.885 Publication
Researcher Affiliations
- Polytechnic Institute of Portalegre, Agrarian School of Elvas, Elvas, Portugal.
- VALORIZA, Research Centre for Endogenous Resources Valorization, Polytechnic Institute of Portalegre, Portalegre, Portugal.
- Laboratory of Equine Reproduction and Equine Spermatology, Universidad de Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Universidad de Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Universidad de Extremadura, Cáceres, Spain.
MeSH Terms
- Male
- Animals
- Horses
- Caspase 3
- Flow Cytometry
- Lipid Peroxidation
- Fluorescent Dyes
- Spermatozoa
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