A simple flow cytometry protocol to determine simultaneously live, dead and apoptotic stallion spermatozoa in fresh and frozen thawed samples.
Abstract: Spermatozoa undergo apoptotic changes during the cryopreservation process. These changes, recently termed spermptosis, resemble the cryopreservation induced delayed onset of cell death observed after thawing of somatic cells. Due to its importance in cryobiology, methods to easily identify spermptotic cells are warranted. In this study, a well-validated method for identification of spermatozoa with caspase 3 activity was compared with use of the combination of Hoechst 33342 (H-42) and ethidium homodimer (Eth-1). Live, dead and apoptotic spermatozoa assessed with each method were compared using descriptive statistics and method agreement analysis. No differences were observed in the percentages of spermatozoa in each of the categories investigated with each method. Moreover the method agreement analysis indicated there were consistent findings using both methods The combination H-42/Eth-1 can be successfully used to determine apoptosis in addition to dead and live spermatozoa. Moreover the intensity of H-42 fluorescence (bright and dim populations) allows for distinguishing of live and dead sperm cells.
Copyright © 2017 Elsevier B.V. All rights reserved.
Publication Date: 2017-12-16 PubMed ID: 29258708DOI: 10.1016/j.anireprosci.2017.12.009Google Scholar: Lookup
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- Journal Article
Summary
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The research investigates the reliability of two methods for identifying live, dead and apoptotic sperm cells in both fresh and frozen/thawed samples, with results suggesting that the combination of Hoechst 33342 and ethidium homodimer can be successfully used for this purpose.
Introduction
- The research focuses on the process sperm cells undergo during cryopreservation, termed spermptosis, which mirrors the delayed onset of cell death found in somatic cells post-thawing. It asserts the necessity of reliable methods to identify spermptotic cells due to its significance in cryobiology.
Methods Used
- The study compares two methods for identifying spermatozoa: one well-validated method that identifies spermatozoa with caspase 3 activity and another that employs the combination of two fluorescent stains, Hoechst 33342 (H-42) and ethidium homodimer (Eth-1).
- The research then assesses live, dead and apoptotic spermatoa using both methods, comparing the results using descriptive statistics and method agreement analysis.
Results
- The research found no significant differences in the percentages of spermatozoa categorized as either live, dead or apoptotic with the use of either method.
- On subsequently analyzing the consistency of the two methods through method agreement analysis, it was found that the findings from both were congruent.
- The study concluded that the combination of H-42 and Eth-1 could be effectively utilized to determine not just live and dead sperm cells, but also apoptotic ones.
- Moreover, the intensity of the H-42 fluorescence was found to allow for a distinction between live and dead sperm cells, separating them into ‘bright’ and ‘dim’ populations.
Significance
- The results of this study offer a methodological alternative for identifying apoptotic sperm cells in addition to live and dead ones.
- This novel insight could potentially enhance procedures in cryobiology related to sperm preservation and fertility treatment methodologies.
Cite This Article
APA
Gil MC, Ferrusola CO, Anel-López L, Ortiz-Rodriguez JM, Alvarez M, de Paz P, Anel L, Peña FJ.
(2017).
A simple flow cytometry protocol to determine simultaneously live, dead and apoptotic stallion spermatozoa in fresh and frozen thawed samples.
Anim Reprod Sci, 189, 69-76.
https://doi.org/10.1016/j.anireprosci.2017.12.009 Publication
Researcher Affiliations
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Department of Animal Medicine and Surgery, University of León, Spain.
- Department of Animal Medicine and Surgery, University of León, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Department of Animal Medicine and Surgery, University of León, Spain.
- Department of Molecular Biology, University of León, Spain.
- Department of Animal Medicine and Surgery, University of León, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain. Electronic address: fjuanpvega@unex.es.
MeSH Terms
- Animals
- Apoptosis
- Cryopreservation / veterinary
- Flow Cytometry / veterinary
- Horses / physiology
- Male
- Semen Analysis / methods
- Semen Analysis / veterinary
- Semen Preservation / veterinary
- Spermatozoa / physiology
Citations
This article has been cited 3 times.- Neila-Montero M, Riesco MF, Alvarez M, Montes-Garrido R, Boixo JC, de Paz P, Anel-Lopez L, Anel L. Centrifugal force assessment in ram sperm: identifying species-specific impact.. Acta Vet Scand 2021 Nov 4;63(1):42.
- 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.
- Ortiz-Rodriguez JM, Balao da Silva C, Masot J, Redondo E, Gazquez A, Tapia JA, Gil C, Ortega-Ferrusola C, Peña FJ. Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3.. PLoS One 2019;14(7):e0211994.
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