Assessment of stallion spermatozoa viability by flow cytometry and light microscope analysis.
Abstract: Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of each aliquot by 1) motility; 2) eosin-nigrosin staining; and 3) dual fluorescent staining. For the latter, aliquots incubated with SYBR-14 and propidium iodide had live and dead spermatozoa quantitated by fluorescent microscope (2 x 100 sperm/sample) and flow cytometry (10,000 sperm/sample). We found a linear relationship between the ratio of live:dead spermatozoa and the percentage of spermatozoa counted as live (P0.75; fluorescent microscope, r>0.76; flow cytometry, r>0.75; motility, r>0.76). To determine viability of cryopreserved equine spermatozoa, we froze 17 fresh ejaculates from 6 stallions in a glycine extender. Each sample was thawed, extended 1:1 with EZ Mixin and evaluated as above. Cryopreserved spermatozoa assessed by flow cytometry tended to be less well correlated (r<0.68) with the other methods, and estimates were significantly higher with eosin-nigrosin staining (P<0.001). This study shows that different methods may equally estimate viability of fresh equine spermatozoa. However, evaluation by flow cytometry appears to be less precise with cryopreserved spermatozoa.
Publication Date: 2001-02-24 PubMed ID: 11192180DOI: 10.1016/s0093-691x(00)00428-3Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study investigates various methods of assessing the viability of stallion spermatozoa and finds that different techniques may produce comparable results for fresh sperm. However, the accuracy of flow cytometry seems to diminish for cryopreserved sperm.
Study Context and Design
- In order to ascertain the viability of spermatozoa, researchers collected two ejaculates from each of four stallions and diluted them to 50×10(6) sperm/mL using a nonfat dried milk solids glucose extender, also called EZ Mixin.
- To provide different ratios of live and dead sperm, half the ejaculate was deliberately freeze-killed by being immersed in liquid nitrogen for ten minutes which formed the following ratios: 100:0, 75:25, 50:50, 25:75, and 0:100.
Methods of Assessment
- Viability of each sperm sample was analyzed through three different methods: motility evaluation, eosin-nigrosin staining, and dual fluorescent staining.
- For dual fluorescent staining, the samples were incubated with SYBR-14 and propidium iodide. These stained samples were then quantified for live and dead sperm using a fluorescent microscope and flow cytometry.
Findings with Fresh Spermatozoa
- The study found a linear relationship between the ratio of live to dead sperm and the percentage of sperm counted as alive. This relationship was found to be statistically significant.
- For fresh spermatozoa, the correlation coefficients of the known live:dead ratio were high for all methods, suggesting that all three analysis techniques were approximately as effective for assessing fresh sperm viability.
Findings with Cryopreserved Spermatozoa
- For the cryopreserved sperm, the study froze 17 fresh ejaculates from six stallions and assessed their viability after thawing using the same methods as above.
- Cryopreserved sperm evaluated by flow cytometry were less correlated with the known live:dead ratio, implying a lower precision of this technique with frozen sperm.
- Estimations of viability were significantly higher when assessed by eosin-nigrosin staining.
Study Conclusion
- The study concludes that different techniques may provide similar evaluations of viability for fresh stallion spermatozoa. However, for cryopreserved sperm, the accuracy of the flow cytometry technique appears to be decreased.
Cite This Article
APA
Merkies K, Chenier T, Plante C, Buhr MM.
(2001).
Assessment of stallion spermatozoa viability by flow cytometry and light microscope analysis.
Theriogenology, 54(8), 1215-1224.
https://doi.org/10.1016/s0093-691x(00)00428-3 Publication
Researcher Affiliations
- Department of Animal and Poultry Science, University of Guelph, Ontario.
MeSH Terms
- Aniline Compounds / chemistry
- Animals
- Cryopreservation / methods
- Cryopreservation / veterinary
- Eosine Yellowish-(YS) / chemistry
- Flow Cytometry / veterinary
- Fluorescent Dyes / chemistry
- Horses / physiology
- Male
- Microscopy, Fluorescence / veterinary
- Organic Chemicals
- Propidium / chemistry
- Semen Preservation / methods
- Semen Preservation / veterinary
- Sperm Motility / physiology
- Spermatozoa / physiology
- Statistics, Nonparametric
Citations
This article has been cited 5 times.- Dziekońska A, Lecewicz M, Partyka A, Niżański W. Fluorescence Microscopy and Flow-Cytometry Assessment of Substructures in European Red Deer Epididymal Spermatozoa after Cryopreservation. Animals (Basel) 2023 Mar 8;13(6).
- Rečková Z, Filipčík R, Soušková K, Kopec T, Hošek M, Pešan V. The efficiency of different types of extenders for semen cooling in stallions. Anim Biosci 2022 May;35(5):670-676.
- Wysokińska A, Szablicka D. Integrity of Sperm Cell Membrane in the Semen of Crossbred and Purebred Boars during Storage at 17 °C: Heterosis Effects. Animals (Basel) 2021 Nov 25;11(12).
- Hernández-Avilés C. Analysis of Motion Characteristics and Plasma Membrane Intactness (Viability) in Sperm from Domestic Animals. Methods Mol Biol 2025;2954:241-259.
- Chacón LJ, Yepes GD, Cardozo J, Rueda F, Castillo V, Torres A, Martins J, Ardila A. Seminal Plasma Proteins Associated with The Fertility of Brahman Bulls in The Colombian Low Tropics. Trop Life Sci Res 2023 Sep;34(3):259-277.
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