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Home / Equine Research Bank / Biol Reprod / Assessment of spermatozoal function using dual fluorescent s...
Biology of reproduction1986; 34(1); 127-138; doi: 10.1095/biolreprod34.1.127

Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses.

Abstract: Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained the green fluorescein fluorophore mainly in the acrosome. A third population, presumed to be degenerate spermatozoa, possessed only red fluorescent nuclei. These populations were quantified using dual parameter flow cytometry in 14 samples of cryopreserved bovine spermatozoa for which fertility and seminal quality data were available. Flow cytometric analyses were highly correlated with other seminal quality measurements. Sequential flow cytometric analyses provided the ability to rapidly quantitate changes in specific fluorescently stained populations. The ability to make rapid quantitative measurements should allow development of new and presumably more reliable information on the functional aspects of spermatozoa.
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Publication Date: 1986-02-01 PubMed ID: 3955132DOI: 10.1095/biolreprod34.1.127Google Scholar: Lookup
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  • Comparative Study
  • Journal Article
  • Research Support
  • Non-U.S. Gov't
  • Research Support
  • U.S. Gov't
  • Non-P.H.S.

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 explored the use of dual fluorescent staining and flow cytometric analyses to assess the functionality of spermatozoa from various species. The study demonstrated a strong correlation between the results from this technique and other seminal quality measurements, suggesting a possible new approach for quick and reliable sperm quality assessment.

Methodology

  • The sperm samples used in this study were sourced from several species including bulls, boars, dogs, horses, mice, and humans.
  • A dual fluorescent stain was used for the examination of spermatozoa. This stain comprised of carboxyfluorescein diacetate (CFDA), a substrate that can diffuse through cell membranes, and propidium iodide (PI), a nuclear stain unable to easily permeate membranes.
  • Three unique populations of spermatozoa became apparent upon microscopic assessment based on fluorescent staining patterns.
  • The research also performed flow cytometric analyses on 14 samples of cryopreserved bovine sperm, which were correlated with fertility and seminal quality data for the same samples.

Findings

  • The study identified three distinct populations of spermatozoa. The first group were thought to be viable sperm due to its motility and retention of products from the fluorescein chromophore (CFDA) throughout the cell.
  • A second population of sperm was identified, their nuclei were stained red by the PI, and were noted to retain the green fluorescein fluorophore mostly in the acrosome, an important part of the sperm cell that contains enzymes required for penetrating the egg during fertilization.
  • The third population presumably consisted of degenerate spermatozoa that only exhibited red fluorescent nuclei, staining positively with PI, indicating compromised membrane integrity.
  • The study found a high correlation between the flow cytometric analyses results and other seminal quality measurements, suggesting that the flow cytometric analyses could quickly provide reliable quantitative information on sperm functionality.

Implications

  • This research suggests that the dual fluorescent staining protocol and flow cytometric analyses may be a reliable, quick method for assessing the functional aspects of spermatozoa, which could potentially be advantageous in various fields such as fertility clinics and animal breeding.

Cite This Article

APA
Garner DL, Pinkel D, Johnson LA, Pace MM. (1986). Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. Biol Reprod, 34(1), 127-138. https://doi.org/10.1095/biolreprod34.1.127

Publication

Biology of reproduction
ISSN: 0006-3363
NlmUniqueID: 0207224
Country: United States
Language: English
Volume: 34
Issue: 1
Pages: 127-138

Researcher Affiliations

Garner, D L
    Pinkel, D
      Johnson, L A
        Pace, M M

          MeSH Terms

          • Acrosome / physiology
          • Animals
          • Cattle
          • Dogs
          • Flow Cytometry / instrumentation
          • Flow Cytometry / methods
          • Fluoresceins
          • Horses
          • Humans
          • Male
          • Species Specificity
          • Sperm Count
          • Spermatozoa / cytology
          • Spermatozoa / physiology
          • Swine

          Citations

          This article has been cited 18 times.
          1. Yániz JL, Palacín I, Silvestre MA, Hidalgo CO, Tamargo C, Santolaria P. Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls. Biology (Basel) 2021 Nov 4;10(11).
            doi: 10.3390/biology10111135pubmed: 34827128google scholar: lookup
          2. Chen PR, Redel BK, Kerns KC, Spate LD, Prather RS. Challenges and Considerations during In Vitro Production of Porcine Embryos. Cells 2021 Oct 15;10(10).
            doi: 10.3390/cells10102770pubmed: 34685749google scholar: lookup
          3. Tanga BM, Qamar AY, Raza S, Bang S, Fang X, Yoon K, Cho J. Semen evaluation: methodological advancements in sperm quality-specific fertility assessment - A review. Anim Biosci 2021 Aug;34(8):1253-1270.
            doi: 10.5713/ab.21.0072pubmed: 33902175google scholar: lookup
          4. Chen YC, Liu HC, Wei LY, Huang JF, Lin CC, Blesbois E, Chen MC. Sperm Quality Parameters and Reproductive Efficiency in Muscovy Duck (Cairina moschata). J Poult Sci 2016 Jul 25;53(3):223-232.
            doi: 10.2141/jpsa.0150162pubmed: 32908388google scholar: lookup
          5. Alquézar-Baeta C, Gimeno-Martos S, Miguel-Jiménez S, Santolaria P, Yániz J, Palacín I, Casao A, Cebrián-Pérez JÁ, Muiño-Blanco T, Pérez-Pé R. OpenCASA: A new open-source and scalable tool for sperm quality analysis. PLoS Comput Biol 2019 Jan;15(1):e1006691.
            doi: 10.1371/journal.pcbi.1006691pubmed: 30657753google scholar: lookup
          6. Sullenberger C, Piqué D, Ogata Y, Mensa-Wilmot K. AEE788 Inhibits Basal Body Assembly and Blocks DNA Replication in the African Trypanosome. Mol Pharmacol 2017 May;91(5):482-498.
            doi: 10.1124/mol.116.106906pubmed: 28246189google scholar: lookup
          7. Chaveiro A, Cerqueira C, Silva J, Franco J, Moreira da Silva F. Evaluation of frozen thawed cauda epididymal sperms and in vitro fertilizing potential of bovine sperm collected from the cauda epididymal. Iran J Vet Res 2015 Spring;16(2):188-93.
            pubmed: 27175174
          8. Hossain MS, Johannisson A, Wallgren M, Nagy S, Siqueira AP, Rodriguez-Martinez H. Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art. Asian J Androl 2011 May;13(3):406-19.
            doi: 10.1038/aja.2011.15pubmed: 21478895google scholar: lookup
          9. Sutkeviciene N, Riskeviciene V, Januskauskas A, Zilinskas H, Andersson M. Assessment of sperm quality traits in relation to fertility in boar semen. Acta Vet Scand 2009 Dec 16;51(1):53.
            doi: 10.1186/1751-0147-51-53pubmed: 20015377google scholar: lookup
          10. Khandwekar AP, Patil DP, Khandwekar V, Shouche YS, Sawant S, Doble M. TecoflexTM functionalization by curdlan and its effect on protein adsorption and bacterial and tissue cell adhesion. J Mater Sci Mater Med 2009 May;20(5):1115-29.
            doi: 10.1007/s10856-008-3655-3pubmed: 19093193google scholar: lookup
          11. López A, Söderquist L, Rodriguez-Martinez H. Sperm viability in ram semen diluted and stored in three different extenders. Acta Vet Scand 1999;40(1):1-9.
            doi: 10.1186/BF03547036pubmed: 10418191google scholar: lookup
          12. de S Cestaro DS, Varela Junior AS, Valverde A, de Souza Votto AP, de Moraes Vaz Batista Filgueira D, Yeste M, Corcini CD. Effects of ultraviolet B radiation and cold storage on Ram sperm morphology and physiology. Int J Biometeorol 2026 Feb 2;70(2):46.
            doi: 10.1007/s00484-025-03082-4pubmed: 41627547google scholar: lookup
          13. de Figueiredo MM, Bueno VC, Royes ICL, Mattos RC, Bastos HBA, Rechsteiner SF. Protamine1, 2 and Catsper1: sperm quality and fertility indicators in Stallions. Anim Reprod 2025;22(4):e20250040.
            doi: 10.1590/1984-3143-AR2025-0040pubmed: 41393904google scholar: lookup
          14. Hernández-Avilés C. Analysis of Motion Characteristics and Plasma Membrane Intactness (Viability) in Sperm from Domestic Animals. Methods Mol Biol 2025;2954:241-259.
            doi: 10.1007/978-1-0716-4698-4_14pubmed: 40601280google scholar: lookup
          15. Vicente-Carrillo A, de Mercado de la Peña E, Martín San Juan A, Nieto-Cristóbal H, Rodríguez-Martínez H, Álvarez-Rodríguez M. Spermatology: Current Methods to Study Sperm Classical Variables. Methods Mol Biol 2025;2897:3-27.
            doi: 10.1007/978-1-0716-4406-5_1pubmed: 40202624google scholar: lookup
          16. Bueno VC, Bastos HBA, Centeno LA, Kretzmann NA, Mattos RC, Rechsteiner SF. PLCζ, WBP2NL and TNF-α expression in spermatozoa is associated with stallion fertility and seminal quality?. Anim Reprod 2024;21(1):e20230088.
            doi: 10.1590/1984-3143-AR2023-0088pubmed: 38628496google scholar: lookup
          17. Lyngdoh ME, Chettri J, Kharchandy VF, Sheel R, Choudhury AR, Sarkar B, Pattanayak A, Deori S, Abedin SN, Kadirvel G. Synthesis of green zinc-oxide nanoparticles and its dose-dependent beneficial effect on spermatozoa during preservation: sperm functional integrity, fertility and antimicrobial activity. Front Bioeng Biotechnol 2024;12:1326143.
            doi: 10.3389/fbioe.2024.1326143pubmed: 38464542google scholar: lookup
          18. Kalo D, Mendelson P, Komsky-Elbaz A, Voet H, Roth Z. The Effect of Mycotoxins and Their Mixtures on Bovine Spermatozoa Characteristics. Toxins (Basel) 2023 Sep 6;15(9).
            doi: 10.3390/toxins15090556pubmed: 37755982google scholar: lookup
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