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Reproduction in domestic animals = Zuchthygiene2022; 58(1); 10-19; doi: 10.1111/rda.14241

Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm.

Abstract: Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n = 5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n = 5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1 μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.
© 2022 Wiley-VCH GmbH.
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Publication Date: 2022-09-14 PubMed ID: 36059066DOI: 10.1111/rda.14241Google Scholar: Lookup
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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 is an investigation into the relationship between abnormal sperm morphologies and reactive oxygen species (ROS) production in stallion sperm, utilizing an innovative technique called imaging flow cytometry.

Objective and Methodology

  • The primary goal of the study was to discover the association between varieties of sperm morphologies and reactive oxygen species (ROS) production in fresh and chilled stallion semen.
  • The study utilized a novel technique known as Imaging Flow Cytometry, which allows the simultaneous gathering of image and flow cytometry data.
  • The researchers collected semen samples from four stallions located at the University of California, Davis for assessing fresh semen. For 24-hour cool-stored semen study, samples were gathered from stallions at Texas A&M University and shipped overnight for evaluation.
  • Samples were co-stained with SytoxGreen™, a nuclear dye for dead cells and dihydroethidium (DHE), a dye used to detect ROS, and analyzed using the Amnis® ImageStream® system.

Findings and Conclusion

  • Abnormal morphology categories included abnormal head, midpiece, tail, proximal cytoplasmic droplet, distal cytoplasmic droplet, and normal cells.
  • In both fresh and chilled semen, ROS production was significantly higher in viable cells with abnormal head, abnormal midpiece, and proximal cytoplasmic droplet.
  • The coiled tail and midpiece also showed significantly higher levels of ROS production in fresh semen.
  • No significant difference in ROS levels was found between normal cells and abnormal tail and distal cytoplasmic droplet cells in either type of semen.
  • The findings suggest that the production of ROS is indicative of abnormal cell morphology and function in stallion sperm.
  • The research confirms that imaging flow cytometry is a valuable tool for assessing stallion semen.

Cite This Article

APA
Bulkeley E, Santistevan AC, Varner D, Meyers S. (2022). Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm. Reprod Domest Anim, 58(1), 10-19. https://doi.org/10.1111/rda.14241

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 58
Issue: 1
Pages: 10-19

Researcher Affiliations

Bulkeley, Evelyn
  • Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California, USA.
Santistevan, Anthony C
  • Department of Psychology, College of Letters and Sciences, University of California, Davis, California, USA.
Varner, Dickson
  • Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Texas A&M University, Texas, USA.
Meyers, Stuart
  • Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California, USA.

MeSH Terms

  • Humans
  • Male
  • Horses
  • Animals
  • Semen / physiology
  • Reactive Oxygen Species
  • Flow Cytometry / veterinary
  • Sperm Motility / physiology
  • Semen Preservation / veterinary
  • Semen Preservation / methods
  • Spermatozoa / physiology

Grant Funding

  • Center of Equine Health, UC Davis
  • State of California Satellite Wagering Fund
  • T32OD010931-11 / National Research Service Award
  • National Institutes of Health (NIH)
  • UC Davis School of Veterinary Medicine Endowment Funds

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Citations

This article has been cited 5 times.
  1. Aragona F, Rizzo M, Giudice E, Fazio F, Costa A, Di Bella B, De Caro S, Arfuso F, Briglia M, Piccione G, Giannetto C. Circadian Oscillation of Leukocyte Subpopulations and Inflammatory Cytokines over a 24-H Period in Horses. Vet Sci 2025 Apr 20;12(4).
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  2. Janarathanam VA, Issac PK, Pan I, Kamaraj N, Ansar S, Kumar YA, Guru A. Investigating antioxidant effects of hamamelitannin-conjugated zinc oxide nanoparticles on oxidative stress-Induced neurotoxicity in zebrafish larvae model. Mol Biol Rep 2024 Oct 22;51(1):1087.
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  3. Johannisson A, Morrell JM, Ntallaris T. A combination of biomarkers for predicting stallion sperm fertility. Vet Res Commun 2024 Aug;48(4):2157-2169.
    doi: 10.1007/s11259-024-10372-6pubmed: 38652412google scholar: lookup
  4. 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).
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  5. Zhao Y, Yang J, Lu D, Zhu Y, Liao K, Tian Y, Yin R. The Loss-Function of KNL1 Causes Oligospermia and Asthenospermia in Mice by Affecting the Assembly and Separation of the Spindle through Flow Cytometry and Immunofluorescence. Sensors (Basel) 2023 Feb 25;23(5).
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