Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA.
Abstract: To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa - including sex sorting - as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry-based assays. After sorting, oxidative stress increased from 26% to 33% in pre- and post-incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post-sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.
© 2015 Blackwell Verlag GmbH.
Publication Date: 2015-11-22 PubMed ID: 26592367DOI: 10.1111/rda.12640Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research focuses on studying whether the commonly used method of spermatozoa selection for chromosomal sex using flow cytometry induces oxidative stress leading to damage in mitochondria and genomic DNA in equine species.
Methodology
- The primary technique used for selecting the chromosomal sex of spermatozoa is the Beltsville sorting technology which deploys flow cytometry.
- This technology was used for inducing possible changes on sperm intactness, particularly focusing on whether it leads to the production of reactive oxygen species (ROS), known for causing oxidative stress.
- The researchers used specific assays based on flow cytometry to identify whether the ROS production resulted in DNA damage in the sorted spermatozoa.
Key Findings
- Post sorting, there was an observed increase in oxidative stress from 26% to 33% in pre- and post-incubation controls, with a further increase to 46% following sex sorting, indicating an induction of oxidative stress by the sorting procedure employed.
- The DNA fragmentation index post sorting was approximately 10% higher, demonstrating potential DNA damage caused by the increased oxidative stress.
- The researchers propose that this increase in oxidative stress could likely be due to the unphysiological extension of sperm, caused by the removal of significant amounts of seminal plasma, further impacted by high pressure on the mitochondria during the sorting procedure.
Conclusion
- The study suggests that the flow cytometric sorting procedure can increase the production of reactive oxygen species (ROS) leading to oxidative stress and potential DNA damage in sorted spermatozoa.
- This raises significant concerns about the use of this method for chromosomal sex selection in spermatozoa and calls for further research to explore safer and less damaging techniques.
Cite This Article
APA
Balao da Silva CM, Ortega-Ferrusola C, Morrell JM, Rodriguez Martínez H, Peña FJ.
(2015).
Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA.
Reprod Domest Anim, 51(1), 18-25.
https://doi.org/10.1111/rda.12640 Publication
Researcher Affiliations
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Caceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Caceres, Spain.
- Division of Reproduction, Faculty of Veterinary Medicine and Animal Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
- Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Caceres, Spain.
MeSH Terms
- Animals
- Cell Separation / methods
- Cell Separation / veterinary
- DNA Damage
- DNA Fragmentation
- DNA, Mitochondrial / genetics
- Flow Cytometry / veterinary
- Horses / genetics
- Male
- Membrane Potential, Mitochondrial
- Oxidative Stress / genetics
- Semen / physiology
- Sex Preselection / methods
- Sex Preselection / veterinary
- Sperm Motility
- Spermatozoa / physiology
Citations
This article has been cited 12 times.- Orsolini MF, Meyers SA, Dini P. An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II. Animals (Basel) 2021 Nov 20;11(11).
- Reese S, Pirez MC, Steele H, Kölle S. The reproductive success of bovine sperm after sex-sorting: a meta-analysis. Sci Rep 2021 Aug 30;11(1):17366.
- Pintus E, Ros-Santaella JL. Impact of Oxidative Stress on Male Reproduction in Domestic and Wild Animals. Antioxidants (Basel) 2021 Jul 20;10(7).
- Gualtieri R, Kalthur G, Barbato V, Longobardi S, Di Rella F, Adiga SK, Talevi R. Sperm Oxidative Stress during In Vitro Manipulation and Its Effects on Sperm Function and Embryo Development. Antioxidants (Basel) 2021 Jun 25;10(7).
- Neculai-Valeanu AS, Ariton AM. Game-Changing Approaches in Sperm Sex-Sorting: Microfluidics and Nanotechnology. Animals (Basel) 2021 Apr 20;11(4).
- Kumaresan A, Das Gupta M, Datta TK, Morrell JM. Sperm DNA Integrity and Male Fertility in Farm Animals: A Review. Front Vet Sci 2020;7:321.
- Steele H, Makri D, Maalouf WE, Reese S, Kölle S. Bovine Sperm Sexing Alters Sperm Morphokinetics and Subsequent Early Embryonic Development. Sci Rep 2020 Apr 10;10(1):6255.
- Peña FJ, O'Flaherty C, Ortiz Rodríguez JM, Martín Cano FE, Gaitskell-Phillips GL, Gil MC, Ortega Ferrusola C. Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa. Antioxidants (Basel) 2019 Nov 19;8(11).
- Li CY, Hao HS, Zhao YH, Zhang PP, Wang HY, Pang YW, Du WH, Zhao SJ, Liu Y, Huang JM, Wang JJ, Ruan WM, Hao T, Reiter RJ, Zhu HB, Zhao XM. Melatonin Improves the Fertilization Capacity of Sex-Sorted Bull Sperm by Inhibiting Apoptosis and Increasing Fertilization Capacitation via MT1. Int J Mol Sci 2019 Aug 12;20(16).
- 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.
- Kalo D, Yaacobi-Artzi S, Manovich S, Michaelov A, Komsky-Elbaz A, Roth Z. Environmental Stress-Induced Alterations in Embryo Developmental Morphokinetics. J Xenobiot 2024 Oct 21;14(4):1613-1637.
- Ngcobo JN, Nedambale TL, Sithole SM, Mtileni B, Mpofu TJ, Ramukhithi FV, Chokoe TC, Nephawe KA. A systematic review on the prospects of X- and Y-sexed semen in ruminant livestock: implications for conservation, a South African perspective. Front Vet Sci 2024;11:1384768.
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