Flow cytometric detection and identification of different leukocyte subpopulations in stallion semen.
Abstract: To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups, 27.85 ± 5.7% of events were positive for CD4, CD8, CD21 or CD172a, while in native semen samples, leukocytes were scarce (0.114 ± 0.134%). The most abundant leukocyte subpopulation in semen was of lymphoid origin (CD4-positive cells [0.015 ± 0.02%]), whereas CD21-positive cells (B cells; 0.001 ± 0.001%) were virtually absent in ejaculates of fertile stallions. This presented flow cytometric assay for the detection and identification of different leukocyte population in equine antioxidant-treated ejaculates can be used as an additional tool for spermatological examination in stallions.
© 2023 The Authors. Reproduction in Domestic Animals published by Wiley-VCH GmbH.
Publication Date: 2023-08-16 PubMed ID: 37587653DOI: 10.1111/rda.14451Google Scholar: Lookup
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- Journal Article
Summary
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The research paper is about the development and implementation of an antibody-based flow cytometric assay to accurately identify and detect different types of leukocyte subpopulations in stallion semen for a more comprehensive evaluation of stallion ejaculates.
Objective of the Study
- The primary goal was to enhance the precision in analyzing stallion ejaculates by establishing a flow cytometric assay. This sophisticated investigation is colour and antibody-based and aids in the detection and identification of various leukocyte subgroups in stallion semen (such as CD4-, CD8-, CD21-, CD172a-positive cells).
Research Methodology
- The assay was established by supplementing native semen with blood leukocytes (20% leukocytes, 80% sperm cells) and then examining it using flow cytometric analysis.
- In order to prevent the rapid demise of lymphoid cells in the collected mixture of sperm and leukocytes, antioxidants (ascorbic acid and butylated hydroxytoluol) were added instantaneously post-collection.
Results and Findings
- The control setups showed that approximately 28% of events tested positive for CD4, CD8, CD21, or CD172a leukocyte subpopulations.
- However, in the native semen samples, leukocytes were rare, constituting just over 0.1%.
- The most common leukocyte subpopulation in semen was of lymphoid origin (specifically the CD4-positive cells), making up around 0.015%, whereas CD21-positive cells (B cells) were virtually absent in the ejaculates of fertile stallions, accounting for around 0.001%.
Conclusion
- The results demonstrated that a flow cytometric assay can be employed to assess the different leukocyte subpopulations present in equine antioxidant-treated ejaculates.
- This methodology provides a new tool for spermatological evaluation in stallions, offering enhanced precision in detecting and analyzing leukocytes in stallion semen.
Cite This Article
APA
Köhne M, Kirch F, Tönissen A, Martinsson G, Rabe U, Sieme H, Schuberth HJ.
(2023).
Flow cytometric detection and identification of different leukocyte subpopulations in stallion semen.
Reprod Domest Anim.
https://doi.org/10.1111/rda.14451 Publication
Researcher Affiliations
- Clinic for Horses - Unit for Reproductive Medicine, University of Veterinary Medicine, Foundation, Hanover, Germany.
- Lower Saxony State Stud, Celle, Germany.
- Clinic for Horses - Unit for Reproductive Medicine, University of Veterinary Medicine, Foundation, Hanover, Germany.
- Lower Saxony State Stud, Celle, Germany.
- Institute of Immunology, University of Veterinary Medicine, Foundation, Hanover, Germany.
- Clinic for Horses - Unit for Reproductive Medicine, University of Veterinary Medicine, Foundation, Hanover, Germany.
- Institute of Immunology, University of Veterinary Medicine, Foundation, Hanover, Germany.
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