Analytical validation of five diagnostic tests for the detection of polymorphonuclear cells in stallion semen.
Abstract: The objectives of this study were to evaluate the ability of five diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and even low concentrations of PMNs affect motility. One ejaculate was obtained from six stallions. Aliquots of 50 × 10 purified sperm were incubated, in triplicate, with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 × 10 PMN/ml, 3) 0.5 × 10 PMN/ml, 4) 2.5 × 10 PMN/ml, 5) 5 × 10 PMN/ml, 6) 10 × 10 PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD13 immunolabeling. Sperm motility was evaluated after 4 h at 38 °C. The number of leucocytes detected with the LEDT differed among treatments (P<0.0001), from negative results in control samples to moderate or large numbers in the samples with the highest PMN concentration. The hemacytometer count and CD13 immunostaining detected differences with the control treatment at the lowest PMN concentration (2.5 × 10 PMN/ml; P<0.001). Sperm motion was lower in samples with ≥5 × 10 PMN/ml (P<0.0001). Thus, a sample was considered leucospermic if it contained ≥5 × 10 PMN/ml. The LEDT had the best sensitivity (100%), followed by cytology (78%), peroxidase test (60%), CD13 immunostaining (56%) and hemacytometer count (47%). The LEDT had the lowest specificity (65%), which was 95% for all other tests. In conclusion, the LEDT was a simple, economic and sensitive stall-side test to screen semen for presence of PMNs. Because of the lower specificity, positive LEDT results should be confirmed with the identification of peroxidase-positive cells or CD13-positive cells.
Copyright © 2022. Published by Elsevier Inc.
Publication Date: 2022-12-02 PubMed ID: 36464032DOI: 10.1016/j.jevs.2022.104185Google Scholar: Lookup
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Summary
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This research article investigates the efficacy of five diagnostic tests in detecting polymorphonuclear cells (PMNs) in stallion semen and how concentrations of these cells impact sperm motility. The study found that all five tests have diagnostic value, and even low concentrations of PMNs can affect sperm motility; however, the Leukocyte Esterase Dipstick Test (LEDT) was found to be the most sensitive of them all.
Objective and Hypothesis
- This study aimed to assess the capability of five diagnostic tests to detect polymorphonuclear cells, a type of white blood cell, in stallion semen.
- The researchers also set out to establish the concentration of PMNs that impacts sperm motility.
- It was hypothesized that all tests would have diagnostic value, and even presence of low levels of PMNs would affect motility.
Methodology
- An ejaculate sample was obtained from each of six stallions.
- Chunks of purified sperm were incubated with six varying concentrations of purified PMNs.
- Quantification of PMNs was done using five different methods: a hemacytometer, cytology, a leukocyte esterase dipstick test (LEDT), a peroxidase test, and CD13 immunolabeling.
- Sperm motility was evaluated after incubation of 4 hours at 38 °C.
Findings
- The results varied among the tests with the LEDT showing different outcomes from the controls to the samples with the highest PMN concentration.
- Differences with the control treatment at the lowest level of PMN were detected through the hemacytometer count and CD13 immunostaining approach.
- Samples with more than 5 x 10 PMN/ml showed reduced sperm movement.
- A sample was considered to contain leukocytes if it contained more than 5 x 10 PMN/ml.
- Sensitivity (or the ability to correctly identify positives) was highest with the LEDT (100%), followed by cytology (78%), peroxidase test (60%), CD13 immunostaining (56%) and hemacytometer count (47%).
- Whereas the LEDT had the lowest specificity (correctly identifying negatives) at 65%, for all other tests it was 95%.
Conclusion
- The LEDT was found to be a simple, economic, and sensitive on-site test to screen stallion semen for the presence of PMNs.
- However, due to its lower specificity, positive LEDT results should be cross-verified with the identification of peroxidase-positive cells or CD13-positive cells.
Cite This Article
APA
Ferrer MS, Hurley DJ, Norton N, Ellerbrock RE.
(2022).
Analytical validation of five diagnostic tests for the detection of polymorphonuclear cells in stallion semen.
J Equine Vet Sci, 122, 104185.
https://doi.org/10.1016/j.jevs.2022.104185 Publication
Researcher Affiliations
- Department of Large Animal Medicine. Electronic address: msferrer@uga.edu.
- Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30605.
- Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30605.
- Department of Large Animal Medicine.
MeSH Terms
- Male
- Horses
- Animals
- Semen
- Sperm Motility
- Spermatozoa
- Diagnostic Tests, Routine
Conflict of Interest Statement
Declaration of Interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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