Related Experiment Video
Updated: May 7, 2026
The Examination of Peroxidase-Positive Leukocytes in Semen
Published on: January 19, 2024
Effect of non-sperm cells removal with single-layer colloidal centrifugation on myeloperoxidase concentration in
Jérôme Ponthier1, Sheila R Teague, Thierry Y Franck
1Equine Clinic, Veterinary Medicine Faculty, ULg, University of Liège, Liège, Belgium.
Abstract:
Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO and its concentration is associated with decreased sperm motility. Recently, MPO concentration in post-thaw semen was shown to be associated with the presence of non-sperm cells (NSC). The objective of this study was to evaluate the effect of a single-layer colloidal centrifugation before cryopreservation on NSC and MPO concentrations in equine semen. The experimental design consisted of freezing semen with or without previous centrifugation through two concentrations of single-layer colloid media. Non-sperm cells and MPO concentrations were assessed in pellet and upper layer at each step of the procedure and MPO was detected in cells by immunocytochemistry. Single-layer colloid centrifugation decreased NSC and MPO concentrations in post-thaw semen. The MPO concentration was correlated with concentration of NSC in the upper layer of the supernatant. In post-thaw semen, with or without previous single-layer colloid centrifugation, MPO concentration was correlated with concentration of NSC. Overall, neutrophils were rarely observed and NSC were mainly epithelial cells or cellular debris, as demonstrated by MPO immunocytochemistry. At all steps of the semen processing and cryopreservation, MPO immunostaining was clearly identified only on NSC. In conclusion, our study shows that NSC present in fresh semen release MPO during freezing.
Insights
Single-layer colloidal centrifugation effectively reduces non-sperm cells (NSC) and myeloperoxidase (MPO) in equine semen before cryopreservation. This method helps maintain semen quality by lowering MPO, an enzyme released by NSC during freezing.
Area of Science:
- Reproductive Biology
- Animal Science
- Biochemistry
Background:
- Myeloperoxidase (MPO) is a pro-oxidant enzyme released by neutrophils.
- Elevated MPO in post-thaw equine semen correlates with reduced sperm motility and the presence of non-sperm cells (NSC).
- NSC, primarily epithelial cells and cellular debris, are implicated in MPO release during cryopreservation.
Purpose of the Study:
- To evaluate the efficacy of single-layer colloidal centrifugation in reducing NSC and MPO concentrations in equine semen prior to cryopreservation.
- To assess the impact of different single-layer colloid concentrations on NSC and MPO levels.
- To investigate the relationship between NSC, MPO, and semen quality during cryopreservation.
Main Methods:
- Equine semen was cryopreserved with or without prior centrifugation through two concentrations of single-layer colloid media.
- Non-sperm cell (NSC) and MPO concentrations were measured at various stages of processing and post-thaw.
- Immunocytochemistry was used to detect MPO within cells, identifying NSC as the primary MPO source.
Main Results:
- Single-layer colloidal centrifugation significantly decreased NSC and MPO concentrations in post-thaw equine semen.
- MPO concentration was positively correlated with NSC concentration in both the supernatant and post-thaw semen.
- Immunocytochemistry confirmed MPO presence predominantly in NSC, not neutrophils, indicating NSC release MPO during freezing.
Conclusions:
- Single-layer colloidal centrifugation is an effective method for reducing NSC and MPO contamination in equine semen before cryopreservation.
- The presence of NSC in fresh semen contributes to MPO release during the freezing process.
- Reducing NSC via centrifugation can mitigate MPO-associated negative effects on equine semen quality.
