Effect of non–sperm cells removal with single-layer colloidal centrifugation on myeloperoxidase concentration in post-thaw equine semen

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.

Concentration, Activity and Biochemical Characterization of Myeloperoxidase in Fresh and Post‐Thaw Equine Semen and their Implication on Freezability

“…Total MPO concentrations observed in fresh semen are comparable with previously published values (Ponthier et al. , ), but very high when compared to MPO concentrations observed in other equine biological fluids (Art et al. ; Grulke et al.…”

“…This report confirms that free MPO is present in stallion fresh semen as previously described . Total MPO concentrations observed in fresh semen are comparable with previously published values (Ponthier et al , 2013, but very high when compared to MPO concentrations observed in other In raw semen, spermatozoa concentration, total motility and total MPO concentration were all correlated. Recently, raw semen concentration has been directly correlated with CASA values (Parrilla-Hernandez et al 2012).…”

“…Recent studies (Ponthier et al. ) confirmed that NSC undergoing cellular damage associated to freezing (Graham‐Pole et al. ; Wang et al.…”

“…Freezing of the sperm‐rich pellet for storage led to cellular lysis and MPO release by non‐sperm cells (Ponthier et al. ). MPO released at this step was more active, but a decrease in sperm‐rich pellet MPO activity was observed during freezing procedures.…”

Flow Cytometry Probes to Evaluate Stallion Spermatozoa

“…Consequently, a better understanding of sperm function in relation to clinical andrology and sperm biotechnologies [1,2] has arisen. Relevant advances in stallion sperm biology include, among others, the following: the understanding of osmotic shock [3][4][5] and its implications on cryopreservation, which promoted the development of new protocols based on more permeant cryoprotectants [6][7][8][9][10]; advances in understanding the role of reactive oxygen species (ROS) [11][12][13][14]; and the development of practical methods for stallion sperm separation and selection through colloidal centrifugation [15][16][17][18][19][20][21]. More recently, the understanding of sperm bioenergetics and mitochondrial functionality have become two hot topics in stallion andrology.…”