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

Abstract

Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extraction followed by enzymatic detection. MPO subunits present in semen were characterized by Western blot. Purified active MPO was added in saline solution and freezing extender to control its activity during freezing procedure. Differences between medians were determined using Kruskal-Wallis test, and correlations were determined using Spearman's test for nonparametric data. Active MPO concentration was low in seminal plasma and thawed semen, but high in pellet (p = 0.0058), as the opposite relation was observed for total MPO concentration (p < 0.0001). In seminal plasma and post-thaw semen, inactive 86-kDa MPO precursor was mainly observed. Purified MPO activity was decreased in the extender (p = 0.0286). MPO activity in pellet was highly correlated with thawed progressive motility (r = -0.5576, p = 0.0086). Inactive MPO precursor and unknown low molecular weight inactive MPO precursor subunits explain low MPO activity in semen. Major MPO activity was observed in pellet, and post-thaw loss of activity is partially explained by MPO inactivation in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor.

Characterization of Myeloperoxidase in the Healthy Equine Endometrium

“…In addition, some molecules seem to be able to inhibit plasma MPO by different mechanisms and at different degrees [74][75][76][77] but, to our knowledge, they have not been studied in uterine fluid yet. Furthermore, in equine sperm supernatant, where high concentrations of total MPO are also observed, Ponthier et al [20] showed an important presence of MPO precursor. Unlike in the human [2], the enzyme precursor is inactive in the equine [38] and is then cleaved into the active subunit of the enzyme.…”

“…In horses, MPO has been identified, both under active and inactive form, in various fluids and tissues [18][19][20][21] and has been shown to be involved in numerous inflammatory processes [18,19,[22][23][24]. Some studies have focused on MPO and its implications in pathological conditions of the endometrium, such as endometritis and endometrosis [24][25][26][27], which are a major cause of infertility in the mare that adversely impact the horse breeding industry.…”

Presence of melatonin‐catabolizing non‐specific enzymes myeloperoxidase and indoleamine 2,3‐dioxygenase in the ram reproductive tract

“…As these lightweight MPO bands were previously detected by Ponthier et al (2014) in horse semen, we decided to determine whether MPO was also present in the epididymal and ejaculated ram spermatozoa. IFI analyses revealed that MPO is located in the postacrosomal region of all spermatozoa, with some of them showing a bright band in the equatorial region.…”

Functional Assessment of Diluent Choice for Semen Cryopreservation from Stallions with High and Low Freezability

“…The approval of artificial insemination usage in horse breeding programs by most breed associations intensified research on this biotechnology, particularly due to its potential for semen storage from valuable stallions for unlimited periods of time. However, fertility rates using horse frozen-thawed semen is consistently lower than in other species [4,30], thus suggesting that horse semen is particularly sensitive to cryoinjury [19,33].…”

“…Semen cryotolerance in horses depends upon genetic factors and cryopreservation protocol [2,38], such as diluent and cryoprotectant choice [2,28]. For these reasons, the effect of individual stallions has been extensively investigated throughout the years, aiming to identify factors associated to semen cryotolerance in horses, especially for stallions classified as holding low semen freezability potential [12,15,18,32,33,39].…”