Association between Myeloperoxidase Concentration in Equine Frozen Semen and Post-Thawing Parameters

Abstract

Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on the impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent, which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty-five straws from different stallions were analysed. Post-thawing spermatozoal concentration, and progressive and total motility were determined by Computer-Assisted Semen Analysis. Freezability was determined according to post-thawing progressive motility (above or below 15%). Percentage of alive spermatozoa and abnormal forms was determined after Eosin-Nigrosin and Diff-Quick(®) staining, respectively. Post-thawing MPO concentration was measured by enzyme-linked immunosorbent assay (ELISA). Our study shows that frozen thawed semen contains large amounts of free MPO. We also observed that post-thawing MPO ELISA assay can be used as an indicator of equine semen freezability. High MPO concentration samples showed lower total and progressive motility. A higher proportion of abnormal head shape associated with acrosome reaction was observed in our late examinations of the high concentration MPO group. Our results show that MPO adversely affects total and progressive motility of equine semen. A negative correlation between normal motile forms and MPO concentration was also observed. The effect of MPO on dead or abnormal forms remains to be precised.

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

“…The larger seminal plasma proportion (10%) in the final freezing medium used for dilution of the sperm-rich pellet could explain this poor correlation. This proportion of seminal plasma present in frozen semen may also explain the lack of correlation between post-thaw total MPO and motility previously described (Ponthier et al 2010b).…”

“…This proportion of seminal plasma present in frozen semen may also explain the lack of correlation between post‐thaw total MPO and motility previously described (Ponthier et al. ).…”

“…Myeloperoxidase ELISA Before analysis, supernatants of each sample were separately centrifuged again (4°C, 15 min, 15 000 9 g) to discard the heavy proteins and the cellular fragments. MPO concentrations in the raw semen supernatant (ELISA-SN), in the supernatant of washed sperm-rich pellet (ELISA-PEL) and in the supernatant of post-thaw semen (ELISA-PT) were determined by a commercial ELISA (Eq/ERK/01; Bioptis, SA, Li ege, Belgium) as previously described (Franck et al 2005;Ponthier et al 2010aPonthier et al , 2010bPonthier et al , 2012. Each sample was assayed twice and the mean value was used.…”

“…), MPO is naturally abundant in equine semen and its concentration is even higher in poor post‐thaw quality semen (Ponthier et al. , , ). The relation between total MPO concentration in equine semen and its activity assayed by specific immmunological extraction followed by enzymatic detection (SIEFED) (Franck et al.…”

“…Since its purification (Mathy-Hartert et al 1998), an enzyme-linked immunosorbent assay (ELISA) has been developed with polyclonal antibodies detecting all MPO subunits (Franck et al 2005). While small MPO amounts have been identified in other biological fluids (Art et al 2006;Grulke et al 2008), MPO is naturally abundant in equine semen and its concentration is even higher in poor post-thaw quality semen (Ponthier et al 2010a(Ponthier et al , 2010b. The relation between total MPO concentration in equine semen and its activity assayed by specific immmunological extraction followed by enzymatic detection (SIEFED) (Franck et al 2006) has not been investigated yet.…”