Myeloperoxidase activity decreases in equine semen freezing extenders

in Amir, Arav (Ed.) Proceedings of the 3rd Cryo Congress (2013, March 23)

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils, and associated with decreased post-thaw motility of equine semen. This study aimed to compare MPO activity in pure ... [more ▼]

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils, and associated with decreased post-thaw motility of equine semen. This study aimed to compare MPO activity in pure equine freezing extender, raw and post-thaw semen. Active MPO Concentration (AMC) was measured with Specific Immunologic Extraction Followed by Enzymatic Detection in 20 ejaculates. Raw semen intra cellular AMC was determined in the supernatant after membrane lysis, each pellet containing 100x106 spermatozoa. AMC was also assayed in supernatants of semen frozen following a conventional method using INRA FreezeTM (IMV, France). Effect of freezing procedure on AMC was tested by experimentally adding 500ng of purified active MPO (Calbiochem, Germany) in 4 samples either with 5ml of PBS or INRA FreezeTM before assay. AMC was higher in sperm-rich pellet (0.306ng/ml) than in post-thaw semen (0.002ng/ml) (p=0.058). After experimental MPO addition, no activity variation was observed during the freezing procedure (after dilution, 1, 2 hours of cooling and post-thawing) within the same medium. Purified MPO activity was decreased in INRA FreezeTM when compared to PBS at all timings of sampling (p=0.0286). When all samples were pooled, remaining activity in INRA FreezeTM was 23.93±13.13%. MPO fixation on large proteins contained in the extender experimentally reduces AMC, as previously observed in plasma. However, AMC decrease observed during semen freezing is more important than after experimental addition. That could be explained by a MPO interaction with seminal plasma, a partial MPO release or a MPO inactivation during equine semen freezing. [less ▲]

A flow cytometric study on the effect of myeloperoxidase on stallion spermatozoal motility and structure

in Journal of Equine Veterinary Science (2012, August), 32(8), 509

Myeloperoxidase (MPO) is a pro-oxidant enzyme that is normally contained in neutrophils. MPO has recently been associated with keratinized cells and with decreased post-thaw motility in stallion semen [1 ... [more ▼]

Myeloperoxidase (MPO) is a pro-oxidant enzyme that is normally contained in neutrophils. MPO has recently been associated with keratinized cells and with decreased post-thaw motility in stallion semen [1]. The aim of the study was to determine effects of experimental addition of active MPO on motility, mitochondrial potential, apoptosis induction, membrane and acrosome integrity in equine semen. Three stallions were used and semen was collected four times. Extended (INRA96TM) semen was processed for density gradient centrifugation (Equipure Bottom Layer®) [2]. Purified pellet was re-extended to 100x106spermatozoa/ml in INRA96TM and divided in 3 samples. One sample was used for control and active human MPO (Calbiochem, Merck) was added in the other two samples to final concentration of 5 or 50 ng/ml. After incubation (2 hours, 20°C), motility was analysed with Computer Assisted Semen Analysis (IVOS, Hamilton-Throne, Beverly, MA, USA) and cytometric analyzes were perfomed with EasyCyte (IMV). Mitochondrial potential and apoptosis were assayed using Guava Mitopotential JC-1 and 7-AAD kit (Millipore). Membrane and acrosome integrity were respectively assayed with PI (Propidium Iodide) (Invitrogen) and PNA (Peanut Agglutinin-Fluorescein Iso Thio Cyanate) (Sigma-Aldrich). Statistical differences (p<0.05) were determined using Kruskall-Wallis test. No effect of the stallions was observed on parameters assayed in this study. Unlike total motility, progressive motility was decreased in both MPO concentrations (p<0.001). MPO addition had no effect on membrane and acrosome integrity. No differences were detected for the percentages of spermatozoa having polarised or depolarised mitochondria. Apoptosis, assayed by 7-AAD fluorescence, was not increased by the treatments. Our results agree with previously published effects of in vitro ROS production systems with xanthine oxidase [3], showing an effect on motility but no influence on mitochondria and membrane or acrosome integrity. However, membrane lipoperoxidation was increased by ROS in this study [3], and it could be linked to the impaired motility also observed in our protocol. Further studies with increasing concentrations of added MPO should be conducted to correlate motility with lipoperoxidation. References [1] Ponthier J, Desvals M, Franck T, de la Rebiere de Pouyade G, Spalart M, Palmer E, Serteyn D, Deleuze S. Myeloperoxidase in equine semen: Concentration and Localization during freezing processing. Journal of Equine Veterinary Science 2012;32: 32-37. [2] Edmond AJ, Teague SR, Brinsko SP, Comerford KL, Waite JA, Mancill SS, Love CC, Varner DD. Effect of density-gradient centrifugation on quality and recovery rate of equine spermatozoa. Animal Reproduction Science 2008;107: 318-318. [3] Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl 2000;21: 895-902. [less ▲]

Effect of artificial insemination site on post-mating endometritis in mares

in Reproduction in Domestic Animals (2012, August), 47(suppl 5), 103

Aim of the study was to determine the effect of artificial insemination (AI) location on post-mating endometrial inflammation 1 and 6 days after AI. Six mares with ages ranging from 12 to 23 years were ... [more ▼]

Aim of the study was to determine the effect of artificial insemination (AI) location on post-mating endometrial inflammation 1 and 6 days after AI. Six mares with ages ranging from 12 to 23 years were inseminated with a same batch of frozen semen for 3 consecutive cycles. Mares were inseminated with the following procedures in a random order: (1) Deep uterine insemination with 4ml of semen; (2) Horn bifurcation with 4ml of semen; (3) Horn bifurcation with 4ml of semen and 6ml of extender. During each cycle, Cotton (C) and Brush (B) swabs were collected at 4 different moments: mid- dioestrus, mares with a 35mm follicle, 24h and 6 days after AI. Swabs were smeared on slides, fixed and stained (Diff-Quick®) before examination under light microscopy. Proportions of inflammatory and epithelial cells were determined and differences were studied with kruskal-wallis test for non-parametric data. Distensions of uterine lumen due to intraluminal fluid observed during ultrasound exams were measured and recorded. Quality of slides was better (p=0.0006) with B swabs than C swabs with 97% versus 65% of slides readable. B swabs were associated with higher percent of endometrial cells retrieved (p=0.0323), making them a better tool for endometritis diagnosis, which is consistent with our previous reports. Volume of intraluminal fluid and percent of inflammatory cells, both on B and C swabs, were not influenced by AI location regardless the timing of sampling. Small volume deep uterine AI did not significantly affect inflammatory response by the endometrium in our experiment. [less ▲]