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 ▲]

Examen andrologique de l'étalon - Badanie andrologiczne ogiera

in Gajewski, Zdzislaw (Ed.) Rosrod Koni II - wybrane problemy (2012, November 24)

QualityThe andrological examination is the main procedure to predict the breeding performance. It includes clinical examination as well as additionnal methods. The general health status, libido and the ... [more ▼]

QualityThe andrological examination is the main procedure to predict the breeding performance. It includes clinical examination as well as additionnal methods. The general health status, libido and the reproductive tract status has to be precisely evaluated. However, the final assessment has to be established trough the results of these analysis. [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 ▲]

Origine et effet de la myéloperoxydase lors de la congélation du sperme d'étalon

Doctoral thesis (2012)

Semen freezing allows worldwide commercialization of equine genetic. Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Currently, post-thaw semen quality is only determined ... [more ▼]

Semen freezing allows worldwide commercialization of equine genetic. Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Currently, post-thaw semen quality is only determined by progressive motility, but its definition is not standardised. Spermatozoa are highly differentiated cells and freezing lesions can occur on DNA, membrane, mitochondria or acrosome. Current research focuses on prediction of freezability, improvement of freezing extenders and prevention of Reactive Oxygen Species (ROS) effects. Under its O2 form, oxygen is inactive and oxidase or oxygenase enzymes are required to produce ROS. Two pathways of ROS production in semen are described: the intrinsic pathway reflecting ROS escaping from the spermatozoon mitochondria and the extrinsic pathway corresponding to ROS produced by inflammatory cells. ROS induce DNA fragmentation, lipid peroxidation, apoptosis and decreased motility of spermatozoa. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. It is responsible for formation of hypochlorous acid, a strong oxidant, which could damage spermatozoa. However, MPO presence and effects have never been investigated in equine semen. The first aim was to assay presence of MPO in equine thawed semen and to determine a relation between MPO concentration and post-thaw semen parameters. 35 straws from different stallions were analyzed. Post-thaw spermatozoa and MPO concentrations, viability, morphology, progressive and total motility were determined. Our study showed that thawed semen contains large amounts of free MPO. High MPO concentration samples showed lower total and progressive motility. Higher proportion of acrosome reaction was observed in late examinations of the high MPO concentration group. As MPO was present in frozen semen and did interfere with its quality, timing and origin of its release was determined during the freezing process. Forty ejaculates were frozen with a classical procedure. MPO ELISA and MPO immunocytochemistries (ICC) were assayed in raw semen, centrifugation supernatant, and after cooling down to 4°C. Post-thaw MPO concentration and spermogram parameters were determined. MPO concentration increased after cooling and thawing when compared to fresh semen. As temperature decreased, MPO was higher in post-thaw poor quality samples. Non-sperm cells (NSC) showed various degrees of MPO-ICC, and were mostly epithelial cells with nuclear picnosis. Elastase, another neutrophil pro-inflammatory enzyme, was also assayed in post-thaw semen. In twenty ejaculates, NSC concentration was determined in fresh semen. Post-thaw motilities were determined by CASA; MPO and elastase concentrations were assayed by ELISA. Post-thaw elastase concentrations were low and there was no difference according to semen quality. NSC or MPO concentrations were not correlated to elastase concentration. NSC concentration was higher in unfreezable semen and correlated to post-thaw MPO concentration. To confirm MPO release by NSC during freezing, the effect of washing semen with density gradient centrifugation (DGC) was then assayed on NSC and MPO concentrations. NSC and MPO concentrations were assessed at each step and MPO localization was performed by ICC. DGC washing decreased NSC and MPO concentrations in post-thaw semen and NSC were mainly remaining in DGC supernatant. MPO concentration was correlated with NSC concentration in the upper layer of the DGC supernatant and in post-thaw semen. NSC were epithelial cells showing MPO-ICC staining. Fresh semen MPO concentration had no effect on fresh or post-thaw semen quality, while post-thaw semen concentrations were correlated with decreased motility. To understand these findings, concentration, activity and structure of MPO present in seminal plasma, sperm-rich pellet and post-thaw semen were assayed. Factor inducing MPO release was determined by adding or not glycerol in samples stored at 4°C or 20°C. Total MPO was high in seminal plasma and thawed semen and low in sperm-rich pellet. Active MPO was high in sperm-rich pellet and low in seminal plasma and post-thaw semen. MPO concentrations were correlated in post-thaw and in semen cooled at 4°C with or without glycerol. Active MPO in sperm-rich pellet and post-thaw progressive motility were highly negatively correlated. MPO present in fresh semen is mainly the native inactive enzyme subunit. To confirm our previous findings, effect of active MPO and fresh or frozen-thawed NSC was assayed on purified spermatozoa motility, mitochondrial potential, membrane and acrosome integrity. Highest MPO concentration tested (50ng/ml) decreased motility. However, highest MPO concentration did not affect mitochondrial potential, membrane or acrosome integrity. Thawed NSC did decrease motility and mitochondrial potential when compared to fresh NSC, suggesting a synergic effect between MPO and other products released by NSC after thawing. Temperature decrease during freezing process increases MPO concentration and post-thaw concentration is negatively associated to post-thaw motilities. ICC slides have shown MPO presence on epithelial keratinized and pycnotic cells while neutrophils were rarely observed. Semen washing with DGC decreases MPO and NSC concentrations in post-thaw semen as NSC and MPO concentrations were positively correlated. MPO present in seminal plasma is native and inactive form while MPO present in sperm-rich pellet is active and negatively correlated to the post-thaw progressive motility. Addition of active MPO in semen decreased motility but had no effect on acrosome integrity, despite what had previously been suggested. Thawed NSC addition to spermatozoa decreased mitochondrial potential, suggesting a synergic effect between MPO and other factors released by NSC. Further studies should investigate the origin of high inactive MPO concentrations in fresh semen. Other studies should be conduced about the origin of epithelial keratinized pyknotic NSC in fresh semen and the pathophysiological mechanism leading to their MPO release during freezing. Large scale studies should be conducted to confirm the use of NSC concentration in fresh semen or active MPO concentration in sperm rich pellet as freezability prognosis. Further studies should also investigate effect of MPO specific inhibitors. [less ▲]

Sperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus).

in Theriogenology (2012)

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offsprings), but they also constitute major tools to ... [more ▼]

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offsprings), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology such as growth, behaviour and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm trait measured differed significantly between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males respectively ranged from 0.92 to 1.33 %, from 1.69 to 2.22 × 10(9) cells mL-1 and from 18’04’’ to 27’32’’. Mean values of total motility and curvilinear velocity 1 min after sperm activation respectively ranged from 53 to 58 % and from 71 to 76 µm s-1 for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13 % of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype. [less ▲]

EQUINE SEMEN FREEZING: STATE OF THE ART AND PERSPECTIVES.

Conference (2011, November 19)

Depuis vingt ans, la congélation du sperme a permis de diffuser la génétique des étalons. Les congélations effectuées en dehors des saisons sportives permettent d’inséminer de nombreuses juments pendant ... [more ▼]

Depuis vingt ans, la congélation du sperme a permis de diffuser la génétique des étalons. Les congélations effectuées en dehors des saisons sportives permettent d’inséminer de nombreuses juments pendant que les étalons concourent dans des lieux éloignés. Les éleveurs ont en permanence accès à des doses d’insémination sans risques sanitaires. Cependant, malgré une bonne qualité de sperme frais, 20% des éjaculats équins ne supportent pas la congélation (Vidament, 2005). Le but de cette présentation est de revoir la physiologie du spermatozoïde et d’en discuter les implications dans le contexte de la congélation. Les améliorations des techniques de congélation présentes ou à venir seront aussi évoquées. [less ▲]

Effect of Deferoxamine Mesylate on Freezability of Blood Supplemented Canine Semen

Poster (2011, September)

Association between Myeloperoxidase concentration in equine frozen semen and post thawing parameters

in Reproduction in Domestic Animals (2010), 45(5), 811-816

Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase ... [more ▼]

Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on 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 analyzed. Post-thawing spermatozoal concentration, progressive and total motility were determined by CASA. Freezability was determined according to post-thawing progressive motility (over or under 15%). Percent of alive spermatozoa and abnormal forms were determined after Eosin-Nigrosin and Diff-Quick® staining respectively. Post-thawing MPO concentration was measured by 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. [less ▲]

Is neutrophil elastase associated with myeloperoxidase concentration and post-thawing parameters in equine frozen semen?

in Animal Reproduction Science (2010), 121S

Non sperm cells concentration is higher in unfreezable semen. A relation between non-sperm cells in fresh semen and MPO and post thawing quality has been observed. Neutrophil Elastase seems to have no ... [more ▼]

Non sperm cells concentration is higher in unfreezable semen. A relation between non-sperm cells in fresh semen and MPO and post thawing quality has been observed. Neutrophil Elastase seems to have no effect on semen characteristics and to be not associated with on-sperm cells and freezability. [less ▲]