References of "Ponthier, Jérôme"
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See detailHigh concentrations of Myeloperoxidase in the equine uterus as an indicator of endometritis
Parrilla Hernandez, Sonia ULg; Ponthier, Jérôme ULg; Franck, Thierry ULg et al

in Theriogenology (in press)

Intra-luminal fluid and excessive abnormal hyper-edema are regularly used for the diagnosis of endometritis in the mare, which is routinely confirmed by the presence of neutrophils on endometrial smears ... [more ▼]

Intra-luminal fluid and excessive abnormal hyper-edema are regularly used for the diagnosis of endometritis in the mare, which is routinely confirmed by the presence of neutrophils on endometrial smears. Studies show a relation between neutrophils and myeloperoxidase (MPO), an enzyme contained in and released by neutrophils during degranulation or after cell lysis. This enzyme has been found in many fluids and tissues and associated with different inflammatory pathologies in the horse. The aims of this study were to assess the presence and concentration of MPO in the equine uterus, and to investigate its relation with neutrophils, and other clinical signs of endometritis. Mares (n=51) were evaluated for presence of intra-luminal fluid and excessive endometrial edema before breeding, and a small volume lavage and cytology samples were obtained. From 69 cycles, supernatant of the uterine flushes was analysed with a specific equine MPO ELISA assay to measure MPO concentration. Cytology samples were used for the diagnosis of endometritis. MPO was present in the uterus of all estrus mares in highly variable concentrations. MPO concentrations were significantly (p<0.05) higher in samples with positive cytologies and in presence of intra-luminal fluid. Occasionally, some samples with negative cytologies showed high MPO concentration, but the opposite was never observed. Cycles presenting hyper-edema weren’t associated to high concentration of MPO, intra-luminal fluid or positive cytology making it a poor diagnostic tool of endometritis. [less ▲]

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See detailConcentration, Activity and Biochemical Characterization of Myeloperoxidase in Fresh and Post-thaw equine semen and their Implication on Freezability
Ponthier, Jérôme ULg; Franck, Thierry ULg; Parrilla Hernandez, Sonia ULg et al

in Reproduction in Domestic Animals (in press)

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 ... [more ▼]

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 3 blot. Purified active MPO was added in PBS 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). Inac- tive 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 inactiva- tion in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor. [less ▲]

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See detailUse of automatic stapling device during castration to prevent (re)occurrence of inguinal hernia in horses with large vaginal rings.
Salciccia, Alexandra ULg; de la Rebière de Pouyade, Geoffroy ULg; Ponthier, Jérôme ULg et al

in Journal of Equine Veterinary Science (2014, January), 34(1), 99-100

(Re)ocurrence of inguinal hernia in case of very large vaginal rings may simply be prevented by closure of the vaginal tunic by TA staples during castration by inguinal approach. This method provides a ... [more ▼]

(Re)ocurrence of inguinal hernia in case of very large vaginal rings may simply be prevented by closure of the vaginal tunic by TA staples during castration by inguinal approach. This method provides a good resistance to internal pressure, appears to be safe, fast and easy to perform and may therefore be an interesting alternative to laparoscopic techniques when castration is considered. [less ▲]

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See detailSperm Motility and Lactate production at different sperm concentrations
Ponthier, Jérôme ULg; De Tullio, Pascal ULg; Blommaert, Didier ULg et al

in Journal of Equine Veterinary Science (2014, January), 34(1), 75-76

Lactate production is associated with total spermatozoa concentration. It negatively affects preservation of total and progressive motility, showing an effect of by-products of anaerobic metabolism on ... [more ▼]

Lactate production is associated with total spermatozoa concentration. It negatively affects preservation of total and progressive motility, showing an effect of by-products of anaerobic metabolism on long-term storage. Moreover, our data show that non-progressive motile spermatozoa are highly associated to lactate concentration, and thus, anaerobic glycolysis. More studies are required to determine relative contributions of aerobiosis and anaerobiosis to spermatozoa motility under different storage conditions. [less ▲]

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See detailEffect of non-sperm cells removal with single layer colloidal centrifugation on myeloperoxidase concentration in post-thaw equine semen
Ponthier, Jérôme ULg; Teague, Sheila; Franck, Thierry ULg et al

in Theriogenology (2013), 80(9), 1082-1087

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 ... [more ▼]

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 presence of non-sperm cells. The aim of this study was to describe the effect of centrifugal fractionation of semen prior to cryopreservation on post-thaw concentrations of non-sperm cells and MPO. The experimental design consisted in 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 non-sperm cells and MPO concentrations in post-thaw semen. The MPO concentration was correlated with concentration of non-sperm cells 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 non-sperm cells. Overall, neutrophils were rarely observed and non-sperm cells were mainly epithelial cells or cellular debris, as demonstrated by MPO immunocytochemistry. Following single layer colloid centrifugation, MPO concentration was decreased, and correlations observed in different samples of the experiments revealed an association between concentrations of MPO and non-sperm cells. At all steps of the semen processing and cryopreservation, MPO immunostaining was clearly identified only on non-sperm cells. Our results demonstrate that non-sperm cells present in fresh semen release MPO during freezing. [less ▲]

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See detailPyovagina caused by pyometra and a persistent hymen in a 9-year-old bitch
Van Den Berghe, Femke ULg; Ponthier, Jérôme ULg; Parrilla Hernandez, Sonia ULg et al

in Reproduction in Domestic Animals (2013, September), 48(suppl1), 114

Pyovagina with a persistent hymen is very uncommon in a 9-year-old intact bitch. Blood discharge during prooestrus phases of previous cycles and the recent purulent vulvar discharge indicate an -at least ... [more ▼]

Pyovagina with a persistent hymen is very uncommon in a 9-year-old intact bitch. Blood discharge during prooestrus phases of previous cycles and the recent purulent vulvar discharge indicate an -at least partially- perforated hymen. For an unknown cause the hymen must have re-sealed after uterine contamination explaining a pyovagina that was bulging in the perineal area [less ▲]

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See detailInfluence of concentration on equine fresh semen conservation
Ponthier, Jérôme ULg; Bloomaert, Didier; Parrilla Hernandez, Sonia ULg et al

in Reproduction in Domestic Animals (2013, September), 48(suppl1), 85

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See detailA case of Longitudinal Vaginal Septum in a mare
Parrilla Hernandez, Sonia ULg; Ponthier, Jérôme ULg; Deleuze, Stefan ULg

in Reproduction in Domestic Animals (2013, September), 48(suppl1), 112-113

LVS occurs when there is failure in the fusion of müllerian ducts or in the regression of the vaginal septum. Primary or secondary cause of infertility, increases the risk of dystocia or alter sport ... [more ▼]

LVS occurs when there is failure in the fusion of müllerian ducts or in the regression of the vaginal septum. Primary or secondary cause of infertility, increases the risk of dystocia or alter sport performance. It is most likely under-diagnosed as it is often asymptomatic and an incidental finding. [less ▲]

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See detailMyeloperoxidase activity decreases in equine semen freezing extenders
Ponthier, Jérôme ULg; Franck, Thierry ULg; Niesten, Ariane ULg et al

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 ▲]

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See detailINFLUENCE OF MYELOPEROXIDASE ACTIVITY ON EQUINE POST-THAW SEMEN QUALITY
Ponthier, Jérôme ULg; Franck, Thierry ULg; Parrilla Hernandez, Sonia ULg et al

in Edeas, Marvin (Ed.) Proceedings of the 2nd ISANH World congress on Fertility and Antioxidants (2012, December 06)

This study confirms that active MPO is associated with cellular fraction of the ejaculate, as previously suggested for total MPO concentration in thawed semen (2). However, active MPO concentrations were ... [more ▼]

This study confirms that active MPO is associated with cellular fraction of the ejaculate, as previously suggested for total MPO concentration in thawed semen (2). However, active MPO concentrations were dramatically lower than total MPO concentrations observed in equine semen (3), which could be explained by presence of inactive MPO subunits in semen. MPO activity in sperm-rich pellet can be used as a predictive marker of post-thaw semen quality. Moreover, methods to inhibit MPO should be investigated in semen. [less ▲]

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See detailExamen andrologique de l'étalon - Badanie andrologiczne ogiera
Ponthier, Jérôme ULg

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 ▲]

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See detailSquamous metaplasia of the prostate and diffuse alopecia in a 13-year-old castrated dog due to chronic ingestion of exogenous estradiol
Van Den Berghe, Femke ULg; Ponthier, Jérôme ULg; Deleuze, Stefan ULg

in Reproduction in Domestic Animals (2012, August), 47(S4), 561

Introduction. A 13-year old whippet with a generalized progressive alopecia since 2 years was referred to our clinic to perform a low-dose dexamethasone stimulation test and abdominal ultrasound to ... [more ▼]

Introduction. A 13-year old whippet with a generalized progressive alopecia since 2 years was referred to our clinic to perform a low-dose dexamethasone stimulation test and abdominal ultrasound to exclude or confirm Cushing disease. The dog had previously been treated with trilostane for 4 months without having any positive effects. Earlier blood analysis showed no significantly abnormalities and ACTH stimulation test was negative. Having bilateral cryptorchidism, the dog was castrated at young age. Clinical findings and treatment. Abdominal ultrasound revealed a slightly enlarged right adrenal gland (diameter of 1 cm at the cranial part). The prostate was enlarged and heterogeneous with multiple anechogenic cavities, indicating hormonal stimulation. Craniad the prostate and ventrally of the bladder, a hypoechogenic, oval structure (3 cm by 1.7 cm) could be seen, and was suspected to be a remaining cryptorchide testicle. Fine needle aspiration however showed that the structure contained a thick purulent liquid, with large amounts of polymorhonuclear white blood cells. Blood analysis revealed a leukocytosis (29370/mm3) with neutrophilia (23202/mm3). Low-dose dexamethasone test definitely excluded Cushing disease (basal cortisol levels 2,17 µg/dl; cortisol at 4 and 8 hours < 1 µg/dl). A prostatic wash was performed and revealed a severe prostatitis and presence of keratinized prostatic cells, indicating a squamous metaplasia of the prostate. Measurement of seric LH levels (LH Witness®, Synbiotics) showed an LH concentration lower than 1 ng/ml, indicating a hormonal negative feedback on the hypothalamo-hypophyso-gonadal axis. After a more thorough anamnesis, it became clear that the dog was licking and ingesting a transdermal estradiol containing cream (Estrogel®) from his owner, since two years, causing his symptoms. The dog was put on antibiotic treatment for the prostatitis (enrofloxacine 10 mg/kg SID) and underwent surgery to excise the abscess cranial of the prostate. The two ductuli deferentes were attached to this structure. Histopathological analysis revealed this tissue to be embryological remanents or a morphological anomaly with a urogenital origin. Control after 4 weeks showed that the prostate slightly decreased in volume, however, anechogenic cavities where still present of which one increased in volume. The alopecia was still present as well, both probably due to a prolonged action of the estrogens. Conclusion. Even though injectable estrogen preparations are not longer available in veterinary medicine in Europe, a thorough anamnesis towards other exogenous estrogen sources is still necessary and can reduce the number of supplementary exams performed. [less ▲]

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See detailA flow cytometric study on the effect of myeloperoxidase on stallion spermatozoal motility and structure
Ponthier, Jérôme ULg; Parrilla Hernandez, Sonia ULg; Van Den Berghe, Femke ULg et al

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 ▲]

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See detailMyeloperoxidase as an indicator of endometritis in the mare: preliminary results
Parrilla Hernandez, Sonia ULg; Franck, Thierry ULg; Ponthier, Jérôme ULg et al

in Reproduction in Domestic Animals (2012, August), 47(suppl 5), 65

Diagnosis of endometritis in the mare is routinely based on the presence of polymorphonuclear cells (PMNs) on endometrial smears. Studies show a relation between PMNs and myeloperoxidase (MPO), an enzyme ... [more ▼]

Diagnosis of endometritis in the mare is routinely based on the presence of polymorphonuclear cells (PMNs) on endometrial smears. Studies show a relation between PMNs and myeloperoxidase (MPO), an enzyme released by PMNs during degranulation or after cell lysis, in many fluids and tissues. The aims of this study were to assess the presence and concentration of MPO in the mare’s uterus, and to investigate its relation with PMNs. Thirty-six cycles from 28 mares (ages ranging from 6 to 22 years) were used. Endometrial cytological samples were obtained with a small volume uterine flush and either a uterine cotton swab or a cytobrush, when a follicle >35 mm was observed by ultrasound. The smears were stained with Diff-QuickÒ and one or more PMNs per field (400·) was diagnosed as endometritis. The supernatant of the flushes was used to measure MPO concentration with a specific equine MPO ELISA assay. Our results showed the presence of MPO in the equine uterus during oestrus (mean = 2839 + 2785). MPO concentrations were signifi- cantly (p < 0.05) higher in samples with positive cytological results. Occasionally, some samples with negative cytological results showed high MPO concentration, but the opposite was never observed. Clinical signs of endometritis are not always present, or they may be delayed. An early diagnostic improves the success of treatment. Our results show that high quantities of MPO in endometrial samples indicate the presence of PMNs. Further studies are needed to determine if MPO concentration could be routinely used as a tool of early detection of endometritis. [less ▲]

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See detailEffect of artificial insemination site on post-mating endometritis in mares
Rodier, Clémentine; Ponthier, Jérôme ULg; Parrilla Hernandez, Sonia ULg et al

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 ▲]

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See detailRaw semen concentration directly influences CASA velocity pathways
Parrilla Hernandez, Sonia ULg; Deleuze, Stefan ULg; Van Den Berghe, Femke ULg et al

in Journal of Equine Veterinary Science (2012, August), 32(8), 505

We observed an influence of the stallion on volume, spermatozoa concentration and all CASA parameters (p<0.0001), which are highly dependant on each other due to geometrical association of these data ... [more ▼]

We observed an influence of the stallion on volume, spermatozoa concentration and all CASA parameters (p<0.0001), which are highly dependant on each other due to geometrical association of these data. That stallion effect may have interfered with the associations we observed as stallions seem to have specific concentration and motility pathways. More studies, with more replicates, will allow comparing results from a same stallion and further establish the correlations we report here. [less ▲]

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See detailOrigine et effet de la myéloperoxydase lors de la congélation du sperme d'étalon
Ponthier, Jérôme ULg

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 ▲]

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See detailMyeloperoxidase in Equine Semen: Concentration and Localization during Freezing Processing
Ponthier, Jérôme ULg; Desvals, Maud; Franck, Thierry ULg et al

in Journal of Equine Veterinary Science (2012), (32), 32-37

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutro- phils during degranulation or after lysis. Post-thaw semen contains MPO, and concen- tration of this enzyme is associated ... [more ▼]

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutro- phils during degranulation or after lysis. Post-thaw semen contains MPO, and concen- tration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozenethawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifuga- tion, and after cooling down to 4 C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4 C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO. [less ▲]

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See detailSperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus).
Gennotte, Vincent ULg; François, Ekniel; Rougeot, Carole ULg et al

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 ▲]

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