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See detailDescriptive study of 32 cases of doxycycline-overdosed calves.
Brihoum, Mounir ULg; Amory, Hélène ULg; Desmecht, Daniel ULg et al

in Journal of Veterinary Internal Medicine (2010), 24(5), 1203-10

BACKGROUND: Reports of doxycycline-induced toxicity are limited despite common use of this antibiotic to treat infectious respiratory disorders in calves. OBJECTIVE: To describe previously unreported ... [more ▼]

BACKGROUND: Reports of doxycycline-induced toxicity are limited despite common use of this antibiotic to treat infectious respiratory disorders in calves. OBJECTIVE: To describe previously unreported kidney lesions and diagnostic test results in doxycycline-overdosed calves and to compare these results with other findings reported previously. ANIMALS: Thirty-two calves that presented with adverse effects after receiving high doses of doxycycline as a treatment for mild respiratory disorders. METHOD: Retrospective review of medical records. RESULTS: Clinical examination identified mainly lethargy, dyspnea, cough, tongue paresia or paralysis associated with dysphagia and sialorrhea, tachycardia, tachypnea, and signs of myopathy. Blood analysis indicated increases in creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and sorbitol dehydrogenase activities and increased serum creatinine and urea concentrations. ECG recordings and Doppler echocardiography examination identified ventricular premature beats and a decrease in left ventricular global and systolic function, respectively. Necropsy and histopathology disclosed necrosis of the myocardium, tongue, and some striated muscles, acute renal tubular necrosis, and fatty degeneration or congestion of the liver. CONCLUSIONS: Most of these findings corroborate previous observations made in doxycycline-overdosed calves, and further suggest myocardial and striated muscular toxicity as well as renal toxicity in doxycycline-overdosed calves. [less ▲]

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See detailIs neutrophil elastase associated with myeloperoxidase concentration and post-thawing parameters in equine frozen semen?
Ponthier, Jérôme ULg; de la Rebière de Pouyade, Geoffroy ULg; Desvals, Maud et al

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 ▲]

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See detailCysteamine supplementation of in vitro maturation media: a review.
Deleuze, Stefan ULg; Goudet, G.

in Reproduction in Domestic Animals (2010), 45(6), 476-82

Under in vitro culture conditions, oxidative modifications of cell components via increased reactive oxygen species (ROS) represent a major culture induced stress. Anti-oxidant systems such as glutathione ... [more ▼]

Under in vitro culture conditions, oxidative modifications of cell components via increased reactive oxygen species (ROS) represent a major culture induced stress. Anti-oxidant systems such as glutathione (GSH) can attenuate the deleterious effects of oxidative stress by scavenging ROS. It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur. Addition of low molecular weight compounds to culture media, such as cysteamine, can increase GSH levels by increasing cysteine uptake. Quite naturally, effects of supplementation of in vitro maturation (IVM) media with low molecular weight thiols have been studied in various species. This article reviews the use of cysteamine supplementation for IVM, its effects on maturation rates and further embryo development. [less ▲]

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See detailInfluence of cysteamine on in vitro maturation, in vitro and in vivo fertilization of equine oocytes
Deleuze, Stefan ULg; Dubois, Clotilde; Caillaud, Maud et al

in Reprod Domest Anim (2010), 45(1), 1-7

Contents The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in ... [more ▼]

Contents The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, IVF or in vivo embryonic development under our conditions. Ovum pick up yields (52%) and maturation rates (control group: 47% and cysteamine group: 55%) were similar to those previously reported. From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and five in the cysteamine group (9%). Those two percentages were not statistically different (p > 0.05). No effect of IVF technique was seen on the success rate (6%) in each group. [less ▲]

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See detailIN VITRO NUCLEAR AND CYTOPLASMIC MATURATION OF THE EQUINE OOCYTE: INFLUENCE OF CYSTEAMINE
Deleuze, Stefan ULg

Doctoral thesis (2009)

Research on in vitro embryo production (IVP) in the equine is impeded by the limited availability of mature oocytes as the mare is mono ovulating and superovulation is still difficult (Dippert and Squires ... [more ▼]

Research on in vitro embryo production (IVP) in the equine is impeded by the limited availability of mature oocytes as the mare is mono ovulating and superovulation is still difficult (Dippert and Squires, 1994; Bezard et al., 1995; Alvarenga et al., 2001b). Despite recent improvement in IVM of equine oocytes, success rates of IVM in that species remain low in all culture media tested compared to other species (Goudet et al., 2000b). However, most studies have focused on the percentage of oocytes reaching the metaphase II stage (nuclear maturation) but few concentrated on the final oocyte competence as measured by its ability to develop into a blastocyst and further establish a pregnancy. Blastocyst production rate is influenced not only by culture environment but also by oocyte maturation conditions. Under in vitro culture conditions, oxidative modifications of cell components via increased ROS represent a major culture induced stress (Johnson and Nasr-Esfahani, 1994). Anti-oxidant systems can attenuate the deleterious effects of oxidative stress by scavenging ROS (Del Corso et al., 1994). Glutathione, a tripeptide thiol, is the major non-protein sulfydryl compound in mammalian cells that plays an important role in protecting the cell from oxidative damage (Meister and Tate, 1976; Meister and Anderson, 1983). It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur (Furnus et al., 1998; de Matos and Furnus, 2000). The addition of GSH synthesis precursors, such as cysteamine, a thiol compound, to IVM media has been shown to improve IVP in various species (Takahashi et al., 1993; de Matos et al., 1995; Grupen et al., 1995; de Matos et al., 2002a; de Matos et al., 2002b; de Matos et al., 2003; Gasparrini et al., 2003; Oyamada and Fukui, 2004; Balasubramanian and Rho, 2007; Anand et al., 2008; Singhal et al., 2008; Zhou et al., 2008). Very little information on the use of thiol compounds in the equine is available. Conventional in vitro fertilization (IVF) has not been successful in the mare, and a repeatable IVF technique has not yet been developed (Alm et al., 2001). To overcome the limitation of conventional IVF procedures, other methods to produce embryos from oocytes, either in vivo or in vitro, have been investigated. Among these, intra cytoplasmic sperm injection (ICSI) has permitted efficient equine in vitro blastocyst production (Galli et al., 2002; Lazzari et al., 2002; Choi et al., 2006a; Choi et al., 2006c). However, ICSI requires specific equipment and skills. Transfer of an immature oocyte into the preovulatory follicle of an inseminated recipient mare (Intra-Follicular Oocyte Transfer, IFOT) has produced embryos but the success rate was low (Hinrichs and Digiorgio, 1991). Similarly, oocyte transfer (OT) into the oviduct of an inseminated recipient mare was investigated (McKinnon et al., 1988; Carnevale, 1996; Hinrichs et al., 1997; Carnevale et al., 2001; Carnevale et al., 2003; Carnevale, 2004), and commercial programs using OT for mares with reproductive abnormalities are now available (Carnevale et al., 2001). Unfortunately, IFOT is poorly documented in the literature and reports of OT have been published by various laboratories and under various conditions, making comparisons between results and choosing among these as substitutive techniques to ICSI or embryo transfer difficult. The first aim of the present work was to investigate if there is an influence of supplementation with 100 µM of cysteamine on conventional IVF success rate. Cumulus oocytes complexes (COCs) retrieved by transvaginal ultrasound guided aspiration were matured in vitro with or without cysteamine supplementation and were then submitted to conventional IVF using either calcium ionophore or heparin as capacitation treatment for spermatozoa. A total of 131 oocytes were evaluated for evidence of sperm penetration. Both techniques (ionophore or heparin) yielded 6% of IVF and results were similar both for the cysteamine and the control group. This success rate of IVF is low compared to some published data (Palmer et al., 1991; Dell'Aquila et al., 1996; McPartlin et al., 2009) but similar to what others reported in the literature (Choi et al., 1994; Dell'Aquila et al., 1997a). Although, it seems likely that cysteamine did not significantly improve IVF rates under our conditions, our general success rates for IVF procedures may be too low for us to conclude definitely about the effect of cysteamine. As ICSI was not available to us, the second aim of this work was to determine what in vivo technique could best bypass the lack of an efficient conventional IVF procedure. We compared embryo production following transfer of in vivo recovered oocytes (1) into a recipient’s oviduct or (2) into her preovulatory follicle either immediately after ovum pick up or (3) after in vitro maturation. Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten days after transfer, rates of embryos collected in excess to the number of ovulations were calculated and compared for each group. Embryo collection rates were 32.5% (13/40), 5.5% (3/55) and 12.8% (6/47) for OT, post-IVM and immediate IFOT respectively. OT significantly yielded more embryos than immediate and post-IVM IFOT did. These results show that, when ICSI is not an option, intra-oviductal oocyte transfer is to be preferred to IFOT, as an in vivo alternative, to bypass the inadequacy of conventional in vitro fertilization and to assess oocyte developmental competence. After it was established that in comparison to IFOT, OT is the most reliable in vivo alternative to in vitro fertilization where ICSI technology is not available, this technique was used to assess the effect of cysteamine supplementation on nuclear maturation and oocyte competence. The third aim of this work was to investigate the influence of supplementation with 100 µM of cysteamine on in vitro nuclear and cytoplasmic maturation by specific DNA staining and the ability of oocytes to undergo in vivo fertilization after OT. Oocytes were collected by transvaginal ultrasound guided aspiration and matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining and the embryo yield following OT were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Overall maturation rate was 52%, which is rates reported in the literature ranging from 40 to 70% in the equine (Goudet et al., 1997a; Bogh et al., 2002; Hinrichs et al., 2005; Galli et al., 2007). Nuclear maturation was not statistically different (p>0.05) between oocytes cultured with or without cysteamine (55% and 47% respectively). From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and 5 in the cysteamine group (9%). Those two percentages were not statistically different (p>0.05). Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, or in vivo embryonic development under our conditions. Under our conditions, the addition of 100 µM of cysteamine to a classic culture medium does not improve equine oocyte maturation or embryonic development after OT. The same dose failed to increase GSH content in the equine (Luciano et al., 2006). However, the effect of cysteamine supplementation is highly species and concentration dependant. The inadequacy of the chosen concentration may explain that equine embryo production has not been increased by the cysteamine under our conditions as opposed to what has been observed in many other species. Alternatively, we can hypothesize that some substances present in the IVM medium can interfere with GSH synthesis. This has been suggested for FSH and estradiol (Bing et al., 2001) and, although our maturation medium is not supplemented with gonadotropins or estradiol, factors contained in fetal calf serum or EGF might also have an effect on GSH synthesis. Considering its beneficial effects in many other species, supplementation with cysteamine to different IVM media should be further investigated in the equine. Ideally combining different concentrations and ICSI or OT in order to determine an optimal concentration and its effects on oocyte developmental competence. [less ▲]

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See detailEfficiency of embryonic development after Intra-Follicular and Intra-Oviductal transfer of in vitro and in vivo matured horse oocytes
Deleuze, Stefan ULg; Goudet, Ghylène; Caillaud, Maud et al

in Theriogenology (2009), 72(2), 203

In vivo techniques, such as intra-oviductal Oocyte Transfer (OT) and Intra-Follicular Oocyte Transfer (IFOT) can be considered as alternatives to bypass the lack of efficient superovulation treatments and ... [more ▼]

In vivo techniques, such as intra-oviductal Oocyte Transfer (OT) and Intra-Follicular Oocyte Transfer (IFOT) can be considered as alternatives to bypass the lack of efficient superovulation treatments and the inadequacy of conventional in vitro fertilization techniques in the horse. We compared embryo production following transfer of in vivo recovered oocytes (1) into a recipient’s oviduct or (2) into her preovulatory follicle either immediately after ovum pick up or (3) after in vitro maturation. Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten days after surgery, rates of embryos collected in excess to the number of ovulations were calculated and compared for each group. Embryo collection rates were 32.5% (13/40), 5.5% (3/55) and 12.8% (6/47) for OT, post-IVM and immediate IFOT respectively. OT significantly yielded more embryos than immediate and post-IVM IFOT did. We also showed that in vitro matured oocytes could succesfully be used for IFOT. Our results also suggest that improvement of the IFOT technique could turn it into an inexpansive and easy to perform procedure that could be an answer to the inefficiency of superovulation treatments in the mare. [less ▲]

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See detailMicroscopic study of equine oocyte maturation within quaternary follicles.
Ponthier, Jérôme ULg; Beckers, Jean-François ULg; Deleuze, Stefan ULg

Conference (2009, February 19)

In various species a relationship between oocyte maturity and morphological characteristics of oocytes has been established. To our knowledge, immature oocyte aspect under light microscopy has been paid ... [more ▼]

In various species a relationship between oocyte maturity and morphological characteristics of oocytes has been established. To our knowledge, immature oocyte aspect under light microscopy has been paid little attention in the equine. The aim of the study is to give a general description and analyse microscopic parameters of immature oocytes. Follicles from abattoir ovaries are punctured after measurement of their diameter and their cumulus-oocyte complexes are recovered. Different characters such as oocyte diameter, cumulus aspect, granulosity and polarity of ooplasm are observed under light microscopy. No correlation between estral activity, follicle maturity, cumulus cells aspect, polarity and granulosity of ooplasm was observed. Thickness of zona pellucida differs between oocytes with polar or non polar ooplasm. No correlation was observed between follicle diameter and oocyte diameter and none of the other studied parameters showed an influence on oocyte diameter. Results show that no character of oocytes observed under light microscopy can be related to follicular origin or to any other character. Further studies have to be done to compare these immature oocytes with mature oocytes and to find the ultra-structural origin of characters observed under light microscopy. [less ▲]

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See detailAffections du système reproducteur et équitation - quand les hormones s'en mêlent
Ponthier, Jérôme ULg; Deleuze, Stefan ULg

Conference given outside the academic context (2009)

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See detailMicroscopic study of equine oocyte maturation within quaternary follicles
Ponthier, Jérôme ULg; Beckers, Jean-François ULg; Deleuze, Stefan ULg

in Comptes Rendus de l'Académie Bulgare des Sciences = Proceeding of the Bulgarian Academy of Sciences (2009), 62(4), 491-498

In various species a relationship between oocyte maturity and morphological characteristics of oocytes has been established. To our knowledge, little attention has been paid to the immature oocyte aspect ... [more ▼]

In various species a relationship between oocyte maturity and morphological characteristics of oocytes has been established. To our knowledge, little attention has been paid to the immature oocyte aspect under light microscopy in the equine. The aim of the study is to give a general description and analysis of the microscopic parameters of immature oocytes. Follicles from abattoir ovaries are punctured after measurement of their diameter and their cumulus-oocyte complexes are recovered. Different characters such as oocyte diameter, cumulus aspect, granulosity and polarity of ooplasm are observed under light microscopy. No correlation between the estral activity, follicle maturity, cumulus cells aspect, polarity and granulosity of ooplasm was observed. The thickness of zona pellucida differs between oocytes with polar or nonpolar ooplasm. No correlation was observed between the follicle and oocyte diameter and none of the other studied parameters showed an influence on the oocyte diameter. Results show that no character of the observed oocytes under light microscopy can be related to the follicular origin or to any other character. Further studies have to be done to compare these immature oocytes with mature ones and to find the ultrastructural origin of the characters observed under light microscopy. [less ▲]

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See detailReproduction assistée dans l’espèce équine : collecte, évaluation, maturation et utilisations d’ovocytes équins
Deleuze, Stefan ULg; Ponthier, Jérôme ULg; Hanzen, Christian ULg

in Annales de Médecine Vétérinaire (2009), 153(1), 22-30

Malgré le faible nombre d’ovocytes équins disponibles pour la recherche, des techniques de reproduction assistée récemment développées dans cette espèce permettent maintenant de produire des embryons en ... [more ▼]

Malgré le faible nombre d’ovocytes équins disponibles pour la recherche, des techniques de reproduction assistée récemment développées dans cette espèce permettent maintenant de produire des embryons en utilisant des étalons et des juments subfertiles ou même de sauvegarder leur potentiel génétique après leur mort. Cette revue de littérature décrit les aspects cliniques de la collecte d’ovocytes ex vivo ou in vivo, leur évaluation, leur maturation in vitro et leur utilisation pour le transfert intra-folliculaire ou salpyngien et l’injection intra-cytoplasmique de spermatozoïde. [less ▲]

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See detailThe secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?
Mugnier, S.; Kervella, M.; Douet, C. et al

in Reproductive biology and endocrinology (2009), 7(1), 129

ABSTRACT: BACKGROUND: Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite ... [more ▼]

ABSTRACT: BACKGROUND: Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Methods & results In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes. In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates. CONCLUSION: Our study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization. [less ▲]

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See detailCardiac Power Output during Dobutamine Stress
Sandersen, Charlotte ULg; Mc Entee, Kathleen ULg; Deleuze, Stefan ULg et al

in Journal of Equine Veterinary Science (2009)

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See detailEquine frozen semen parameters in relation with total myeloperoxidase concentration
Ponthier, Jérôme ULg; Franck, Thierry ULg; Mottart, Evelyne et al

in Animal Reproduction Science (2008, September 15)

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See detailPregnancy diagnosis in the mare by semi quantitative relaxin quick assay kit
Ponthier, Jérôme ULg; van de Weerdt, marie-Lys; Deleuze, Stefan ULg

in Reproduction in Domestic Animals (2008, July), 43(s3), 111

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See detailLes maladies vénériennes chez le cheval
Ponthier, Jérôme ULg; Deleuze, Stefan ULg

in sBs Magazine (2008)

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See detailPhysiopathology and therapy of chronic heart failure in the equine species
Amory, Hélène ULg; Deleuze, Stefan ULg; Sandersen, Charlotte ULg

in Proceedings of the Second Resident Meeting of the European College of Equine Internal medicine (ECEIM) (2008)

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See detailMorphological echocardiographic measurements: reference values as a function of body size in equids
Al Haidar, A; Sandersen, Charlotte ULg; Van Erck, Emmanuelle et al

Poster (2008)

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See detailBiomarkers of heart disease
Amory, Hélène ULg; Deleuze, Stefan ULg; Sandersen, Charlotte ULg

in Proceedings of the Second Resident Meeting of the European College of Equine Internal medicine (ECEIM) (2008)

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See detailDiagnostic de gestation chez la jument par un test rapide de dosage semi-quantitatif de la Relaxine
Ponthier, Jérôme ULg; van de Weerdt, Marie-Lys; Deleuze, Stefan ULg

Poster (2007, November)

Introduction. Chez la jument, les méthodes hormonales de diagnostic de gestation sont peu sensibles et/ou manquent de précocité [2]. Le dosage de la progestérone, à considérer comme un diagnostic de non ... [more ▼]

Introduction. Chez la jument, les méthodes hormonales de diagnostic de gestation sont peu sensibles et/ou manquent de précocité [2]. Le dosage de la progestérone, à considérer comme un diagnostic de non gestation [2], est peu sensible, non réalisable après le 120ème jour et ne donne pas d’indication sur la viabilité fœtale. Le dosage de l’eCG (ou PMSG, Pregnant Mare Serum Gonadotrphin) ne tient pas compte de la mortalité embryonnaire et n’est réalisable que pendant une courte période (du 40ème au 140ème jour) [2]. Le dosage des oestrogènes urinaires nécessite souvent un sondage urinaire et est peu utilisé en raison de son caractère tardif [2]. Le dosage de l’oestrone sulfate permet d’estimer la viabilité fœtale mais il est peu précoce (réalisable dès le 100ème jour) [2]. La relaxine, un polypeptide organisé en 2 sous unités et essentiellement synthétisé par le placenta chez la brebis, la jument, la chienne et la chatte [1,4,6,7,9] est une candidate pour le diagnostic de gestation et de viabilité foetale. Chez la chienne et la chatte, il existe un dosage immunologique semi-quantitatif de la relaxine sanguine utilisable sur le terrain dès le 28ème jour chez la chienne et le 31ème jour chez la chatte [notice technique du Relaxin Witness®]. Chez la chienne, les taux de relaxine obtenus par dosages immunologiques sont de 1ng/ml à 4 semaines et atteignent un pic à 4ng/ml à 6 semaines [4]. Chez le chatte, le dosage de la relaxine est aux alentours de 5ng/ml à 31 jours pour atteindre un pic autour de 9ng/ml au 40ème jour [6]. Chez la jument gestante, le taux de relaxine par dosage chromatographique est basal jusqu’au 80ème jour pour ensuite augmenter brutalement et atteindre 80ng/ml chez la jument et 10ng/ml chez la ponette [5,8]. Ensuite, les concentrations vont continuer à augmenter chez la ponette alors qu’elles vont diminuer lentement chez la jument [8]. Dans les deux cas, les valeurs de relaxine chutent brusquement lors du part [8]. On trouve aussi de la relaxine dans le follicule et dans le corps jaune mais l’effet de cette production sur les concentrations sanguines n’est pas connu [3]. Objectifs du travail. Le but est d’étudier la Sensibilité, la Spécificité, la Valeur Prédictive Positive et Négative d’un test immunologique de dosage semi-quantitatif de la Relaxine réalisable sur le terrain comme moyen de diagnostic de gestation. Matériel et méthode. Les sérums de 10 juments (Ponettes, juments de selle ou de trait) sont soumis au test Witness Relaxin® (Synbiotics Corporation, Lyon, France), un test ELISA Sandwich : l’échantillon est mis en contact avec un anticorps, le complexe migre sur une membrane et est capturé sur une zone où est fixée le second anticorps. La fixation fait l’apparaître une bande colorée, une bande de contrôle atteste de la réussite du test. Le diagnostic et le stade de gestation sont confirmés par échographie transrectale ou transabdominale. Le degré de signification est obtenu par le test exact de Fischer. La sensibilité, la spécificité, la Valeur Prédictive Positive et Négative sont calculées. Résultats. Test + Test - Total Gestante 0 6 6 Non Gestante 0 4 4 Total 0 10 10 Dans l’échantillon, 4 juments n’étaient pas gestantes et 6 étaient gestantes de plus de 100 jours. Aucun des tests Witness Relaxine® n’a été positif : ni pour les juments vides, ni pour les juments pleines. La sensibilité est de 0%, la spécificité est de 100%, la Valeur Prédictive Positive du test est non calculable et la Valeur Prédictive Négative du test est de 40% pour une valeur du Test Exact de Fischer significative à100%. Les sérums des 6 juments pleines ont été envoyés au laboratoire Synbiotics pour un ELISA classique. Aucun échantillon n’a répondu aux anticorps du test ELISA. Discussion. Même si les anticorps développés pour le test d’immunomigration ou l’ELISA répondent dans de nombreuses espèces (chien, chat, souris), ils ne réagissent pas chez le cheval. Pourtant, les concentrations en relaxine observées chez le cheval sont plus importantes que celles observées chez les autres espèces [4,5,6,8]. Une première hypothèse fut celle de la sursaturation des anticorps et d’une non reconnaissance de ceux-ci. Cependant, à des dilutions amenant la concentration en relaxine à des titres dix fois inférieurs, le test Witness Relaxin (Synbiotics Corporation, Lyon, France) n’a pas répondu. Afin de soumettre les échantillons à un ELISA classique, les échantillons ont été envoyés au laboratoire Synbiotics. Aux différentes dilutions testées (1 ; 0,1), aucune reconnaissance de la relaxine par les anticorps n’a été notée. Malgré une structure en deux sous-unités (A et B) et un poids moléculaire semblable à celle des autres espèces [7], l’unité B de la relaxine équine n’est pas reconnue par les anticorps du test. Conclusions. Les kits rapides de dosage semi-quantitatif de la relaxine commercialisés ne détectent pas la relaxine équine. Des études doivent développer des anticorps spécifiques à la sous-unité bêta des équidés. Remerciements. Nos remerciements vont à la société Synbiotics (Lyon, France) pour la réalisation des tests ELISA Relaxine classiques et leur collaboration scientifique. Références. 1. KLONISCH T., MATHIAS S., CAMBRIDGE G., HOMBACH-KLONISCH S., RYAN P.L., ALLEN W.R. (1997): Placental localization of Relaxin in the Pregnant Mare. Placenta, 18:121-128. 2. McKINNON A.O. (1993): Diagnosis of pregnancy. in: McKinnon A.O., Voss J.L.: Equine Reproduction. Lea &Febiger, Philadelphia, London: 501-508. 3. RYAN P.L., KLONISCH T., YAMASHIRO S., RENAUD R.L., WASNIDGE C., PORTER D.G. (1997): Expression and localization of relaxin in the ovary of the mare. Journal of Reproduction and Fertility, 110:329-338. 4. STEINETZ B.G., GOLDSMITH L.T., LUST G. (1987): Plasma Relaxin Levels in Pregnant and Lactating dogs. Biology of Reproduction, 37:719-725. 5. STEWART D.R., STABENFELDT G.H. (1981): Relaxin Activity in the Pregnant Mare. Biology of Reproduction, 25:281-289. 6. STEWART D.R., STABENFELDT G.H. (1985): Relaxin Activity in the Pregnant Cat. Biology of Reproduction, 32:848-854. 7. STEWART D.R., PAPKOFF H. (1986): Purification and Characterization of Equine Relaxin. Endocrinology, 119(3):1093-1099. 8. STEWART D.R., ADDIEGO L.A., PASCOE D.R., HALUSKA G.J., PASHEN R. (1992): Breed Differences in Circulating Equine Relaxin. Biology of Reproduction, 46:648-652. 9. WATHES D.C., REES J.M., PORTER D.G. (1988): Identification of relaxin in the placenta of the ewe. Journal of Reproduction and Fertility, 84:247-257. [less ▲]

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