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 recipients 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.

Equine coital exanthema (ECE), caused by equid herpesvirus 3 (EHV-3), is a contagious venereal disease characterised by the formation of painful papules, vesicles, pustules and ulcers on the external genitalia of both mares and stallions. EHV-3 is an alphaherpesvirus, distinct from the other equine herpesviruses, endemic in most horse breeding populations worldwide. EHV-3 is primarily transmitted through coitus, although there is also evidence supporting the possibility of non-coital spreading through infected fomites and contacts other than coitus. The infection does not usually result in systemic illness. Epidemiological observations and serological monitoring suggest the existence of latently infected animals from which EHV-3 is periodically reactivated and transmitted to cohorts but latency of EHV-3 has not been formerly demonstrated. The negative impacts of ECE on equine breeding enterprises are the forced, temporary disruption of the mating activities of mares and stallions, the additional care and supportive treatment in affected horses, and the risk of virus spread by either fresh or frozen semen as well as by artificial insemination and embryo transfer practices. In intensively managed stud operations, which have heavily-scheduled breeding dates for thoroughbred stallions, breeding disruptions may translate into significant end-of-season decreases in the number of entries into the mare book of affected stallions. Also, delayed foaling dates and/or reduced pregnancy rates may occur in mares that miss breeding opportunities due to the disease. Similarly, in the face of an ECE outbreak in artificial insemination and embryo transfer centres, both donor and recipient affected mares show such discomfort that they are reluctant to be inspected, inseminated or transferred, with the consequent loss of opportunity to become pregnant. The additional time and necessary precautions required to manage the donor and receptor mares due to the presence of the disease also have a substantial negative impact. Because ECE is a not a notifiable disease and the diagnosis is made on the basis of typical clinical signs, most cases and outbreaks of ECE remain unnoticed and its true prevalence and economic impact is difficult to assess and is probably underestimated. Therefore, as several aspects of EHV-3 infection are largely unknown and it has severe economic consequences to the horse industry, the general aim of this doctoral study was to increase the knowledge about the biology of EHV-3 infection. The specific objectives were to investigate the iatrogenic transmission of infection and to set up a protocol for experimental reproduction of the disease, to study the reactivation and re-excretion patterns from latency, to evaluate the epidemiological importance of subclinical infections, and to hypothesise about the ECE economic consequences in the current context of the equine industry. During the occurrence of an outbreak of ECE in an embryo transfer centre, approximately 32% (n=35) of the donor mares and 25% (n=125) of the recipient mares showed typical ECE lesions around the anus and on the perineal skin, discomfort, and anorectal lymphadenopathy. EHV-3 was detected in 7 (58%) of the affected mares and specific antibodies in 23 (88%) of the convalescent mares. Since no natural breeding had taken place on the affected mares, it could be hypothesised that the virus spread was a consequence of contamination by means of the gloves or the ultrasonography scanner used. Lymphadenopathy provides a new concern associated with ECE. EHV-3 was isolated from nasal swabs obtained during an outbreak of unilateral rhinitis affecting approximately 40 out of 2000 thoroughbred horses. The fact that an endoscopic examination had been performed in the week previous to the onset of the lesions to evaluate the respiratory tract function was a common finding in all the horses affected. EHV-3 was demonstrated as the etiologic agent of the unilateral rhinitis observed in those 40 thoroughbred horses. The endoscope used for respiratory tract examination was identified as the most likely cause of the spread of the infection. This case is an example of non-genital iatrogenic transmission and reinforces the importance of strict application of hygienic measures in order to reduce the risk of spread of infectious diseases. The virus isolated from this field outbreak as well as that isolated from others were characterized by means of restriction endonuclease (RE) fragment patterns, plaque size and gG gene partial nucleotide sequencing. In the 25 isolates included in the study, different RE patterns were found: two with BamHI (one of them identical to the one of the reference strain), two with Hind III (both different from the one of the reference strain) and one with Eco RI (different from the one of the reference strain). The plaque size was homogeneous between the isolates, and 1.64 and 2.88 times larger than that of the reference strain. Three base substitutions in the gG gene were found at positions 904, 1103 and 1264, which resulted in strains CAT (Australia), AAT (the United States and Brazil), CAG (Argentina) and ACT (Argentina). The RE pattern and the nucleotide sequence of the gG gene obtained revealed that there are genetically distinguishable EHV-3 strains in circulation. Not only the RE patterns, as previously described, but also the nucleotide sequence of the gG gene, could be useful tools for epidemiological studies. The biological implications of these changes are still unknown. Two sets of experimental infection with EHV-3 were carried out under controlled conditions. In the first experiment, two seronegative mares were topically inoculated in the vagina and perineal area with EHV-3. The same protocol was followed in the second experiment in two seronegative and two seropositive mares (the mares which had been included in the first experiment were used six months later). Clinical samples consisted of swabs from the vagina and perineal area, and blood samples were obtained for virological, serological and haematological studies. A scoring system was designed and used for daily clinical evaluation and rectal temperature records from each mare. Neither hyperthermia nor haematological changes were recorded in the mares analyzed. Typical ECE lesions were observed in seronegative animals: the clinical score was 172 and 90 (average score: 131) for the mares included in the first experiment and 160 and 92 (average score: 124) for the mares included in the second experiment. Only slight lesions were observed in the seropositive mares, being the clinical score 53 and 41 (average score: 47). Also, differences were detected in the duration and intensity of virus shedding, being 15 and 9 days (duration) and 105 versus 104 (the highest virus load detected) in the seropositive and seronegative mares, respectively. In one study designed to demonstrate EHV-3 latency and to study reactivation and re-excretion patterns, virus shedding, seroconversion and the presence of a small ECE lesion were observed in one out of two previously naturally infected mares after corticosteroid treatment. EHV-3 was isolated from perineal vaginal swabs of one of the mares, both on day 14 after corticosteroid treatment and along the following 10 days. A small and rounded area of erosion was observed on the left labia of the vulva of the same mare on day 19 after corticosteroid treatment and 5 days after the virus shedding was detected. A significant (four-fold) increase in the antibody titre was found in the mare which shed the virus 28 days after corticosteroid treatment and 14 days after the beginning of virus re-excretion. In concordance with epidemiological observation and serological studies, and in common with other members of the subfamily Alphaherpesvirinae, this study indicates that a state of latency is established after natural infection of EHV-3. A study was carried out to estimate the prevalence of excretion of EHV-3 under field conditions. The virus was detected in perineal-vaginal swabs by real time PCR and specific antibodies were identified by seroneutralization in 14 (6%) and 105 (48%) respectively of 220 thoroughbred mares without clinical signs at the time of breeding. In order to assess the re-excretion patterns of spontaneous reactivation, two seropositive (presumably latently infected) polo mares were kept in isolation for 11 months. Virological investigations on perineal vaginal swabs obtained on a daily basis revealed re-excretion of EHV-3 on two occasions, 3 months apart (each for a 3-day interval) in one of the mares, and on only 1 day in the other mare. Antibodies against EHV-3 were detected with only slight variation during the entire period in both mares. Clear evidence of the existence of EHV-3 shedders in a healthy mare population under both field and isolation conditions is provided. Furthermore, despite the small number of animals included (only two), the study in mares kept in isolation demonstrated that at least two periods of EHV-3 spontaneous reactivation and re-excretion in the presence of serum antibodies were possible in the same animal in an 11-month interval. In conclusion, EHV-3 infections and ECE are still a threat for the equine industry. In the present study, EHV-3 was found in several field outbreaks of ECE in thoroughbred breeding farms; the disease was reported by the veterinarians as a true sanitary problem and thus demands additional preventive measures. In addition, EHV-3 was detected as a not rare event in clinically healthy mares, which constitutes the most relevant finding from an epidemiological perspective. ECE is also a sanitary problem of concern for embryo transfer and routine veterinary practices. The population of EHV-3 latently infected mares which reach up to 50% at the time of breeding deserves special attention. Reactivation of the latent virus is not preventable and those mares can spontaneously reactivate the virus and become a source of infection for highly valuable horses like « shuttle stallions », with the consequent economically negative impact on the equine enterprises. Finally, there is an important need for finding out additional preventive measures including « pen side » diagnostic tools that allow the detection of subclinically EHV-3 shedding mares in order to segregate them from natural breeding and give them an appropriate antiviral treatment before being covered by stallions.

Poor athletic performance of racehorses is a major and significant problem in the racing industry. Determining the definitive reason for poor-performance is however a real diagnostic challenge since many of the causative conditions are multifactorial and may only be manifested during exercise. A retrospective study, including various breeds of horses, confirmed musculoskeletal, cardiovascular and upper respiratory tract clinical problems to be the most frequently implicated in reducing athletic performance. Evaluation of the lower respiratory tract was though not performed in this study. The aim of the first part of this work were thus to determine the prevalence of different sub-clinical diseases in a population of poorly-performing Standardbred trotters, and to evaluate the sportive repercussions by comparing their physiological response to exercise with control horses. Fifty horses underwent thorough clinical and ancillary examinations, including haematological et biochemical evaluation, Doppler echocardiography, standardised exercise tests on treadmill et racetrack, treadmill video-endoscopy et collection of respiratory fluids. Most of the poorly-performing horses exhibited many concomitant diseases. The most frequently diagnosed sub-clinical problems involved the lower and upper respiratory tract. Poor-performers also exhibited higher values of blood lactate and heart rate, as well as lower values of haematological parameters and anti-oxidants, compared to control horses. Inflammatory airway disease being mostly present in poorly-performing horses, the second part of this work will mainly focus on this syndrome. The negative impact of inflammatory airway disease, as diagnosed by cytological evaluation of bronchoalveolar lavage fluid, has previously been described on respiratory function using either forced expiration or forced oscillations techniques. Sedation or bronchoprovocation were however usually required. On the other hand, the clinical significance of tracheal inflammation remains currently controversial. The aim was therefore to exhibit and define the respiratory dysfunctions present in horses subclinically suffering from inflammatory airway disease. Respiratory function was evaluated at rest by IOS in 34 Standardbred trotters, whereas tracheal mucus score, and both tracheal and bronchoalveolar lavages were performed 60 min post-exercise. According to the cytology of bronchoalveolar lavage fluid, the inflammatory group included 19 horses and 15 horses were used as control. A significant correlation was found between both cytological evaluations concerning neutrophil counts, whereas no association was found between tracheal mucus and any cytology. A significant increase of respiratory resistance at the lower frequencies (1 10 Hz) as well as a significant decrease of respiratory reactance beyond 5 Hz was observed in inflammatory compared to control horses. Both parameters were also significantly different between inspiration and expiration in the inflammatory group only. Both eosinophil and mast cell counts of the bronchoalveolar lavage fluid were significantly correlated with respectively respiratory resistance and reactance. The present work involved intensive clinical and functional evaluation of control and asymptomatic poorly-performing horses. The different studies allowed establishing the prevalence of medical subclinical diseases in these latter and evaluating its sportive impact considering the associated physiological responses to exercise. The presence of respiratory dysfunctions in horses with lower airway inflammation, the major trouble associated with disappointing performance, were also exhibited by impulse oscillometry./ La contre-performance est un problème majeur dans lindustrie des courses. En déterminer la cause exacte reste néanmoins un défi diagnostic puisque la plupart des affections présentes sont souvent subcliniques, multifactorielles et peuvent ne se manifester que pendant lexercice. Une étude rétrospective, incluant des chevaux de différentes races et disciplines, a ainsi confirmé les affections cliniques des voies respiratoires supérieures, musculo-squelettiques et cardiovasculaires comme étant les plus fréquemment impliquées dans la réduction des performances athlétiques. Cependant, lévaluation des voies respiratoires profondes navait pas été effectuée chez ces différents chevaux. Lobjectif de la première partie de ce travail était donc de déterminer la prévalence des différentes affections sub-cliniques induisant une contre-performance chez des Trotteurs Français, et den évaluer les répercussions sportives par la comparaison des réponses physiologiques à lexercice avec celle de chevaux contrôles. Cinquante chevaux ont respectivement été soumis à un examen clinique complet, une prise de sang pour analyse hémato-biochimique au repos et 60 minutes après chaque test deffort, une échocardiographie Doppler, des tests deffort standardisés sur piste et tapis roulant, une endoscopie à leffort, une évaluation locomotrice à grande vitesse, ainsi quun lavage trachéal et broncho-alvéolaire réalisés 60 minutes post-effort. La plupart des chevaux contre-performants ou intolérants à leffort présentaient plusieurs affections concomitantes. Les troubles sub-cliniques les plus fréquemment diagnostiqués concernaient respectivement les voies respiratoires profondes et supérieures. Ces chevaux présentaient par ailleurs des paramètres hématologiques (taux dhémoglobine et volume globulaire moyen) et anti-oxydants significativement inférieurs, et des paramètres pro-oxydants significativement supérieurs aux chevaux contrôles. De plus, les valeurs de fréquence cardiaque et lactatémie étaient, lors des différents tests deffort, significativement supérieures à celles des chevaux contrôles, Linflammation des voies respiratoires profondes étant majoritairement présente chez ces chevaux présentant des performances décevantes, la deuxième partie de ce travail se concentre plus spécifiquement sur ce syndrome. Limpact négatif sur la fonction respiratoire de cette affection, telle que diagnostiquée par lévaluation cytologique du liquide de lavage broncho-alvéolaire, a précédemment été décrite à laide de techniques dexpiration forcée ou doscillations forcées. Une sédation ou une bronchoprovocation étaient cependant généralement requises pour la réalisation de ces tests. Parallèlement, la signification clinique de linflammation trachéale reste actuellement controversée. Lobjectif était ainsi de mettre en évidence et définir les dysfonctions respiratoires présentes chez des chevaux souffrant sub-cliniquement de maladie inflammatoire des voies respiratoires. La fonction respiratoire a été évaluée au repos par oscillométrie à impulsions chez 34 Trotteurs Français asymptomatiques, alors que le score de mucus trachéal et les différents lavages ont été évalués 60 minutes post-effort. Sur base de la cytologie broncho-alvéolaire, le groupe inflammatoire comprenait 19 chevaux et 15 ont été utilisés comme contrôles. Une corrélation significative était observée entre les cytologies concernant le taux de neutrophiles, alors quaucune association nétait présente entre score de mucus trachéal et cytologies des différents lavages. Une augmentation significative de la résistance respiratoire aux faibles fréquences (1 à 10 Hz) et une diminution de la réactance respiratoire au-delà de 5Hz a été observée chez les chevaux inflammatoires comparativement aux contrôles. Ces deux paramètres étaient également significativement différents entre inspiration et expiration dans le groupe inflammatoire uniquement. La résistance et la réactance respiratoire étaient par ailleurs respectivement corrélées aux taux déosinophiles et de mastocytes du lavage broncho-alvéolaire. Ce travail comprenait une évaluation clinique et fonctionnelle intensive chez des chevaux contrôles et des chevaux contre-performants. Les études menées ont permis détablir la prévalence des affections médicales sub-cliniques chez ces derniers et den évaluer limpact sportif par lintermédiaire des réponses physiologiques à lexercice. La présence de dysfonctions respiratoires chez les chevaux avec inflammation des voies respiratoires profondes, premier trouble associé à des performances décevantes, a également pu être mise en évidence à laide de loscillométrie à impulsion.