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Steroidome and metabolome analysis in gilt saliva to identify biomarkers of boar effect receptivity

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G. Goudet (INRA) et al., 70th Annual Meeting of the European Federation Animal Science (EAAP), 26-30 août 2019, Ghent, Belgique, p. 489, poster

Our objective was to develop alternatives to hormonal treatments to synchronize oestrus of gilts. Before puberty gilts exhibit a pre-puberty period during which boar exposure could induce and synchronize first ovulation. To develop practical non-invasive tools to identify this period an  improve detection of the gilts to stimulate, we searched for salivary biomarkers of the pre-puberty period. Saliva samples were collected from 30 Large-White × Landrace crossbred gilts from 140 to 175 days of age. Gilts were exposed to a boar twice a day and subjected to oestrus detection from 150 to 175 days of age. Among the 30 gilts, 10 were detected in oestrus 4 to 7 days after introduction of the boar and were considered receptive to the boar effect, 14 were detected in oestrus more than 8 days after boar introduction, 6 did not show oestrus and were considered non-receptive. Saliva samples from 6 receptive and 6 non-receptive gilts were analysed for steroidome using GC-MS/MS and for metabolome using 1H-NMR spectroscopy. Four saliva samples per gilt were analysed: 26 days and 11 days before boar introduction (BI-26 and BI-11), the day of boar introduction (BI), 3 days later for receptive gilts (BI+3) or 7 days later for nonreceptive gilts (BI+7). Data were analysed using repeated measures one-way ANOVA and orthogonal partial least squares discriminant analysis. Thirty steroids and 35 metabolites were detected in gilt saliva. The concentrations of 6 steroids were higher (P<0.05) in receptive gilts than in non-receptive gilts at BI-26, BI-11 and BI. The concentration of 2 metabolites were lower (P<0.05) in receptive gilts than in non-receptive gilts at BI-11. These candidates could be potential salivary biomarkers to detect receptive gilts. However, their low and variable concentrations in saliva require expensive analysis and limit their use in pig farms.

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2019

Evolution of steroid concentrations in saliva from immature to pubertal gilts for the identification of biomarkers of gilts receptivity to boar effect

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Gylène Goudet et al., Livestock science, 2019 volume 228, octobre, p. 5-17

Estrus synchronization is necessary for management of gilt reproduction in pig farms. It is usually achieved by using synthetic progestagens, but there is increasing demand for non-hormonal alternative tools with the prospect of sustainability of livestock production. Moreover, in organic farms, synthetic hormones are not allowed. Before reaching puberty, gilts exhibit a “waiting period” during which external stimulations, such as boar exposure, could trigger and synchronize the first ovulation. However, practical non-invasive tools for detection of the “waiting period” in pig farms are lacking. During this period, estrone levels in urine are high, but urine sampling is difficult in group-housed females. Our objective was to identify among steroids potential biomarkers of this “waiting period” through saliva monitoring from immature to pubertal gilts using gas chromatography coupled to tandem mass spectrometry.

Starting between 144 to 147 days of age, six Large White gilts were submitted to ultrasound puberty diagnosis 3 times a week until first ovulation. Urine and saliva samples were collected to analyze weekly estrone and steroidome respectively, until puberty. Urinary estrone concentration significantly increased 2 weeks before first ovulation occurring between 182 and 192 days of age. The period with increasing estrone levels was considered as the “waiting period”. Steroidome analysis allowed identifying and quantifying 28 steroids in 500 µl of gilts saliva. Significant decrease of dehydroepiandrosterone and significant increase of 5α-dihydroprogesterone and 17β-estradiol were detected 2 weeks before puberty, suggesting that these steroids could be potential biomarkers of the “waiting period”.

These results show that painless sampling of saliva could be a non-invasive welfare-friendly tool for the identification of the physiological hormonal status of the gilts and possibly the optimal time for application of the boar effect, a solution to synchronize puberty without exogenous hormones.

2019

Analyse des stéroïdes dans la salive de cochettes pour identifier des biomarqueurs de la période de réceptivité à l’effet mâle

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51es Journées de la Recherche Porcine, 5 et 6 février 2019, Paris, p. 43-44, par Ghylène Goudet (INRA) et al., poster

Poster. 

L’élevage porcin conventionnel se caractérise par une conduite en bandes qui présente de nombreux avantages pour la gestion des animaux (inséminations, surveillance des mises-bas, ajustement de la taille des portées, soins aux porcelets), l’organisation de l’élevage (utilisation optimale des bâtiments, nettoyage des locaux entre bandes) et la production de lots de porcelets homogènes pour l’engraissement et l’abattage. La conduite en bandes nécessite la synchronisation des cycles des femelles, réalisée par administration d’agonistes de synthèse de la progestérone chez une majorité d’éleveurs. Les effets négatifs des résidus hormonaux sur la santé humaine et l’environnement conduisent à mettre en place de nouvelles pratiques d’élevage. Notre objectif à long terme est de développer des alternatives aux traitements hormonaux pour la synchronisation des oestrus des cochettes, notamment lors de l’entrée dans la première bande. Avant la puberté, les cochettes atteignent une phase de pré-puberté au cours de laquelle une stimulation externe peut déclencher la première ovulation (Prunier, 1989). L’exposition au verrat (appelée effet mâle) pourrait favoriser le déclenchement et la synchronisation de la première ovulation si elle est appliquée pendant cette phase de pré-puberté. Notre objectif est d’améliorer le repérage des femelles à stimuler en identifiant des biomarqueurs de la phase de pré-puberté. Ces biomarqueurs sont recherchés dans la salive des cochettes car les prélèvements de salive sont non-invasifs.

Analysis of steroids in gilt saliva to identify biomarkers of the period of receptivity to the boar effect

Our objective was to develop alternatives to hormonal treatments to synchronize oestrus of gilts. Before puberty, gilts experience a pre-puberty period during which boar exposure could induce and synchronize the first ovulation. To develop practical non-invasive tools to identify this period and improve detection of the gilts to stimulate, we searched for salivary biomarkers of the pre-puberty period. Saliva samples were collected from 30 Large-White x Landrace crossbred gilts from 140 to 175 days of age. Gilts were exposed to a boar twice a day and subjected to oestrus detection from 150 to 175 days of age. They were then slaughtered to ascertain puberty. Among the 30 gilts, 10 were detected in oestrus 4 to 7 days after introduction of the boar and were considered receptive to the boar effect, 14 were detected in oestrus more than 8 days after boar introduction, six did not show oestrus before slaughter and were considered non-receptive. Salivary steroidome analysis was performed for six receptive and six non-receptive gilts using gas chromatography coupled to tandem mass spectrometry. Four saliva samples per gilt were analysed: 26 days and 11 days before boar introduction (day0-26 and day0-11), the day of boar introduction (day0), 3 days later for receptive gilts (day0+3) or 7 days later for non-receptive gilts (day0+7). Saliva analysis detected 30 steroids. The concentrations of six of them were higher (P < 0.05) in receptive gilts than in non-receptive gilts at day0-26 (progesterone), day0-11 (3520-hexahydroprogesterone, 3520-hexahydroprogesterone, dehydroepiandrosterone, androstenediol) and day0 (35-tetrahydroprogesterone). Their low and variable concentrations in saliva require expensive analysis and limit their use in pig farms. However, progesterone could be an interesting biomarker of the pre-puberty period.

2019

Analyse des stéroïdes dans la salive de cochettes pour identifier des biomarqueurs de la période de réceptivité à l'effet mâle

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Poster présenté par Ghylène Goudet (INRA) et al., aux 51es Journées de la Recherche Porcine, 5 et 6 février 2019

En élevage porcin,la conduite en bandes présente des avantages pour la gestion des animaux et l'organistion de l'élevage. Pour synchroniser les cycles des cochettes et les intégrer dans les bandes, la majorité des éleveurs administre un agoniste de synthèse de la progestérone. Afin de limiter le recours aux traitements hormonaux, notre objectif à long terme est de développer des alternatives à l'utilisation des hormones.  Avant la puberté, les cochettes  atteignent un stade de pré-puberté au cours duquel une exposition au verrat (effet mâle) favoriserait le déclenchement et la synchronisation de la première ovulation.  L'objectif de cette étude est d'améliorer le repérage des femelles à exposer au verrat en identifiant dans la la salive des biomarqueurs de la phase de pré-puberté.

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2019

Salivary and urinary metabolome analysis for pre-puberty-related biomarkers identification in porcine

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Gylène Gaudet et al., Animal, 2018, 5 septembre, 11 pages

Estrus synchronization is important for optimal management of gilt reproduction in pig farms. Hormonal treatments, such as synthetic progestogens, are used on a routine basis, but there is a growing demand for non-hormonal alternative breeding tools. Before puberty, gilts exhibit a 'waiting period,' related to the ovarian development and gonadotrophin secretions, during which external stimulations, such as boar exposure, could induce and synchronize first ovulation. Practical non-invasive tools for identification of this period in farms are lacking. During this period, urinary oestrone levels are high, but urine sampling is difficult in group-housed females. The aim of this work was to search for specific biomarkers of the 'waiting period' in saliva and urine. In total, nine 144- to 147-day-old Large White gilts were subjected to trans-abdominal ultrasonography three times a week for 5 weeks until puberty detection (week -5 to week -1 before puberty). Urine and saliva samples were collected for oestrone assay to detect the 'waiting period' and for metabolome analysis using 1H-nuclear magnetic resonance spectroscopy to detect potential biomarkers of the 'waiting period.' Gilts were slaughtered 7 days after puberty detection for puberty confirmation. Results were consistent with ultrasonography data for six gilts. Urine and saliva samples from these six gilts were analyzed. Urinary estrone concentration significantly increased 2 weeks before puberty detection. Metabolome analysis of urine samples allowed the identification of 78 spectral bins, among them, 42 low-molecular-weight metabolites were identified. Metabolome analysis of salivary samples allowed the identification of 59 spectral bins, among them, 23 low-molecular-weight metabolites were detected and 17 were identified. No potential biomarker was identified in urinary samples. In saliva, butyrate and 2HOvalerate, 5.79 ppm (putatively uridine), formate, malonate and propionate could be biomarker candidates to ascertain the pre-puberty period in gilt reproduction. These results confirm that non-invasive salivary samples could allow the identification of the physiological status of the gilts and presumably the optimal time for application of the boar effect. This could contribute to synchronize puberty onset and hence to develop non-hormonal breeding tools.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/FA6DFF10CB490FA7F83C89B8F3696779/S1751731118002161a.pdf/salivary_and_urinary_metabolome_analysis_for_prepubertyrelated_biomarkers_identification_in_porcine.pdf

2018

Mesure des concentrations des stéroïdes dans la salive de cochettes immatures, pré-pubères et pubères

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Ghylène Goudet et al., 49es Journées de la Recherche Porcine, Paris, 31 janvier et 1er février 2017, p. 175-176, poster

L’élevage porcin conventionnel se caractérise par une conduite en bandes qui présente de nombreux avantages techniques (surveillance des mises-bas, ajustement de la taille des portées, gestion des porcelets…) et sanitaires (nettoyage-désinfection des locaux entre bandes). Une majorité d’éleveurs administrent des agonistes de synthèse de la progestérone pour synchroniser les cycles des cochettes de renouvellement et les intégrer dans les bandes (Boulot et al., 2005). Les effets négatifs des résidus hormonaux sur la santé humaine et l’environnement conduisent à mettre en place de nouvelles pratiques d’élevage. Notre objectif à long terme est de développer des alternatives aux traitements hormonaux pour la synchronisation des oestrus des cochettes, notamment lors de l’entrée dans la première bande.
Avant la première ovulation, les cochettes atteignent un stade physiologique de pré-puberté au cours duquel une stimulation externe peut déclencher la première ovulation. L’exposition au verrat (appelée effet mâle) pourrait favoriser le déclenchement et la synchronisation de la puberté si elle est appliquée pendant cette période de pré-puberté (Prunier, 1989). Cette pratique est très peu utilisée en élevage, car le moment optimal et les modalités d’exposition au verrat ne sont pas clairement définis. Notre objectif est de mieux caractériser la phase de pré-puberté et de rechercher des biomarqueurs de cette phase à l’aide de techniques non-invasives.
Pendant la phase de pré-puberté, les concentrations d’oestrone urinaire augmentent (Camous et al., 1985). En conditions d’élevage avec logement en groupe, des prélèvements réguliers d’urine sont difficilement envisageables. Les dosages hormonaux classiques reposent sur des prélèvements sanguins trop invasifs. En revanche, le suivi de biomarqueurs salivaires est non invasif et relativement facile à mettre en place. La recherche de biomarqueurs salivaires de la phase de prépuberté permettra d’améliorer le repérage des femelles à stimuler et de diminuer le nombre de femelles mises à la reproduction alors qu’elles sont impubères.

ENG

Evaluation of steroids concentrations in the saliva of immature, prepubertal and pubertal gilts

Synchronizing oestrus in gilts is an important tool for pig producers to optimize the management of reproduction. Synthetic progestogens are used routinely in pig farms for this purpose, but there is a need for alternative non-hormonal breeding tools. Before puberty, gilts exhibit a “waiting period”, related to ovarian development and gonadotrophin secretions, during which an external stimulation, such as boar exposure, could induce and synchronize the first ovulation. Our aim was to characterize this “waiting period” and search for biomarkers of this period using non-invasive tools. During this period, an increase of urinary oestrone concentration has been observed, but urinary sampling is difficult in group-housed females. Salivary samples are non-invasive and easier to perform. Thus, the aim of this study was to evaluate steroid concentrations in the saliva of immature to pubertal gilts in order to search for biomarkers of the “waiting period”. Trans-abdominal ultrasonographies were performed over a period of 5 weeks on six 140-day-old Large White gilts until puberty detection. Urinary samples were collected for oestrone assay to detect the “waiting period”. Salivary samples were collected for steroidome analysis using gas chromatography coupled with tandem mass spectrometry to detect potential biomarkers of the “waiting period”. Urinary oestrone concentration increased 2 weeks before puberty. Steroidome analysis allowed the quantification of 28 steroids, 13 of them showing significant differences between weeks. Dehydroepiandrosterone concentrations decreased and estradiol-17 concentrations increased significantly just before the “waiting period”. These steroids could be biomarkers of this period. Present results confirm that non-invasive salivary samples could allow the identification of the physiological status of the gilts and the optimal time for application of the boar effect.

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2017

Evaluation of steroid concentrations in the saliva of pre-pubertal gilts for the identification of biomarkers of the pubertal stage of maturity

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Ghylène Goudet et al., ESDAR, 24-26 août 2017, Bern, Suisse, abstract

Estrus synchronization is important for optimal management of gilt reproduction in farms. Synthetic progestagens are used for this purpose, but there is growing demand for non-hormonal alternatives. Before puberty, gilts exhibit a “waiting period”, related to ovarian development and gonadotrophin secretions, during which external stimulations, such as boar exposure, could induce and synchronize first ovulation. Practical non-invasive tools for identification of this period in farms are lacking. During this period, urinary estrone levels are high, but urine sampling is difficult in group-housed females. Our aim was to search for steroidal biomarkers of this “waiting period” from immature to pubertal gilts through saliva monitoring.
Six 140-day-old Large White gilts were subjected to ultrasound puberty diagnosis 3 times a week for 5 weeks until first ovulation. Urine and saliva samples were collected at the same frequency for estrone assay and steroidome analysis respectively. Urinary estrone concentration significantly increased 2 weeks before puberty (detected at 182-192 days). Steroidome analysis quantified 28 steroids in saliva. Significant variations were detected within 2 weeks before puberty for dehydroepiandrosterone (decrease) and estradiol-17&a538; (increase). These steroids could be biomarkers of the “waiting period”.
These results confirm that non-invasive salivary sampling could allow the identification of the physiological status of the gilts and presumably the optimal time for application of the boar effect.

Document réservé Espace Pro, veuillez vous identifier
2017

Exactitude d'un kit de dosage rapide de la progestérone chez la truie

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Despite new applications of ultrasonography, blood progesterone is still a reference diagnostic tool for the assessment of reproductive status in many species, including the sow (2, 5). Techniques based on saliva or fecal samples (3), have been investigated, but only serum semi-quantitative ELISA (1) can be implemented at low costs. This study was designed to validate a new ELISA kit, for rapid field determination of sow progesterone.
PDF icon Exactitude d'un kit de dosage rapide de la progestérone chez la truie
2007