Spermatogenesis in the turkey (\u3ci\u3eMeleagris gallopavo\u3c/i\u3e): Quantitative approach in immature and adult males subjected to various photoperiods

The objectives of this study were to identify and quantitate the germ cell populations of the testes in sexually mature male turkeys (Trial 1), determine the duration of meiosis based on BrdU labeling and stereological analyses (Trial 2), and examine the impact of various photoperiods on germinal and somatic cell populations in immature and adult males (Trial 3). In Trial 1, both testes within a male had similar stereological components (P \u3e 0.05) for all parameters analyzed. In Trial 2, the duration of Type-1 spermatocytes and round spermatids in turkeys lasted 4.5 ± 0.5 and 2.0 ± 0.5 days, respectively. In Trial 3, the short photoperiod (7L:17D) delayed testicular growth (in the stereological parameters analyzed). In contrast, the effect of a moderately short photoperiod (10.5L:13.5D) was comparable to the effect of a long (14L:10D) or increasing photoperiod (7L:17D to 14L:10D) on the stereological parameters examined. With the exception of the short photoperiod, all other photoperiods used in this study induced comparable early testicular maturation, with maximum testi

Effect of various photoperiods on testicular weight, weekly sperm output and plasma levels of LH and testosterone over the reproductive season in male turkeys

The effects of duration and variation in photoperiod on testis weight, testicular sperm production, semen output, and hormone status over the reproductive season in male turkeys were investigated. In Experiment 1, four groups of males raised from 17 to 23 wk of age under a constant short photoperiod were subjected to a constant short (Group 1: 7L:17D; Group 2: 10.5L:13.5D), constant long (Group 3: 14L:10D) or progressively increasing photoperiod (Group 4: 7L:17D to 14L:10D) up to 60 wk of age. In Experiment 2, four groups of males first raised as in Experiment 1 up to 23 wk of age were placed under a constant short (Group 5: 10.5L:13.5D), constant long (Group 6: 14L:10D), or night-interrupted photoperiod (Group 7: 6L:2.5D:1L:14.5D, referred to as subjective 9.5L:14.5D; Group 8: 6L:3.5D:1L:13.5D), referred to as subjective 10.5L:13.5D) up to 60 wk of age. Males in Groups 2–4 had similar reproductive characteristics, whereas sexual maturity was delayed from 29 to 49 wk in males from Group 1. In Experiment 2, males in Groups 5 and 8 had similar reproductive characteristics, whereas sexual maturity was delayed in males in Group 7 in a manner similar to that observed in Group 1. In both experiments, plasma LH and testosterone concentrations were poor indicators of testis development and semen production, irrespective of age and photoperiod. We conclude that a moderately short photoperiod such as 10.5L:13.5D or subjective 10.5L:13.5D may stimulate reproductive characteristics of male turkeys in a manner comparable to constant long or increasing photoperiods. We inferred the existence of a threshold of photosensitivity in male turkeys for photoperiods longer than 9.5L:14.5D, but shorter than or equal to 10.5L:13.5D

Rearing conditions during the force-feeding period in male mule ducks and their impact upon stress and welfare

The aim of the first experiment conducted was to further characterise HPA axis functionality in male mule ducks during the force-feeding period, by measuring corticosterone levels (Exp. 1). The objectives of the two other experiments were to investigate the impact of different rearing conditions on stress response (Exp. 2) and behaviour patterns (Exp. 3) in male mule ducks. The rearing conditions examined comprised individual (Exps. 1–3) and collective battery cages (Exps. 2, 3), as well as collective floor pens (Exps. 2, 3). The ducks were then fed (Exps. 1–3) by force-feeding for foie gras production or ad libitum (Exp. 1). The highest levels of corticosterone (up to 100 ng$\cdot$mL$^{-1})$ were measured after initial capture and handling in a large collective rearing pen, transfer to a different environment, initial placement in a net for 10 min and injection of 5 $\mu$g$\cdot$kg$^{-1}$ BW of 1-24 ACTH agonist. Both force-fed and non-force fed male mule ducks responded to a first physical constraint in a net by a large increase in corticosterone levels. Their HPA axis was therefore functional although the effect quickly vanished, which was interpreted as an indication that habituation took place. Most often, corticosterone levels measured before and after force-feeding during the force-feeding period did not differ significantly ($P > 0.05$) when the ducks were raised in individual cages, even on the first occurrence. A significant increase in corticosterone levels was observed after the first force-fed meal for both groups of ducks raised collectively, i.e. in cages or floor pens, when the practice involved capture and handling. Following the injection of 0.625 and 5 $\mu$g$\cdot$kg$^{-1}$ BW doses of 1-24 ACTH, cortico-adrenal responses were significantly ($P < 0.05$) higher and lower for ducks raised in collective floor pens compared to those raised in individual cages, respectively. This hypersensitivity and lower maximal capacity may result from a chronic stressful state related to repeated acute stress (i.e. capture and handling twice daily). Ducks raised in cages (individual or collective) spent more time standing (less lying) and less time inactive i.e. expressing passive behaviour patterns, which suggest that they were not presenting signs of passive coping or learned helplessness. Behaviour observations did not provide any indication of stereotyped behaviour. From these results, it could have been concluded that placement in individual battery cages the limited period of force-feeding are not detrimental in terms of welfare. However, they cannot achieve full wing stretching or express a full range of social behaviours as required by the European Council recommendation (Scientific Committee on Animal Health and Animal Welfare, Welfare aspects of the production of foie gras in ducks and geese, CEC, DGXXIV/B3/AW/R06, 1998, 94 p.). They may also have more difficulty in thermoregulating as indicated by the fact that they had higher frequencies of both panting and watering. In terms of welfare, since signs of acute and possibly chronic stress were observed when the force-feeding procedure involved capture and handling, there is a need to set up new models of collective cages and better define the optimal group size and density to be used in future rearing conditions.Impacts sur les réponses de stress et le bien-être des conditions d'élevage du canard mulard mâle durant la période de gavage. Dans une première expérience (Exp. 1), la fonctionnalité de l'axe hypothalamus-hypophyse-surrénales (HPA) a été caractérisée durant la période de gavage chez le canard mulard, par mesure de la corticostéronémie (Exp. 1). Les objectifs des autres expériences réalisées étaient d'analyser les effets des conditions d'hébergement sur les réponses de stress (Exp. 2) et les comportements exprimés (Exp. 3). Après avoir été élevés collectivement au sol, les canards ont été transférés en cages batteries individuelles (Exps. 1–3), ou collectives (Exps. 2, 3), ou en loges collectives au sol (Exps. 2, 3). Les canards s'alimentent spontanément ad libitum (Exp. 1) ou reçoivent une alimentation par gavage pour la production de foie gras (Exps. 1–3). Les corticostéronémies les plus élevées (jusqu'à 100 ng$\cdot$mL$^{-1}$ plasma) ont été mesurées avant transfert lors d'une première capture, immédiatement après transfert, après une première contention dans un filet pendant 10 minutes et après l'injection de 5 $\mu$g$\cdot$kg$^{-1}$ PV de 1-24 ACTH. Les canards, gavés ou non, ont répondu par une élévation significative de leur corticostéronémie après une première contention dans un filet pendant 10 minutes. La réponse s'est progressivement estompée en dépit du fait que l'axe HPA était fonctionnel ; résultats qui suggèrent la mise en place d'un processus d'habituation. Au cours de la période expérimentale, l'acte de gavage n'induit généralement pas d'élévation ($P > 0,05$) de la corticostéronémie, chez les canards placés en cage individuelle. Ce résultat suggère que l'acte de gavage n'est pas perçu comme un stress aigu majeur par le canard mulard dans ces conditions expérimentales. Une augmentation significative de la corticostéronémie a été observée après le premier repas de gavage pour les canards des deux groupes élevés collectivement (c.a.d. cage et loge au sol) ; conditions dans lesquelles la pratique de l'acte nécessite une capture et une contention. Les réponses en corticostérone mesurées après injection i.m. de doses de 0,625 ou 5 $\mu$g$\cdot$kg$^{-1}$ PV de 1-24 ACTH, pour ces mêmes groupes de canards, suggèrent un état d'hypersensibilité et une diminution de la capacité de réponse maximale des surrénales, qui caractérisent potentiellement un état de stress chronique. Cet état de fait a pu être engendré par la répétition de stress aigus liés à l'acte de gavage dans ces conditions expérimentales impliquant pour la pratique de l'acte gavage : capture, manipulation et contention deux fois par jour. Les observations comportementales n'ont pas permis de mettre en évidence de comportements stéréotypés chez le canard mulard. Les canards placés en cages batteries ont passé plus de temps debout et sont plus actifs ; résultat qui suggèrent qu'ils n'expriment pas de signes d'adaptation passive ou de passivité acquise. Nous pourrions donc en conclure que le placement en cage individuelle durant la période de gavage n'a pas de conséquence négative en termes de bien-être. Les canards ne peuvent toutefois dans ces conditions expérimentales réaliser l'étirement complet des ailes ainsi que divers comportements sociaux qui sont stipulés dans la recommandation du Conseil de l'Europe (Scientific Committee on Animal Health and Animal Welfare, Welfare aspects of the production of foie gras in ducks and geese, CEC, DGXXIV/B3/AW/R06, 1998, 94 p.). En outre, ils ont sans doute des besoins supérieurs en termes de thermorégulation, comme le suggère l'observation de fréquences supérieures d'halètement et d'abreuvement. Des signes de stress aigu et éventuellement chronique ayant été mis en évidence lorsque la pratique du gavage exige une capture et une contention, il est nécessaire de concevoir de nouveaux modèles de cage collective et aussi de mieux définir les tailles de groupe et les densités optimales à utiliser dans ces conditions d'hébergement

The Polyphenol Fisetin Protects Bone by Repressing NF-κB and MKP-1-Dependent Signaling Pathways in Osteoclasts

International audienceOsteoporosis is a bone pathology leading to increase fractures risk and challenging quality of life. Since current treatments could exhibit deleterious side effects, the use of food compounds derived from plants represents a promising innovative alternative due to their potential therapeutic and preventive activities against human diseases. In this study, we investigated the ability of the polyphenol fisetin to counter osteoporosis and analyzed the cellular and molecular mechanisms involved. In vivo, fisetin consumption significantly prevented bone loss in estrogen deficiency and inflammation mice osteoporosis models. Indeed, bone mineral density, micro-architecture parameters and bone markers were positively modulated by fisetin. Consistent with in vivo results, we showed that fisetin represses RANKL-induced osteoclast differentiation and activity as demonstrated by an inhibition of multinucleated cells formation, TRAP activity and differentiation genes expression. The signaling pathways NF-kB, p38 MAPK, JNK and the key transcription factors c-Fos and NFATc1 expressions induced by RANKL, were negatively regulated by fisetin. We further showed that fisetin inhibits the constitutive proteasomal degradation of MKP-1, the phosphatase that deactivates p38 and JNK. Consistently, using shRNA stable cell lines, we demonstrated that impairment of MKP-1 decreases fisetin potency. Taken together, these results strongly support that fisetin should be further considered as a bone protective agent

La fisétine protège le tissu osseux en ciblant les voies de signalisation NF-kB et MKP-1 dans les ostéoclastes et l’activité transcriptionnelle de Runx2 dans les ostéoblastes

Cette année les assises sont en partenariat avec le Pôle de compétitivité Lyonbiopôle et le cluster Nutravita, des symposiums thématiques portant sur les interactions entre la recherche académique, l’innovation et l’industrie seront proposésL’ostéoporose est une pathologie osseuse induisant une augmentation des risques de fractures et altérant la qualité de vie des patients. Les traitements actuels peuvent présenter des effets secondaires délétères. C’est pourquoi la recherche d’alternatives, notamment préventives est importante. Dans cette étude, nous avons analysé la capacité d’un polyphénol, la fisétine, à préserver la santé osseuse et les mécanismes cellulaires et moléculaires impliqués. In vivo, nous avons démontré que la consommation de fisétine prévient la perte osseuse induite par privation estrogénique ou inflammation chez la souris. En effet, la consommation de fisétine se traduit par une modulation positive de la densité minérale osseuse, de la microarchitecture osseuse et des taux de marqueurs osseux sériques. De façon intéressante, la fisétine module aussi bien les cellules ostéformatrices et sur les cellules ostéorésorbantes. Nous avons démontré qu’elle réprime la différenciation et l’activité des ostéoclastes induites par RANKL : inhibition de la formation de cellules géantes multinucléées, de leur activité TRAP et de l’expression de gènes de différenciation (CTR, TRAP, MMP9, Cathepsine K). Les voies de signalisation réprimées sont les voies NF- B, p38 MAPK, JNK ainsi que l’expression génique et protéique des facteurs de transcription clés c-Fos et NFATc1. Le mécanisme d’action de la fisétine passe par un blocage de la dégradation constitutive de MKP-1, une phosphatase inhibitrice de p38 and JNK et l’invalidation de MKP-1 par shRNA prévient l’action inhibitrice de la fisétine. Inversement, dans des préostéoblastes primaires en culture, la fisétine stimule la formation de nodules de minéralisation, leur activité phosphatase alcaline et l’expression de marqueurs de différenciation. Alors que le niveau d’expression du facteur de transcription Runx2 n’est pas modulé par la fisétine, son activité transcriptionnelle est augmentée. En effet, elle stimule l’activité luciférase de gène rapporteurs dont le promoteur porte des éléments de réponse à Runx2 et induit l’expression de gènes cibles tels que l’ostéocalcine ou le collagène de type I. Ces résultats suggèrent que la fisétine pourrait constituer un candidat intéressant dans le cadre d’une stratégie de prévention nutritionnelle de l’ostéoporos

Fisetin inhibits RANKL-induced NF-κB activity.

<p>(A). Raw264.7 osteoclast precursors were pre-incubated with DMSO as control (-) or fisetin (5 µM) for 3 hours, then induced for 5 to 60 minutes with RANKL in the presence of DMSO as control (-) or fisetin (5 µM). Total protein extracts were analyzed by western-blotting for the indicated proteins. Blots are representative of 3 independent experiments. (B). Similar experiments were conducted with increasing doses of fisetin (1 to 10 µM), for a 15 minutes incubation with RANKL. Blots are representative of 3 independent experiments. (C). Raw264.7 were transfected with NF-κB-luc reporter for 6 hours, pretreated with DMSO as control (fisetin 0 µM) or fisetin (1 to 5 µM) for 3 hours then with RANKL and DMSO as control (fisetin 0 µM) or fisetin (1 to 5 µM) for 48 additional hours before RLU measurement. RLU was related to the total protein concentration for each point. (n = 3 wells, representative of 3 independent experiments). (D). Raw264.7 were pretreated with DMSO as control (fisetin 0 µM) or fisetin (5 µM) for 3 hours, then induced with RANKL and DMSO as control (fisetin 0 µM) or fisetin (5 µM) for 6 hours and the indicated mRNAs were analyzed by RT qPCR. (n = 3 wells, representative of 3 independent experiments). For all data, (*) significantly different from control, p<0.05, (#) significantly different from RANKL-fisetin 0 µM, p<0.05.</p

Fisetin significantly prevents ovariectomy-induced bone loss.

<p>(A). Study design. One week prior ovariectomy, mice (n = 12/group) received by gavage vehicle or fisetin at 5 and 25 mg/kg. The animals were subjected to sham operation (SH) or ovariectomy (OVX), then vehicle or fisetin was administrated by gavage for 4 weeks. At the end of the experiment, the uterus were weighed (B), the femurs were analyzed for trabecular bone mineral density (BMD) (C) and micro-architecture (D and E: OVX and OVX+fisetin 25 mg/kg). BV/TV: bone volume/total volume, Tb.Th: trabecular thickness, Tb.N: trabecular number, Tb.Sp: trabecular spaces. (F). Serum CTX1 and osteocalcin were analyzed by ELISA. For all data, (*) significantly different from SH, p<0.05, (#) significantly different from OVX-fisetin 0 mg/kg, p<0.05.</p

Fisetin represses RANKL-induced osteoclast differentiation.

<p>(A). Primary bone marrow cultures cells (BMC) and osteoclasts precursors Raw264.7 were pre-incubated with DMSO as control (fisetin 0 µM) or different doses of fisetin (1 to 5 µM) for 3 hours, then induced to differentiate in the presence of RANKL and DMSO as control (fisetin 0 µM) or fisetin (1 to 5 µM). After, 7 days (BMC) or 4 days (Raw264.7), TRAP staining was performed. Scale bars correspond to 500 µm. (n = 3 wells, representative of 3 independent experiments). (B). Giant TRAP (+) multinucleated cells (MNC: more than 3 nuclei) were counted at the end of the differentiation process. (C). Raw264.7 TRAP activity was measured. (n = 3 wells, representative of 3 independent experiments). (D). Osteoclast precursors Raw264.7 were cultured for 48 hours in the presence of DMSO as control (fisetin 0 µM) or different doses of fisetin (1 to 5 µM) and the relative viability was measured by an XTT assay. (n = 8 wells, representative of 3 independent experiments). (E). Indicated mRNAs of Raw264.7 were analyzed by RT qPCR after a terminal differentiation process performed as in A. (n = 3 wells, representative of 3 independent experiments). For all data, (*) significantly different from control, p<0.05, (#) significantly different from RANKL-fisetin 0 µM, p<0.05.</p

Molecular mechanisms by which fisetin controls osteoclast differentiation and activity.

<p>Fisetin negatively controls osteoclasts differentiation process by inhibiting RANKL-induced NF-κB signaling (a), stabilizing MKP-1 (b), the phosphatase that negatively controls p38 MAPK and JNK signaling pathways, thus inhibiting their RANKL-induced activation (b’) and counteracting the RANKL-induced c-Fos expression (c). These actions result in a transcriptional repression of the key transcription factor NFATc1 and a subsequent inhibition of its target genes: CTR, TRAP, MMP9 or cathepsin K.</p