| The present study first demonstrated the difference in hypothalamic expression of genes regulating appetite and energy homeostasis, such as corticotropin-releasing hormone (CRH), glucocorticoid receptor (GR) and fat mass and obesity associated (FTO) gene, between layer and broiler chicks. Secondly, the leptin and corticosterone contents in the egg were also different between breeds, which were higher in the yolk of broilers. To test the hypothesis that maternal leptin play an important role in posthatch growth and energy homeostasis, leptin and its antagonist were administered in ovo to investigate the programming effect on early posthatch growth, as well as hypothalamic expression of genes regulating appetite and energy homeostasis, especially CRH and GR.1 Comparative studies of the appetite and energy homeostasis regulation genes in the hypothalamus of layer and broiler chicksLayer and broiler chickens demonstrate striking differences in body weight and body composition. However, the mechanism underlying such difference is still elusive. The body weight and food intake were already significantly higher in broiler chicks at day (D) 7 posthatching, but the food intake relative to body weight gain (food conversion ration, FCR) was actually higher in layer chicks. No breed differences were observed for hypothalamic expression of neuropeptide Y (NPY), agouti-related protein (AGRP), pro-opiomelanocortin (POMC), orexin (ORX), leptin receptor (LEPR) and 20-hydroxysteroid dehydrogenase (20HSD) at D7 by in situ hybridization (ISH) and/or real-time RT-PCR. A tendency higher expression of hypothalamic acetyl CoA carboxylase (ACC) or fatty acid synthase (FAS) mRNA was found in broilers than layers (P<0.10). Broiler chicks expressed lower CRH mRNA (P<0.05) yet higher accumulation of CRH peptide in hypothalamus, which means the different levels in transcription, translation and release of CRH. However, broiler chicks expressed significantly higher GR mRNA (P<0.05) and protein (P<0.01) in hypothalamus compared to layer chicks, suggesting an augmented GR-mediated negative feedback regulation of CRH transcription and release in broiler chicks. Furthermore, FTO gene was also more highly expressed in hypothalamus of broiler chicks (P<0.05). These results suggest that the genes related to energy balance and obesity, such as GR, CRH and FTO, rather than orexigenic neuropeptides, are impacted by the genetic selection practices and play a role in the regulation of body weight and energy homostasis in chicken.2 Analysis of the methylation levels of CRH and GR gene promoters in hypothalamus and egg hormone contents in layer and broler chicksTo investigate whether the gene expression differences were related to the DNA methylation and associated with breed dfferences in maternal hormone deposition in the egg, the methylation levels of CRH and GR gene promoter in the hypothalamus, and the leptin and corticosterone contents in the egg were determined in layer and broiler chickens. No difference was observed in the hypothalamic CRH or GR gene promoter methylation levels (P>0.05) between layer and broiler chicks by methylation-sensitive restriction enzyme PCR. Radioimmunoassay (RIA) and enzyme immunoassay (EIA) methods showed higher levels of yolk leptin and corticosterone in broiler eggs than layer eggs (P<0.05). The results suggested that the maternal hormones, such as leptin and corticosteron, are different in breeds. However the further research should be continued to identify the relationship between maternal hormones and breed differences.3 Effects of in ovo leptin administration on the appetite and energy homeostasis regulation genes in the hypothalamus of broiler chicksLeptin in ovo administration was employed to investigate the effect of leptin deposited in eggs on the expression of appetite and energy homeostasis regulation genes in the hypothalamus of broiler chicks. Breeder eggs were randomly allocated to three groups and injected with 0.5μg (low leptin, LL),5.0μg (high leptin, HL) of recombinant mice leptin in 100μL of PBS or 100μL PBS (Control, Con) before incubation. The hatchings were raised under the same condition till 21 days (D21) of age when the samples were collected for analysis. The early posthatch growth rate of broiler chicks was influenced by leptin which showed gender specific and dose dependent. Chicks treated with high or low doses of leptin in ovo had lower hatch weight than the control group, especially the female chicks in low leptin group (P<0.01). The posthatch growth rate was higher in both leptin-treated groups compared to control group. At D21, chicks treated with leptin in ovo demonstrated significantly higher body weight than control group (P=0.042, P=0.003). The hypothalamic gene and protein levels of 21-old-day chicks were measured by real-time RT-PCR or Western blotting. Except for the CRH mRNA, the expressions of hypothalamic LEPR, FAS and GR mRNA were decreased in male chicks of HL group (P<0.05). Significantly higher FTO mRNA (P<0.01) levels were detected in the hypothalamus of male chicks in both leptin groups. In the hypothalamus of female chicks, GR mRNA levels were decreased in LL group (P<0.05). The CRH protein levels were higher in the hypothalamus of male chicks in HL group (P<0.05). Hypothalamic GR protein levels were significantly elevated by both low and high doses of leptin in ovo (P<0.01). No changes were found in female chicks. The GR mRNA was widely distributed in the brain of D21 broilers by in situ hybridization, mainly in the corpus striatum, nucleus rotundus (ROT), dorsolateral nucleus (DLN), nucleus ovoidalis (OV), nucleus reticularis superior (Rsd) and the preoptic area (POA) of hypothalamus. The GR mRNA signals was also detected in lateral hypothalamus (LHy), paraventricular nucleus (PVN), periventricular nucleus (PHN), ventromedial nucleus (VMN), infundibular nucleus (IN) and hippocampus (Hp). In high or low doses of leptin group, the expressions of GR mRNA were found down regulated in PVN, PHN and VMN (P<0.05) of D21 broiler chicks. However, no difference was found in LHy, IN or Hp of D21 broiler chicks among groups (P>0.05). These results indicate that in ovo administration of leptin influence the early posthatch development of broiler chicks, and the expressions of appetite and energy homeostasis regulation genes, especially the GR.4 Effects of in ovo leptin and its antagonist administration on the appetite and energy homeostasis regulation genes in the hypothalamus of broiler chicksLeptin and its antagonist in ovo administration were employed to further investigate the effect and mechanism of leptin deposited in eggs on the appetite and energy homeostasis regulation genes in the hypothalamus of broiler chicks. Breeder eggs were randomly allocated to four groups and injected with recombinant mice leptin (0.5μg, L), recombinant mice leptin antagonist (0.5μg, LA), leptin (0.5μg) plus leptin antagonist (0.5μg) (L+LA) in 100μL of PBS or 100μL PBS (Control, Con) before incubation. The hatchings were raised under the same condition till 21 days (D21) of age when the samples were collected for analysis. The early posthatch growth rate of broiler chicks was influenced by leptin and its antagonist which showed gender specific. All treated group chicks had no difference on hatch weight with the control group (P>0.05). Compared with other groups, the posthatch body weight and relative body weight gain were elevated in leptin group of both male and female broiler chicks (P<0.05). The food intake of broiler chicks was increased in leptin group, and significantly higher than control group (P<0.05). However, the antagonist had no effect on the body weight and food intake compared with control group chicks. Using RIA, higher serum leptin levels were detected in female chicks of both LA and L+LA group than contol and leptin group at D21 (P<0.05). The expressions of LEPR, CRH and GR mRNA or protein in the hypothalamus of broiler chicks were measured by real-time RT-PCR and Western blotting. At D21, female chicks in leptin group showed lower expressions of GR mRNA than control group (P<0.05). The LEPR mRNA levels were higher in male chicks of L+LA group than control group, wheras the CRH mRNA levels were lower. Only the GR protein levels were found elevated in male chicks of leptin group than control (P<0.01) and L+LA group (P<0.05). Furthermore, at D21 female chicks, the GR mRNA levels were decreased in PVN and PHN of leptin group compared with control group (P<0.05), wheras the GR mRNA levels were down regulated in PHN (P<0.01) and LHy (P<0.05) of L+LA group by fluorescence in situ hybridization (FISH). These results indicate that leptin has the effect on the early posthatch development of broiler chickens. CRH and GR play an important role in the programming effect of leptin, and regulate the body weight and energy homeostasis of chickens. |
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