Bone marrow mesenchymal stem cells(BMSCs) could differentiate into various cell types including adipocytes and myocytes, which had important scientific significance not only in the field of tissue regeneration, but also in the field of agricultural science. In an attempt to exhibit the characterization and differentiation into adipocytes and myocytes of porcine BMSCs, we isolated and purified porcine BMSCs by red blood cell lysis method and percoll gradient centrifugation. The purified cells presented a stretched fibroblast-like phenotype when adhered to the culture plate. The results of flow cytometry analysis and immunofluorescence staining demonstrated that the isolated cells were positive for mesenchymal surface markers CD29, CD44 and negative for hematopoietic markers CD45 and the adhesion molecules CD31. Cells were induced to differentiate into adipocytes with adipogenic medium containing insulin, dexamethasone, oleate and octanoate. Oil Red O staining demonstrated that the porcine BMSCs successfully differentiated to adipocytes. Moreover, the findings of real-time PCR and Western blotting indicated that the induced cells expressed adipogenic marker genes(PPAR-γ, C/EBP-α, perilipin, aP2) mRNA or proteins(PPAR-γ, perilipin, aP2). On the other hand, porcine BMSCs were induced into myoctyes with myogenic medium supplemented with 5-azacytidine, basic fibroblast growth factor, chick embryo extract and horse serum. Mor phological observation by hochest 33342 staining showed that the induced cells presented as multi-nucleus muscular tube structure. And myogenic marker genes(Myf5, desmin) mRNA or proteins(Myf5, MyoD, myogenin, desmin) were found in the induced cells. In addition, the results of immunofluorescence staining revealed that myogenic marker(Myf5, MyoD, myogenin, desmin, S-MyHC) proteins was positive in the induced cells. Above all, these results suggested that the isolated porcine BMSCs were not only consistent with the characterization of mesenchymal stem cells, but also exhibited the multipotential
Feed intake control is vital to ensuring optimal nutrition and achieving full potential for growth and development in poultry. The aim of the present study was to investigate the effects of L-leucine, L-glutamate, L-tryptophan and L-arginine on feed intake and the mRNA expression levels of hypothalamic Neuropeptide involved in feed intake regulation in broiler chicks. Leucine, glutamate, tryptophan or arginine was intra-cerebroventricularly (ICV) administrated to 4d-old broiler chicks respectively and the feed intake were recorded at various time points. Quantitative PCR was performed to determine the hypothalamic mRNA expression levels of Neuropeptide Y (NPY), agouti related protein (AgRP), pro-opiomelanocortin (POMC), melanocortin receptor 4 (MC4R) and corticotrophin releasing factor (CRF). Our results showed that ICV administration of L-leucine (0.15 or 1.5 μmol) significantly (P<0.05) increased feed intake up to 2 h post-administration period and elevated both hypothalamic NPY and AgRP mRNA expression levels. In contrast, ICV administration of L-glutamate (1.6μmol) significantly (P<0.05) decreased feed intake 0.25, 0.5 and 2 h post-injection, and increased hypothalamic CRF and MC4R mRNA expression levels. Meanwhile, both L-tryptophan (10 or 100μg) and L-arginine (20 or 200μg) had no significant effect on feed intake. These findings suggested that L-leucine and L-glutamate could act within the hypothalamus to influence food intake, and that both orexigenic and anorexigenic Neuropeptide genes might contribute directly to these effects.
Different amino acids have been shown to affect feed intake when injected directly into the central nervous system of birds. In the present study, we investigated the effects of L-glutamine and L-alanine on feed intake and the mRNA expression levels of hypothalamic neuropeptides involved in feed intake regulation in broiler chicks. L-Glutamine or L- alanine was intra-cerebroventricularly (ICV) administered to 4-d-old broiler chicks and the feed intake were recorded at various time points. Quantitative PCR was performed to determine the hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti related protein (AgRP), pro-opiomelanocortin (POMC), melanocortin receptor 4 (MC4R) and corticotropin releasing factor (CRF). Our results showed that ICV administration of L-glutamine (0.55 or 5.5 μmol) significantly increased feed intake up to 2 h post-administration period and the hypothalamic NPY mRNA expression levels, while it markedly decreased hypothalamic POMC and CRF mRNA expression levels. In contrast, ICV administration of L-alanine (4 μmol) significantly decreased feed intake for the first 0.5 h post-administration period, and reduced the hypothalamic AgRP mRNA expression levels, while it remarkablely enhanced the mRNA expression levels of MC4R and CRF. These findings suggested that L-glutamine and L-alanine could act within the hypothalamus to influence feed intake in broiler chicks, and that both orexigenic and anorexigenic neuropeptide genes might contribute directly to these effects.
