Effects of “Bioactive” amino acids leucine, glutamate, arginine and tryptophan on feed intake and mRNA expression of relative neuropeptides in broiler chicks

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 （1.6 μmol） 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 100 μg） and L-arginine （20 or 200 μg） 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.

Effect of Down-regulated Expression of cPouV and cNanog on Pluripotency of Chicken(Gallus gallus) Embryonic Stem Cells(cESCs)

To explore the pluripotency maintenance and update the functional influence of pluripotency genes cNanog and cPouV in chicken （ C,a/lus gallus） embry- onic stem cells （ cESCs）, the stable RNAi vectors pSuper-cNanog and pSuper-cPouV constructed previously were used to transfect cESCs. The mRNA levels of two target genes were detected with real- time PCR. These two genes were down-regulated since the 48^th and the down-reg-lation continued with the extension of time, the interference efficiency reached 65% at 96^th hour （P 〈0.05）. With the down-regulation of cNanog or cPouV gene, cESCs showed differentiation and prolifera- tion rate of these cells slowed down, the domed colony of these cells disappeared gradually when the edge of colony became irregular. At 96^th hour after transfection, the alkline phosphatase （AKP） and stage-specific embryonic antigen-1 （ SSEA-1 ） were not be detected in cNanog gene-knecked out eESCs, but it was done in that with cPouV gene -knocked out. The cPouV-suppressing cESCs were again transfected with pSuper-cNanog, the pluripotency markers AKP and SSEA-1 were both not found expressing at the 48^th hour. The results showed that cPouV and cNartog genes played an important role in maintaining pluripotency and self- renewal in cESCs, and cNanog gene was dominant. To sum up, our results may provide insights into the molecular regulation mechanism of avian during development.

Chicken Mesenchymal Stem Cells as Feeder Cells Facilitate the Cultivation of Primordial Germ Cells from Circulating Blood and Gonadal Ridge

Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture;however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.

Dual-bionic regenerative microenvironment for peripheral nerve repair

Autologous nerve grafting serves is considered the gold standard treatment for peripheral nerve defects;however,limited availability and donor area destruction restrict its widespread clinical application.Although the performance of allogeneic decellularized nerve implants has been explored,challenges such as insufficient human donors have been a major drawback to its clinical use.Tissue-engineered neural regeneration materials have been developed over the years,and researchers have explored strategies to mimic the peripheral neural microenvironment during the design of nerve catheter grafts,namely the extracellular matrix(ECM),which includes mechanical,physical,and biochemical signals that support nerve regeneration.In this study,polycaprolactone/silk fibroin(PCL/SF)-aligned electrospun material was modified with ECM derived from human umbilical cord mesenchymal stem cells(hUMSCs),and a dual-bionic nerve regeneration material was successfully fabricated.The results indicated that the developed biomimetic material had excellent biological properties,providing sufficient anchorage for Schwann cells and subsequent axon regeneration and angiogenesis processes.Moreover,the dual-bionic material exerted a similar effect to that of autologous nerve transplantation in bridging peripheral nerve defects in rats.In conclusion,this study provides a new concept for designing neural regeneration materials,and the prepared dual-bionic repair materials have excellent auxiliary regenerative ability and further preclinical testing is warranted to evaluate its clinical application potential.