Effect of Muscle Extract and Graphene Oxide on Muscle Structure of Chicken Embryos.

The effects of CEME and it complex with GO injected in ovo on the growth and development of chicken embryo hindlimb muscle were investigated. First, the preliminary in vitro study on primary muscle precursor cell culture obtained from a nine-day-old chicken embryo was performed to assess toxicity (MTT assay) of CEME, GO (100 ppm) and it complex with different concentrations (1, 2, 5, and 10 wt.%). The effect on cell proliferation was investigated by BrdU assay. CEME at concentrations 1-5% increased cell proliferation, but not the complex with GO. In vitro cytotoxicity was highest in 10% and GO groups. Next, the main experiment with chicken embryos was performed with CEME, GO and it complex injected in ovo on day one of embryogenesis. On day 20 of embryogenesis survival, morphological development, histological structure of the muscle, and biochemical parameters of blood serum of the embryos were measured. No negative effect on mortality, body weight, or biochemistry of blood after use of CEME or GO-CEME complexes was observed. Interestingly, the slight toxicity of GO, observed in in vitro studies, was not observed in vivo. The use of CEME at the levels of 2% and 5% improved the structure of the lower limb muscle by increasing the number of cells, and the administration of 2% CEME increased the number of nuclei visible in the stained cross-section of the muscle. The complex GO-CEME did not further improve the muscle structure. The results indicate that CEME can be applied as an in ovo enhancer of muscle development in broilers.

Effects of Graphene Oxide Nanofilm and Chicken Embryo Muscle Extract on Muscle Progenitor Cell Differentiation and Contraction.

Finding an effective muscle regeneration technique is a priority for regenerative medicine. It is known that the key factors determining tissue formation include cells, capable of proliferating and/or differentiating, a niche (surface) allowing their colonization and growth factors. The interaction between these factors, especially between the surface of the artificial niche and growth factors, is not entirely clear. Moreover, it seems that the use of a complex of complementary growth factors instead of a few strictly defined ones could increase the effectiveness of tissue maturation, including muscle tissue. In this study, we evaluated whether graphene oxide (GO) nanofilm, chicken embryo muscle extract (CEME), and GO combined with CEME would affect the differentiation and functional maturation of muscle precursor cells, as well as the ability to spontaneously contract a pseudo-tissue muscle. CEME was extracted on day 18 of embryogenesis. Muscle cells obtained from an 8-day-old chicken embryo limb bud were treated with GO and CEME. Cell morphology and differentiation were observed using different microscopy methods. Cytotoxicity and viability of cells were measured by lactate dehydrogenase and Vybrant Cell Proliferation assays. Gene expression of myogenic regulatory genes was measured by Real-Time PCR. Our results demonstrate that CEME, independent of the culture surface, was the main factor influencing the intense differentiation of muscle progenitor cells. The present results, for the first time, clearly demonstrated that the cultured tissue-like structure was capable of inducing contractions without externally applied impulses. It has been indicated that a small amount of CEME in media (about 1%) allows the culture of pseudo-tissue muscle capable of spontaneous contraction. The study showed that the graphene oxide may be used as a niche for differentiating muscle cells, but the decisive influence on the maturation of muscle tissue, especially muscle contractions, depends on the complexity of the applied growth factors.

Effect of taurine and gold nanoparticles on the morphological and molecular characteristics of muscle development during chicken embryogenesis.

The objective of the present investigation was to evaluate the effects of taurine and Au nanoparticles on the expression of genes related to embryonic muscle development and on the morphological characteristics of muscles. Fertilised chicken eggs (n = 160) were randomly divided into four groups: without injection (Control) and with injection of Au nanoparticles (NanoAu), taurine (Tau) or Au nanoparticles with taurine (NanoAu + Tau). The experimental solutions were given in ovo, on the third day of incubation, by injecting 0.3 ml of the experimental solution into the air sack. The embryos were evaluated on the 20th day of incubation. The methods included gene expression at the mRNA and protein levels, immunohistochemistry, histology and microscopy. In groups NanoAu, Tau and NanoAu + Tau, the muscle structure and the number of muscle cells were affected. Furthermore, taurine increased fibre diameter, the total number of nuclei, the proportion of proliferating cell nuclear antigen (PCNA)-positive cells and the total cell number. Also, gene expression of basic fibroblast growth factor-2 and PCNA was downregulated. There were no significant interactions between NanoAu and taurine, indicating that NanoAu did not enhance the effects of taurine. It may be concluded that 20 days after injection, NanoAu affected some parameters of muscle development, but the most profound effects were those of taurine.

Effect of heparan sulfate and gold nanoparticles on muscle development during embryogenesis.

PURPOSE: It was hypothesized that heparan sulfate (HS) as an essential compound for myogenesis and nanoparticles of gold (nano-Au) as highly reactive compounds can affect muscle development as a consequence of molecular regulation of muscle cell formation, and that these effects may be enhanced by a complex of HS conjugated with nano-Au. The objective of the present study was to determine the effect of administration of nano-Au, HS, and a nano-Au+HS complex on the morphological and molecular characteristics of breast muscle during embryogenesis. METHODS: Chicken embryos were used as in vivo model. Fertilized chicken eggs (n = 350) were randomly divided into the control group and the groups treated with nano-Au, HS, and nano-Au+HS. The experimental solutions were given in ovo on the first day of incubation and the embryos were evaluated on day 20 of incubation. The methods included biochemical indices in blood, immunohistochemistry, microscopy (transmission electron microscopy, scanning electron microscopy, confocal), and gene expression at the messenger ribonucleic acid and protein levels. RESULTS: The treatments did not adversely affect mortality, organ weight, and homeostasis of the embryos. HS stimulated the development and maturation of breast muscle by increasing the number of nuclei, satellite cells, and muscle fibers and affected the expression of basic fibroblast growth factor-2 and paired-box transcription factor-7. Furthermore, the nano-Au+HS complex contributed to the increased number of myocytes and nuclei in chicken embryo muscles. CONCLUSION: The results indicate that the administration of HS and nano-Au affects muscle development and that this effect is enhanced by conjugating HS with nano-Au.

Influence of hydrocolloidal silver nanoparticles on gastrointestinal microflora and morphology of enterocytes of quails.

The objective of the present study was to examine the effects of hydrocolloidal silver nanoparticles (Ag-nano) on microbial profile of caecum and morphology of enterocytes in duodenum of Japanese quail, as a model animal for poultry. Quails (Coturnix coturnix japonica) (10 d old) were randomly divided into four groups (15 quails each) and located into four cages for 12 days. Quails were fed with granulated diets given ad libitum and had free access to drinking water. Ag-nano were added to drinking water at concentrations of 0, 5, 15 and 25 mg/kg. At the end of the experiment, the animals were killed and samples of duodenum and caeca microflora were collected. This initial investigation demonstrated that silver nanoparticles did not influence emphatically microflora of quail caecum; however, water containing 25 mg/kg of Ag-nano significantly increased the population of lactic acid bacteria. Furthermore, Ag-nano did not show any damaging properties on enterocytes of duodenal villi.