In biological research, chicken embryos are a classic experimental model for the exploration of the embryonic development and cell differentiation.  Transferring exogenous substances into chicken embryos for producing medical antibodies has been widely used in the production practice.  However, there are few studies about the effect of the different injection site and dosage on chicken embryos.  The aim of this study was to explore the effects of different injection sites and dosages on chicken embryo hatching rate and development, so as to provide a basis for further studies using the chicken embryo model.  Freshly laid eggs (Rugao yellow chicken) were injected with different doses of saline at the tip, equatorial plane and the blunt end of the egg shell, respectively.  Egg hatching rate was recorded and compared among injection sites and different doses.  A trypan blue stain was also injected at the aforementioned sites and the growth of chicken embryos was observed.  The SPSS (statistical package for the social science) software was used to analyze the relationship between the chicken eggs hatching rate and the different injection sites or the different dosages.  The experimental results showed that there were significant differences on egg hatching rates among the different injection sites and doses (P<0.05).  The hatchability of the blunt end injection group was significantly higher than that of the other two sites.  The egg hatching rate decreased with increased saline doses.  The egg hatching rate of the 100 µL saline injection group was higher than the 200 and 300 µL dosage groups.  Ultimately, we suggest that the optimal chicken embryo injection process is during early development, at the blunt end site with a dose less than 100 µL to minimize damage to the egg.

Spermatogonial stem cells (SSCs) form the foundation for spermatogenesis and sustain male fertility. To explore the regulatory mechanisms of chicken SSCs generation, we obtained highly purified chicken embryonic stem cells (ESCs), primordial germ cells (PGCs) and SSCs by fluorescence-activated cell sorting (FACS). High-throughput analysis methods (RNA-Seq) were used to sequence the transcriptome level of these cells. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment were used to analyze RNA-Seq results. BMP4 was used to induce chicken ESCs differentiation to SSCs-like cells in vitro. The quantitative real-time (qRT)-PCR was used to detect the expression changes of the key genes. The results showed that 22 relevant critical pathways were found by RNA-Seq, one of them was the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. Total of 103 related genes were detected in this pathway. Protein-protein interactions analysis found that 87 proteins were significantly related to 19 key proteins in this pathway. These 87 proteins were enriched in 21 biological processes and 18 signaling pathways. Moreover, during the differentiation of chicken ESCs to SSCs-like cells induced by BMP4 in vitro, JAK2 and STAT3 were activated. The qRT-PCR results showed that the expression trends of JAK2 and STAT3 were basically the same as in vivo. We concluded that JAK/STAT signaling pathway plays an important role in the process of chicken SSCs generation both in vivo and in vitro; it may achieve its function through multiple biological processes and other related pathways.