Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.

In mammals, Myostatin (MSTN) is a known negative regulator of muscle growth and development, but its role in birds is poorly understood. To investigate the molecular mechanism of MSTN on muscle growth and development in chickens, we knocked out MSTN in chicken fetal myoblasts (CFMs) and sequenced the mRNA transcriptomes. The amplicon sequencing results show that the editing efficiency of the cells was 76%. The transcriptomic results showed that 296 differentially expressed genes were generated after down-regulation of MSTN, including angiotensin I converting enzyme (ACE), extracellular fatty acid-binding protein (EXFABP) and troponin T1, slow skeletal type (TNNT1). These genes are closely associated with myoblast differentiation, muscle growth and energy metabolism. Subsequent enrichment analysis showed that DEGs of CFMs were related to MAPK, Pl3K/Akt, and STAT3 signaling pathways. The MAPK and Pl3K/Akt signaling pathways are two of the three known signaling pathways involved in the biological effects of MSTN in mammals, and the STAT3 pathway is also significantly enriched in MSTN knock out chicken leg muscles. The results of this study will help to understand the possible molecular mechanism of MSTN regulating the early differentiation of CFMs and lay a foundation for further research on the molecular mechanism of MSTN involvement in muscle growth and development.

Despite the notable progress made in recent years, the understanding of the genetic control of gonadal sex differentiation and asymmetrical ovariogenesis in chicken during embryonic development remains incomplete. This study aimed to identify potential key genes and speculate about the mechanisms associated with ovary and testis development via an analysis of the results of PacBio and Illumina transcriptome sequencing of embryonic chicken gonads at the initiation of sexual differentiation (E4.5, E5.5, and E6.5). PacBio sequencing detected 328 and 233 significantly up-regulated transcript isoforms in females and males at E4.5, respectively. Illumina sequencing detected 95, 296 and 445 DEGs at E4.5, E5.5, and E6.5, respectively. Moreover, both sexes showed asymmetrical expression in gonads, and more DEGs were detected on the left side. There were 12 DEGs involved in cell proliferation shared between males and females in the left gonads. GO analysis suggested that coagulation pathways may be involved in the degradation of the right gonad in females and that blood oxygen transport pathways may be involved in preventing the degradation of the right gonad in males. These results provide a comprehensive expression profile of chicken embryo gonads at the initiation of sexual differentiation, which can serve as a theoretical basis for further understanding the mechanism of bird sex determination and its evolutionary process.

Pullorum disease, an intestinal disease in chickens caused by Salmonella enterica serovar pullorum (S. Pullorum), is a significant threat to the poultry industry and results in substantial economic losses. The bacteria’s transmission, both vertical and horizontal, makes it difficult to completely eliminate it. Control strategies for pullorum disease primarily involve stringent eradication programs that cull infected birds and employ antibiotics for treatment. However, eradication programs are costly, and antibiotic use is restricted. Therefore, developing alternative control strategies is essential. Increasingly, studies are focusing on modulating the gut microbiota to control intestinal diseases. Modulating the chicken gut microbiota may offer a novel strategy for preventing and controlling pullorum disease in poultry. However, the impact of S. Pullorum on the chicken gut microbiota has not been well established, prompting our exploration of the relationship between S. Pullorum and the chicken gut microbiota in this study. In this study, we initially analyzed the dynamic distribution of the gut microbiota in chickens infected with S. Pullorum. Alpha diversity analysis revealed a decrease in observed OTUs and the Shannon diversity index in the infected group, suggesting a reduction in the richness of the chicken gut microbiota due to S. Pullorum infection. Principal coordinate analysis (PCoA) showed distinct clusters between the gut microbiota of infected and uninfected groups, indicating S. Pullorum infection changed the chicken gut microbiota structure. Specifically, S. Pullorum infection enriched the relative abundance of the genera Escherichia-Shigella (65% in infected vs. 40.6% in uninfected groups) and Enterococcus (10.8% vs. 3.7%) while reducing the abundance of Lactobacillus (9.9% vs. 32%) in the chicken microbiota. Additionally, based on the observed changes in the chicken gut microbiota, we isolated microorganisms, including Bifidobacterium pseudolongum, Streptococcus equi and Lacticaseibacillus paracasei (L. paracasei), which were decreased by S. Pullorum infection. Notably, the L. paracasei Lp02 strain was found to effectively inhibit S. Pullorum proliferation in vitro and alleviate its infection in vivo. We found that S. Pullorum infection reduced the richness of the chicken gut microbiota and enriched the relative abundance of the genera Escherichia-Shigella and Enterococcus while decreasing the abundance of the anaerobic genus Lactobacillus. Furthermore, microbiota analysis enabled the isolation of several antimicrobial microorganisms from healthy chicken feces, with a L. paracasei strain notably inhibiting S. Pullorum proliferation in vitro and alleviating its infection in vivo. Overall, this research enhances our understanding of the interaction between gut microbiota and pathogen infection, as well as offers new perspectives and strategies for modulating the chicken gut microbiota to control pullorum disease.

In the avian species, genetic modification by cell nuclear transfer is infeasible due to its unique reproductive system. The in vitro primordial germ cell modification approach is difficult and cumbersome, although it is the main method of genetic modification in chickens. In the present study, the adenoviral CRISPR/Cas9 vector was directly microinjected into the dorsal aorta of chicken embryos to achieve in vivo genetic modification. The results demonstrated that keratin 75-like 4 (KRT75L4), a candidate gene crucial for feather development, was widely knocked out, and an 8bp deletion was the predominant mutation that occurred in multiple tissues in chimeras, particularly in the gonad (2.63–11.57%). As we expected, significant modification was detected in the sperm of G0 (0.16–4.85%), confirming the potential to generate homozygous chickens and establishing this vector as a simple and effective method for genetic modification in avian species.

Primordial germ cells (PGCs) are the earliest precursors of gametes and essential cellular materials for poultry germplasm conservation and genetic modification. In this study, PGCs isolated from 2.5-day male and female Silkie chicken embryos were defined as circulating PGCs (cPGC_M and cPGC_F), whereas PGCs isolated from 8.5-day male and female embryos were defined as gonad-derived PGCs (gPGC_M and gPGC_F). RNA sequencing with three biological replicates per group was performed to characterize developmental-stage- and sex-associated transcriptional programs. Comparative transcriptomic analyses identified stage-associated differences in pathways related to cell–matrix interaction, focal adhesion, PI3K–Akt signaling, and stem cell pluripotency, with more differentially expressed genes detected in the female than in the male stage comparison. Sex-biased expression was detectable at the circulating stage, mainly reflecting W- and Z-linked expression differences, and the number of sex-biased genes was greater after gonadal colonization, primarily because more autosomal differentially expressed genes were detected. Candidate genes and pathways, including HINTW, DMRT1, SOX2, EDNRB, LPAR4, GFRA3, ECM–receptor interaction, ribosome-related modules, and calcium signaling, were associated with these comparisons and are presented as targets for future validation rather than as confirmed regulators. Because the study used three biological replicates per group, RT-qPCR corroboration was limited to six female stage-associated genes, and no functional perturbation assays were performed, the findings are descriptive and do not establish causal mechanisms of PGC migration, gonadal colonization, or sex differentiation. Within these limitations, the dataset provides an exploratory resource for subsequent studies of chicken PGC biology and biotechnology.