Kaixuan Yang

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.

Pullorum disease (PD) is one of the common infectious diseases in the poultry industry in the world. Our previous study showed that gut bacterial structure has a significant difference between positive and negative hens. However, the gut bacterial basis of intergenerational transmission of PD continues to elude a scientific explanation. The present study carried out fecal microbiota transplantation (FMT) in chicks of a negative group, then fecal samples of the chicks in the control team (CT), Salmonella pullorum (S. pullorum)-negative transplantation team (PN) and S. pullorum-positive transplantation team (PP) were separately collected to be analyzed for microbial structure and prediction functions. Microbial diversity results revealed that there was a large difference in the gut microbiota of these three groups. Prevotella and Parasutterella with higher abundance in PN (p < 0.05) were transplanted from gut bacteria of S. pullorum-negative hens. Furthermore, the differences of the most major microbial functions (top 100) were similar in hens and chicks, including a pentose phosphate pathway and oxidative phosphorylation. The data provided a reference for exploring the intergenerational transmission and genetic mechanisms of gut microbiota associated with S. pullorum in poultry, as well as a theoretical basis for improving intestinal health through the rational regulation of microbiota-host interactions.

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.