The gastrointestinal tract is populated by a complex and vast microbial network, with a composition that reflects the relationships of the symbiosis, co-metabolism, and co-evolution of these microorganisms with their host. The mechanism that underlies such interactions between the genetics of the host and gut microbiota remains elusive. To understand how genetic variation of the host shapes the gut microbiota and interacts with it to affect the metabolic phenotype of the host, we compared the abundance of microbial taxa and their functional performance between two lines of chickens (fat and lean) that had undergone long-term divergent selection for abdominal fat pad weight, which resulted in a 4.5-fold increase in the fat line compared to the lean line. Our analysis revealed that the proportions of Fusobacteria and Proteobacteria differed significantly between the two lines (8 vs. 18% and 33 vs. 24%, respectively) at the phylum level. Eight bacterial genera and 11 species were also substantially influenced by the host genotype. Differences between the two lines in the frequency of host alleles at loci that influence accumulation of abdominal fat were associated with differences in the abundance and composition of the gut microbiota. Moreover, microbial genome functional analysis showed that the gut microbiota was involved in pathways that are associated with fat metabolism such as lipid and glycan biosynthesis, as well as amino acid and energy metabolism. Interestingly, citrate cycle and peroxisome proliferator activated receptor (PPAR) signaling pathways that play important roles in lipid storage and metabolism were more prevalent in the fat line than in the lean line. Our study demonstrates that long-term divergent selection not only alters the composition of the gut microbiota, but also influences its functional performance by enriching its relative abundance in microbial taxa. These results support the hypothesis that the host and gut microbiota interact at the genetic level and that these interactions result in their co-evolution.

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.

Accurate quantification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-induced insertions and deletions (indels) is essential in chicken genome-editing studies, where low editing efficiencies and mosaicism can complicate the assessment of target-site modifications. Existing assays often involve a trade-off between affordability and the sensitivity orsequence information needed for detailed analysis. Here, we introduce IndelQuant, a cost-efficient workflow integrating sample-specific barcode PCR, pooled amplicon library construction, AdapterRemoval3-based read processing, and CRISPResso2-based indel quantification. The workflow provides graphical and tabularsummaries of editing efficiency, indel positions, indel-size distributions, and sample-level mutation profiles. We evaluated the workflow in TYR-edited DF-1 cells and genome-edited chickens. In edited chickens, IndelQuant detected tissue-specific indel signals, most below 5%, while negative controls showed background frequencies from 0 to 0.03%. These findings indicate that IndelQuant retains the sequence-level information provided by amplicon sequencing while simplifying sample processing and analysis, offering a practical approach to routine screening for low-frequency and mosaic indels in poultry genome-editing studies.