The in ovo feeding (IOF) of L-arginine (L-Arg) to chick embryos is a viable method for improving early intestinal development, subsequently leading to an acceleration in growth rate during the posthatch stage. However, the liver, being the pivotal organ for energy metabolism in poultry, the precise effects and mechanisms of L-Arg on the liver development and metabolism remain unclear. To elucidate these, the present study injected 2 doses of L-Arg (10 mg/egg and 15 mg/egg) into the embryos of Hongyao chickens at 17.5 d of incubation, subsequently incubating them until d 19 for further analysis. IOF of 15 mg L-Arg/egg significantly increased the organ indices of liver and small intestine, as well as the duodenal villus height/crypt depth. RNA-Seq analysis of liver tissues showed that the metabolism of xenobiotics, amino acid metabolism, and the fatty acid metabolism were significantly enriched in L-Arg injection group. The core differentially expressed genes (DEGs) were primarily involved in cell proliferation and fatty acid metabolism. The CCK8 assays revealed that supplemental L-Arg significantly enhanced the proliferation of primary embryo hepatocytes and leghorn male hepatoma (LMH) cells. Upregulation of core DEGs, including HBEGF, HES4, NEK3, EGR1, and USP2, significantly promoted the proliferation of liver cells. Additionally, analysis of triglyceride and total cholesterol content, as well as oil red O staining, indicated that supplemental L-Arg effectively reduced lipid accumulation. Overall, L-Arg supplementation in late chick embryos may promote early liver and small intestine development by reducing liver lipid deposition and enhancing energy efficiency, necessitating further experimental validation. This study provides profound insights into the molecular regulatory network of L-Arg in promoting the development of chicken embryos. The identified DEGs that promote cell proliferation and lipid metabolism can serve as novel targets for further developing methods to enhance early development of chicken embryos.

Research on hepatic steatosis in animal husbandry has been a prominent area of study. Developing an appropriate in vitro cellular steatosis model is crucial for comprehensively investigating the mechanisms involved in liver lipid deposition in poultry and for identifying potential interventions to address abnormalities in lipid metabolism. The research on the methods of in vitro liver steatosis in chickens, particularly the effects of different fat mixtures, is still lacking. In this study, LMH cells were utilized to investigate the effects of OA, SO, PA, SP, and their pairwise combinations on steatosis development, with the aim of identifying the optimal conditions for inducing steatosis. Analysis of triglyceride (TG) content in LMH cells revealed that OA and SP had limited efficacy in increasing TG content, while a combination of SO and PA in a 1:2 ratio exhibited the highest TG content. Moreover, Oil Red O staining results in LMH cells demonstrated that the combination treatment had a more pronounced induction effect compared to 0.375 mM SO. Additionally, RNA-seq analysis showed that 0.375 mM SO significantly influenced the expression of genes associated with fatty acid metabolism compared to the control group, whereas the combination of SO and PA led to an enrichment of key GO terms associated with programmed cell death. These findings suggest that varying conditions of cellular steatosis could lead to distinct disruptions in gene expression. The optimal conditions for inducing steatosis in LMH cells were also tested on chicken embryonic liver cells and embryos. TG detection and Oil Red O staining assays showed that the combination of SO and PA successfully induced steatosis. However, the gene expression pattern differed from that of LMH cells. This study lays the foundations for further investigations into avian hepatic steatosis.

Objective: Microtubule dynamics regulator protein 2 (RMDN2) plays a crucial role in cell division, cytoskeleton maintenance, and various cellular processes, thereby establishing it as a candidate gene influencing chicken follicle development in our previous studies. This research aims to explore single-nucleotide polymorphisms (SNPs), perform phylogenetic analysis, and assess sequence characteristics of RMDN2, offering valuable insights for molecular marker-assisted breeding and enhancing the understanding of its regulatory mechanisms.Methods: SNPs within the RMDN2 coding sequence region were identified in an F2 resource population. Bioinformatics tools were employed to investigate the effects of SNP mutations on the structure and function of RMDN2 protein. Additionally, a phylogenetic tree was constructed to elucidate the potential mechanisms underlying the role of RMDN2 in chicken laying traits.Results: Four novel exonic SNPs were identified: SNP1 (c.250G>A, p.Val84Ile), SNP2 (c.270G>C, p.Lys90Asn), SNP3 (c.533G>T, p.Gly178Val), and SNP4 (c.606G>A). The heterozygous genotypes of SNP1, SNP3, and SNP4 showed a significant association with increased egg number at 66 weeks (p<0.05). In contrast, the heterozygous genotype of SNP2 is associated with higher body weight at first egg (BWFE) (p<0.05). Notably, the H1H1 haplotype combination demonstrated a significant association with reduced BWFE, body weight at 105 days, and first egg weight (p<0.05). Missense mutations in SNP1, SNP2, and SNP3 may influence the hydrophilic/hydrophobic properties, transmembrane regions, functional domains, and secondary structure of the RMDN2 protein, potentially reducing its stability. Phylogenetic analysis demonstrated complete sequence homology between chicken and quail, indicating substantial conservation within species of the same order, while showing a marked decrease across different taxonomic orders.Conclusion: These findings enrich the candidate gene pool associated with the regulation of laying traits in chickens. However, further validation through in vivo and in vitro experiments remains necessary to strengthen the theoretical foundation for molecular breeding strategies.