Probiotics improve eggshell quality via regulating microbial composition in the uterine and cecum

Probiotics benefit the health and production performance of chickens, but their impact on egg and eggshell quality, particularly in the later stage, remains unclear. Here, 1-day-old Tianfu green shell-laying hens were fed either non-probiotics feed (n = 180) or feed supplemented with 100 mg / kg probiotics (n = 180). 16S rDNA sequencing indicated that dietary probiotics decreased the distribution of uterine p_Firmicutes, g_Fusobacterium, and s_Fusobacterium_unclassified, while increased p_Proteobacteria, g_Ralstonia, and s_Ralstonia_unclassified. PICRUSt2 and Bug Base analysis revealed enrichment in fatty acid metabolism, thiamine metabolism, vitamin B6 metabolism pathways and increased relative abundance of Proteobacteria, Firmicutes, Bacteroidetes. With LDA > 4.5, 35 and 25 marker bacterial taxa were identified in the uterus and cecum, respectively. Probiotics significantly increased uterine villi length and width, and the expressions of ATP2B2,SLC26A9,TF,OC-17,OCX-32, and OVAL in the uterus at the early and peak laying stage. Meantime, probiotics improved egg quality, pore density of eggshell barrier layer, and levels of Ca²⁺, Na⁺, and Mg²⁺, whereas dropped levels of P^3-, S^2- and K⁺ in eggshell. In serum, Ca²⁺, K⁺, Na⁺ had a response to dietary probiotics at different laying stages, except Cl^-. Furthermore, the changes of these phenotypes are closely related to the microbial structure of the uterus and cecum. Overall, the data suggest that dietary probiotics improved uterine and cecal microbiota, optimized egg quality, eggshell quality, uterus development, and regulated mineralization gene expression and ion content in serum and eggshell, thereby improving productivity of laying hens. These results provide reference for the application of probiotics in the laying industry.