This study aimed to evaluate the effects of fermented corn–soybean meal mixed feed on intestinal barrier function and cecal microbiota in laying hens. A total of 360 Jingfen No.6 laying hens (22 wk-old) were assigned to 4 dietary treatments, which were offered basal diets (without antibiotics) containing 0, 4, 6 and 8% of fermented mixed feed respectively. The results showed that the pH value and anti-nutritional factor concentrations in fermented mixed feed were lower than those in unfermented feed (p < 0.05). Moreover, fermentation in the feed significantly increased the crude protein content (p < 0.05). Supplementation with fermented feed significantly reduced the crypt depth and increased the villi height:crypt depth ratio of duodenum and jejunum (p < 0.05). Meanwhile, fermented feed increased the secretory immunoglobulin A content and MUC2 mRNA expression of jejunum (p < 0.05). These beneficial effects were exhibited at the addition level ≥6% and microbial composition of caeca in the control, and so 6% fermented feed groups were analyzed. The structure of the gut microbiota was remarkably altered by additions, characterized by increased abundances of some health-promoting bacteria, such as Parasutterella, Butyricicoccus and Erysipelotrichaceae (p < 0.05). In summary, fermented mixed feed modulated cecal flora, subsequently contributing to improvements in intestinal morphology and barrier functions in laying hens.

This experiment was conducted to investigate the effects of different compound probiotics on the performance, cecal microflora, and intestinal immunity of laying hens. A total of 270 Jing Fen No.6 (22-week-old) were randomly divided into 3 groups: basal diet (CON); basal diet supplemented with 6% fermented feed A by Bacillussubtilis,Lactobacillus, and Yeast (FA); and with 6% fermented feed B by C. butyricum and L. salivarius (FB). Phytic acid, trypsin inhibitor, β-glucan concentrations, and pH value in fermented feed were lower than the CON group (p < 0.05). The feed conversion ratio (FCR) in the experimental groups was decreased, while albumen height and Haugh unit were increased, compared with the CON group (p < 0.05). Fermented feed could upregulate the expression of the signal pathway (TLR4/MyD88/NF-κB) to inhibit mRNA expression of pro-inflammatory cytokines (p < 0.05). Fermented feed promoted the level of Romboutsia (in the FA group) Butyricicoccus (in the FB group), and other beneficial bacteria, and reduced opportunistic pathogens, such as Enterocooccus (p < 0.05). Spearman’s correlations showed that the above bacteria were closely related to albumen height and intestinal immunity. In summary, fermented feed can decrease the feed conversion ratio, and improve the performance and intestinal immunity of laying hens, which may be related to the improvement of the cecal microflora structure.

Biological macromolecules identified in albumen were found benefit to intestinal health, whether albumen contains exosomes and function of their cargos in intestinal inflammation remain unknown. This study aimed to investigate characteristics and cargos of albumen exosomes, as well as their potential roles in alleviating inflammation in intestinal epithelial cells. Our results demonstrated that albumen contains exosomes that are cup-shaped morphology vesicles with diameter ranging from 50 to 200 nm. There were 278 miRNAs and 45 proteins with higher expression levels in albumen exosomes, and they were mainly involved in immune responses and programmed cell death pathways, including apoptosis and p53 signaling pathway. LPS induced overexpression of pro-inflammatory cytokines IL-1β and TNF-α and excessive apoptosis, which could be reversed by albumen exosomes. The beneficial effects of exosomes could be mainly attributed to miRNA cargos and their inhibition on inflammatory response signaling pathways (p53 and NF-κB pathways). Mechanically, exosome miR-22 targeted ATM and inhibited p53/NF-κB pathway, alleviating LPS-induced overexpression of Caspase-3 and Bax, and inflammatory response. Collectively, albumen exosomes alleviate inflammation of intestinal epithelial cells via miR-22/ATM/p53/NF-κB axis and these findings may provide theoretical basis to the potential application of albumen exosomes for intestinal inflammation.

The objective of this study was to investigate the effects of starch structure (Amylopectin/Amylose, AP/AM) in a low-protein diet on production performance, nitrogen utilization efficiency, and cecal flora in laying hens. Four hundred eighty 45-wk-age Hy-Line Gray laying hens were randomly allocated to five dietary groups and subjected to a 12-wk feeding trial. The AP/AM ratios of the five experiment diets were 1.0, 1.5, 2.0, 3.0, and 4.0. The results indicated that compared to other groups, laying hens fed with AP/AM 4.0 diets showed significantly improved average egg weight and feed conversion ratio (P < 0.05). Furthermore, as the AP/AM ratio increased, there was a significant linear enhancement in intestinal amino acids apparent digestibility, apparent metabolizable energy, and villus area (P < 0.05). Compared to the high AP groups, high-AM diets significantly increased eggshell thickness, crude protein digestibility, and reduced energy supply from amino acid oxidation in ileum (P < 0.05). Additionally, moderate-AM diets enriched with short-chain fatty acid-producing bacteria in the cecum, such as Lactobacillus, Rikenellaceae_RC9_gut_group, and Christensenellaceae_R-7_group, which are associated with the promoting nitrogen utilization. These findings may offer useful information on optimizing starch structure for the design of food products and relevant therapies due to the potential effects on nutrient metabolism and gut homeostasis.

Background: Higher embryonic mortality, especially in aged breeding hens, is associated with insufficient hepatic functionality in maintaining redox homeostasis. Our previous study demonstrated that egg exosome-derived miRNAs may play a key role in modulating embryonic oxidation-reduction process, whereas the exact function and mechanism were still poorly understood. The present study aimed to investigate the roles of egg exosome miRNAs in maintaining dynamic equilibrium of free radicals and peroxide agents in embryonic liver, as well as demonstrate the specific mechanism using oxidative stress-challenged hepatocytes. Results: Compared to 36-week-old breeding hens, decreased hatchability and increased embryonic mortality were observed in 65-week-old breeding hens. Meanwhile, the older group showed the increased MDA levels and decreased SOD and GSH-Px activities in embryonic liver, muscle and serum. Embryonic mortality was significantly positively correlated with MDA level and negatively correlated with GSH-Px activity in embryonic liver. In addition, 363 differentially expressed genes (DEGs) were identified in embryonic liver, 13 differentially expressed miRNAs (DE-miRNAs) were identified in egg exosomes. These DEGs and DE-miRNAs were involved in oxidoreductase activity, glutathione metabolic process, MAPK signaling pathway, apoptosis and autophagy. miRNA-mRNA network analysis further found that DEGs targeted by DE-miRNAs were mainly enriched in programmed cell death, such as apoptosis and autophagy. Wherein, MAPK10 with highest MCC and AUC values was significantly related to GSH-Px activity and MDA level, and served as the target gene of miR-145-5p based on dual luciferase reporter experiment and correlation analysis. Bioinformatics analysis found that miR-145-5p/MAPK10 axis might alleviate peroxide generation and apoptosis. In primary hepatocytes of chick embryos, miR-145-5p transfection significantly reversed H₂O₂-induced mitochondrial ROS increase, MAPK10, BAX and CASP3 overexpression and excessive apoptosis. Conclusion: Exosome miR-145-5p in eggs could target MAPK10 and decrease mitochondrial ROS, attenuating oxidative damage and apoptosis in hepatocytes of chick embryos. These findings may provide new theoretical basis for the improvement of maternal physiological status to maintain embryonic redox homeostasis by nutritional or genetic modifications. Graphical

Apidaecin (Api), an antimicrobial peptide, exhibits in vitro efficacy against Salmonella Typhimurium (S. Typhimurium) and enhances broiler growth performance via modulation of intestinal barrier function. This study evaluated the effects of dietary apidaecin on intestinal health in laying hens and its potential to mitigate S. Typhimurium infection and egg contamination. A total of 288 Hy-Line grey layers (45-wk-old) were randomly allocated to 4 dietary treatments (6 replicates per treatment, 12 birds per replicate). Basal diet supplemented with 0 (Con), 300 (Api300), 500 (Api500), or 700 (Api700) mg/kg apidaecin for 12 wk. Based on intestinal barrier function assessment, 18 hens each from the Con and Api 500 groups were selected for S. Typhimurium challenge. Apidaecin’s inhibitory effects on S. Typhimurium adhesion and invasion were further assessed using an in vitro intestinal epithelial cell model. Data were analyzed using one-way ANOVA with Duncan’s post-hoc test for multi-group comparisons and independent t-tests for 2-group comparisons. Results indicated that daily feed intake, crypt depth and villus height to crypt depth ratio (V/C) of intestinal tract showed a quadratic response (P < 0.05) to increasing levels of apidaecin supplementation. Apidaecin (500 mg/kg) significantly reduced feed intake (1.76%), jejunal (29.04%), and ileal crypt depth (31.04%), while increasing duodenal (24.41%), jejunal (45.15%), and ileal V/C (36.39%) vs. control (P < 0.05). Apidaecin significantly increased (P < 0.05) the relative abundance of beneficial short-chain fatty acid-producing bacteria (e.g. Phascolarctobacterium and Desulfovibrio), while reducing (P < 0.05) the relative abundance of enteritis-associated pathogens (e.g. Actinobacillus) of cecum. These beneficial effects were maximized at the 500 mg/kg diet level. Post-challenge, Api 500 hens exhibited increased (P < 0.05) jejunal and ileal V/C ratios and ileal secretory IgA expression compared to controls. Furthermore, apidaecin supplementation significantly reduced (P < 0.05) S. Typhimurium loads in the ileum, spleen, liver and on eggshells. In vitro assays confirmed apidaecin’s direct inhibition (P < 0.05) of S. Typhimurium adhesion to and invasion of intestinal epithelial cells. Collectively, these findings support the potential of dietary apidaecin supplementation in poultry feed formulations to enhance intestinal health and improve the food safety of egg products by reducing Salmonella contamination.