Quercetin, a well-known flavonoid, has been demonstrated to exert beneficial effects on intestinal functions and gut microbiota in birds. In this study, we investigated the effects of quercetin supplementation on inflammatory responses, intestinal barrier functions and gut microbial community in LPS-challenged laying hens. A total of two hundred eighty-eight 32-wk-old Jingfen No.6 laying hens were randomly assigned to 3 groups, the CON group, the LC group and the LQ group. LQ group was fed with 0.4 mg/kg quercetin and at the end of 12 wk, LC and LQ groups were challenged intraperitoneally with lipopolysaccharide (LPS). After LPS challenge, 8 birds of each group were randomly selected and sampled. LPS challenge induced an obvious intestinal mucosal injury, necrosis and shedding, while quercetin intervention maintained its structure. Quercetin significantly decreased the elevated malondialdehyde contents (P < 0.05), and increased the activity of total antioxidant capacity and glutathione peroxidase (P < 0.05) in intestinal mucosa of LPS-challenged laying hens. Quercetin rescued the LPS-induced decreases in goblet cell density and mucin2 expression levels (P < 0.05). There was a significant decline (P < 0.05) in the mRNA expression of Claudin1 and Occludin in intestinal mucosa of LPS-challenged layers, which could be alleviated (P < 0.05) by dietary quercetin. LPS challenge induced the increased expression levels (P < 0.05) of IL-1β and TLR-4 in intestinal mucosa, while these rises could be reversed (P < 0.05) following dietary quercetin supplementation. LPS challenge induced a shift in gut microenvironment, and quercetin addition could elevate the relative abundance of some short chain fatty acids (SCFA)-producing or health-promoting bacteria such as Phascolarctobacterium, Negativicutes, Selenomonadales, Megamonas, Prevotellaceae, and Bacteroides_salanitronis. In conclusion, dietary quercetin addition ameliorated the LPS challenge-induced intestinal inflammation and improved intestinal functions, possibly associated with its modulation on gut microbiota, particularly the increased population of SCFA-producing bacteria.

The aim of this study was to investigate the effects of different probiotic fermented diets on production performance and intestinal health of laying hens. A total of 360 healthy 22-wk-age Jingfen No. 6 layers were randomly divided into 4 treatments: basal diet (CON); supplemented with 6% Clostridium butyricum fermented feed (CB); supplemented with 6% Lactobacillus crispatus fermented feed (LC); supplemented with 6% Lactobacillus salivarius fermented feed (LS). The experiment lasted for 8 wk. The results showed that the levels of crude fiber, β-glucan and pH of feed decreased significantly after fermentation (P < 0.05). Compared with CON group, the feed conversion ratio (FCR) was decreased significantly (P < 0.05), and albumen height and Haugh unit in LC group and LS group were increased significantly (P < 0.05). Fermented feed supplementation significantly improved villus height (VH) of the jejunum and the ratio of villus height to crypt depth (VH/CD) of the ileum (P < 0.05). Additionally, the VH and VH/CD of the duodenum were significantly increased in LS group (P < 0.05). Furthermore, the ACE and chao1 indexes in LS group were extremely significant higher than that in the other 3 groups (P < 0.05). In addition, compared with CON group, the abundance of Rikenellaceae and Methanobacteriaceae was significantly decreased at the family level in LC group and LS group (P < 0.05), while the abundance of Ruminocaceae was significantly higher (P < 0.05). Collectively, feeding Lactobacillus salivarius and Lactobacillus crispatus fermented feed improved the FCR, albumen height and Haugh unit of laying hens, and Lactobacillus salivarius fermented feed supplementation could improve intestinal health by ameliorating intestinal morphology, altering microbial composition and enhancing microbial community richness.

Fatty liver hemorrhagic syndrome is a widespread metabolic disease in laying hens that decreases egg production and even causes death in severe cases. Many traditional Chinese medicine ingredients, such as saikosaponin a (SSa), have been shown to alleviate fatty liver, but the underlying mechanisms remain unclear. In this study, we aimed to explore the alleviation of dietary SSa on excessive hepatic lipid deposition and the interactions between intestinal microbiota and bile acid (BA) in laying hens. Fifty-four 35-wk-old laying hens were randomly allocated into 3 treatment groups with 6 replicates (3 birds per replicate) and fed with a basal diet (CON), high-energy and low-protein diet (HELP), and HELP diet with 30 mg/kg SSa (HELP + SSa). SSa reversed diet-induced egg production rate decrease (P < 0.05). SSa could potently ameliorate HELP-induced accumulation of hepatic cholesterol and liver injury via the increase (P < 0.05) of mRNA expression of BA synthesis gene, such as cholesterol 7 alpha-hydroxylase 1. SSa treatment alleviated gut dysbiosis, especially reducing (P < 0.05) the relative abundance of bile salt hydrolase (BSH)-producing bacteria such as Lactobacillus, Bifidobacterium, and Turicibacter. Ileal BA metabolomic analysis revealed that SSa increased (P < 0.05) the content of tauro-conjugated BAs, mainly taurochenodeoxycholic acid and tauro-α-muricholic acid. The mRNA expression of farnesoid X receptor (FXR) and fibroblast growth factor 19 were decreased (P < 0.05) in intestine, which was associated with increased gene expression of enzymes in the BA synthesis that reduced the levels of cholesterol. Moreover, SSa treatment inhibited intestinal BA reabsorption via decreasing (P < 0.05) the mRNA expression of apical sodium-dependent bile acid transporter. Our findings indicated that SSa reduced liver cholesterol accumulation and alleviated fatty liver in laying hens through microbiota-BA-intestinal FXR crosstalk.

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 decline in eggshell quality resulting from aging hens poses a threat to the financial benefits of the egg industry. The deterioration of eggshell quality with age can be attributed to changes in its ultrastructure and chemical composition. Specific matrix proteins in eggshells have a role in controlling crystal growth and regulating structural organization. However, the variations in ultrastructure and organic matrix of eggshells in aging hens remain poorly understood. This study assessed the physical traits, mechanical quality, chemical content, as well as the microstructural and nanostructural properties of eggs from Jing Tint 6 hens at 38, 58, 78, and 108 wk of age. Subsequently, a quantitative proteomic analysis was conducted to identify differences in protein abundance in eggshells between the ages of 38 and 108 wk. The results indicated a notable decline in shell thickness, breaking strength, index, fracture toughness, and stiffness in the 108-wk-age group compared to the other groups (P < 0.05). The ultrastructure variations primarily involved an increased ratio of the mammillary layer and a reduced thickness of the effective layer of eggshell in the 108-wk-age group (P < 0.05). However, no significant differences in eggshell compositions were observed among the various age groups (P > 0.05). Proteomic analysis revealed the identification of 76 differentially expressed proteins (DEPs) in the eggshells of the 38-wk-age group and 108-wk-age group, which comprised proteins associated with biomineralization, calcium ion binding, immunity, as well as protein synthesis and folding. The downregulation of ovocleidin-116, osteopontin, and calcium-ion-related proteins, together with the upregulation of ovalbumin, lysozyme C, and antimicrobial proteins, has the potential to influence the structural organization of the eggshell. Therefore, the deterioration of eggshell quality with age may be attributed to the alterations in ultrastructure and the abundance of matrix proteins.

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.