Background: Post-weaning diarrhea in piglets reduces growth performance and increases mortality, thereby causing serious economic losses. The intestinal epithelial cells and microbiota reciprocally regulate each other in order to maintain intestinal homeostasis and control inflammation. However, a relative paucity of research has been focused on the host-derived regulatory network that controls mucin O-glycans and thereby changes gut microbiota during diarrhea in infancy. At the development stage just after birth, the ontogeny of intestinal epithelium, immune system, and gut microbiota appear similar in piglets and human infants. Here, we investigated the changes of mucin O-glycans associated with gut microbiota using a diarrheal post-weaned piglet model. Results: We found that diarrhea disrupted the colonic mucus layer and caused aberrant mucin O-glycans, including reduced acidic glycans and truncated glycans, leading to an impaired gut microenvironment. Subsequently, the onset of diarrhea, changes in microbiota and bacterial translocation, resulting in compromised epithelial barrier integrity, enhanced susceptibility to inflammation, and mild growth faltering. Furthermore, we found the activation of NLRP3 inflammasome complexes in the diarrheal piglets when compared to the healthy counterparts, triggered the release of proinflammatory cytokines IL-1β and IL-18, and diminished autophagosome formation, specifically the defective conversion of LC3A/B I into LC3A/B II and the accumulation of p62. Additionally, selective blocking of the autophagy pathway by 3-MA led to the reduction in goblet cell-specific gene transcript levels in vitro. Conclusions: We observed that diarrheal piglets exhibited colonic microbiota dysbiosis and mucosal barrier dysfunction. Our data demonstrated that diarrhea resulted in the activation of inflammasomes and autophagy restriction along with aberrant mucin O-glycans including reduced acidic glycans and truncated glycans. The results suggested the mucin O-glycans-microbiota axis is likely associated with diarrheal pathogenesis. Our study provides novel insights into the pathophysiology of early-weaning-induced diarrheal disease in piglets and potentially understanding of disease mechanisms of diarrhea for human infants. Understanding the molecular pathology and pathogenesis of diarrhea is a prerequisite for the development of novel and effective therapies. Our data suggest that facilitating O-glycan elongation, modifying the microbiota, and developing specific inhibitors to some key inflammasomes could be the options for therapy of diarrhea including human infants. Graphical

Glycerol was generally added to the inoculum as a cryoprotectant. However, it was also a suitable substrate for microbial fermentation, which may produce more SCFAs, thereby decreased pH of the fermentation broth. This study investigated the effect of supplementing glycerol to inoculum on in vitro fermentation and whether an enhanced buffer capacity of medium could maintain the pH stability during in vitro batch fermentation, subsequently improving the accuracy of short chain fatty acids (SCFAs) determination, especially propionate. Two ileal digesta were fermented by pig fecal inoculum with or without glycerol (served as anti-frozen inoculum or frozen inoculum) in standard buffer or enhanced buffer solution (served as normal or modified medium). Along with the fermentation, adding glycerol decreased the pH of fermentation broth (p < 0.05). However, modified medium could alleviate the pH decrement compared with normal medium (p < 0.05). The concentration of total propionic acid production was much higher than that of other SCFAs in anti-frozen inoculum fermentation at 24 and 36 h, thereby increasing the variation (SD) of net production of propionate. The α-diversity analysis showed that adding glycerol decreased Chao1 and Shannon index under normal medium fermentation (p < 0.05) compared to modified medium (p < 0.05) along with fermentation. PCoA showed that all groups were clustered differently (p < 0.01). Adding glycerol improved the relative abundances of Firmicutes, Anaerovibrio, unclassified_f_Selenomonadaceae, and decreased the relative abundance of Proteobacteria (p < 0.05). The relative abundances of Firmicutes, such as Lactobacillus, Blautia and Eubacterium_Ruminantium_group in modified medium with frozen inoculum fermentation were higher than (p < 0.05) those in normal medium at 36 h of incubation. These results showed that adding glycerol in inoculum changed the fermentation patterns, regardless of substrate and medium, and suggested fermentation using frozen inoculum with modified medium could maintain stability of pH, improve the accuracy of SCFA determination, as well as maintain a balanced microbial community.

Broilers face challenges in improving intestinal health and growth performance. Glutamine (Gln), a functional amino acid, exerts beneficial effects in promoting intestinal development and immunity. This study investigated the effects of graded levels of dietary Gln supplementation on the growth performance, intestinal morphology, immune indices, and cecal microbiota of broilers. Three hundred one-day-old Arbor Acres broilers (51.67±1.42 g) were assigned to five groups in a completely random design, with six replicates each group and 10 broilers in each replicate. The control group was fed the basal diet (Con), while the experimental groups were fed the basal diet containing 4 g/kg (Gln1), 6 g/kg (Gln2), 8 g/kg (Gln3), and 10 g/kg Gln (Gln4), respectively. The results showed that dietary Gln supplementation had quadratic effect on ADG, F/G, and final BW of broilers (P < 0.01), with higher average daily gain (ADG), final body weight (BW) in Gln2 group (P < 0.05). Dietary Gln supplementation quadratically improved serum immune indices of broilers (P < 0.01), with higher IgA, IgG, and IgM levels in Gln1 and Gln2 groups (P < 0.05). The Gln1 and Gln2 groups increased Bursa of Fabricius index at both d21 and d42 (P < 0.05). There was a quadratic effect of Gln on intestinal development (P < 0.01). Gln1 and Gln2 groups had higher duodenal VH and V/C, and the Gln2 group increased jejunal VH and V/C, and ileal VH at 21d (P < 0.05). Duodenal V/C improved in the Gln1 and Gln2 groups, and duodenal CD reduced in Gln1 group at 42d (P < 0.05). Gln1 and Gln2 groups increased cecal α-diversity (ACE, Chao1) at d21 (P < 0.05). PCoA revealed distinct cecal microbiota profiles across groups, with Gln-supplemented groups reducing pathogenic Escherichia-Shigella and Mucispirillum, while enriching beneficial genera (e.g., Ruminococcaceae_UCG-014, Barnesiella, and Butyricimonas) (P < 0.05). Collectively, dietary Gln-supplemented at 4-6 g/kg optimally enhances growth, immunity, and intestinal development in broilers, which is related to modulate cecal microbiota toward a beneficial composition.

Background: Tryptophan (Trp) is a limiting amino acid in poultry nutrition. Dietary supplementation of Trp not only enhances production performance, but also supports intestinal barrier integrity, alleviates stress, and boosts immunity, in which the derivatives from gut Trp-metabolizing commensal microbes play crucial roles. However, research on how excessive Trp affects poultry growth, metabolism, and gut microbiota composition remains limited. Methods: In this study, we investigated the effects of varying Trp levels (0.23%, 0.29%, 0.35%) on broiler production performance, immune function, and antioxidant levels through controlled feeding trials. These host responses were further correlated with cecal microbiota metagenomic sequencing data using multivariate analysis. Results: Compared with the basal 0.23% Trp level, a 0.35% of Trp addition significantly impaired broiler body weight gain and feed intake, and 0.29% Trp could increase thymus index and serum superoxide dismutase (SOD) level without affecting the growth performance; neither of these two levels affected the feed conversion rate. The cecal microbial metagenomic data further revealed that Trp supplementation reduced the abundance of harmful bacteria, while increasing the abundance of beneficial bacteria and Trp-metabolizing microorganisms. Correlation analysis showed that Trp supplementation was negatively correlated with body weight (BW) but positively correlated with thymus index and SOD level, with similar trends observed between the abundance of specific Trp-utilizing microorganisms and these indicators. Functional analysis revealed an increase in the abundance of KEGG orthology (KO) related to Trp metabolism from the aforementioned microbes. Conclusions: An appropriate addition of Trp (0.29%) can enhance certain metabolic levels without affecting production performance, which might be achieved through relevant metabolic pathways of intestinal microorganisms.

This study was conducted to evaluate the effect of corn particle size on the particle size of intestinal digesta or feces and nutrient digestibility of corn–soybean meal diets. Twenty-four growing barrows (initial BW: 21.9 ± 1.62 kg) were randomly divided into 4 groups of 6 pigs. A T-cannula was surgically placed in the anterior duodenum (about 50 cm from pylorus) of pigs in Groups 1 and 2 or in the distal ileum of pigs in Groups 3 and 4. Corn used to formulate diets had mean particle size (MPS) of 365 µm (Corn 1) or 682 µm (Corn 2), resulting in diets with MPS of 390 µm (Diet 1) or 511 μm (Diet 2). Diet 1 or 2 were randomly assigned within pig Groups 1 or 2 and 3 or 4. The digestive enzyme activities of duodenal fluid, particle size of intestinal digesta and feces, as well as nutrient digestibility, were determined for each pig as the experiment unit. The MPS of duodenal digesta (181 vs. 287 µm, p < 0.01), ileal digesta (253 vs. 331 µm, p < 0.01), and feces (195 vs. 293 µm, p < 0.01) was significantly reduced for pigs fed Diet 1 vs. Diet 2, respectively. Compared with Diet 2, Diet 1 significantly reduced the proportion of particles above 0.5 mm, but significantly increased the proportion of particles between 0.072 and 0.5 mm (p < 0.01) in digesta and feces (p < 0.01). Diet 1 significantly increased solubles percentage (<0.072 mm) in duodenal digesta (p < 0.05) but did not affect solubles percentage in ileal digesta and feces. The MPS of diet did not affect the activities of amylase, trypsin, and chymotrypsin in the duodenal fluid and the apparent total tract digestibility (ATTD) of dry matter, gross energy, crude protein, ether extract, neutral detergent fiber (NDF) and acid detergent fiber (ADF) in pigs offered Diet 1 compared to Diet 2. The in vitro digestible energy (IVDE) (3706 vs. 3641 kcal/kg; p = 0.03) was greater for Corn 1 vs. Corn 2. However, no significant difference was observed in IVDE (3574 vs. 3561 kcal/kg; p = 0.47) for Diet 1 vs. Diet 2. In conclusion, the particle size of digesta and feces was dependent on the dietary particle size. However, the digestive enzyme activities of duodenal fluid and ATTD of energy and nutrients were not affected by reducing dietary MPS from 511 to 390 µm.

Conjugated linoleic acid (CLA) isomers have been reported to reduce body weight and promote glycolipid metabolism in animals. In a preliminary study, we revealed that trans-10, cis-12-CLA (10,12-CLA) plays an important role in modulating lipid metabolism in chickens. However, the underlying mechanism remains unclear. In this study, we constructed an isolated in vitro model with primary chicken hepatocytes to investigate the effect of 10,12-CLA on lipid metabolism. 10,12-CLA inhibited lipid accumulation by decreasing the mRNA expression of sterol regulatory element-binding protein-1c (SREBP-1c), SREBP2, 3‑hydroxy-3-methylglutaryl-CoA reductase (HMGCR), fatty acid synthase (FAS), adipose triacylglyceride lipase (ACC), and lipoprotein lipase (LPL) and increasing the mRNA expression of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1 (CPT1) and adipose triacylglyceride lipase (ATGL). Furthermore, 10,12-CLA treatment activated the protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2) and AMP-activated protein kinase (AMPK), whereas treatment with the ERK1/2 inhibitor U0126 reversed the inhibitory effects of 10,12-CLA on lipid accumulation by blocking the ERK1/2-AMPK pathway, leading to increased lipid accumulation and triglyceride content in primary chicken hepatocytes. These findings suggest that in chicken hepatocytes, 10,12-CLA alleviates hepatocyte lipid deposition by activating the ERK1/2-AMPK pathway, promoting fatty acid oxidation and reducing lipid synthesis, revealing the potential mechanism through which 10,12-CLA regulates hepatic lipid metabolism in chickens.