This study investigated cecal bacterial community profile, cecal and serum metabolites, and its biosynthesis pathway in late-phase laying hens during 6 weeks feeding restriction (FR), using 16S rDNA as gene sequencing and non-targeted LC-MS/MS as metabolomics approach. We used three groups (ad libitum, FR20, and FR40). FR can reduce excessive fat in late-phase laying hens, while egg production rate is not affected, except for the FR40 group. In phylum level, FR20 had more population of Bacteriodetes and Firmicutes amongst groups. The same result is at genus level, FR20 were higher of the predominant genus (Bacteroides and Rikenellaceae_RC9_gut_group). Both of FR20 and FR40 reduced Proteobacteria as potential pathogenic bacteria. Non-targeted metabolomic analysis revealed that FR20 modified 20 metabolites in cecal and 10 metabolites in serum of laying hens, whereas 48 cecal metabolites and 31 serum metabolites has revealed in FR40. KEGG assay showed FR20 and FR40 upregulated lipid, carbohydrate, amino acid, nucleic acid pathway, and FR40 modified steroid metabolism in cecal analysis. In serum, only FR40 modified lipid, amino acid pathway, and carbohydrate biosynthesis were shown. This study showed that FR during late-phase laying hens altered the microbiome composition, modified metabolites profile and biosynthesis of the cecal as well as serum.

Beeswax and resin are the main components of propolis, both of which are hydrophobic. The use of emulsifiers helps to improve the extraction of active propolis compounds and makes them more widely used. In this study, we investigated the optimal parameters for the emulsification of Taiwanese green propolis (TGP) using different polysorbates (polysorbate-20, polysorbate-60, and polysorbate-80) and evaluated the effects on the immunomodulatory response in broilers. The results showed that 4 mg/mL of TGP in combination with 2% polysorbate-60 at 60 °C for 60 min significantly decreased the undissolved particle size of ethanol extract of TGP during the emulsification. The bioactive compounds of TGP, the propolins (C, D, F, G, and H), were also detected after emulsification. Supplementation of emulsified TGP (eTGP) in the drinking water of broilers before and after vaccination significantly enhanced the antibody titer response to infectious bronchitis virus at 28 days of age. In the lipopolysaccharide-challenged model, supplementation of eTGP in the drinking water of broilers decreased pro-inflammatory gene expression and increased anti-inflammatory gene expression. These results together suggested that the polysorbate-60 could effectively emulsify the ethanol extract of TGP. Moreover, eTGP could be used as a vaccine adjuvant and an immunomodulator to improve the immune response of broilers.

With the global shortage of feed ingredients, the use of agricultural by-products has become an alternative to animal feed. Okara, a by-product of soymilk and tofu processing, is high in nutrients but contains non-starch polysaccharides (NSP) and has a high-water content, which are disadvantages in animal feed. Herein, we mixed okara and copra meal (CM) with probiotics (Lactobacillus species and Clostridium butyricum) and NSP enzymes (NSPases) for solid-state fermentation (SSF) to enhance okara feed value; the optimal parameters of fermented okara and CM (FOCM) and their effects on broiler growth performance and ileal microbiota were investigated. The result showed that FOCM in combination with NSPases and probiotics at 55% of the initial moisture content and 60 h fermentation time at 37 °C were able to degrade NSP and reduce sugar content. After fermentation, the total viable counts, lactic acid, and butyric acid contents in the FOCM were 8.6 log CFU/g, 3.7%, and 17.15%, respectively. During the fifth week of the feeding period and over the whole feeding period, broilers fed with 1.25% and 2.5% FOCM had a better feed conversion ratio (p < 0.05); both groups increased the duodenal villus length (p < 0.05). Further microbiome analysis suggested that FOCM significantly increased the abundance of the phylum Firmicutes and reduced the abundance of Escherichia-Shigella in the ileal content (p < 0.05). Collectively, supplementation of probiotics and enzymes during SSF was found to be effective in enhancing the nutritional value of FOCM. Moreover, dietary supplementation of FOCM improved the broiler feed conversion ratio, gut morphology, and ileal microbiota.

Substrate availability and microbial accessibility are critical determinants in the conversion of okara into animal feed through solid-state fermentation (SSF). This study comprised two experiments to (1) optimize Lactobacillus SSF of okara using rice bran for moisture adjustment and (2) evaluate the feasibility of the resulting okara and rice bran fermented product (ORFP) for broiler feeding. Both experiments were interpreted using microbiota and metabolomic analyses. In Experiment 1, okara and rice bran were inoculated with three homofermentative Lactobacillus species (Lactobacillus acidophilus, L. delbrueckii, and L. salivarius) and fermented at four moisture levels (45, 50, 55, and 60%) for 0, 48, 60, and 72 h. The optimal SSF condition was identified at 60% moisture and 72 h, as indicated by increased lactic acid production and degradation of major soybean storage proteins. Fermentation reduced the abundance of Proteobacteria-associated genera (Pantoea, Pseudomonas, and Enterobacter) and increased Lactobacillus and Streptococcus, accompanied by changes in metabolites related to nucleotide, amino acid, and plant-derived compound metabolism. In Experiment 2, 112 one-day-old Arbor Acres broilers were assigned to a basal diet or diets containing 1.25, 2.5, or 5.0% ORFP. Broilers fed 2.5% ORFP maintained feed conversion ratio and increased ileal villus height. Ileal microbiota analysis revealed an increased abundance of the carbohydrate-utilizing bacterium Romboutsia timonensis in the 1.25 and 2.5% ORFP groups. Functional prediction indicated enhanced substrate utilization at these inclusion levels, with broader metabolic enrichment observed at 2.5% ORFP, whereas fewer functional pathways were observed in the 5.0% ORFP group. Further metabolomic analysis indicated enrichment of amino acid- and sulfur-related pathways in the 1.25 and 2.5% ORFP groups. These changes were associated with mucosal-associated metabolism, as shown by the positive association between R. timonensis and glucosamine-6-sulfate. Overall, these results indicate that Lactobacillus SSF combined with rice bran modulates substrate availability and microbial accessibility of okara, with 2.5% ORFP associated with improved intestinal morphology and changes in microbiota and metabolite profiles in broilers.