This study evaluated the effects of dietary L-Ornithine L-Aspartate (OA) supplementation on growth performance, immune response, intestinal health, and cecal microbiota in broilers challenged with lipopolysaccharide (LPS). A total of 624 one-day-old Arbor Acres broilers were assigned to a 4 × 2 factorial arrangement with four dietary OA levels (0, 400, 600, and 800 mg/kg) and two challenge treatments (saline or LPS). Birds were intraperitoneally injected with LPS (1 mg/kg BW) or sterile saline on d 23, 25, and 27, and the experiment lasted 42 d. LPS challenge impaired growth performance, induced systemic inflammation, reduced microbial diversity, and disrupted intestinal homeostasis in broilers. Dietary OA supplementation improved growth performance, attenuated inflammatory responses as evidenced by reduced serum proinflammatory cytokine levels, with significant OA × LPS interactions observed for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and by suppressing splenic expression of inflammatory cytokines, toll-like receptor 4 (TLR4), and myeloid differentiation factor 88 (MyD88). Moreover, OA supplementation improved intestinal barrier function as shown by reduced serum diamine oxidase activity, enhanced jejunal morphology, increased secretory immunoglobulin A (sIgA) expression, and the abundance of beneficial bacterial taxa in the cecum. In conclusion, dietary OA supplementation could improve growth performance in broilers by alleviating inflammation, improving immune homeostasis and intestinal health, which may be associated with modulation of the splenic TLR4/MyD88 signaling pathway. Based on a comprehensive evaluation of growth performance, immune regulation, intestinal health, and inflammatory responses, 400 mg/kg was identified as the optimal dietary inclusion level under the conditions of the present study.

This study aimed to determine the available energy values of sorghum for Arbor Acres (AA) broilers and to develop and compare prediction equations using multivariate linear stepwise regression (MLSR) and machine learning-based linear regression (LR). It is important to clarify that the LR used in this study is fundamentally a linear regression model. The primary difference from MLSR lies in the variable selection process and optimization algorithm. Ten sorghum samples with varied chemical compositions were used. Test diets were formulated by replacing 40% of the energy-yielding components of a reference diet with individual sorghum. A total of 7929-day-old and 39623-day-old male AA broilers were used in two balance trials. Apparent metabolizable energy (AME), nitrogen-corrected AME (AMEn), and net energy (NE) were determined using the substitution method and respiratory calorimetry. Correlation analyses between energy values and chemical components were performed. Prediction equations for AME and NE were developed using both MLSR (in SPSS) and LR (in Python scikit-learn). The AME, AMEn, and NE values for sorghum were significantly higher in 26-28-day-old broilers compared to 12-14-day-old broilers (P < 0.05). Tannin (TN) content showed significant negative correlations with AME and AMEn. Accurate prediction equations for AME and NE were established for both age stages using key predictors such as acid detergent fiber (ADF), TN, phytic acid (PA), dry matter (DM), and crude protein (CP). Equations developed with both MLSR and LR methods demonstrated a good fit to the development data. Validation results indicated that the predictive accuracy and performance of the two methods were comparable. This study provides definitive available energy values of sorghum for broilers at different ages. The developed prediction equations, whether based on MLSR or LR, are effective tools for estimating the energy content of sorghum. The findings support the use of sorghum as a viable alternative energy source in broiler diets and provide a scientific basis for its precise application in feed formulation.

This study investigated the effects of a dietary supplement containing a gallic acid-usnic acid complex (GU) on broilers challenged with necrotic enteritis (NE). A total of 324 one-day-old Arbor Acres broilers were randomly assigned to three groups with 6 replicates of 18 broilers each: (1) basal diet (NC); (2) basal diet + NE challenge (PC); (3) basal diet + NE challenge + 500 mg/kg GU (GU). The NE challenge was induced by oral administration of a coccidial quadrivalent vaccine on day 12, followed by Clostridium perfringens type A on days 17-20. The results showed that GU supplementation significantly improved growth performance, as evidenced by increased body weight and average daily gain (ADG) compared to the PC group at 21, 28, and 42 days of age (P < 0.05). GU effectively inhibited key pathogenic microbes through its antimicrobial and antiparasitic actions, as evidenced by reduced fecal coccidial oocyst shedding and decreased cecal Clostridium perfringens colonization compared to the PC group. It enhanced intestinal barrier function via improved ileal villus structure, increased goblet cell density, and upregulated the expression of the tight junction protein Claudin-1. GU significantly reduced the concentrations of interleukin-1β (IL-1β) and lipopolysaccharide (LPS) in the ileum and serum (P < 0.05), downregulated the mRNA expression of nuclear factor-kappa B (NF-κB) and myeloid differentiation primary response 88 (MyD88) (P < 0.05). Furthermore, GU modulated the gut microbiota by increasing α-diversity and enriching beneficial bacteria including Cyanobacteria, Bacteroides coprocola, and Parabacteroides merdae. In conclusion, GU alleviates NE in broilers by modulating the gut microbiota, inhibiting pathogenic microbes, strengthening the intestinal barrier, and mitigating inflammation and oxidative stress, thus presenting a promising nutritional strategy for sustainable broiler production.

Maternal nutrition is a key determinant of offspring growth, as poultry embryonic muscle depends entirely on yolk-derived nutrients. Methionine (Met), a vital amino acid and methyl donor in one-carbon metabolism, regulates myogenesis, yet its epigenetic mechanisms remain poorly understood. Two independent maternal experiments and corresponding offspring trials were performed. For the maternal trials, Experiment 1 included 720 WOD188 broiler breeder hens (41-week-old, initial body weight [BW]: 4500 ± 300 g) randomly assigned to 3 treatments with 8 replicates of 30 birds each, and fed a basal diet and basal diets supplemented with 0.15% DL-Met or 0.17% 2-hydroxy-4-(methylthio)-butanoate (HMTBA) for 7 weeks. Experiment 2 used 480 41-week-old hens (initial BW: 4100 ± 285 g) randomly allocated to 3 treatments with 8 replicates of 20 birds each, and fed a basal diet and the basal diets supplemented with 0.15% DL-Met or 0.09% choline chloride for 5 weeks. For the offspring trials, fertilized eggs were incubated in both experiments. A total of 240 1-d-old male chicks (initial BW: 42.5 ± 2.5 g) per trial were grouped by maternal treatment and reared for 6 weeks (Exp. 1) or 14 d (Exp. 2, for N6-methyladenosine [m6A] analysis). Results showed that maternal Met supplementation, compared with HMTBA, significantly enhanced Met metabolism by increasing serum methionine adenosyltransferases (MATs) activity and S-adenosylmethionine (SAM) concentrations (P < 0.05), upregulating MAT2A relative mRNA expression in liver and ovarian (P < 0.05). Compared with the CON group, Met supplementation also increased yolk Met and SAM deposition (P < 0.05). Maternal Met supplementation promoted embryonic breast muscle development at embryonic d 19 (E19), as evidenced by higher muscle index and fiber number (P < 0.05), together with increased METTL3 relative mRNA expression and global m6A methylation levels compared with the CON group (P < 0.05). These effects persisted at d 14, with the Met group showing higher body weight, breast muscle index, SAM concentrations, and m6A levels than the CON group (P < 0.05). To investigate the regulatory role of m6A modification in offspring muscle development, d 14 samples from the control and Met groups were analyzed by RNA-Seq and MeRIP-Seq. The results demonstrated that IGF2 and CDK1 transcripts displayed METTL3-dependent m6A hypermethylation and were significantly upregulated in the Met group compared with the CON group (P < 0.05). RNA immunoprecipitation assays confirmed that METTL3 enhanced their stability and activated the IGF2/PI3K/CDK1 signaling pathway. Moreover, primary myoblast experiments demonstrated that Met and SAM promoted proliferation and accelerated the cell cycle through METTL3-mediated m6A modifications. Collectively, these findings demonstrate that maternal Met supplementation improves embryonic and offspring skeletal muscle development through METTL3-dependent RNA methylation, highlighting m6A modification as a key epigenetic mechanism and providing a theoretical basis for maternal nutritional strategies to optimize muscle growth in poultry production.

Low-protein diets supplemented with crystalline amino acids are widely used to reduce nitrogen excretion and feed cost in broiler production, but their metabolic consequences and the role of dietary net energy remain unclear. This study evaluated the effects of low-protein diets on nutrient utilization and metabolic responses in broilers and further examined whether increasing dietary net energy could compensate for protein reduction. To clarify the metabolic consequences of replacing intact protein with crystalline amino acids under low-protein conditions, 180 AA broilers with an average initial body weight of 330 g at 11 days of age were assigned to two dietary treatments: a standard-protein diet and a low-protein diet in which casein was partially replaced by crystalline amino acids. Growth performance, respiratory calorimetry, serum biochemical indices, and liver metabolomics were determined (P < 0.05). To further determine whether dietary net energy could compensate for crude protein reduction, 144 AA broilers with similar initial body weight at 11 days of age were allocated to a 2 × 2 factorial arrangement with two crude protein levels and two net energy levels to evaluate growth performance, carcass traits, serum biochemical indices, and intestinal nutrient transporter expression. In Experiment 1, the low-protein diet reduced final body weight and average daily gain, increased feed conversion ratio, and decreased net energy intake, energy retention, and nitrogen retention. Serum albumin increased, HDL decreased, and AST showed an increasing trend (P < 0.05). Liver metabolomics showed clear separation between the two groups, with differential metabolites mainly enriched in sphingolipid metabolism, the citrate cycle, glycolysis/gluconeogenesis, and pyruvate metabolism. Among these metabolites, 8-Oxodecanoylcarnitine and 1-Stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine were identified as potential biomarkers for evaluating glucose and lipid metabolism in broilers under low-protein conditions. In Experiment 2, significant crude protein × net energy interactions were observed for final body weight and average daily gain. Reducing crude protein altered intestinal amino acid transporter expression by increasing several free amino acid transporters and decreasing PepT1 expression (P < 0.05). Importantly, increasing dietary net energy by approximately 0.5 MJ/kg for each 1% reduction in crude protein helped maintain carcass traits and avoid excessive fat deposition. These findings indicate that low-protein diets should be formulated based on coordinated energy–nitrogen supply, and that increasing net energy by approximately 0.5 MJ/kg for every 1% reduction in crude protein may be an effective strategy for optimizing low-protein diets in broilers.​

Enteric methane (CH4) emissions from ruminants are a major source of agricultural greenhouse gases and represent an energy loss to the host. Methyl-coenzyme M reductase (MCR) is the terminal enzyme in methanogenesis and represents a key target for CH4 mitigation. This study integrated computational screening, in vitro fermentation, and in vivo experiments to identify plant-derived compounds capable of reducing enteric CH4. Molecular docking of 3,900 phytochemicals identified proanthocyanidins (PAC) as top candidate, exhibiting strong predicted affinity to the MCR active site (-8.150 kcal/mol). In vitro rumen fermentation assays showed that PAC supplementation reduced CH4 production by 22

Background Clostridium perfringens degrades the intestinal mucus layer, further contributing to intestinal infections and diarrhea. Gallotannins protect the intestinal tract and alleviate diarrhea, but the mechanism involved is unknown. Objective This study aimed to elucidate the mechanisms by which pentagalloylglucose (PGG) and tannic acid (TA) inhibit C. perfringens, protect the intestinal mucus layer, and alleviate diarrhea. Methods The bactericidal activity and biofilm disruption were studied via proteomics, Raman spectroscopy, and Zeta potential. The mucus layer was analyzed through atomic force microscopy, viscoelasticity assays, and molecular dynamics simulations. The protective effects of PGG and TA on epithelial cells were studied using a C. perfringens-mucus-epithelial cell model. A C. perfringens mouse model was established to investigate the protective effects of PGG and TA on mice. Results PGG and TA displayed strong bactericidal activity at 125 µg/ml, inhibiting bacterial sliding, disrupting biofilm formation, and inducing bacterial aggregation. Membrane protein analysis revealed structural alterations and upregulation of pathways related to membrane permeability, quorum sensing, oxidative phosphorylation, and ABC transporters. These effects increased bacterial stress and accelerated cell death. Molecular docking confirmed that PGG and TA interact with membrane proteins through specific intermolecular forces. C. perfringens infection reduces mucus viscoelasticity and increases roughness. At 7.8 µg/ml, PGG and TA bound to the cysteine-rich region of mucin 2 via intermolecular forces. This enhances viscoelasticity, improving cross-linking and reducing surface roughness. In a coculture model, PGG or TA regulated the expression of inflammatory cytokines and improved cell viability. In C. perfringens-infected mice, PGG and TA significantly alleviated weight loss and effectively restored the inner mucus layer thickness and goblet cell counts (p < 0.05), while concurrently suppressing the overexpression of pro-inflammatory cytokines (IL-1β and IL-6;p<0.05) and downregulated abnormally elevated intestinal barrier and proliferation markers (Ascl2 and Lgr5;p<0.05). Correlation analysis further revealed an inverse relationship, suggesting that the compromised physical mucus defense triggers a compensatory upregulation of mucosal immune signaling and epithelial renewal mechanisms. Conclusion This study reveals that PGG and TA exert antidiarrheal effects through a dual mechanism—inhibiting C. perfringens proliferation and enhancing intestinal mucus barrier integrity—and first elucidates the molecular regulatory pathways underlying the interaction between tannins and the pathogen-mucus layer interplay, offering novel targeted strategies for the treatment of infectious diarrhea.

INTRODUCTION:Dysregulation of hepatic nitrogen metabolism precipitates ammonia (NH3) accumulation, severely compromising broiler growth performance and muscle protein homeostasis via the liver-muscle axis. While L-ornithine-L-aspartate (OA) enhances NH3 clearance and mitigates sarcopenia in human liver disease, its effects and mechanisms on nitrogen metabolism and muscle development in broilers remain limited. OBJECTIVES:This study investigated the impact and underlying mechanisms of dietary OA supplementation on white-feathered broilers. METHODS:Four experiments were conducted using Arbor Acres broilers. Experiments 1 and 2 evaluated dietary OA (0-750 mg/kg) to determine the optimal inclusion rate (400 mg/kg). Experiment 3 established a stable hyperammonemia model via intraperitoneal injection of ammonium acetate. Experiment 4 utilized a 2 × 2 factorial design (0 or 400 mg/kg OA × saline or ammonium acetate challenge) to elucidate OA's protective mechanisms against NH3-induced toxicity. RESULTS:OA supplementation (optimally 400 mg/kg) significantly increased body weight and feed conversion efficiency. OA enhanced Gln metabolism by regulating hepatic nitrogen metabolism. Specifically, it promoted Gln synthesis (increased glutamine synthetase activity and Gln levels) and NH3 detoxification, thereby improving amino acid utilization. Furthermore, OA promoted protein synthesis by activating the hepatic mTOR signaling pathway. At 42 days, 400 mg/kg OA significantly increased breast muscle percentage, improving both growth and slaughter performance. Additionally, OA significantly increased levels of Gln in breast muscle, and effectively reduced NH3 and myostatin levels, thereby alleviating the suppression of muscle growth. OA also promoted muscle development by increasing protein synthesis and deposition through up-regulating the mTOR signaling pathway and down-regulating NF-κB expression. CONCLUSION:OA safely improves broiler growth and slaughter performance by enhancing Gln production and NH3 detoxification through the liver-muscle axis, and increasing protein synthesis and deposition. With a confirmed 5-fold safety margin, OA serves as a safe and novel feed additive to optimize nitrogen metabolism in poultry production.

Background Embryonic muscle development is a highly dynamic and complex process, coordinated by numerous genes and transcriptional regulators such as small RNAs. Results Here, we profiled transcriptomic dynamics in breast muscle from Arbor Acres (AA) broilers and TaoYuan (TY) chickens at three embryonic time points (E9, E13, and E18). While developmentally regulated genes enriched similar biological pathways in both breeds, the architecture of microRNA (miRNA)-mRNA interaction networks was distinct. Integrative analysis combining weighted gene co-expression network analysis, differential expression analysis, and time-series clustering identified 161 candidate genes, including a subset of 39 showing progressively decreasing expression. Differential expression analysis of miRNA revealed that gga-miR-1744-3p was uniquely up regulated at E13 and E18 in TY chickens. By integrating predictions from TargetScan and miRDB, we further identified two core genes (USP8 and ZBTB38) from the set of 39 candidate genes, which are predicted targets of gga-miR-1744-3p. This gene was differentially expressed in the heart, breast muscle, and adipose tissue, and exhibited significantly higher expression in TY chickens. Functional assays confirmed that gga-miR-1744-3p promotes proliferation of chicken primary myoblasts. Conclusions This study provides a comprehensive understanding of the development patterns and molecular mechanisms of breast muscles in broilers during embryogenesis.

The experiment was conducted to clarify the dynamic requirement of tryptophan (Trp) in broilers under low-protein diets and construct a prediction model based on the Trp requirement. A total of 1080 male and 1080 female Arbor Acres broilers at 0 d old were randomly assigned to 6 treatments and allotted to 3 stages: 0 to 14 d starter, 15 to 28 d grower, and 29 to 42 d finisher. The birds in the control group (group NC) were fed with conventional protein diets with nutrient levels formulated to breeder recommendations. The experimental groups (groups Ⅰ to Ⅴ) were fed low-protein diets with different Trp levels which were 80% (group Ⅰ), 90% (group Ⅱ), 100% (group Ⅲ), 110% (group Ⅳ) and 120% (group Ⅴ) of NC group, respectively. The results showed that the optimal response dose of Dig.Trp/Dig.Lys was 0.172 to 0.175 for female broilers, 0.163 to 0.170 for male broilers in starter stage; 0.167 to 0.175 for female broilers, 0.168 to 0.176 for male broilers in grower stage; 0.175 to 0.177 for female broilers in finisher stage. The dynamic model of Dig.Trp/Dig.Lys daily requirement on average metabolic BW and ADG was y = 0.835 × BW0.75-1.745 × ADG-24.181 for females and y = -0.054 × BW0.75 + 0.434 × ADG-0.107 for males in starter phase; y = -0.208 × BW0.75 + 0.759 × ADG + 12.686 for females and y = -0.028 × BW0.75 + 0.523 × ADG-11.082 for males in grower phase; y = 0.393 × BW0.75-0.286 × ADG-91.961 for females and y = 0.146 × BW0.75 + 0.323 × ADG-55.300 for males in finisher phase. The optimal response Trp level in low-protein diets for growth performance can also positively regulate the development of immune organs, serum ALT, AST, immunoglobulins and hormone levels. In conclusion, optimal dietary Trp supplementation in broilers fed low-protein diets resulted in increased growth performance along with improved immune system and stress status. This research provides precise nutritional strategies to enhance poultry performance and efficiency.

The phenomenon where excessive activation of branched-chain amino acid (BCAA) degrading enzymes caused by high concentrations of leucine (Leu) leads to a decrease in the overall concentration of BCAA [including isoleucine (Ile) and valine (Val)] is called BCAA antagonism. Although this phenomenon has long been widely studied, the specific mechanism of its occurrence is still poorly understood. In this study, we investigated the specific mechanism by which Val and Ile alleviate the antagonistic effect caused by high concentrations of Leu through influencing insulin function. First, the ratios of Ile and Val in the low-protein diet were adjusted up and down by 15

Fatty liver hemorrhagic syndrome (FLHS) is a critical disease threatening the laying hen industry. Inulin, a widely used prebiotic, has shown promise in alleviating metabolic disorders, but its role in mitigating FLHS in laying hens is not fully understood. Here, we investigated the effects and underlying mechanisms of inulin-mediated alleviation of FLHS in a high-carbohydrate low-protein diet (HCD)-induced laying hen model. We found that inulin supplementation significantly ameliorated HCD-induced hyperlipidemia, hyperglycemia, hepatic steatosis, liver injury, and oxidative stress. These phenotypic improvements were accompanied by enhanced fatty acid oxidation and suppressed lipid synthesis and inflammation. Microbiota analysis revealed that inulin reshaped the HCD-perturbed cecal microbiota, with Faecalibacterium identified as the only dominant genus substantially depleted by HCD and restored by inulin. Targeted metabolomics showed that inulin elevated cecal spermidine levels, which strongly correlated with Faecalibacterium abundance and improved metabolic traits. Fecal microbiota transplantation (FMT) from inulin-treated donors replicated the protective effects, confirming the causal role of gut microbiota in mediating inulin's anti-FLHS activity. Further mechanistic investigation using the representative species Faecalibacterium prausnitzii demonstrated that inulin enhanced spermidine production through transcriptional activation of the spermidine biosynthetic pathway. Spermidine, in turn, upregulated hepatic ALDH1A2 expression, enhancing retinoic acid synthesis and activating the AMPK-SIRT1 axis, thereby reducing lipid accumulation in hepatocytes. Collectively, these findings establish a novel Faecalibacterium-spermidine-ALDH1A2-retinoic acid-AMPK-SIRT1 axis through which inulin alleviates FLHS, highlighting inulin as a dietary intervention targeting the gut-liver axis and offering novel therapeutic avenues for preventing this disorder in laying hens.

Chicken feather waste poses significant environmental challenges due to its highly recalcitrant keratin structure. Here, we report the isolation and characterization of a novel feather-degrading bacterium, Bacillus sp. A6, isolated from chicken intestinal contents. Following systematic optimization of the fermentation conditions, a feather degradation efficiency of 92.98% was achieved under the optimized conditions (5 g/L inulin, 4% inoculum, initial pH 9, 2% feathers, and 37℃). Furthermore, scale-up in a 5-L fermenter achieved a comparable degradation efficiency of 91.65% within 36 h. The resulting hydrolysate was rich in soluble proteins, peptides, and essential amino acids and exhibited enhanced antioxidant activity. Integrated genomic and transcriptomic analyses suggested that efficient keratin utilization is associated with coordinated metabolic reprogramming involving enhanced energy metabolism, iron acquisition, lipid barrier removal, disulfide bond reduction, extracellular proteolysis, biosurfactant-mediated substrate accessibility, and adaptive nutrient assimilation. These findings further suggest that keratin degradation may represent a coordinated physiological process rather than a single-enzyme reaction. Collectively, this study provides mechanistic insights into microbial feather degradation and offers a potentially scalable strategy for sustainable valorization of feather waste into value-added biomolecules.

The interactive effects of microencapsulated essential oils-organic acids preparation (EOA) and non-starch polysaccharide (NSP) enzymes on intestinal health of broilers fed wheat-based diets remain poorly elucidated. This study aimed to investigate the effects of dietary supplementation with EOA alone and in combination with NSP enzymes on growth performance, slaughter characteristics, serum biochemical indexes and intestinal microenvironment of broilers fed wheat-based diets. A total of 360 one-day-old male Arbor Acres broilers were randomly allocated to five treatment groups for 42 days (n = 6 replicates/treatment, 12 birds/replicate): the basal diet supplemented with 200 mg/kg EOA (A), 400 mg/kg EOA (B), 200 mg/kg NSP enzymes (C), along with 200 mg/kg NSP enzymes plus 200 mg/kg EOA (D), and 200 mg/kg NSP enzymes plus 400 mg/kg EOA (E), respectively. Growth performance was monitored on d 1, 21, and 42; slaughter characteristics, serum biochemical indexes and jejunal digestive enzyme activities were assessed on d 42; and jejunal villus morphology on d 21 and 42 was measured using hematoxylin and eosin (HE) staining. Additionally, on d 42, jejunal gene expression was analyzed by qPCR, cecal short-chain fatty acids (SCFAs) content was determined using gas chromatography (GC), and cecal microbiota composition was characterized via 16S rRNA gene sequencing. Data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test, and statistical significance was set at P < 0.05. Results showed that compared with the single enzymes-added group (group C), dietary addition of EOA alone and combined with NSP enzymes had no significant impacts on the growth performance, slaughter characteristics and serum biochemical indexes of broilers (P > 0.05). However, group D exhibited a 23% decrease in jejunal crypt depth and a 37% increase in villus height to crypt depth ratio (VH/CD) on d 21, and a 29% decrease in crypt depth with a 36% increase in VH/CD on d 42 compared with group C (P < 0.05). Single EOA supplementation significantly decreased jejunal trypsin activity, with 53% and 50% reductions in group A and group B, respectively, relative to group C (P = 0.035). For cecal SCFAs, group B showed a 69% decrease in isovaleric acid content (P = 0.012), while group D had a 73% increase in butyric acid content and a 34% increase in total SCFAs content compared with group C (P < 0.05). In group E, the jejunal mRNA expression of IFN-γ was upregulated 2.0-fold, and the relative abundance of Bacteroides_fragilis in the cecum was reduced by 42% when compared with group C (P = 0.001 and P = 0.034, respectively). Collectively, these findings indicate that the combined application of NSP enzymes with low-dose EOA (200 mg/kg) in wheat-based diets can optimize the intestinal microenvironment of broilers by reducing crypt depth, elevating the VH/CD ratio, and increasing the content of cecal butyric acid and total SCFAs, without compromising growth performance. In contrast, the combination of NSP enzymes with high-dose EOA (400 mg/kg) triggers intestinal microbial dysbiosis and intestinal inflammatory responses in broilers. Therefore, high-dose EOA is not recommended for combined supplementation with NSP enzymes in wheat-based broiler diets under conventional conditions.

BACKGROUND:The efficacy of low-protein diets, an effective approach to the high-quality protein feed shortage, relies on precisely meeting the amino acid requirements of broilers. OBJECTIVE:This study aimed to determine the arginine requirement of WOD188 broilers fed low-protein diets and to assess the effects of arginine on growth performance, intestinal function, and nitrogen metabolism. METHODS:Experiment 1: WOD188 broilers (29-d-old, male, n = 300) were randomly assigned to a normal-protein diet or 4 low-protein diets with standardized ileal digestible arginine-to-lysine ratios (SID Arg:Lys) of 96%, 107%, 118%, and 128% (6 cages/treatment, 10 broilers/cage). After 13 d, growth performance was recorded and regarded as the primary outcome for evaluating the arginine requirement. Samples of blood, breast muscle, intestine, and chyme were collected for the analysis of serum parameters, meat quality, intestinal morphology, gene/protein expression, and intestinal microbiota. Experiment 2: A total of 90 broilers (35-37-d-old) were fed the same 5 diets and collected excreta to measure nitrogen content. Data were analyzed by one-way ANOVA and regression modeling. RESULTS:A dynamic requirement model for SID arginine intake (Y) was established as Y = 1.703 × BW0.70 + 0.750 × body weight gain (BWG). The optimal growth performance and peak nitrogen utilization efficiency were achieved at SID Arg:Lys 107%-115%. Low-protein diets with arginine supplementation decreased meat pH, fecal nitrogen content, serum total protein (TP) and blood urea nitrogen (BUN) concentrations (P < 0.05), while increasing intestinal villus height and NO concentrations (P < 0.05). It also upregulated intestinal CAT-2 mRNA expression, barrier-related protein (ZO-1/ Occludin) expression, and decreased interleukin 10 inflammatory factor concentrations (P < 0.05). The low-protein diet significantly reduced bacterial α-diversity (ACE and Chao1 index, P < 0.05), linear discriminant analysis (LDA) effect size analysis further identified Lactobacillus as key biomarkers LDA score > 2.0, false discovery rate (FDR)-adjusted P < 0.05). CONCLUSIONS:This study established the dynamic arginine requirement model of WOD188 broilers fed the low-protein diet. Dietary arginine enhanced growth performance, carcass traits, and meat quality, while improving intestinal microbiota composition, barrier, and transport function, ultimately elevating nitrogen utilization efficiency.

The liver is the major organ of lipid metabolism in broiler chickens, and disruptions in lipid metabolism can lead to oxidative stress and inflammatory responses, which in turn can cause liver damage, seriously impacting the health of broiler chickens. The following experiment was conducted to investigate the potential effects and related underlying mechanism of l-Ornithine l-Aspartate (OA) on lipid metabolism and inflammatory responses in the livers of broiler chickens fed with a corn-soybean meal basal diet. Dietary addition of OA at 500 mg/kg modulated the AMPK/SREBP1C signaling pathway and down-regulated the relative expression of genes related to lipid synthesis, such as FAS, ACC1, and SCD1, and then reduced the synthesis of fatty acids in the liver and decreased the abdominal fat index, as compared to that of the control group fed with the basal diet. In addition, OA increased mitochondrial membrane oxygen consumption rate, mitigated liver mitochondrial dysfunction, reduced reactive oxygen species production, and decreased inflammatory responses by inhibiting the expression of NF-κB signaling pathway and lowering the levels of inflammatory factors, including IL-1β and TNF-α. In conclusion, dietary OA addition would be not only a therapeutic measure for liver health but also a potential strategy to prevent abdominal fat deposition.

This study elucidated how synchronized glucose and amino acid availability regulates muscle deposition. 540 21 day-old broilers followed a 3 × 3 factorial design: AM/AP ratios (0.19, 0.29, 0.41) and SID Lys levels (1.00%, 1.20%, 1.40%). Significant interactions between AM/AP ratios and SID Lys levels on BW, BWG, breast and thigh muscle percentage, and protein deposition, 0.19 AM/AP with 1.20% SID Lys was highest, whereas 0.41 AM/AP with 1.40% SID Lys was lowest. These represented synchronized (RDS) and asynchronous (SDS) model, respectively. Compared to SDS, RDS increased serum glucose and IGF-1 levels, nutrient digestibility, and transporter expression (GLUT2, SGLT1, CAT1). Metabolomics revealed RDS optimized glycolytic and TCA flux while attenuating Lys catabolism (AASS, ALDH7A1, AADAT). Molecularly, RDS activated AKT/mTOR-mediated synthesis and suppressed AMPK/FoxO3a-mediated degradation. Collectively, synchronizing nutrients via rapidly digestible starch (0.19 AM/AP) optimizes metabolic flux and mitigates amino acid wastage, offering a critical strategy for enhancing performance in low-protein diets.

L-Ornithine-L-aspartate (OA) is a stable salt formed by the ionic bonding of ornithine and aspartic acid. While OA is known to regulate nitrogen metabolism and ammonia (NH3) detoxification more effectively than its individual components in clinical settings, their specific effects and mechanisms in broiler chickens remain unexplored. Crucially, it is unknown whether OA exerts superior biological effects compared to a physical mixture of ornithine and aspartic acid in broiler chickens. Therefore, this study selected white-feathered broilers to investigate the comparative effects of dietary supplementation with OA versus a mixture of ornithine and aspartic acid on growth performance, nitrogen metabolism, and intestinal health. The objective of this study was to elucidate the mechanisms underlying the potential superiority of the salt form (OA) over the mixture. Compared to the basal diet, both supplementation groups improved growth performance and slaughter performance by promoting glutamine (Gln) synthesis and NH3 detoxification, thereby enhancing protein deposition. Specifically, supplementation significantly reduced the feed conversion ratio from d 1 to 21 and increased body weight and breast muscle percentage at d 42 (P < 0.05). Notably, OA demonstrated superior efficacy compared to the mixture. Mechanistically, OA significantly increased hepatic ornithine aminotransferase (OAT) activity at d 21, facilitating ornithine transamination for Gln synthesis. In the gut, OA uniquely reduced duodenal crypt depth (CD) and up-regulated the mRNA expression of key amino acid (AA) transporters (SLC1A5, SLC25A15, and SLC38A2) in the jejunum, leading to significantly higher apparent ileal digestibility of AAs (P < 0.05). Metabolomic and microbiomic analyses revealed that, compared to the mixture, OA significantly modulated arginine biosynthesis pathways (gga00220) and down-regulated L-ornithine (C00077) abundance in ileal chyme. Furthermore, OA improved the cecal microbiota by increasing the relative abundance of butyrate-producing Agathobaculum, reducing pathogenic Escherichia, and up-regulating energy metabolism pathways. In summary, while both forms are beneficial, OA is superior to the physical mixture of ornithine and aspartic acid in improving broiler performance. This advantage of the OA was correlated with the up-regulated intestinal AA transporters, improved the digestibility of nutrients such as ornithine, and the modulated gut microbiota towards a butyrate-producing profile, coupled with increased hepatic OAT activity.

The poultry industry faces challenges due to its high reliance on imported soybean meal (SBM) and the rising costs of conventional protein ingredients, prompting interest in using miscellaneous meals as alternative protein sources. However, low-protein diets formulated with these meals often impair broiler growth performance and protein digestion. In the present study, the effects of Bacillus velezensis CML532 supplementation on growth performance, protein digestion, and gut health were investigated in broilers fed corn-miscellaneous meal-based diets without SBM during the grower and finisher phases. A total of 252 one-d-old healthy male Shengze 901 plus broiler chicks (initial body weight 43.76 ± 0.61 g) were randomly assigned to 3 dietary treatments, each comprising 6 replicates of 14 birds: control group, corn-miscellaneous meal-based normal-protein diet (CM); corn-miscellaneous meal-based low-protein diet (CML) group; and CML diet with supplemental 5 × 109 colony-forming unit (CFU)/kg B. velezensis CML532 (CMLB) group. The experiment lasted 42 d. The results showed that, compared with the CM group, the lower crude protein level (-2%) in the CML group significantly increased feed conversion ratio (FCR), and decreased half-eviscerated rate, eviscerated rate, and breast muscle rate (P < 0.05). Meanwhile, the mRNA expression of calcium sensing receptor (CaSR) and cationic amino acid transporter 1 (CAT1), as well as the concentration of anti-inflammatory factor interleukin-10 (IL-10), were significantly downregulated (P < 0.05). Compared with the CML group, B. velezensis supplementation significantly decreased FCR, improved slaughter traits, increased apparent protein and amino acid digestibility (P < 0.05), and reduced serum uric acid levels (P < 0.001). Notably, growth performance in the CMLB group in terms of final body weight and FCR of the whole growth period was comparable to that in the CM group (P > 0.05). In the jejunum, the CMLB group significantly increased the expression of intestinal epithelial barrier proteins, and elevated the activities of α-amylase and chymotrypsin in the digesta compared with both the CM and CML groups (P < 0.05). Furthermore, the mRNA expression levels of amino acid sensing receptors CaSR, taste receptor type 1 member 1 (T1R1), G protein-coupled receptor class C group 6 member A (GPRC6A), and transporter CAT1 were markedly upregulated in the CMLB group (P < 0.05). Ileal microbial analysis revealed a decreased relative abundance of uncultured_bacterium_g_Lactobacillus in the CML group, whereas B. velezensis supplementation increased the enrichment of Coriobacteriaceae _bacterium_CHKCI002 and B. velezensis, accompanied by enhanced His metabolism and biosynthesis pathways. These results indicated that B. velezensis CML532 supplementation improved protein digestion and utilization by enhancing digestive enzyme activity and modulating gut microbial composition, thereby promoting growth performance and intestinal health in broilers fed with low-protein corn-miscellaneous meal-based diets to levels comparable with those fed normal protein diets.

Clostridium perfringens is a pathogen that secretes multiple toxins, impacting humans and animals. It can cause intestinal diseases such as necrotic enteritis. Although tannins inhibit C. perfringens proliferation, the precise underlying mechanisms are unclear. This study integrated transcriptomics and metabolomics to systematically investigate the mechanism by which tannins, specifically pentagalloylglucose (PGG) and tannic acid (TA), inhibit C. perfringens and potential pathways to alleviate infection in vivo. Ion concentration measurements, flow cytometric analysis, and transmission electron microscopy revealed that PGG and TA damaged the cell membrane structure of C. perfringens, triggering cytoplasmic content leakage. Additionally, PGG and TA significantly affected C. perfringens at the transcriptional and metabolic levels. Bioinformatics analysis revealed that PGG and TA induced amino acid restriction, disrupted energy metabolism, and impeded the ability of C. perfringens to sense and respond to the external environment. In an in vitro C. perfringens-infected intestinal cell model, PGG and TA bound α toxin, significantly reduced the mRNA expression of inflammatory factors, and improved intestinal barrier function and cell viability. Compared to PGG, TA exhibited stronger inhibitory activity against C. perfringens and binding to α toxin. In vivo, PGG and TA alleviated C. perfringens-induced weight loss in mice, improved intestinal villi morphology, and reduced intestinal inflammation and tight junction gene dysregulation. These findings indicate that tannins inhibit C. perfringens, improve gut tissue integrity and reduce inflammation, demonstrating their multi-target effects of resisting intestinal diseases caused by harmful bacteria. This offers new insights for plant polyphenol-based strategies against necrotic enteritis.