The experiment aimed to evaluate the effects of different ratios of Clostridium autoethanogenum protein (CAP) used in the diets on the growth performance, muscle quality, serum indexes, and mTOR pathway of white feather broilers. Four hundred and eighty 1-day-old Arbor Acres (AA) broilers, comprising equal numbers of males and females, were randomly assigned to one of four treatments, and each treatment consisted of 12 replicates of 10 birds. Four diets were formulated based on isoenergetic and isonitrogenous principles. The control group (CAP 0) did not receive any CAP, while the experimental groups received 2% (CAP 2), 3% (CAP 3), and 4% (CAP 4) of CAP for six weeks. Compared with the CAP0, (1) The feed conversion ratio (FCR) was lower (p < 0.05), and the leg muscle yield was higher (p < 0.05) in the CAP3 and CAP4; (2) The serum levels of TP, ALB, T-AOC, and SOD were improved in the CAP3 (p < 0.05); (3) The expression of Lipin-1 gene was down-regulated and AMPKɑ2, Akt, and 4E-BP1 genes were up-regulated in the experiment group (p < 0.05); (4) The inclusion of 3% CAP in the diet increased the levels of 4E-BP1, S6K1, Akt, and AMPKɑ2 phosphorylation by modulating the mTOR signaling pathway (p < 0.05). In conclusion, broiler diets containing 3% CAP can activate the mTOR signaling pathway to promote muscle synthesis and improve growth performance.

Intestinal health is vital for poultry production, and protein plays a key role in intestinal nutrition. The present study used 16S rRNA gene sequencing and serum metabolomics to investigate the effect of CAP on the cecal microflora structure and serum metabolites in 42-day-old broiler chickens. A total of 480 one-day-old Arbor Acres broiler chickens were randomly divided into four treatments with twelve replicates comprising 10 chickens each, evenly divided by sex. The four groups were basal diet group (CAP0), treatment group 1 (CAP2), treatment group 2 (CAP3), and treatment group 3 (CAP4). The broilers in the CAP0 group were fed a basal diet (without CAP), while those in the CAP2, CAP3, and CAP4 groups received diets containing 2%, 3%, and 4% CAP, respectively. Growth performance results showed that dietary CAP supplementation significantly ameliorated the feed conversion rate (FCR) of broilers at 42 days in the CAP3 and CAP4 groups (p < 0.05). Microbial results revealed that CAP did not alter the dominant microorganisms in the cecum at the phylum, family, and genus levels. LEfSe analysis showed significantly higher relative abundances of p_Desulfobacterota, f_Desulfovibrionaceae, and g_Ruminococcus in the CAP3 group compared to the CAP0 and CAP4 groups. Metabolomic analyses indicated that the effect of incorporating CAP into the diet on serum metabolites primarily focused on organic acids and their derivatives, small peptides, amino acid derivatives, and oxidized lipids. The addition of 3% or 4% CAP to the diet can enhance metabolic pathways such as the citrate cycle (TCA cycle) and arginine and proline metabolism. In summary, incorporating CAP into the diet can increase the relative abundance of beneficial bacteria in the cecum and improve the feed conversion efficiency of broilers by enhancing amino acid and energy metabolism.

1. A study used gene synthesis to obtain the functional domains of chicken epidermal growth factor (cEGF) and examined their impact on broiler growth performance, small intestinal morphology, digestive enzyme activities in the intestinal contents and the structure of duodenal microflora. 2. The pET-32a-cEGF recombinant expression vector was constructed. The specific band at 26 KDa was shown by SDS-PAGE analysis and WB results. The purified protein content was shown to be 1687 mu g/ml by assay. 3. A total of 180 healthy, one-day-old Arbor Acres male, white-feathered broilers were randomly divided into three dietary treatment groups (six replicate pens, 10 birds per replicate): A control diet (ND); cEGF diet (cEGF), control supplemented with 250 mg/kg cEGF and the control diet (CD) supplemented with 250 mg/kg chlortetracycline. 4. The results showed that feeding the cEGF and CD diet reduced FCR of broilers aged 1-21 d, average daily feed intake (ADFI) at 22-42 d, and the FCR in the whole period (1-42 d; p < 0.05). Compared with the ND group, the cEGF diet increased duodenal alpha-amylase and alkaline phosphatase activities in the 1-21 d, duodenal lipase, alkaline phosphatase, and ileal alkaline phosphatase activities in the post-period and increased villus height in the duodenum and ileum (p < 0.05). In addition, the ACE and Chao1 index for the birds fed cEGF were higher than the ND group (p < 0.05). At the phyla level, Firmicutes and Proteobacteria were dominant in all groups. At the genus level, the dominant genus was Lactobacillus. The LEfSe analysis showed that the cEGF group was enriched by 11 species including Brevibacillus, Eisenbergiella, Cloacibacterium, Butyricoccus spp. 5. The addition of 250 mg/kg cEGF to the diet can increase growth performance by improving intestinal development and digestive enzyme activity, which may be related to the duodenal intestinal microflora. Therefore, cEGF is an effective alternative to antibiotics in broiler farming.

This study was conducted to evaluate the impacts of a probiotic-enzyme complex (PEC) on laying performance, egg quality, intestinal health, and serum metabolite levels in laying hens fed a low-protein diet. A total of 720 Hy-Line Brown laying hens aged 45 weeks were randomly allocated into three groups, with six replicates per group and 40 hens per replicate. The experiment lasted for 10 weeks. The high-protein (HP, 17% CP) group was fed a high-protein diet, whereas the low-protein (LP, 15% CP) group received a low-protein diet, and the PEC group was fed the same LP diet supplemented with the 0.1% PEC (consisting of Bacillus subtilis K104, Lactobacillus plantarum A490, Clostridium butyricum DS142, and acid protease). Results indicated that the PEC group exhibited significantly higher egg production rate and average daily egg yield compared to the LP group ( p < 0.05), while the FCR was significantly lower than that of the LP group ( p < 0.05). The eggshell strength, jejunal villus height, and villus height-to-crypt depth ratio in the PEC group were significantly higher than those in the LP group ( p < 0.05). Additionally, the probiotic-enzyme complex enhanced the biosynthesis of arginine and cofactor pathway in laying hens, increasing the relative abundance of beneficial bacteria such as Lactobacillus and Bacillus. In conclusion, supplementing laying hen diets with the probiotic-enzyme complex improves gut health and metabolic function, thereby enhancing laying performance and eggshell quality, and mitigating the adverse effects of low-protein diets in laying hens.

Few studies have investigated the effects of L-valine (L-Val) supplementation on in vitro rumen fermentation parameters and methane (CH₄) production in low-nitrogen diets for ruminants. Therefore, we examined the impact of L-Val supplementation in low-protein diets on in vitro rumen fermentation parameters, CH₄ production, and microbial community structure. Two crude protein (CP) levels and 4 L-Val levels were tested as follows: CON group (Control group with 14.05% CP), LD group (low-nitrogen diets with 11.26% CP), LVA group (LD group + 0.25% L-Val), LVB group (LD group + 0.5% L-Val), LVC group (LD group + 0.75% L-Val), and LVD group (LD group + 1% L-Val). The experiment was conducted at five time points (2 h, 4 h, 8 h, 12 h, 24 h), with three replicates per treatment at each time point. Results indicated the following: (1) Fermentation pH decreased overall with time; at each time point, the LVB group exhibited the highest pH, significantly higher than the LD, LVC, and LVD groups (p < 0.05). (2) Ammonia nitrogen (NH₃-N) concentration increased over time, with LVA~LVD groups showing higher levels than the LD group at 24 h, while showing no difference compared to the CON group (p > 0.05). (3) Microbial protein (MCP) trends aligned with NH₃-N, with the LVB group exhibiting higher MCP than the LD group, while showing no difference compared to the CON group (p > 0.05). (4) Compared to the LD group, adding 0.5~1% L-Val increased acetic acid, total VFA (TVFA), and isobutyric acid concentrations at 4 h, 8 h, and 24 h fermentation (p < 0.05). (5) The LVB group exhibited higher proportions of protozoa and Fibrobacter succinogenes (F. succinogenes) compared to the LD group (p < 0.05). The proportion of F. succinogenes showed no significant difference from the CON group (p > 0.05), while the proportion of Butyrivibrio fibrisolvens (B. fibrisolvens) decreased when L-Val addition exceeded 0.5%. (6) Correlation analysis revealed positive correlation between protozoa and TVFA (R = 0.512, p = 0.030). Isobutyric acid showed positive correlations with protozoa, B. fibrisolvens, and F. succinogenes (p < 0.05). In summary, under the present experimental conditions, the addition of 0.5% L-Val to a low-nitrogen diet did not affect predicted CH₄ production, but improved other in vitro rumen fermentation parameters, including acetate, isobutyrate and MCP. Meanwhile, it favored the growth and proliferation of the fibrolytic bacteria (B. fibrisolvens and F. succinogenes). This provides a theoretical basis for the rational formulation of low-nitrogen diets for sheep.

Antimicrobial peptides (AMPs) are a diverse class of bioactive molecules that exert their effects through multiple mechanisms, including membrane disruption, inhibition of cell wall synthesis, interference with intracellular targets, and binding to nucleic acids or proteins. They exhibit broad-spectrum antimicrobial activity and a low propensity to induce drug resistance, positioning them as promising candidates for combating multidrug-resistant infections. However, their clinical translation faces significant challenges, including protease susceptibility, potential cytotoxicity, high production costs, and suboptimal pharmacokinetic properties. In this study, a strain of Bacillus licheniformis SAM-D318 isolated from Hu sheep intestine was used as the peptide-producing strain; genomic analysis confirmed the absence of classical virulence toxin genes despite intrinsic multidrug resistance. A previously unreported cationic α-helical antimicrobial peptide, SAM-KB40 (4176.92 Da, pI 10.886), was obtained from the fermentation supernatant of B. licheniformis SAM-D318 and purified using cation-exchange chromatography and C18 reversed-phase chromatography, achieving a 70.45-fold increase in specific activity. Structural prediction by PEP-FOLD4 suggested an α-helical propensity in the C-terminal region, while circular dichroism spectroscopy indicated that SAM-KB40 adopts a predominantly random coil conformation in aqueous solution. The peptide exhibited potent antibacterial activity against Clostridium perfringens (MIC = 0.5 µg/mL, MBC = 4 µg/mL) and showed selective inhibition against Staphylococcus aureus and Listeria monocytogenes. SAM-KB40 demonstrated exceptional thermal stability (> 97% activity retained at 100 °C for 60 min), good tolerance to acidic conditions (pH 3–4) and bile salts (up to 0.50%), and remarkably low hemolytic activity (1.96% at 1000 µg/mL). Notably, the peptide neutralized recombinant C. perfringens α-toxin at 16 µg/mL in a Vero cell-based assay and exhibited no cytotoxicity at concentrations up to 128 µg/mL, demonstrating a favorable in vitro safety profile.Sequence homology analysis against UniProt, NCBI, APD, and BACTIBASE databases confirmed that SAM-KB40 has not been previously reported. While the peptide shows promising in vitro dual antibacterial and toxin-neutralizing capacity, its high sensitivity to proteinase K (39.78% residual activity) limits oral application. These findings position SAM-KB40 as a promising lead candidate for further development as a feed additive or therapeutic agent against C. perfringens-associated diseases, pending subsequent in vivo validation.