High levels of zinc oxide (ZnO) and copper sulfate are widely used as alternative growth promoters in postweaning pig diet. However, excessive exposure to these metals may drive co-selection for heavy metal (HMR) and antibiotic resistance (AMR). Nursery diets also contain abundant iron to offset the low bioavailability of plant-derived iron, yet how dietary iron influence gut dysbiosis and microbial resistance in postweaning pigs remains unclear. This exploratory study examined the effects of dietary iron and metal-based growth promoters on the fecal resistome of postweaning pigs using shotgun metagenomics and whole-genome sequencing (WGS).

Fifty weanling pigs were stratified and randomly assigned to five dietary treatments for 24 d. Experimental diets included a control diet (Con) containing 25, 139, and 141 mg/kg of Cu, Fe, and Zn, respectively, a low-iron diet (LFe, 19 mg Fe/kg), a high-iron diet (HFe, 1,219 mg Fe/kg), a high-copper diet (HCu, 257 mg Cu/kg), and a high-zinc diet (HZn, 2,631 mg Zn/kg, including 2,490 mg Zn/kg from ZnO). All pigs were orally administered with F18 enterotoxigenic Escherichia coli (ETEC) on d 13-16. Metagenome sequencing were performed on d 24 fecal DNA (n = 24) to identify HMR genes (BacMet Predicted database) and AMR genes (CARD database). Functional annotation was performed using HUMAnN3. Whole genome sequencing (WGS) was conducted on 120 E. coli isolates from fecal cultures on d 1, 12, and 24, and AMR and virulence genes were identified from contig assemblies using ABRicate.

Dietary metal treatments significantly altered β-diversity of HMR genes compared with Con, with HZn differing from both HCu and LFe (P < 0.05). Fecal iron levels correlated with sodB (ρ = 0.64, P = 0.075), an iron-containing superoxide dismutase, while fecal copper levels correlated with pcoC (ρ = 0.66, P = 0.075), a plasmid-mediated copper resistance gene. Across metagenomes, 172 AMR genes were identified, dominated by glycopeptide and tetracycline resistance. While dietary iron had minimal effects on fecal AMR profile, HZn induced the largest shifts in resistome, including increases of ant(9)-la, conferring aminoglycoside resistance on mobile genetic elements, and adeF, encoding a multidrug efflux pump (P < 0.05). Functional profiling revealed enrichment of carbohydrate metabolism pathways in HZn group (P < 0.05). WGS of E. coli isolates showed distinct AMR profiles under HZn on d 24 and distinct virulence profile under LFe on d 12, exhibiting increased prevalence of exotoxin and T3SS genes (P < 0.05).

Dietary iron restriction enhanced E. coli virulence genes, whereas excessive ZnO induced the most pronounced changes in the gut resistome and microbial metabolism, highlighting a risk for AMR co-selection and marked influence on gut microbiota.

Amuc_1100, the most abundant outer membrane protein of Akkermansia muciniphila, alleviates high-fat diet (HFD)-induced hepatic lipid accumulation and modulates gut microbiota in fish; however, its mechanism and mediators remain unknown. Using zebrafish model, this study aims to determine the mechanism by which Amuc_1100 reduces HFD-induced hepatic lipid accumulation through modulation of gut microbiota.

In main study, 1-month-old zebrafish were fed a low-fat diet (LFD), HFD, or HFD supplemented with 0.01% Amuc_1100 (AM0.01) for 4 weeks. Body weight gain, hepatic lipid accumulation, microbial translocation, and gut microbiota composition were evaluated. In parallel, larvae at 5 d post-fertilization were fed the same diets for 7 d and analyzed by Oil Red O staining. In validation experiments, germ-free (GF) zebrafish received microbiota transplants from donor fish fed HFD or AM0.01. Antibiotics (ABS)-treated zebrafish were fed LFD, HFD, or AM0.01 for 4 weeks. Intestinal protein interacting with Amuc_1100 was identified via pull-down and co-immunoprecipitation, and its role was confirmed using protein-protein interaction (PPI) inhibitor BV02 and gene knockdown. Data were analyzed by Student's t-test or one-way ANOVA.

Compared with HFD group, zebrafish in AM0.01 group showed lower body weight gain, reduced hepatic lipid accumulation, and decreased microbial translocation (P < 0.05). AM0.01 feeding increased Bacillus abundance while reducing Acinetobacter, Plesiomonas and Aeromonas abundances relative to HFD (P < 0.05). GF zebrafish colonized with microbiota from AM0.01-fed donors showed less hepatic lipid accumulation than those receiving microbiota from HFD-fed donors (P < 0.05). In contrast, ABS-treated zebrafish showed no significant difference in hepatic triacylglycerol content between HFD and AM0.01 groups (P > 0.05). Using pull-down assays with intestinal proteins from LFD-fed zebrafish, we identified 14-3-3β/α-A as an interacting protein of Amuc_1100. When 14-3-3β/α-A PPI was inhibited by BV02, Amuc_1100 failed to alter the HFD-induced gut microbiota profile in 1-month-old zebrafish (P > 0.05). Moreover, either BV02 treatment or 14-3-3β/α-A knockdown abolished the protective effect of Amuc_1100 against hepatic lipid accumulation in conventional and GF zebrafish (P < 0.05).

Amuc_1100 reduces hepatic lipid accumulation by modulating gut microbiota through intestinal 14-3-3β/α-A, highlighting its potential as a therapeutic target.

Fibro-adipogenic progenitors (FAPs) serve as the developmental origin of intramuscular adipocytes in skeletal muscle. Enhancing their adipogenic transition could increase intramuscular fat (IMF) deposition, thereby improving meat flavor in chickens. However, critical aspects of FAPs including their cellular composition, dynamic changes during skeletal muscle growth, and associated regulatory mechanisms remain poorly understood. Therefore, we comprehensively characterized FAPs in Langshan chickens from d 1 to d 98 using both in vivo and in vitro approaches combined with single-nucleus RNA sequencing (snRNA-seq) analysis.

Our analysis of IMF contents and adipogenesis-related gene expression in the pectoralis major muscle of Langshan chickens revealed that the adipogenic properties of FAPs peaked at d 1, reached its lowest point at d 14, and subsequently increased until d 98. The snRNA-seq analysis successfully identified the population of FAPs along with their 5 subtypes including the pre-adipogenic, adipogenic, and fibrotic FAPs. The ratio of the pre-adipogenic subtype decreased from d 14 to d 98, which was reversely correlated with the changes of the adipogenic subtype, suggesting a differentiating process. Furthermore, RNA velocity and pseudo-trajectory analysis revealed that the initial FAPs had superior fibrotic capacity but decreased over time which contrasted with their enhanced adipogenic capacity with development. Notably, BMPER was identified as an important regulator for the adipogenic differentiation of FAPs, which was also confirmed by in vitro over-expression studies. In addition, the expression of BMPER in the adipogenic portion of FAPs was found to be highly conserved across human and mouse skeletal muscles.

Our study provides the first comprehensive atlas of FAPs in the skeletal muscle of chickens and identifies BMPER as a key regulator for the adipogenic differentiation of FAPs. The findings will not only provide novel targets for breeding chickens with high IMF content but also offer significant insights into understanding the cell fate decision of FAPs under both physiological and pathological conditions across species.

The bovine liver is a key organ governing nutrient metabolism, immune regulation, and growth. However, the effects of birth season and growth potential on hepatic protein expression remain poorly understood.

This study investigated the liver proteome of steer calves born during the spring and fall 2023 calving seasons (n = 5-6 steer/growth trait/season). Using a comparative label-free quantitative proteomics approach, 2,133 proteins were identified and quantified following feedlot entry. Principal component and hierarchical clustering analyses revealed distinct segregation of protein expression profiles according to both birth season (spring vs. fall) and growth trait (high vs. moderate), with calving season exerting the stronger overall influence. Bioinformatic and pathway enrichment analyses identified significant growth- and season-independent proteins. Growth-associated proteins were primarily involved in immune signaling, including antigen processing and presentation, as well as glutathione-CYP detoxification pathways. In contrast, season-independent proteins were enriched in pathways related to circadian rhythm, hormonal regulation, and muscle contraction.

These results demonstrate that both growth trajectory and season of birth independently modulate the bovine liver proteome, with stronger seasonal effects, providing novel insight into the metabolic and immune mechanisms underlying variation in calf growth performance.