The Impact of Dietary Calcium Concentration and Source, and Fiber Type and Source on Broiler Intestinal Histopathology, Microbiome Modulation, and Transcriptomic Response under Subclinical Enteric Infection

Abstract

Understanding the complex host-pathogen-microbiome interactions during enteric challenges is essential for developing sustainable nutrition-based gut health strategies in antibiotic-free broiler production. The removal of antibiotic growth promoters has exacerbated multifactorial diseases like necrotic enteritis (NE), creating a need to understand how nutrition-based strategies modulate the gut environment. The present research utilized a multi-omics approach incorporating histopathology, metagenomics, and transcriptomics within an Eimeria spp. and Clostridium perfringens co-infection model to investigate the mechanistic impacts of two nutritional strategies: (1) modulation of dietary calcium (Ca) concentration and limestone particle size (PS) and (2) manipulation of dietary fiber (DF) type and concentration. Dietary Ca is an essential macromineral in broiler nutrition; however excessive inclusion, now a common industry practice, can elevate digesta pH, impair nutrient digestibility, and thereby increase the risk of NE. The first nutritional strategy evaluated the interactive effects of limestone PS and dietary Ca concentration on intestinal pathophysiology. Broilers were assigned to a 2 × 3 + 1 factorial arrangement, including an unchallenged control and six challenged groups fed diets with two limestone PS (coarse, 910 μm; fine, 200 μm) and three Ca concentrations (adequate, reduced, low). The challenge induced significant jejunal villus atrophy. Reducing Ca concentration in fine limestone diets decreased the abundance of Ruminococcaceae in the jejunum and upregulated host cell cycle pathways, while the same reduction in coarse limestone diets suppressed the immune-modulating bacterium Candidatus Arthromitus. Furthermore, coarse limestone at adequate Ca promoted jejunal inflammatory pathways, whereas at a reduced Ca concentration, it suppressed this pathway. These results demonstrate that limestone PS is a critical factor that modifies the host’s biological and microbial response to dietary Ca concentration. The second nutritional strategy explored the complex role of DF, which has dual functions. Insoluble fibers improve digestive function while soluble fibers can enhance microbial fermentation, yet excessive fiber inclusion can also be a predisposing factor for NE by increasing digesta viscosity and slowing feed passage rate. This study investigated how different fiber sources at varying concentrations modulate gut health across the jejunum and ceca. Broilers were assigned to one of 8 treatments, including an unchallenged control, a challenged control, and six challenged groups fed diets with oat hulls (OH) or soy hulls (SH) alone, or in combination with wheat middlings (WM) or sugar beet pulp (SBP). In the jejunum, specific insoluble-soluble fiber combinations including OH-WM and OH-SBP significantly reduced cumulative pathology scores. Host transcriptomic analysis revealed that fiber supplementation suppressed inflammatory pathways while upregulating cell cycle and DNA repair pathways. In the cecum, the enteric challenge reduced microbial diversity, depleted key butyrate-producing bacterium Faecalibacterium prausnitzii, and induced a strong inflammatory host response with the upregulation of TNFα and NF-κB. The OH-WM combination notably reversed the effect by enriching butyrate-producers, reducing C. perfringens abundance, and downregulating host inflammatory signaling. These findings demonstrate that specific fiber combinations, particularly those with OH, enhance gut resilience through complementary, segment-specific mechanisms. Ultimately, this multi-omics research highlights that optimizing nutritional strategies particularly under enteric challenge conditions requires a detailed understanding of the complex interactions between nutritional components, gut microbiome and host transcriptome.