Mixed fungal polysaccharides enhance intestinal health, antioxidant capacity, and microbiota diversity in broiler chickens

Abstract

Poultry production faces escalating challenges from intensive farming practices, where stressors, including high stocking density, pathogen exposure, and dietary fluctuations, disrupt intestinal integrity, microbiota balance, and antioxidant defenses. These disruptions impair nutrient absorption, growth performance, and immune function, leading to significant economic losses. Although antibiotics have historically mitigated such issues, growing restrictions due to antimicrobial resistance necessitate natural alternatives. Fungal polysaccharides (FP)—notably lentinan (LNT) from Lentinula edodes and polysaccharide from Ganoderma lucidum (GLP), are promising candidates owing to their immunomodulatory, antioxidant, and prebiotic properties. However, existing research focuses predominantly on individual FP, neglecting potential synergies in blended formulations. Structurally, LNT (β-(1→3)-D-glucan backbone) enhances rumen volatile fatty acid production and fiber degradation, whereas GLP (heterogeneous α/β-glycans) potently activates the Nrf2/HO-1 antioxidant pathway and modulates Th1/Th2 immunity. These divergent mechanisms imply complementary effects when combined. In our previous experiments on broiler feeding, we reported that a combination of GLP (68.32% polysaccharide content, composed of mannose, glucose, arabinose, rhamnose, and galactose at a molar ratio of 1.00:16.37:18.82:1.42:17.42) and LNT (76.52% polysaccharide content, composed of mannose, galacturonic acid, arabinose, galactose, glucose, and rhamnose at a molar ratio of 1.00:15.22:8.23:2.05:1.78:4.26) at a 1:1 ratio maximally promoted broiler growth (unpublished data), but their impacts on intestinal morphology, antioxidant signaling, and the microbiota remain uncharacterized. We therefore hypothesize that mixed FP synergistically may enhance intestinal health by simultaneously improving nutrient absorption, activating antioxidant pathways, and stabilizing microbial ecosystems. To investigate the effects of mixed FP on intestinal development, 240 one-day-old Arbor Acres male broilers were randomly assigned to the 0 mg/kg FP (Control), 200 mg/kg FP (Group I), 400 mg/kg FP (Group II), and 600 mg/kg FP (Group III) groups. Broilers were housed in three-tier battery cages (0.7 m × 0.7 m × 0.4 m; 12 broilers/cage), with five replicate cages per experimental group maintained under identical conditions. The experiments were approved by the College of Animal Science and Technology in Anhui Agricultural University (approval number: SYXK 2016-007). All the cages were subjected to a 16 h light: 8 h dark cycle with ad libitum access to water and twice-daily feeding (09:00/16:00) of basal diets (Supplementary Table S1). On day 42, the duodenum, jejunum, and ileum segments were collected, fixed in 4% paraformaldehyde, sectioned at 5 μm, and stained with hematoxylin-eosin. Villus height (VH), crypt depth (CD), and VH/CD ratios were measured via Case Viewer software. The results revealed that Group II significantly increased VH and VH/CD across all the intestinal segments while reducing CD (Figure 1A; P < 0.05 vs the control); these findings suggest enhanced nutrient absorption capacity and intestinal health. To evaluate antioxidant capacity and signaling pathway activation, intestinal tissues were homogenized in PBS (1:9, w/v). The total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) activities were determined via commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)). For gene expression analysis, total RNA was extracted and reverse-transcribed. The qPCR was performed via specific primers for HO-1, NQO1, CAT, Nrf2, and Keap1, with β-actin used as the reference gene (primer sequences and product sizes are listed in Supplementary Table S2). The results demonstrated that Group II significantly elevated antioxidant enzyme activities (P < 0.05), upregulated HO-1, NQO1, CAT, and Nrf2, and

1. Introduction

Poultry production faces escalating challenges from intensive farming practices, where stressors, including high stocking density, pathogen exposure, and dietary fluctuations, disrupt intestinal integrity, microbiota balance, and antioxidant defenses [1]. These disruptions impair nutrient absorption, growth performance, and immune function, leading to significant economic losses [2]. Although antibiotics have historically mitigated such issues, growing restrictions due to antimicrobial resistance necessitate natural alternatives [1].

Fungal polysaccharides (FP)—notably lentinan (LNT) from Lentinula edodes and polysaccharide from Ganoderma lucidum (GLP), are promising candidates owing to their immunomodulatory, antioxidant, and prebiotic properties [3]. However, existing research focuses predominantly on individual FP, neglecting potential synergies in blended formulations. Structurally, LNT (β-(1→3)-D-glucan backbone) enhances rumen volatile fatty acid production and fiber degradation [4], whereas GLP (heterogeneous α/β-glycans) potently activates the Nrf2/HO-1 antioxidant pathway and modulates Th1/Th2 immunity [5]. These divergent mechanisms imply complementary effects when combined.

In our previous experiments on broiler feeding, we reported that a combination of GLP (68.32% polysaccharide content, composed of mannose, glucose, arabinose, rhamnose, and galactose at a molar ratio of 1.00:16.37:18.82:1.42:17.42) and LNT (76.52% polysaccharide content, composed of mannose, galacturonic acid, arabinose, galactose, glucose, and rhamnose at a molar ratio of 1.00:15.22:8.23:2.05:1.78:4.26) at a 1:1 ratio maximally promoted broiler growth (unpublished data), but their impacts on intestinal morphology, antioxidant signaling, and the microbiota remain uncharacterized. We therefore hypothesize that mixed FP synergistically may enhance intestinal health by simultaneously improving nutrient absorption, activating antioxidant pathways, and stabilizing microbial ecosystems.