Mycobiome temporal and functional dynamics in broilers: Ecological perspective on bacterial-fungal correlations and the effect of feed additives

The gut mycobiome (the fungal component of the microbiome) of chickens, though less abundant than bacterial populations, plays a vital role in gut ecology, yet remains underexplored. This study investigated the temporal, dietary, and ecological factors shaping the broiler chicken excreta-associated fungal communities and their correlation with bacterial microbiota. A total of 320 Cobb 500 (1-day-old) chicks were raised for 21 days in 32 randomly allocated cages. Treatments consisted of four experimental diets: a Basal Diet, a Basal Diet with an Antibiotic (bacitracin methylene disalicylate), an Essential oils blend (oregano oil, rosemary, and red pepper), or a Probiotic (Bacillus subtilis). Shotgun metagenomic sequencing was performed on excreta samples collected at days 1, 10, and 21 to evaluate fungal diversity, composition, cross-kingdom correlation and functional profiling. The fungal community was dominated by Ascomycota and Basidiomycota across all treatments and time points. While alpha diversity metrics did not differ significantly between treatments (P > 0.05), fungal richness and evenness increased significantly over time (P < 0.05), indicating age-driven ecological succession. Beta diversity analysis revealed distinct age-related clustering patterns, with early dominance by Candida albicans and later shifts toward genera such as Fusarium and Malassezia. Feed additives exerted limited influence on fungal composition or diversity metrics, although clustering patterns suggested subtle treatment-specific effects over time. Cross-kingdom correlation analysis identified co-occurring temporal dynamics between the two microbial communities. Candida was positively correlated with Streptococcus and Escherichia/Shigella but negatively associated with beneficial genera like Bifidobacterium and Faecalibacterium. Additionally, microbial functional characteristics were observed in each treatment exhibiting metabolic features. Overall, this study demonstrates that excreta fungal succession in the broiler gut is primarily driven by host age and highlights the temporal plasticity of concurrent changes in fungal and bacteria communities. The findings underscore the importance of multi-kingdom ecological approaches to better understand gut health in poultry production.