Dietary muramidase leads to the downregulation of peptidoglycan biosynthesis and to caecal microbial modulation in laying hens

Background: In-feed muramidase enzyme has been linked to numerous advantages in several animal species. In the past years, muramidase has been shown to be effective in hydrolyzing peptidoglycan fragments, especially at small-intestine level in broilers, and to improve digestibility and performance. Moreover, previous studies also showed a possible anti-inflammatory effect of some secondary metabolites derived from the hydrolysis of peptidoglycan. Although a major effort has been carried out to unravel the in vivo mechanism of action of muramidase, there is currently little information on its metabolic interactions in laying hens, especially considering the fundamental differences with broilers in terms of microbiota and host genetics. Therefore, we conducted a 20-week study, testing five different levels of inclusion of muramidase, from 0 mg/kg to 600 mg/kg. We analyzed dry matter and nitrogen digestibility, apparent metabolizable energy, body weight gain, caecal microbiota and microbiome. Results: The intervention with muramidase (Balancius ^®, DSM Nutritional Products Ltd., Basel, Switzerland) led to a drop in α-diversity (Shannon index; P < 0.05) and to microbial composition changes, with a decrease in Lactobacillus and an increase in Collinsella, amongst others (Q < 0.05), at all the muramidase concentrations compared to 0 mg/kg. In parallel, we found that muramidase led to an increased protein digestibility as revealed by the increased nitrogen retention, together with a dose-dependent amelioration of body weight, dry matter digestibility and metabolizable energy (P < 0.05). At functional gene level, we observed a net decrease in the microbial potential to metabolize amino acids, likely as a direct consequence of the lower amino acid availability at caecal level, as linked to the increased nitrogen retention. Moreover, muramidase also led to a decreased microbial functional potential to synthesize peptidoglycan. Conclusion: This study is the first to investigate the effects of dietary muramidase supplementation on nutrient digestibility and metagenomics in laying hens. Our findings align perfectly with the previous studies in broilers, especially in terms of increased protein digestibility. Moreover, for the first time, a direct correlation between the observed phenotype and both microbiota and microbiome has allowed us to gain further insights into the mechanism of action of muramidase in laying hens.