Effect of reused litter on early cecal microbiota succession and the immune cells distribution in layer chickens.

Abstract

This study was designed to evaluate early immune development and gut microbiome establishment in layer chicks reared on reused or fresh litter. A total of 160 newly hatched female Hy-Line W80 layer were housed on fresh or used litter (n = 80/group). Body weight was recorded weekly, ceca, ileum and spleen samples were collected at days 7 and 35 for gut microbiome analysis and intraepithelial lymphocytes (IELs) study. Results showed that layer chicks reared on fresh litter exhibited significantly higher body weight compared with layer chicks reared on used litter from day 14 to day 35 (P < 0.05), while mortality remained low and was not significantly different between groups. Cecal microbiota analysis revealed significant litter-associated differences in alpha and beta diversity at day 7 (P < 0.05). However, by day 35, microbial richness and overall community composition were no longer significantly different between treatments. At the phylum level, Bacillota was the dominant phylum in both litter groups at days 7 and 35. Pseudomonadota was the second most abundant phylum at day 7, whereas Bacteroidota became the second most abundant phylum by day 35. This shift indicates age related maturation of the cecal microbial community. In terms of immune responses, no significant difference was found in IEL subsets at day 7 between litter groups. However, by 35 days, the used litter exposure resulted in a significant expansion of TCR ⁻ , TCRγδ + , TCRαβ, TCRαβ ⁺ CD8αβ⁺ and TCRαβ + CD8αα + IEL subsets (P < 0.05). Similarly, splenic TCRγδ+ and TCRαβ + CD8αβ+ populations were significantly elevated in used litter layer chicks at day 35. These findings highlight that exposure to different litter environment was associated with distinct cecal microbial profiles and immune-cell distributions in layer chicks. Given the limited information available in laying hens, these results provide novel baseline data and support further studies investigating the role of litter-associated microbial exposure in shaping early immune development.