Age- and sex-dependent morphological alterations of rectal mucosa-associated lymphoid tissue in chickens (Gallus gallus domesticus)

The gut mucosal immune system plays a pivotal role in maintaining intestinal homeostasis, defending against pathogens, and shaping microbiota composition in both mammals and birds that develop mucosa-associated lymphoid tissue (MALT). Here, we characterized the formation and maturation of rectal-MALT (RMALT) in chickens. Given that this maturation critically affects immune competence and health, this study aimed to characterize the developmental dynamics of chicken RMALT, considering sex differences. We investigated RMALT in both sexes across three age groups (1 day, 1 month, and 1 year) using integrated histomorphological and functional analyses. Histologically, RMALT in both sexes increased in size and progressed from diffuse lymphoid aggregations to solitary follicles with age. Immunohistochemistry showed that RMALT is composed of CD3⁺ T cells, Bu-1⁺ B cells, MHCII⁺ antigen-presenting cells, and KUL01⁺ monocyte/macrophage. Notably, 1-year-old females possessed larger RMALT sizes than their male counterparts. Both sexes showed >90% PCNA⁺ proliferating cells in B-cell regions, but females had significantly more proliferating cells in T-cell areas, suggesting enhanced cellular activity. Furthermore, we examined the antigen uptake pathway in the rectal epithelium by focusing on goblet and microfold cell-like cells. In both sexes of 1-year-old chickens, the follicle-associated epithelium showed fewer goblet cells than the non-follicle-associated epithelium regions. Moreover, Jacalin^- CSF1R⁺ microfold cell-like cells in the follicle-associated epithelium significantly increased with aging. Thus, the age-related progression of RMALT suggests a pivotal role in maintaining local immunological conditions in the rectum, particularly in females. These findings provide foundational insights into avian mucosal immunity, which could inform strategies for improving poultry health management.