At day 21 of age, Ross-308 broilers were orally gavaged with 7.5 × 10⁶ CFU/mL S. Typhimurium (n = 30), and another 30 birds were kept as the control. The body weight of birds was recorded on days 0, 2, 7, and 14 days post-infection (dpi) to calculate body weight gains (BWGs). At each time point, seven birds per group were euthanized for sample collection to acquire IELs and lymphocytes from the ileum and spleen for flow cytometric analysis. A reduction in BWGs of the infected groups compared to the control group was observed only at 2 dpi. Additionally, there were no changes in the expression of IFN-γ, IL-1β, and TNF-α in the ileum at 2 and 7 dpi. The number of IELs increased significantly following Salmonella infection in the ileum at 2 and 7 dpi without any changes in spleen lymphocytes. The increase in the total number of IELs was derived from the elevated numbers of conventional CD8αβ⁺TCRαβ⁺ and natural IEL populations (CD4⁻CD8⁻TCRαβ⁺, CD8αα⁺TCRαβ⁺, TCRγδ⁺, non-T cells (TCR^neg, and iCD8α cells)). The increase in regulatory IELs and the stable expression of proinflammatory cytokine genes during the first week of infection suggests the potential role of IELs in modulating intestinal inflammation.

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.

To evaluate the effect of in-ovo supplementation of bovine milk-derived Osteopontin (bmOPN) in chicks, fertilized specific-pathogen-free eggs were randomly allocated into six treatment groups (n = 26 eggs/group) on embryonic day 18. The hatchability was collected from six independent experiments. Eggs in each treatment received a 200 μL in-ovo injection of PBS containing 0 mg, 0.1 mg, 1 mg, 10 mg, 25 mg, or 50 mg of bmOPN. On day of hatch (DOH), hatchability and chick quality parameters were assessed. Body weights were recorded on DOH, 7, and 14 days of age. On day 7 of age, intestinal histomorphometric parameters, including villus height (VH), crypt depth (CD), and VH:CD ratio, were measured in the 0, 1, and 25 mg groups. Peripheral blood mononuclear cells (PBMCs) and intraepithelial lymphocytes (IELs) were isolated from 0 and 1 mg groups for flow cytometry analysis. Data were analyzed using Generalized Linear Mixed Model, one-way ANOVA, Kruskal-Wallis or Mann-Whitney U test followed by Tukey HSD/Dunn test. The hatchability increased by approximately 8% in the 1 mg and 25 mg groups compared to the control, reaching 93.6% and 93.9%, respectively. We observed dose-dependent decreases in chick length and residual yolk percentage, along with an increase in navel score up to 25 mg bmOPN. Hatched chick body weights increased slightly (1-2 g) in the 1, 10, and 25 mg groups, and by day 14, chicks in the 1 mg and 10 mg groups maintained higher body weights and body weight gains. On day 7, bmOPN administration increased the number of intestinal T-cell IELs (TCRαβ⁺ subsets and TCRγδ⁺), as well as non-T IELs (including iCD8α⁺ cells), while no changes were observed in peripheral blood mononuclear cells. These findings suggest that in-ovo supplementation of bmOPN enhances hatchability, chick quality, growth performance, and mucosal immune development. However, further studies are warranted to investigate the effects of bmOPN administered through different routes on intestinal function and immune responses during inflammation in both broiler and layer chickens.