Effects of Sodium Diformate on Immune Organs, Inflammatory Cytokines, Intestinal Morphology, and Cecal Microbiota of Hy-Line Brown Laying Hens

Background: Organic acidifiers are promising alternatives to in-feed antibiotics, but the effects of sodium diformate (NaDF) on intestinal health and cecal microbiota in laying hens remain unclear. Objective: This study aimed to investigate the effects of dietary NaDF supplementation on immune organ indices, pro-inflammatory cytokines, intestinal morphology, and cecal microbiota in Hy-Line Brown laying hens. Methods: A total of 720 twenty-one-week-old hens were randomly assigned to 6 groups (6 replicates of 20 hens each). Hens were fed a basal diet supplemented with 0 g/kg, 1 g/kg, 2 g/kg, 3 g/kg, 4 g/kg, or 5 g/kg NaDF for 24 weeks following a 1-week pre-feeding period. Results: Dietary NaDF supplementation showed no significant effect on spleen coefficient or the expression of pro-inflammatory cytokines across intestinal segments (P > 0.05). Notably, 1 g/kg to 4 g/kg NaDF significantly increased duodenal villus height (P < 0.05). The ratio of villus height to crypt depth was not significantly affected, with a linear increasing trend observed (P < 0.05), peaking at 4 g/kg. Ileal villus height was significantly increased (P < 0.05), with linear and quadratic responses (P = 0.028, P = 0.004). Cecal microbiota analysis revealed no significant changes in α diversity or microbial composition at the phylum level (P > 0.05). However, supplementation with 4 g/kg NaDF significantly reduced the abundance of Parabacteroides and Mordavella massiliensis (P < 0.05), while Alistipes and Prevotella showed increasing trends (P = 0.083, P = 0.055). Conclusion: Dietary NaDF supplementation improved duodenal and ileal morphology and exerted minor effects on specific bacterial genera, thereby promoting intestinal health in laying hens. However, it did not significantly affect the spleen index or inflammatory cytokine expression. The 4 g/kg level showed the most consistent improvements in intestinal morphology.