Diet Addition with Pomegranate and Onion Extract, Either in an Aqueous or Cyclodextrin-Encapsulated Form, Alters Intestinal Microbiota, Morphometry, and Tight Junction Gene Expression in Broilers

Abstract This study investigated the effects of dietary supplementation with pomegranate peel and onion leaf extracts, administered either as an aqueous solution or in cyclodextrin-encapsulated form, on broiler chickens. 120 day-old Ross 308 broiler chicks were allocated to three groups: non-supplemented (CONTROL), supplemented with aqueous (AQPOMON) and cyclodextrin (CDPOMON) extracts (0.1%/kg DM). Intestinal morphometry was assessed by measuring crypt depth (CD), villus length (VL), and VL:CD ratio in the mid-duodenum, mid-jejunum, and mid-ileum. Tight junction (TJ) protein expression in the mid-duodenum, mid-ileum and mid-jejunum, especially claudin-3, was assessed through immunohistochemistry (IHC), and gene expression of TJ proteins (zonula occludens 1 ( TJP1 ), claudin-1 ( CLDN1 ) , claudin-3 ( CLDN3 ), and occludin ( OCLDN )) via qPCR in the ileum and cecum. Additionally, 16S rRNA gene sequencing was used to study bacterial diversity in the ileum and cecum. Results showed that VL and CD were variably affected by extract supplementation resulting in decreased VL:CD in all intestinal segments. by the aqueous extract (P<0.05). The AQPOMON diet increased TJ protein gene expression and enhanced the intestinal barrier. Firmicutes , Proteobacteria , and Bacteroidota were the dominant phyla across all groups, with differentially abundant taxa like Campylobacteria (ileum) and Clostridia (cecum) in AQPOMON, and Lactobacillaceae (ileum) and Methanobacteriaceae (cecum) in CDPOMON. The AQPOMON diet proved more effective than the CDPOMON in improving aspects of broiler intestinal health.