Effect of dietary vitamin D₃ on growth performance and functional homeostasis of particular intestinal segments in grass carp fingerlings

The intestine is the principal digestive organ which is generally divided into three segments as the proximal intestine (PI), the mid intestine (MI), and the distal intestine (DI) in fish. In this study, the role of dietary vitamin D₃ (VD₃) in regulating the function and health of different intestinal segments was evaluated in grass carp (Ctenopharyngodon idella) fingerlings through the supplementation with the increasing levels of dietary VD₃ (0, 500, 1000, 1500, 2000, and 2500 IU/kg). The results showed that VD₃ supplementation (1000–2000 IU/kg) significantly enhanced the fish growth performance, and the correlation analysis suggested that this effect was positively correlated with intestinal somatic index (ISI) and intestinal length index (ILI) of MI and DI except PI. In support of this finding, dietary VD₃ enhanced the activities of digestive and brush border enzymes in the fish mainly in MI and DI rather than PI. Furthermore, the intestinal microbiota analysis revealed that VD₃ increased the abundance of intestinal flora at the genus level rather than the phylum level. Meanwhile, hematoxylin and eosin staining presented that VD₃ supplementation reduced epithelial goblet cell hyperplasia and lamina propria edema compared with the VD₃ deficiency group in the MI and DI. Further studies showed that feeding VD₃ decreased malondialdehyde levels but increased total antioxidant capacity, glutathione levels, and total superoxidase activity in MI or DI but not PI, suggesting the antioxidant potential of VD₃ in two intestinal segments. Moreover, in MI and DI but not PI, dietary VD₃ suppressed pro-inflammatory cytokine (il1 \(\beta\) $\beta$ and tnf \(\alpha\) $\alpha$ ) but enhanced antimicrobial peptide (hepcidin and βdefensin) mRNA levels and increased the activities of immune-related enzymes (lysozyme and acid phosphatase), highlighting the ability to improve fish immunity. Corresponding to these effects, dietary VD₃ was able to mediate NF-E2-related factor 2 (Nrf2) and NF-κB pathways in MI and DI. Finally, quadratic regression models based on weight gain (PWG), feed efficiency (PE), ISI, and ILI suggested that the appropriate VD₃ requirements for grass carp fingerlings were 1233.70–1387.11 IU/kg. Taken together, our findings strengthened the potential of dietary VD₃ in managing the functions and health of particular intestinal segments and provided valuable reference data for VD₃ requirements of fingerling fish.