Regulatory effects of hawthorn leaf flavonoids and stevioside on the uterine function and eggshell quality in laying...

This study aimed to investigate the effects of hawthorn leaf flavonoids (HF) and stevioside (ST) on eggshell quality, uterine function of the oviduct, and microbial composition in laying hens. A total of 630 Jing Tint 6 laying hens aged 66 weeks with similar body weights (1.8 ± 0.2 kg) were randomly divided into 7 groups with 6 replicates of 15 hens each. The control group (CON) was fed a basal diet, while the treatment groups were supplemented with 120 mg/kg HF (HF120), 120 mg/kg ST (ST120), 120 mg/kg HF + 120 mg/kg ST (HF120+ST120), 120 mg/kg HF + 60 mg/kg ST (HF120+ST60), 60 mg/kg HF + 120 mg/kg ST (HF60+ST120), and 60 mg/kg HF + 60 mg/kg ST (HF60+ST60) for 8 weeks. The results indicated that the combined dietary supplementation of HF and ST significantly improved eggshell strength, thickness, and fracture toughness (P < 0.05), with the greatest enhancement observed in the HF120+ST120 group. The CON, HF120, ST120, and HF120+ST120 groups were selected for further analyses. All treatments significantly increased the total thickness of the eggshell ultrastructure (P = 0.003). Particularly, the HF120+ST120 group significantly increased the frequency of in mammilla early fusion (P = 0.001), and reduced both the frequency of B-type mammillae (P = 0.034) and late fusion (P = 0.008). Serum calcium levels were significantly higher in the HF120+ST120 group compared to the CON group (P = 0.020). Histological analysis revealed that dietary supplementation with HF and ST enhanced the duodenal structure (increased villus height and villus-to-crypt ratio) and uterine structure (increased villus fold height), thereby promoting calcium absorption (P < 0.05). Furthermore, the HF120+ST120 treatment significantly increased antioxidant capacity and levels of anti-inflammatory cytokines (interleukin-4 and interleukin-10) in the uterus compared to the CON group (P < 0.05). Transcriptomic analysis of the uterus showed that the HF120+ST120 treatment influenced the expression of genes involved in calcium transport and matrix protein synthesis, including 13 transporter genes related to eggshell mineralization (CALB1, ATP2B1, CA4, etc.) and 5 genes encoding matrix proteins (MEPE, BPIFB3, SPP1, SPP1, and CLU; P < 0.05). Additionally, the abundance of Proteobacteria and Mycoplasma was significantly reduced in the HF120+ST120 treatment group (P < 0.05), with Rikenellaceae_RC9_gut_group and Ruminococcus dominating in this group. In conclusion, dietary supplementation with HF and ST could improve the intestinal and uterine villus morphology, enhance uterine antioxidant and anti-inflammatory capability, balance microecology, and improve calcium transport and matrix protein synthesis in the uterus, ultimately improving the eggshell quality of laying hens. This study provides a novel strategy for improving eggshell quality using plant extract combination, which may help to reduce economic losses in laying hen farming.