摘要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).Tran-scriptomic 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 abun-dance 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.