摘要Background Dietary supplementation of xylooligosaccharides(XOS)has been found to influence gut health by manipulating cecal microbiota and producing microbe-origin metabolites.But no study investigated and com-pared the effect of in ovo feeding of xylobiose(XOS2)and xylotriose(XOS3)in chickens.This study investigated the effect of in ovo feeding of these XOS compounds on post-hatch gut health parameters in chickens.A total of 144 fertilized chicken eggs were divided into three groups:a)non-injected control(CON),b)XOS2,and c)XOS3.On the 17th embryonic day,the eggs of the XOS2 and XOS3 groups were injected with 3 mg of XOS2 and XOS3 diluted in 0.5 mL of 0.85％normal saline through the amniotic sac.After hatching,the chicks were raised for 21 d.Blood was collected on d 14 to measure plasma immunoglobulin.Cecal digesta were collected for measuring short-chain fatty acids(SCFA)on d 14 and 21,and for microbial ecology and microbial metabolic pathway analyses on d 7 and 21.Results The results were considered significantly different at P＜0.05.ELISA quantified plasma IgA and IgG on d 14 chickens,revealing no differences among the treatments.Gas chromatography results showed no significant differ-ences in the concentrations of cecal SCFAs on d 14 but significant differences on d 21.However,the SCFA concentra-tions were lower in the XOS3 than in the CON group on d 21.The cecal metagenomics data showed that the abun-dance of the family Clostridiaceae significantly decreased on d 7,and the abundance of the family Oscillospiraceae increased on d 21 in the XOS2 compared to the CON.There was a reduction in the relative abundance of genus Clostridium sensu stricto 1 in the XOS2 compared to the CON on d 7 and the genus Ruminococcus torques in both XOS2 and XOS3 groups compared to the CON on d 21.The XOS2 and XOS3 groups reduced the genes for chondroitin sulfate degradation Ⅰ and L-histidine degradation Ⅰ pathways,which contribute to improved gut health,respectively,in the microbiome on d 7.In contrast,on d 21,the XOS2 and XOS3 groups enriched the thiamin salvage Ⅱ,L-isoleu-cine biosynthesis Ⅳ,and O-antigen building blocks biosynthesis(E.coli)pathways,which are indicative of improved gut health.Unlike the XOS3 and CON,the microbiome enriched the pathways associated with energy enhancement,including flavin biosynthesis Ⅰ,sucrose degradation Ⅲ,and Calvin-Benson-Bassham cycle pathways,in the XOS2 group on d 21.Conclusion In ovo XOS2 and XOS3 feeding promoted benefiicial bacterial growth and reduced harmful bacteria at the family and genus levels.The metagenomic-based microbial metabolic pathway profiling predicted a favorable change in the availability of cecal metabolites in the XOS2 and XOS3 groups.The modulation of microbiota and meta-bolic pathways suggests that in ovo XOS2 and XOS3 feeding improved gut health during the post-hatch period of broilers.