Abstract: [Objective] To explore the differences in serum biochemical indices, oviducts and intestinal microbiota between laying hens producing dark-spotted eggs and normal eggs. [Method] A total of 120 60-week-old Dawu Jinfeng laying hens were selected and divided into the normal egg group and the dark-spotted egg group. The differences of serum antioxidant indices, lipid metabolism indices, oviduct length and weight, and intestinal microbiota structure of laying hens were compared. [Result] Compared with the normal egg group, the activities of catalase （CAT）, glutathione peroxidase （GSH-Px）, superoxide dismutase (SOD), total antioxidant capacity （T-AOC） and malondialdehyde （MDA） content in serum of laying hens in the dark-spotted egg group increased to varying degrees, but the differences were not significant （P>0.05）. Serum alanine aminotransferase （ALT） activity significantly increased （P<0.05）, whereas there were no significant differences （P>0.05） in the contents of high-density lipoprotein cholesterol （HDL-C）, low-density lipoprotein cholesterol （LDL-C）, triglyceride （TG）, total cholesterol （TC）, aspartate aminotransferase （AST） activity or other indices. Oviduct weight significantly decreased （P<0.05）, while there was no significant difference （P>0.05） in oviduct length. There were no significant differences in the richness or phylum-level diversity of the intestinal microbiota （P>0.05）; at the genus level, the relative abundance of unclassified Bacteroidetes significantly increased （P<0.05）. [Conclusion] The occurrence of dark-spotted eggs is not related to the body′s antioxidant capacity and lipid metabolism level, but the relationship with liver health, oviduct development status and intestinal microbiota composition deserves attention.

Key words: laying hens, dark-spotted eggs, antioxidant capacity, oviduct development, intestinal microbiota composition