Owing to the practical interest in understanding duration of fertility (DF) to reduce the cost of producing hatching eggs by decreasing the frequency of artificial insemination, as well to uncover the mechanism of the estrogen-gut microbiome axis, elucidating the interaction between the maternal microbiome and the function of sperm storage tubules (SST) has become important for revealing the DF in laying hens. In this study, we investigated the compositional, structural, and functional differences in gut microbiomes between hens with high (HSST, n = 8) and low SST activity (LSST, n = 10) by performing phenotypic selection from approximately 400 individual hens based on their DFs. Their cecal microbial communities were analyzed by sequencing the V4 region of the 16S rRNA gene. The microbiome abundance estimators from the ceca of HSST and LSST hens were not significantly different at the phylum and genus …