The sperm storage tubules located in the mucosal folds of the uterovaginal junction ( UVJ ) are the primary site of sperm storage in chicken hens after natural mating or artificial insemination ( AI ). The short-term sperm storage (24 h after mating or AI) in hens was highly associated with immunity and pH-related pathway genes. However, the underlying mechanism of longer duration of sperm storage in female birds remains largely unclear. In the present study, transcriptome analysis was applied to uncover the dynamic gene expression changes in chicken UVJ tissues at two time points (day 3 and day 9) after AI. A total of 574 differentially expressed genes ( DEG ) were enriched, including 266 upregulated and 308 downregulated DEG. The validation of 5 DEG using quantitative PCR showed a similar expression tendency with RNA sequencing results. The gene ontology terms of DEG were highly enriched in heparin binding (9 genes including COMP , CTGF , and IMPG2 ), glycosaminoglycan binding (10 genes including PCOLCE , POSTN , and RSPO3 ), and response to estradiol and ion transport ( AREG , RAMP3 , SFRP1 , and SSTR1 ). Kyoto encyclopedia of genes and genomes pathway-enrichment analyses of DEG revealed 10 significant pathways ( P < 0.05) represented by calcium signaling pathway (7 genes including CACNA1G , PDE1C , PDGFRB , and SLC8A1 ) and glycosaminoglycan biosynthesis ( B3GNT7 , CSGALNACT1 , GLCE , and ST3GAL1 ). Protein-protein interaction network of DEG established the connection-regulating epithelial cell or cell-matrix adhesion and migration. The enriched pathways and genes were highly correlated with temporary sperm storage in and possibly sequential sperm release from chicken UVJ overtime after AI. Of these, HIP1 , PDE1C , and calcium-related genes were the most interesting candidates associated with sperm storage duration. This report provided a global gene expression profile of the chicken UVJ regarding the capacity of sperm storage overtime after AI. The outcome of this study will contribute to further understanding of the long-term sperm maintenance in avian females and eventually improving the duration of fertile egg performance by selected chicken breeding.

Avian sperm storage tubules ( SSTs ), which are located in the uterovaginal junction ( UVJ ) of the oviduct, are primary sperm storage sites after mating or artificial insemination. The mechanism underlying reduced sperm storage efficiency of SSTs which is highly correlated with decreased fertility rates in aged laying breeders remains largely unclear. Here, comparative transcriptomic analysis between the aged and young White Leghorn hens (120 vs. 30 wk) was applied to identify gene expression changes of UVJs containing SSTs. Bioinformatics analysis revealed 567 upregulated and 1998 downregulated differentially expressed genes. Gene ontology analysis was highly enriched in terms of immune system, cell adhesion, and cytoskeleton proteins. Kyoto Encyclopedia of Genes and Genomes analysis revealed 5 significant ( P < 0.05) pathways including inositol phosphate and glycerophospholipid metabolism. β-Galactosidase staining of chicken UVJ sections suggested increased cell senescence via aging. Oil Red O staining and immunohistochemistry detection of ADFP both confirmed distribution of lipid droplets in SST cells with increased intensity in aged breeders. The lipid synthesis and metabolism-related genes represented by TFAP2 and PLD1 were differentially expressed in aged laying breeders. The upregulation of IL15 and downregulation of a large number of immune-related genes in aged breeders indicate altered immune homeostasis in UVJs and SSTs. The increased accumulation of lipids, and altered immunity homeostasis, combined with other factors ( TJP1 , MYL9 , AFDN , and RPL13 , etc.) are potentially dominant effectors to decrease the sperm storage efficiency and egg fertility in aged laying breeders.

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 taxonomic levels, although the relative abundances for the phylum Firmicutes and the genus Lactobacillus were higher in the HSST group. Furthermore, some taxonomic levels of bacteria expressing the components of several metabolic pathways differed between the HSST and LSST groups. Moreover, predicting functional microbiomes by Kyoto Encyclopedia of Genes and Genomes ( KEGG ) revealed that certain pathways, such as the metabolism of carbohydrates and protein, cellular processes, and organismal systems, of the HSST group exhibited higher expression of genes associated with bioactivity and energy biosynthesis than those in the LSST group. Our results may provide insights into hen-microbe interactions with respect to DF and will be useful in establishing a strategy for new research to uncover the functional regulation of SST in laying hens.

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