Inflammatory uterine microenvironment in long-term infertility repeat breeder cows compared with normal fertile cows

The reproductive performance of lactating dairy cows is gradually declining, and one of the causes of this problem is the presence of long-term infertility repeat breeder cows (RBCs). The causes of RBCs are largely thought to be maternal factors, including the uterine environment. This study aimed to accurately investigate the uterine environment of RBCs using uterine tissue and fluid. Next, we investigated the effect of nobiletin in bovine endometrial epithelial cells to explore the possibility of improving the uterine environment of RBCs. Uterine fluid was collected by flushing the uterus and endometrial tissues were collected by biopsy on day 7 of the estrous cycle from both normal fertile cows and RBCs (n = 5 in each group). A comprehensive analysis of the uterus revealed that gene expression and altered pathways differed between normal fertile cows and RBCs. Especially, pathways of natural killer cell-mediated cytotoxicity, cell cycle, and calcium signaling pathway were picked up in the uterine tissues of RBCs. In the uterine fluid, the levels of lipopolysaccharide were higher in the RBC than in normal group (P = 0.08). In in vitro experiment, treatment with the uterine fluid from RBCs upregulated inflammation-related pathways and molecules such as interleukin-8 (IL-8) in bovine endometrial epithelial cells. The treatment with nobiletin suppressed IL-8 induced by the treatment with uterine fluid. In conclusion, the uterine environment of RBCs was found to be in inflammatory condition, causing the lower reproductive performance. It is necessary to develop methods to improve to the anti-inflammatory state in the uterine environment of RBCs.