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.

Acetoacetate is a trigger of NLRP3 inflammasome activation in bovine peripheral blood mononuclear cells.

Repeat breeding, which is non-pregnancy following three or more breeding attempts, is a serious reproductive disorder in cattle. In the present study, metabolomic profiling was used to identify metabolites in the blood plasma of repeat breeder cows (RBCs) and non-RBCs. Metabolomic analysis showed that acetoacetate (AcAc), a ketone body, was detected in RBCs, but not in non-RBCs. In contrast, ß-hydroxybutyrate (BHB) was at similar levels in both RBCs and non-RBCs. We hypothesized that an imbalance of AcAc and BHB induces abnormal inflammatory conditions, especially the NLRP3 inflammasome, which regulates sterile inflammation to control interleukin (IL)-1ß secretion, and may be associated with repeat breeding in cattle. To investigate this hypothesis, blood samples were collected from both non-RBCs and RBCs on day 7 of the estrous cycle. The mRNA expression of IL1B in peripheral blood mononuclear cells (PBMCs) was observed to be higher in RBCs than in non-RBCs. To test the effects of AcAc and BHB on inflammatory responses, blood samples were collected from healthy cows and PBMCs were isolated. PBMCs were treated with AcAc and BHB to investigate the activation of the NLRP3 inflammasome (complex of NLRP3, ASC, and caspase-1) and IL-1ß secretion. AcAc treatment resulted in higher protein and/or mRNA expression of NLRP3 and IL-1ß in PBMCs. Moreover, AcAc increased the co-localization of NLRP3 and ASC and stimulated caspase-1 activation, indicating the formation of the platform of the NLRP3 inflammasome. Addition of specific NLRP3 inhibitor, MCC950, suppressed AcAc stimulation-induced IL-1ß secretion. Contrary to the effects of AcAc, BHB treatment suppressed the activation of NLRP3 inflammasome and IL-1ß secretion in response to AcAc and typical NLRP3 inflammasome triggers. These findings demonstrate that AcAc can potentially trigger NLRP3 inflammasome activation, resulting in IL-1ß secretion, and that these inflammatory responses are suppressed by BHB in bovine PBMCs. In addition, the imbalance between AcAc and BHB with higher levels of IL-1ß may be associated with repeat breeding in cattle.

Metabolomic profiles of plasma and uterine luminal fluids from healthy and repeat breeder Holstein cows.

BACKGROUND: Repeat breeding is a critical reproductive disorder in cattle. The problem of repeat breeder cattle remains largely unmanageable due to a lack of informative biomarkers. Here, we utilized metabolomic profiling in an attempt to identify metabolites in the blood plasma and uterine luminal fluids. We collected blood and uterine fluid from repeat breeder and healthy cows on day 7 of the estrous cycle. RESULTS: Metabolomic analysis identified 17 plasma metabolites detected at concentrations that distinguished between the two groups, including decreased various bile acids among the repeat breeders. However, no metabolites that varied significantly were detected in the uterine luminal fluids between two groups. Among the plasma samples, kynurenine was identified as undergoing the most significant variation. Kynurenine is a metabolite produced from tryptophan via the actions of indoleamine 2,3-dioxygenase (IDO). As IDO is key for maternal immune tolerance and induced in response to interferon tau (IFNT, ruminant maternal recognition of pregnancy factor), we examined the responsiveness to IFNT on peripheral blood mononuclear cells (PBMC) isolated from healthy and repeat breeder cows. The mRNA expression of IFNT-response makers (ISG15 and MX2) were significantly increased by IFNT treatment in a dose-dependent manner in both groups. Although treatment with IFNT promoted the expression of IDO in PBMCs from both groups, it did so at a substantially reduced rate among the repeat breeder cows, suggesting that decreased levels of kynurenine may relate to the reduced IDO expression in repeat breeder cows. CONCLUSIONS: These findings provide valuable information towards the identification of critical biomarkers for repeat breeding syndrome in cattle.