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In cattle, artificial insemination (AI) is a technique that allows breeding by depositing frozen-thawed and extended semen into the female reproductive tract. The semen contains sperm with various motility patterns including dead, progressive and hyperactivated. Sperm hyperactivation is high amplitude, asymmetrical beating of sperm tail which usually occurs in the oviduct as part of the capacitation process, but it can also be induced by cryopreservation. After insemination, sperm enter the uterine glands and trigger a pro-inflammatory response in the uterus. Hyperactivated sperm, stimulated by sperm-Toll-like receptor 2 (TLR2), penetrates the mucus and uterine glands more efficiently and enhances the immune response. This facilitates the clearance of excess and dead sperm from the uterus. Some sperm escape the immune response and reach the oviduct either before or after the immune response is initiated. In the oviduct, sperm bind to the epithelium and form a reservoir. This triggers an anti-inflammatory response and preserves the fertilization potential of sperm. Hyperactivation facilitates sperm detaching from the epithelium, swimming through the viscous mucus and cumulus cells, and penetrating the egg's zona pellucida. Sperm-TLR2 activation enhances Ca 2+ -influx and acrosome reaction, which enables sperm to penetrate and fertilize oocytes during in vitro fertilization. Altogether, post-AI in cattle, sperm and maternal immunity interact differentially depending upon the site of sperm hyperactivation - whether it occurs within the uterus or oviduct. Specifically, hyperactivated sperm that enter the uterus after AI or are triggered via sperm-TLR2 activation or other stimuli contribute to sperm-induced uterine inflammation. Such hyperactivated sperm may impede their capacity to ascend to the oviduct. Conversely, sperm that become hyperactivated within the oviduct modulate their interactions with the oviduct and oocytes, which is pivotal during fertilization process. Indeed, the location and timing of sperm hyperactivation partially via TLR2 activation are critical determinants of their different influence on fertility., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites., Competing Interests: Declaration of competing interest The authors declare that they have no financial or competing interests concerning this study., (Copyright © 2024. Published by Elsevier B.V.)

The female reproductive tract (FRT), including the uterus and oviduct (Fallopian tube), is responsible for maintaining an optimal microenvironment for reproductive processes, such as gamete activation and transportation, sperm capacitation, fertilization, and early embryonic and fetal development. The mucosal surface of the FRT may be exposed to pathogens and sexually transmitted microorganisms due to the opening of the cervix during mating. Pathogens and endotoxins may also reach the oviduct through the peritoneal fluid. To maintain an optimum reproductive environment while recognizing and killing pathogenic bacterial and viral agents, the oviduct and uterus should be equipped with an efficient and rigorously controlled immune system. Ovarian sex steroids can affect epithelial cells and underlying stromal cells, which have been shown to mediate innate and adaptive immune responses. This, in turn, protects against potential infections while maintaining an optimal milieu for reproductive events, highlighting the homeostatic involvement of ovarian sex steroids and reproductive epithelial cells. This article will discuss how ovarian sex steroids affect the immune reactions elicited by the epithelial cells of the non-pregnant uterus and oviduct in the bovine, murine, and human species. Finally, we propose that there are regional and species-specific differences in the immune responses in FRT., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Toll-like receptor 2 (TLR2) is a major membrane-bound receptor with ligand and species specificity that activates the host immune response. Heterodimerization of TLR2 with TLR1 (TLR2/1) or TLR6 (TLR2/6), triggered by ligand binding, is essential to initiating the signaling pathway. Bovine TLR2 (bTLR2) heterodimerization has not been defined yet compared with human and mouse TLR2s (hTLR2 and mTLR2). The aim of the present study was to model bovine TLRs (TLRs 1, 2 and 6) and create the heterodimeric forms of the bovine TLR2 using molecular dynamics (MD) simulations. We compared the intermolecular interactions in bTLR2/1-PAM3 and bTLR2/6-PAM2 with the hTLR2 and mTLR2 complexes through docking simulations and subsequent MD analyses. The present computational findings showed that bTLR2 dimerization could have a biological function and activate the immune response, similar to hTLR2 and mTLR2. Agonists and antagonists that are designed for hTLR2 and mTLR2 can target bTLR2. However, the experimental approaches to comparing the functional immune response of TLR2 across species were missing in the present study. This computational study provides a structural analysis of the bTLR2 interaction with bTLR1 and bTLR6 in the presence of an agonist/antagonist and reveals the three-dimensional structure of bTLR2 dimerization. The present findings could guide future experimental studies targeting bTLR2 with different ligands and lipopeptides.

It is known that sperm and seminal plasma (SP) affect uterine immunity. In cattle, artificial insemination enables breeding by depositing frozen and largely diluted sperm with a negligible amount of SP into the uterus. Thus, the present study focused on the impact of frozen-thawed sperm on bovine uterine immunity. We have previously shown that in the bovine uterus, sperm swim smoothly over the luminal epithelium and some sperm interact with uterine glands to induce a weak inflammatory response mainly via the endometrial Toll-like receptor 2 (TLR2) signaling. However, the process by which sperm is encountered in the uterine glands is not completely clear. The present study intended to evaluate the role of sperm-TLR2 in sperm-uterine mucus penetration for reaching the glandular epithelium to induce the uterine immune response. To activate and block sperm-TLR2, they were treated with TLR2 agonist and antagonist, respectively. TLR2 activation enhanced sperm hyperactivation and improved its capacity to penetrate the artificial viscoelastic fluid and estrous-uterine-mucus. In contrast, TLR2-blocked sperm showed completely opposite effects. It is noteworthy, that the TLR2-activated sperm that penetrated the uterine mucus exhibited increased motile activity with hyperactivation. In the sperm-endometrial ex-vivo model, a greater amount of TLR2-activated sperm entered the uterine glands with an immune response, which was seen as the upregulation of mRNA expression for TNFA , IL1B , IL8 , PGES , and TLR2 similar to those in control sperm. On the other hand, a lesser amount of TLR2-blocked sperm entered the uterine glands and weakened the sperm-induced increase only in PGES , suggesting that penetration of a certain number of sperm in the uterine gland is necessary enough to trigger the inflammatory response. Altogether, the present findings indicate that activation of sperm-TLR2 promotes their hyperactivation and mucus penetration with greater motility, allowing them to enter into the uterine glands more. This further suggests that the hyperactivated sperm contributes to triggering the pro-inflammatory cascade partly via TLR2 in the uterus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Akthar, Kim, Umehara, Kanno, Sasaki, Marey, Yousef, Haneda, Shimada and Miyamoto.)

Recently, we reported that sperm induce cluster of differentiation 44 (CD44) expression and Toll-like receptor 2 (TLR2)-mediated inflammatory response in bovine uterus. In the present study, we hypothesized that the interaction between CD44 of bovine endometrial epithelial cells (BEECs) and hyaluronan (HA) affects sperm attachment and thereby enhancing TLR2-mediated inflammation. To test our hypothesis, at first, in-silico approaches were employed to define the binding affinity of HA for CD44 and TLR2. Further, an in-vitro experiment using the sperm-BEECs co-culture model was applied to investigate the effect of HA on sperm attachment and inflammatory response. Here, low molecular weight (LMW) HA at different concentrations (0, 0.1, 1, or 10 µg/mL) was incubated with BEECs for 2 h followed by the co-culture without- or with non-capacitated washed sperm (10 6 /ml) for additional 3 h was performed. The present in-silico model clarified that CD44 is a high-affinity receptor for HA. Moreover, TLR2 interactions with HA oligomer (4- and 8-mers) target a different subdomain (h-bonds) compared to TLR2-agonist (PAM3) which targets a central hydrophobic pocket. However, the interaction of LMW HA (32-mers) with TLR2 revealed no stability of HA at any pocket of TLR2. Notably, the immunofluorescence analysis revealed the HA localization in both endometrial stroma and epithelia of ex-vivo endometrial explant. Moreover, ELISA showed significant levels of HA in BEECs culture media. Importantly, BEECs pretreatment with HA prior to sperm exposure increased the number of attached sperm to BEECs, and upregulated the transcriptional levels of pro-inflammatory genes ( TNFA, IL-1B, IL-8, and PGES ) in BEECs in response to sperm. However, BEECs treated with HA only (no sperm exposure) did not show any significant effect on the transcript abundance of pro-inflammatory genes when compared to the non-treated BEECs. Altogether, our findings strongly suggest a possible cross-talk between sperm and endometrial epithelial cells via HA and HA binding receptors (CD44 and TLR2) to induce a pro-inflammatory response in bovine uterus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ezz, Mansouri, Akthar, Yousef, Kowsar and Miyamoto.)

Toll-like receptor 2 (TLR2) signaling pathway is involved in the sperm-triggered uterine inflammatory response at insemination, but its precise mechanism at molecular-level remains unknown. According to the ligand specificity, TLR2 forms a heterodimer with TLR1 or TLR6 as an initial step to mediate intracellular signaling, leading to a specific type of immune response. Hence, the present study aimed to identify the active TLR2 heterodimer (TLR2/1 or TLR2/6) that is involved in sperm-uterine immune crosstalk in bovine using various models. First, in-vitro (bovine endometrial epithelial cells, BEECs) and ex-vivo (bovine uterine explant) models were employed to test different TLR2 dimerization pathways in endometrial epithelia after exposure to sperm or TLR2 agonists; PAM3 (TLR2/1 agonist), and PAM2 (TLR2/6 agonist). Additionally, in-silico approaches were performed to confirm the dimer stability using de novo protein structure prediction model for bovine TLRs. The in-vitro approach revealed that sperm triggered the mRNA and protein expression of TLR1 and TLR2 but not TLR6 in BEECs. Moreover, this model disclosed that activation of TLR2/6 heterodimer, triggers a much stronger inflammatory response than TLR2/1 and sperm in bovine uterine epithelia. In the ex - vivo model that mimics the intact uterine tissue at insemination, sperm also induced the protein expression of both TLR1 and TLR2, but not TLR6, in bovine endometrium, particularly in uterine glands. Importantly, PAM3 and sperm induced similar and low mRNA expression of pro-inflammatory cytokines and TNFA protein to a lesser extent than PAM2 in endometrial epithelia. This implied that sperm might trigger a weak inflammatory response via TLR2/TLR1 activation which is similar to that of PAM3. Additionally, the in-silico analyses showed that the existence of bridging ligands is essential for heterodimer stability in bovine TLR2 with either TLR1 or TLR6. Altogether, the present findings revealed that sperm utilize TLR2/1, but not TLR2/6, heterodimerization to trigger a weak physiological inflammatory response in the bovine uterus. This might be the way to remove excess dead sperm remaining in the uterine lumen without tissue damage for providing an ideal uterine environment for early embryo reception and implantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mansouri, Yousef, Kowsar, Usui, Akthar and Miyamoto.)

This in vivo study aimed to investigate local and systemic immune responses induced by sperm in cows after artificial insemination (AI). Initially, 12 multiparous Japanese Black cows were subjected to intrauterine AI (AI group, n = 6) or saline infusion (control group, n = 6). The uterine body and horn ipsilateral to the ovulatory follicle were mini-flushed with 2 ml of RPMI-1640 medium at different time points (0, 1, 6, 10, 24, 48 h, and 7 days after AI), centrifuged, and the sediments were examined under a light microscope. Vaginal smears were prepared at 0, 1, 6, and 10 h after AI to investigate the sperm backflow. Subsequently, another experiment was conducted by assigning cows to three groups: intrauterine AI (AI group, n = 5), heat-inactivated AI (Heat-AI group, n = 5), or saline infusion (control group, n = 5). Blood samples were collected, and polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated and analyzed for gene expression using real-time PCR. The results showed that most sperm were rapidly transported either forward into the uterine horn or backward into the vagina within 1 h after AI. The PMNs migrated into the uterine lumen 6 hours after AI. Only active sperm-induced proinflammatory responses in PMNs and PBMCs via upregulation of TNFa, IL8, IL1B, and PGES and downregulation of IL10 at 6 h after AI. These data provide evidence that sperm generate transient proinflammatory responses locally in the uterus and systemically in the peripheral immune cells, which may be prerequisites for uterine clearance, embryo receptivity, and implantation in cows.

The negative impact of zearalenone (ZEN; potent estrogenic mycotoxin) exposure on buffalo embryo production has not yet been determined. In the current study, buffalo sperm and oocytes were exposed to ZEN at different concentrations during maturation. Sperms (with and without ZEN exposure) were incubated for 2 h and evaluated for motility, viability, acrosome integrity, normality, and ultrastructure. Matured oocytes exposed to ZEN were stained to determine their nuclear maturation. Further, their developmental ability was evaluated after in vitro fertilization. Our results showed the toxic effects of ZEN at high concentrations (2000 ng/mL) on different buffalo sperm parameters. The number of acrosome-intact sperm was reduced at 0 h after exposure to a concentration of ≥ 100 ng/mL. Furthermore, the maturation rate of buffalo oocytes (telophase I + metaphase II) was significantly decreased in ZEN-treated oocytes with a higher degeneration rate. Oocytes matured in 1000 ng/mL ZEN and subsequently exhibited considerable reduction in cleavage rate and blastocyst formation compared with control oocytes (2.6% vs. 13.1%). Moreover, the morula rate was decreased (p < 0.001) in ZEN-treated oocytes at concentrations of ≥ 10 ng/mL. Overall, the adverse effects of in vitro ZEN exposure on buffalo sperm parameters and oocyte meiotic progression with a notable reduction in cleavage, morula, and blastocyst rates were defined by these results. Altogether, buffaloes should be considered sensitive to ZEN exposure with respect to their reproductive function., (Copyright © 2023 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

We previously reported that interferon-tau (IFNT), derived from day-7 blastocyst, generates anti-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. However, the real in vivo impact of early embryo-derived IFNT on the uterine proteomic profile is mostly unknown. This study aimed to investigate proteomic changes of uterine flush (UF) when infused with a low physiological level of IFNT without embryo on day-8 post-estrus and its possible impact on the uterine immunological microenvironment. First, a fresh medium was infused into the uterine lumen on day-6, from which UF was obtained 24 h later, and this procedure was repeated on day-7 (control UF). On day-8, this procedure was done with a medium containing recombinant bovine IFNT (100 pg/ml) (IFNT-supplemented UF). Control and IFNT-supplemented UF were tested for immune responses in peripheral blood mononuclear cells (PBMCs). Real-time PCR results revealed that IFNT-supplemented UF downregulated pro-inflammatory cytokines (TNFA, IL1B) and upregulated anti-inflammatory cytokine (TGFB1) and PTGES in PBMCs. Through 2-D PAGE, followed by TOF/TOF mass spectrometer, apolipoprotein-A1 (Apo-A1) protein was identified in the IFNT-supplemented UF, which was confirmed by ELISA analysis. Proteomic analysis revealed again that the in vitro stimulation of BEECs by IFNT upregulated Apo-A1 expression. Further, stimulation of PBMCs with recombinant bovine Apo-A1 downregulated TNFA and NFKB and upregulated TGFB1 and PTGES in PBMCs. Altogether, our results suggest that minute amounts of IFNT alone, normally secreted from bovine blastocyst, stimulate Apo-A1 secretion from the endometrial epithelium in the absence of embryo that initiates an anti-inflammatory environment, which could pave the way for the acceptance of early embryo in the uterus., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Progesterone has been shown to be a potent suppressor of several inflammatory pathways. During pregnancy, progesterone levels increase, allowing for normal pregnancy establishment and maintenance. The dysregulation of progesterone, as well as inflammation, leads to poor pregnancy outcomes. However, it is unclear how progesterone imbalance could impact inflammatory responses in the oviduct and subsequently result in early pregnancy loss. Therefore, in this review, we describe the role of progesterone signaling in regulating the inflammatory response, with a focus on the oviduct and pathological conditions in the Fallopian tubes.

Cumulus cells of ovulated cumulus-oocyte complexes (COCs) express Toll-like receptor 2 (TLR2), pathogen recognition receptors, to recognize and react to sperm signals during fertilization. Sperm also express TLR2, but its contribution to the sperm-oocytes crosstalk is still unclear. Here, we adapted the in vitro fertilization (IVF) model to characterize the potential relevance of sperm TLR2 in sperm-oocytes interactions during fertilization in bovine. The IVF results showed that the ligation of sperm TLR2 with its specific antagonist/agonist resulted in down/up-regulation of the cleavage and blastocyst rates either in COCs or cumulus-free oocytes, but not in zona pellucida (ZP)-free oocytes. The computer-assisted sperm analysis (CASA) system revealed that sperm motility parameters were not affected in TLR2 antagonist/agonist-treated sperm. However, fluorescence imaging of sperm-ZP interactions revealed that the blockage or activation of the TLR2 system in sperm reduced or enhanced both binding and penetration abilities of sperm to ZP compared to control, respectively. Flow cytometrical analysis of acrosome reaction (AR) demonstrated that the TLR2 system adjusted the occurrence of AR in ZP-attached sperm, suggesting that sperm TLR2 plays physiological impacts on the sperm-oocyte crosstalk via regulating ZP-triggered AR in sperm. Given that calcium (Ca 2+ ) influx is a pre-requisite step for the induction of AR, we investigated the impact of the TLR2 system on the ionophore A23187-induced Ca 2+ influx into sperm. Notably, the exposure of sperm to TLR2 antagonist/agonist reduced/increased the intracellular Ca 2+ level in sperm. Together, these findings shed new light that the TLR2 system is involved in sperm AR induction which enables sperm to penetrate and fertilize oocytes during the fertilization, at least in vitro , in cows. This suggests that sperm possibly developed a quite flexible sensing mechanism simultaneously against pathogens as well as COCs toward fertilization with the same TLR2 of the innate immune system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ma, Marey, Shimada and Miyamoto.)

The novel coronavirus disease (COVID-19) is currently a big concern around the world. Recent reports show that the disease severity and mortality of COVID-19 infected patients may vary from gender to gender with a very high risk of death for seniors. In addition, some steroid structures have been reported to affect coronavirus, SARS-CoV-2, function and activity. The entry of SARS-CoV-2 into host cells depends on the binding of coronavirus spike protein to angiotensin converting enzyme-2 (ACE2). Viral main protease is essential for the replication of SARS-CoV-2. It was hypothesized that steroid molecules (e.g., estradiol, progesterone, testosterone, dexamethasone, hydrocortisone, prednisone and calcitriol) could occupy the active site of the protease and could alter the interaction of spike protein with ACE2. Computational data showed that estradiol interacted more strongly with the main protease active site. In the presence of calcitriol, the binding energy of the spike protein to ACE2 was increased, and transferring Apo to Locked S conformer of spike trimer was facilitated. Together, the interaction between spike protein and ACE2 can be disrupted by calcitriol. Potential use of estradiol and calcitriol to reduce virus invasion and replication needs clinical investigation., (© 2022. The Author(s).)

Zearalenone (ZEN)-contaminated diets induce detrimental effects on the bovine reproduction. Recently, we reported that active sperm induce pro-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. This study aimed to investigate the impact of presence of ZEN on the sperm-uterine crosstalk in vitro. BEECs monolayers were stimulated by ZEN (10, 100, and 1000 ng/mL) for 0, 3, 6, 12, or 24 h and gene expressions were analyzed by real-time PCR. Moreover, BEECs were pre-exposed to ZEN (10, 100, and 1000 ng/mL) for 24 h then, co-incubated with sperm for 6 h. Conditioned media (CM) from a sperm-BEECs co-culture, after pre-exposure to ZEN, were harvested and exploited to challenge either polymorphonuclear cells (PMNs) or sperm. Both PMNs phagocytic activity toward sperm and sperm motility parameters were then assessed. Results showed that ZEN alone induced pro-inflammatory responses in BEECs through the induction of mRNA expressions of pro-inflammatory cytokines (TNFA and IL1B) and PGES1 at different time points. Pre-exposure of BEECs to ZEN, amplified the sperm-triggered upregulation of pro-inflammatory cytokines (TNFA and IL1B) and chemokine IL8 mRNA abundance in BEECs. Sperm-BEECs conditioned media, primed by ZEN, stimulated the PMNs phagocytosis for sperm whereas suppressed sperm motility parameters. Taken together, these findings indicate that the presence of ZEN augments the pro-inflammatory cascade triggered by sperm in BEECs, provokes PMNs phagocytosis for sperm, and reduces sperm motility parameters. Such immunological reactions may create a hostile environment for sperm competence and survival in the bovine uterus, thus impair fertility., (Copyright © 2021 Elsevier Inc. All rights reserved.)

During the passage through the female reproductive tract, sperm interact with various compartments and their immune systems. The immune system that protects the female against pathogens also could destroy sperm or prevent them from reaching the site of fertilisation. In particular, the uterine innate immune response is crucial from the perspectives of both the sperm and the uterus. Following insemination, sperm immediately start to trigger inflammation in the uterus by entering uterine glands and activating an innate immune response. In cattle, the activation occurs mainly via TLR2 signalling, if not the only one, between sperm and the uterine epithelium lining the glands. This acute immune response is manifested as the upregulation of mRNA expression of IL8, TNFA, IL1B , and PGES . As a consequence, many sperm are trapped by polymorphonuclear neutrophils, the first and major component of innate immunity. The sperm-induced uterine innate immune responses apparently serve to clear the uterus of excess sperm and, importantly, prepare the endometrium for implantation. Pathophysiological conditions in the uterus seriously disrupt this phenomenon, and thus could directly decrease fertility.

The interaction between early embryo and maternal immune system for the establishment of pregnancy is the focus of several studies; however, it remains unclear. The maternal immune response needs to keep a balance between avoiding any damage to the conceptus and maintaining its function in combating microbes as well. When conceptus-maternal crosstalk cannot achieve this balance, pregnancy losses might occur. Intercommunication between mother and conceptus is fundamental during early pregnancy to dictate the outcome of pregnancy. In ruminants, the embryo reacts with the maternal system mainly via interferon tau (IFNT) release. IFNT can act locally on the embryo and endometrial cells and systemically in several tissues and cells to regulate their response via the expression of interferon-stimulated genes (ISGs). Also, IFNT can induce the expression of inflammatory-related genes in immune cells. Day 7 embryo induces a shift in the maternal immune response towards anti-inflammatory (Th2) immune responses. During maternal recognition of pregnancy, peripheral mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) express markers that configure an anti-inflammatory response. However, PMNs response is more sensitive to the effects of IFNT. PMNs are more likely to express interferon-stimulated genes (ISGs), transforming growth factor-beta (TGFB), interleukin 10 (IL10), and arginase-1 (ARG1), configuring one of the most rapid immune responses to early pregnancy. This review focus on the local and peripheral immune responses during early pregnancy in ruminants, mainly the PMNs function in the immune system., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

This study aimed to characterize proteins and exosomal microRNAs (miRNAs) in the uterine flushings (UF) of cows associated with Day 7 (D7) pregnancy using the embryo donor cows of the embryo transfer program. Superovulated cows either were inseminated (AI cows) or remained non-inseminated (Ctrl cows). UF was collected on D7 in the presence of multiple embryos (AI cows) or without embryos (Ctrl cows) and subjected to isobaric tags for relative and absolute quantification protein analysis. A total of 336 proteins were identified, of which 260 proteins were more than 2-fold higher in AI cows than Ctrl cows. Gene ontology analysis revealed that many differentially expressed proteins were involved in "neutrophil-related" and "extracellular vesicular exosome-related" terms. In silico analysis of proteins with higher concentrations in the UF of AI identified 18 uniquely expressed proteins. Exosomes were isolated from the UF, from which RNA was subjected to miRNA-seq, identifying 37 miRNAs. Of these, three miRNAs were lower, and six miRNAs were higher in the UF of AI cows than those of Ctrl ones. The principal component analysis displayed a close association in miRNA and protein between bta-miR-29a, bta-miR-199b, SUGT1, and PPID. In addition, the receiver operating characteristic curve analysis showed that SUGT1 was the best predictor for the presence of embryos in the uterus. These findings suggest that the presence of multiple D7 embryos in the uterus can lead to significant changes in the protein composition and exosomal miRNA contents of UF, which could mediate innate immunological interactions between the pre-hatching embryo and the uterus in cows., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kusama, Rashid, Kowsar, Marey, Talukder, Nagaoka, Shimada, Khatib, Imakawa and Miyamoto.)

Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Growth hormone (GH) and insulin-like growth factor 1 (IGF1) are crucial for female reproductive functions. The cyclic regulation of the local GH/IGF1 axis in the oviduct and its involvement in oviductal contraction in cattle has not been investigated. Thus, the messenger RNA (mRNA) expression for GH receptor (GHR), IGF1, IGF1 receptor (IGF1R) in the whole oviducts, as well as in cultured bovine oviductal epithelial cells (BOECs) were evaluated. The GHR, IGF1, and IGF1R mRNA expression was significantly higher during postovulatory phase. The luteinizing hormone (LH), estradiol-17β (E2), and LH + E2 treatments significantly increased GHR and IGF1 mRNA expression in cultured BOECs. Further, GH and combination of GH with LH and E2 upregulated IGF1 mRNA expression in the BOECs. Moreover, IGF1 + LH and combined IGF1 + LH + E2 treatments significantly increased prostaglandin synthesis cascade enzyme mRNA expression in the BOECs. An ex vivo microdialysis assay revealed that GH and IGF1 induced the release of oviductal contraction related prostaglandins, endothelin-1, and angiotensin II in follicular and postovulatory phases. Together, the findings strongly suggest that the presence of the active GH/IGF1 axis during the peri-ovulatory period, regulating the local system for the release of oviductal contraction related substances, which may provide the optimal oviductal environment for gametes and early embryo., (© 2021 Wiley Periodicals LLC.)

Upon insemination, sperm cells are exposed to components of the female reproductive tract (FRT) fluids, such as urea and epidermal growth factor (EGF). It has been shown that both urea and EGF use EGF receptor signaling and produce reactive oxygen species (ROS) that are required at certain levels for sperm capacitation and acrosome reaction. We therefore hypothesized that during bovine sperm capacitation, a high level of urea and EGF could interfere with sperm function through overproduction of ROS. High-level urea (40 mg/dl urea is equal to 18.8 mg/dl of blood urea nitrogen) significantly increased ROS production and TUNEL-positive sperm (sperm DNA fragmentation, sDF) percentage, but decreased HOS test score, progressive motility, acrosome reaction and capacitation. The EGF reversed the negative effects of urea on all sperm parameters, with the exception of ROS production and DNA fragmentation, which were higher in urea-EGF-incubated sperm than in control-sperm. The developmental competence of oocytes inseminated with urea-EGF-incubated sperm was significantly reduced compared to the control. A close association of ROS production or sDF with 0-pronuclear and sperm non-capacitation rates was found in the network analysis. In conclusion, EGF enhanced urea-reduced sperm motility; however, it failed to reduce urea-increased sperm ROS or sDF levels and to enhance subsequent oocyte competence. The data suggests that any study to improve sperm quality should be followed by a follow-up assessment of the fertilization outcome.

In mammals, the uterine mucosal immune system simultaneously recognizes and reacts to most bacteria as well as allogenic sperm mainly through the Toll-like receptors (TLR)2/4 signaling pathway. Here, we characterized the impact of pathogen-derived TLR2/4 ligands (peptidoglycan (PGN)/lipopolysaccharide (LPS)) on the immune crosstalk of sperm with the bovine endometrial epithelium. The real-time PCR analysis showed that the presence of low levels of PGN, but not LPS, blocked the sperm-induced inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro . Immunoblotting analysis revealed that PGN prevented the sperm-induced phosphorylation of JNK in BEECs. Activation or blockade of the TLR2 system in the endometrial epithelium verified that TLR2 signaling acts as a commonly-shared pathway for PGN and sperm recognition. The impairment of endometrial sperm recognition, induced by PGN, subsequently inhibited sperm phagocytosis by polymorphonuclear neutrophils (PMNs). Moreover, using an ex vivo endometrial explant that more closely resembles those in vivo conditions, showed that sperm provoked a mild and reversible endometrial tissue injury and triggered PMN recruitment into uterine glands, while PGN inhibited these events. Of note, PGN markedly increased the sperm attachment to uterine glands, and relatively so in the surface epithelium. However, addition of the anti-CD44 antibody into a PGN-sperm-explant co-culture completely blocked sperm attachment into glands and surface epithelia, indicating that the CD44 adhesion molecule is involved in the PGN-triggered sperm attachment to the endometrial epithelium. Together, these findings demonstrate that, the presence of PGN residues disrupts sperm immune recognition and prevents the physiological inflammation induced by sperm in the endometrial epithelium via the MyD88-dependent pathway of TLR2 signaling, possibly leading to impairment of uterine clearance and subsequent embryo receptivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Elesh, Marey, Zinnah, Akthar, Kawai, Naim, Goda, Rawash, Sasaki, Shimada and Miyamoto.)

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudging the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We previously reported that sperm binding to cultured bovine oviduct epithelial cells induces an anti-inflammatory immune response. Now we have developed a differentiated explant model to focus on the oviductal ampulla, where fertilization occurs, and to study the effect of sperm capacitation on the immune response. We used heparin to stimulate bovine sperm capacitation. Fluorescence imaging showed that 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide-labeled sperm pretreated with (Hep (+) ) or without (Hep ( -) ) heparin rapidly attached to the explant ciliated epithelium in similar numbers. However, only Hep (+) sperm upregulated explant messenger RNA (mRNA) transcription of TLR2, IL8, TGFB1, and PGES, without changes in TNFA and IL-10 expression, while Hep ( -) sperm only upregulated PGES. The responses were primarily anti-inflammatory, with a greater response produced by Hep (+) sperm, which also produced a substantial increase in TLR2 protein expression in the epithelium. The addition of TLR1/2 (toll-like receptor 1/2) antagonist to the Hep (+) and (Hep ( -) ) sperm-explant coincubations reduced sperm attachment to the epithelium and inhibited TLR2 protein expression and some of the Hep (+) sperm-induced mRNA transcription. Our observations suggest that the ampullar epithelium immunologically reacts more strongly to sperm that have undergone heparin stimulation of capacitation. This anti-inflammatory response could serve to protect capacitated sperm as they approach the oocyte in the ampulla., (© 2020 Wiley Periodicals LLC.)

High-protein diets contribute to an increase in urea follicular concentrations associated with decreased fertility. Urea has been shown to interfere with the epidermal growth factor (EGF)/EGFR system, which has been shown to have a beneficial effect during in vitro maturation (IVM) of oocytes. Of note, the number of cumulus-oocyte complexes (COCs) in the maturation medium can change the maturation and the developmental competence of COCs. Therefore, it was hypothesized that, the presence of urea and EGF may have a differential effect on the depletion/appearance of AAs and competence of COCs matured individually (I-IVM system) or in groups (G-IVM system). In the G-IVM system, COCs increased consumption (depletion) of AAs compared with other groups in the presence of high-level urea (40 mg/dl) + EGF (10 ng/ml). In the I-IVM system, the non-cleaved COCs depleted more AAs than the cleaved COCs, in particular in the presence of urea. The combination of urea and EGF increased the depletion of AAs in the G-IVM system. However, the EGF abrogated the urea-induced depletion of AAs by the I-IVM COCs. The use of N-acetyl-L-cysteine as an EGFR inhibitor canceled urea-induced depletion of AAs. This shows the inhibiting effect of urea over the EGF/EGFR system. In the presence of urea + EGF, COCs had a lower degree of developmental competence than control in both I- and G-IVM systems. Arginine had the best predictive power to identify highly competent COCs in the G-IVM system, while glutamine was the best predictor of the cleavage in the I-IVM system. In conclusion, this multi-level study shows that COCs matured individually or in groups may have different association with AAs metabolism. These findings provide new insights into the relationships between AA metabolism and the subsequent developmental competence of COCs.

The first 7 days post-insemination are critical for establishment of pregnancy. The pre-ovulatory luteinizing hormone (LH) surge induces ovulation through disruption of the follicle structure that elucidates pro-inflammatory (Th1) responses. Various types of immune cells are recruited into the corpus luteum (CL) to regulate luteal angiogenesis and progesterone (P4) secretion into the circulation to establish pregnancy. The active sperm-uterine crosstalk also induces Th1 responses, mainly via Toll-like receptor (TLR) 2/4 signaling pathway in vitro. The endometrial glands serve as sensors for sperm signals, which trigger Th1 responses. Conversely, the sperm-oviduct binding generates anti-inflammatory (Th2) responses to support sperm survival until fertilization. It is well-established that embryo-maternal crosstalk starts after the embryo hatches out from the zona pellucida (ZP). However most recently, it was shown that the 16-cell stage bovine embryo starts to secrete interferon-tau (IFNT) that induces Th2 immune responses in the oviduct. Once developing embryos descend into the uterine horn, they induce Th2 responses with interferon-stimulated genes (ISGs) expression in the uterine epithelium and local immune cells mainly via IFNT release. Likewise, multiple embryos in the uterus of superovulated donor cows on D7 post-insemination induce Th2 immune responses with ISGs expressions in circulating immune cells. These findings strongly suggest that the maternal immune system reacts to the embryo during the first 7 days post-insemination to induce fetal tolerance. It became evident that the innate immunity of the developing CL, oviduct, and uterus works together to provide optimal conditions for fertilization and early embryonic development during the first 7 days post-insemination., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Previously, we reported that polymorphonuclear neutrophils (PMNs) are constantly existent in the bovine oviduct fluid during the pre-ovulatory stage under physiological conditions. Moreover, incubation of PMNs with bovine oviduct epithelial cells-conditioned medium (BOEC-CM) resulted in suppression of their phagocytic activity for sperm. During pathophysiological conditions, cows may be inseminated by infected semen which exposes oviductal PMNs to allogenic sperm simultaneously with pathogens. This study aimed to visually investigate the role of oviduct epithelium in regulating the phagocytic behavior of PMNs toward sperm as a physiological stimulus, with Escherichia coli (E. coli) as a pathological stimulus. In our experiment, PMNs were incubated for 2 h in BOEC-CM. Phagocytosis was then assayed by co-incubation of these PMNs either with sperm, E. coli, or latex beads. BOEC-CM significantly suppressed the direct phagocytosis of PMNs for sperm, but did not affect their phagocytic activity for E. coli or latex beads. Additionally, an investigation with scanning electron microscopy revealed that BOEC-CM suppressed the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. BOEC-CM did not alter NETs formation towards E. coli. A quantification of NETs formation using an immunofluorescence microscopy showed that the areas of NETs formation for E. coli were significantly larger than those formed for sperm. Our data clearly show that the bovine oviduct, through secretions, protects sperm from phagocytosis by PMNs and eliminates bacterial dissemination through maintaining the phagocytic activity of PMNs towards bacteria.

Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate FOXL2 expression. Therefore, in the present study, we examined whether the ovarian steroid hormones that orchestrate implantation regulate FOXL2 gene expression in ruminants. In sheep, we confirmed that FOXL2 mRNA and protein was expressed in the endometrium across the oestrous cycle (day 4 to day 15 post- oestrus ). Similar to the bovine endometrium, ovine FOXL2 endometrial expression was low during the luteal phase of the oestrous cycle (4 to 12 days post- oestrus ) and at implantation (15 days post- oestrus ) while mRNA and protein expression significantly increased during the luteolytic phase (day 15 post- oestrus in cycle). In pregnant ewes, inhibition of progesterone production by trilostane during the day 5 to 16 period prevented the rise in progesterone concentrations and led to a significant increase of FOXL2 expression in caruncles compared with the control group (1.4-fold, p < 0.05). Ovariectomized ewes or cows that were supplemented with exogenous progesterone for 12 days or 6 days, respectively, had lower endometrial FOXL2 expression compared with control ovariectomized females (sheep, mRNA, 1.8-fold; protein, 2.4-fold; cattle; mRNA, 2.2-fold; p < 0.05). Exogenous oestradiol treatments for 12 days in sheep or 2 days in cattle did not affect FOXL2 endometrial expression compared with control ovariectomized females, except at the protein level in both endometrial areas in the sheep. Moreover, treating bovine endometrial explants with exogenous progesterone for 48h reduced FOXL2 expression. Using in vitro assays with COS7 cells we also demonstrated that progesterone regulates the FOXL2 promoter activity through the progesterone receptor. Collectively, our findings imply that endometrial FOXL2 is, as a direct target of progesterone, involved in early pregnancy and implantation.

We previously reported that sperm binding to cultured monolayers of bovine uterine epithelial cells induces an acute inflammatory response involving the Toll-like receptor (TLR2) signaling pathway. This response serves to clear the uterus of sperm and thereby prepares the endometrium for implantation. The endometrium is lined by surface epithelial cells; however, epithelial cells also line uterine glands. To investigate the source of the immune response, we used an explant model. Explants of bovine endometrium were incubated with bull sperm illuminated by JC1 fluorescent labeling in their mitochondria. The sperm glided over the surface epithelium until they encountered and entered uterine glands, where they remained. Scanning electron microscopy of explants revealed polymorphonuclear neutrophils (PMNs) in uterine glands along with sperm. In the absence of sperm, PMNs were not seen in glands. The incubation of sperm with explants resulted in an acute inflammatory response, seen as the upregulation of mRNA expression of IL8, TNFA, IL1B, PGES and TLR2 in whole explants, as well as increased TNFA protein expression in uterine glands. TLR1/2 antagonist reduced sperm numbers in the glands and inhibited the increase of TNFA. Our observations suggest that uterine glands serve as a site where sperm interact with the uterine epithelium to trigger the innate immune response to clear excess sperm from the uterus.

We have recently shown that the conditioned media from bovine oviductal epithelial cell culture suppress sperm phagocytosis by neutrophils, suggesting that the oviduct around oestrus supplies the anti-inflammatory microenvironment. To investigate the immune response of neutrophils toward the sperm at ovulation in the buffalo oviduct, we examined (a) a detailed distribution of neutrophils in the oviduct in buffaloes, (b) the effect of ovulatory follicular fluid (FF) and oviductal fluid (OF) on sperm phagocytosis by neutrophils, and (c) the interaction of the ovulatory FF with OF on sperm phagocytosis by neutrophils in vitro. Buffalo oviducts were collected from healthy reproductive tracts at a local slaughterhouse. A detailed observation by histological examination and transmission electron microscopy revealed that neutrophils exist in the oviduct epithelium and lumen throughout the oestrous cycle in buffaloes. The number of neutrophils at the oestrus stage was higher in ampulla compared with those in isthmus, whereas they remained relatively constant at the dioestrus stage. Two hours of preincubation of neutrophils with FF enhanced sperm phagocytosis through the formation of neutrophil extracellular traps (NETs) together with H 2 O 2 production, whereas OF around oestrus (eOF) suppressed sperm phagocytosis, NETs formation, and H 2 O 2 production and relieved the above FF-induced inflammatory response. Our findings show that neutrophils exist in the healthy cyclic oviduct across bovine species, and the OF supplies a strong anti-inflammatory environment that could minimize the inflammatory effect of the FF that flows into the oviduct lumen after ovulation and supports the occurrence of fertilization., (© 2019 Wiley Periodicals, Inc.)

We have recently shown that sperm attachment to bovine endometrial epithelial cells (BEECs) triggers uterine local innate immunity with induction of a pro-inflammatory response in vitro, however details of the mechanism remain unknown. Here, we investigated the involvement of Toll-like receptor 2/4 (TLR2/4) pathway in mediating sperm-BEECs inflammatory process. Immunohistochemistry of the uterine tissue revealed that TLR2 and TLR4 proteins were present in the luminal and glandular epithelia of bovine endometrium. Moreover, BEECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h, followed by evaluating mRNA expression of the pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs using a real-time PCR. Both Pam and LPS treatments showed a dose-dependent stimulation of mRNA expression of the pro-inflammatory genes. To elucidate the functional role of TLR2/4 in sperm-BEECs interaction, BEECs monolayers were incubated with either TLR2 antagonist or TLR4 antibody for 2 h prior to the co-culture with sperm for 3 h. Importantly, pre-incubation of BEECs with TLR2 antagonist or TLR4 antibody prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs. Furthermore, sperm increased the phosphorylation levels of TLR2/4 downstream targets (p38MAPK and JNK) in BEECs within 1 h of the co-culture. Treatment of BEECs with TLR2 antagonist prior to sperm addition inhibited JNK phosphorylation, while TLR4 antibody inhibited the phosphorylation of both p38MAPK and JNK. In conclusion, the present in vitro findings strongly suggest that bovine endometrial epithelial cells respond to sperm via TLR2/4 signal transduction., Competing Interests: The authors declare that there is no conflict of interest that could be preserved as prejudicing the impartiality of the research reported.

Increased urea concentration is a major cause of low fertility in dairy cows fed high-protein diets. A strong correlation exists between the urea concentration in the blood and oviduct fluid of dairy cows. In this study, bovine oviduct epithelial cells (BOECs) were incubated with varying concentrations of urea (0, 20, 40, and 80 mg/dL) in the absence of ovarian sex steroids (estradiol and progesterone) and luteinizing hormone. The 80 mg/dL urea reduced the cell viability, and thus was excluded in further analysis. Compared to the control (U0), the 20 mg/dL urea (U20) increased the mRNA expression of Toll-like receptor (TLR) 4, interleukin (IL) 10, IL4, and prostaglandin (PG) E synthase (mPGES) but decreased the mRNA expression of tumor necrosis factor α (TNFA). Compared to U0, the 40 mg/dL urea (U40) decreased the mRNA expression of TNFA and increased alpha-1-acid glycoprotein (AGP). U40 also increased TLR2, IL10, and IL4 mRNA expression compared to U0. In addition, compared to U20, the U40 decreased the mRNA expression of TLR4 and IL1B but increased that of AGP and TLR2. Subsequently, the mRNA expression data were then projected into the Reactome database. The Reactome analysis showed that pathways, including cytokine signaling in the immune system (i.e., TNFs bind their physiological receptors) and death receptor signaling (i.e., TNF signaling), were down-regulated in the presence of urea compared to the U0 group. These in vitro data implied that high urea level can alter the balance between pro- and anti-inflammatory responses in BOECs, thus providing a suboptimal environment for the early reproductive events or a weakened innate immune system, predisposing the oviduct to infections., (Copyright © 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

An understanding gene-gene interaction helps users to design the next experiments efficiently and (if applicable) to make a better decision of drugs application based on the different biological conditions of the patients. This study aimed to identify changes in the hidden relationships between pro- and anti-inflammatory cytokine genes in the bovine oviduct epithelial cells (BOECs) under various experimental conditions using a multilayer response surface method. It was noted that under physiological conditions (BOECs with sperm or sex hormones, such as ovarian sex steroids and LH), the mRNA expressions of IL10, IL1B, TNFA, TLR4, and TNFA were associated with IL1B, TNFA, TLR4, IL4, and IL10, respectively. Under pathophysiological + physiological conditions (BOECs with lipopolysaccharide + hormones, alpha-1-acid glycoprotein + hormones, zearalenone + hormones, or urea + hormones), the relationship among genes was changed. For example, the expression of IL10 and TNFA was associated with (IL1B, TNFA, or IL4) and TLR4 expression, respectively. Furthermore, under physiological conditions, the co-expression of IL10 + TNFA, TLR4 + IL4, TNFA + IL4, TNFA + IL4, or IL10 + IL1B and under pathophysiological + physiological conditions, the co-expression of IL10 + IL4, IL4 + IL10, TNFA + IL10, TNFA + TLR4, or IL10 + IL1B were associated with IL1B, TNFA, TLR4, IL10, or IL4 expression, respectively. Collectively, the relationships between pro- and anti-inflammatory cytokine genes can be changed with respect to the presence/absence of toxins, sex hormones, sperm, and co-expression of other gene pairs in BOECs, suggesting that considerable cautions are needed in interpreting the results obtained from such narrowly focused in vitro studies.

Problem: Recently, we demonstrated both in vitro and in vivo that an immunological crosstalk between Day-7 embryo and immune cells exists locally in the uterus in cows. The peripheral immune response to early embryos at Day-7 of pregnancy in cows remains largely unknown. Thus, we aimed to investigate the response of peripheral blood immune cells in the presence of multiple Day-7 embryos in the uterus in donor cows of embryo transfer program., Method of Study: Superovulated cows were either inseminated (n = 13) at 12-18 hours post-estrus or remained without inseminations (n = 6). Blood was collected following insemination (Day-1) and immediately after uterine flushing (Day-7). Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood for analysis of gene expression., Results: Interferon-stimulated genes (ISGs: ISG15 and OAS1) were increased in both PMNs and PBMCs, with up-regulation of PTGES and anti-inflammatory cytokines (TGFB1 and IL10) expressions at Day-7 of post-inseminations in cows, when compared to those at Day-7 in non-inseminated cows., Conclusion: The findings indicate that the presence of multiple embryos in the uterus generates an anti-inflammatory immune response in peripheral blood immune cells at Day-7 of pregnancy in cows, which might help to accept semi-allogenic embryo in the uterus., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Decreased fertility associated with maternal ageing is a well-known critical problem, and progesterone (P4) concentration decreases during the menopause transition in women. The corpus luteum (CL) secretes P4, thereby supporting the implantation and maintenance of pregnancy. It is proposed that a bovine model is suitable for studying age-associated decline of fertility in women because the physiology of cows is similar to that of women and cows have a greater longevity compared with other animal models. Thus, we investigated the age-dependent qualitative changes and inflammatory responses in the bovine CL. In vivo experiment: Cows were divided into three groups, namely, young (mean age: 34.8 months), middle (80.1 months) and aged (188.9 months). Blood samples were collected on days 7 and 12 during the estrous cycle. In vitro experiments: Cows were divided into young (mean age: 27.6 months) and aged (183.1 months). The CL tissues of these groups were collected from a local slaughterhouse and used for tissue culture experiments. An in vivo experiment, plasma P4 concentration in aged cows was significantly lower than that in young cows, whereas no difference was found regarding the area of CL. An in vitro examination in the bovine CL tissues showed that the luteal P4 concentration, P4 secretion, and mRNA expression of StAR and 3β-HSD were lower in aged cows compared with young cows, especially in the early luteal phase. However, no differences were detected in the mRNA expression of inflammation- and senescence-related factors and inflammatory responses to lipopolysaccharides between the CL tissues from young and aged cows, indicating that an age-dependent increase in inflammation is not involved in the luteal function. P4 production and secretion from the bovine CL diminish in old cows, especially during the early luteal phase, suggesting that senescence may affect the luteal function in cows., (© 2018 Blackwell Verlag GmbH.)

The relationship between vaginal discharge characteristics and metabolic status has been studied in postpartum dairy cows. Vaginal discharges at 2-6 weeks postpartum were scored weekly on a 1 (clear) to 5 (purulent material with stench) scale using the Metricheck device and classified into three groups as follows: Endometritis group (n = 19), greater than or equal to score 4 until 3 weeks postpartum; Delayed involution group (n = 9), score 2 or 3 after having score 1; Healthy group (n = 23), cows except those belonging to the Delayed involution and Endometritis groups. The uterus and cervix were assessed using ultrasonography. Blood samples were collected twice weekly from 1 to 6 weeks postpartum from all cows and were additionally obtained for 13,14-dihydro-15-keto-prostaglandin F2α (PGFM) measurements from multiparous cows. Cows with delayed involution had delayed onset of luteal activity (p < 0.05) and higher serum urea nitrogen (BUN) concentrations (p < 0.01) than healthy animals. Cows with endometritis had more peripartum diseases (p < 0.05), lower milk yield (p < 0.05), longer cervix diameter (p < 0.05), and lower serum albumin (p < 0.01), total cholesterol (p < 0.01), and magnesium (p < 0.01) concentrations, and higher aspartate aminotransferase levels (p < 0.05) than healthy cows. Plasma PGFM concentrations tended to be higher in the Delayed involution group (p = 0.083) and lower in the Endometritis group (p = 0.085) than in the Healthy group. In summary, delayed involution was shown to be associated with higher BUN concentration, and endometritis may have been caused by peripartum diseases and hepatic dysfunction., (© 2018 Blackwell Verlag GmbH.)

In dairy cows, retained fetal membranes (RFM) affect reproductive performance. The aim of this study was to examine the leukocyte counts and the gene expression of tumour necrosis factor α (TNFα), interleukin 1β (IL-1β), IL-8, and IL-10 in polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) in cows with (n = 5) or without (n = 5) RFM during the peripartum period. The lymphocyte counts in RFM cows were higher than those in control cows throughout the experiment (p < .05). The expression of IL-8 in PMNs of control cows was higher (p < .05) compared with that of RFM cows postpartum. In cows with RFM, IL-1β expression was higher (p < .05) in PMNs at 6 weeks postpartum whereas the expression of IL-1β was lower (p < .05) in PBMCs at 4 weeks postpartum. The expression of IL-10 in PBMCs of control cows was higher (p < .05) than that of RFM cows at 2 weeks prepartum and 4 weeks postpartum. Taken together, our data indicate that changes of gene expression of pro- and anti-inflammatory cytokines in RFM cows might be associated with the delayed placental separation and development of uterine inflammation in RFM cows., (© 2018 Japanese Society of Animal Science.)

High-protein diets often lead to an increase in urea concentration in follicular fluid of dairy cows, which may reduce oocyte competence. In the present study, maturation media were supplemented with urea (0, 20, 40 mg/dl), and amino acids (AAs) turnover was evaluated in the 24-h spent media of specimens (cell types), bovine cumulus-oocyte complexes (COCs), cumulus cells (CCs), or denuded oocytes (DOs). The main effects of urea and cell type, and their interaction were significant on the individual turnover (expect threonine, glycine, and tyrosine) and total turnover, depletion, and appearance of AAs. The results showed a high level of urea and DOs increased the depletion of all AAs and that of essential and non-AAs, respectively. Sensitivity analysis revealed the highest sensitivity of isoleucine, lysine, and tryptophan to urea, especially in DOs. Principal component analysis (PCA) evaluated the strong correlations between the turnover of: (1) glutamine, aspartic acid or glycine, and developmental competence and fertilization of COCs; (2) serine, isoleucine, valine or glutamic acid, and cleavage rate of DOs; and (3) serine, glutamine, aspartic acid or alanine, and CCs viability. In conclusion, urea significantly changed the turnover of AAs by COCs, CCs and DOs, and reduced the subsequent developmental competence of bovine oocytes.

In ovarian cystic follicles, molecular changes in the growing follicle may have a local action and contribute to anovulation and cystic formation. One of the candidate molecules that affect the steroid and gonadotropin signaling systems of cystic follicles is lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. To examine the molecular characteristics of bovine cystic follicles, we analyzed LPS concentration in follicular fluid of cystic follicles, and the expression of LPS receptors and steroidogenesis-related genes in granulosa and theca cells. Cystic follicles were categorized as estradiol (E2)-active (EACF) and E2-inactive cystic follicle (EICF). Overall, LPS concentration in follicular fluid of EACF and EICF was higher compared with healthy preovulatory follicles (POF). Expression of luteinizing hormone receptor (LHR) in granulosa and thecal cells was decreased in EACF and EICF compared with POF. Expression of CYP19 in granulosa cells of EACF and EICF was lower than POF. High expression of StAR in granulosa and thecal cells was observed in EICF. In granulosa cells, the expression of TLR4 and TLR2 mRNA was higher in EICF than other follicles. By contrast, higher expression of TLR2 in thecal cells was observed in EICF. Thus, high LPS concentration in follicular fluid of cystic follicles may be associated with the regulation of expression of steroidogenesis-related genes in granulosa and theca cells. These finding revealed the molecular characteristics of bovine ovarian cysts and possible involvement of LPS in the pathology of cystic follicle diseases., (Copyright © 2018. Published by Elsevier B.V.)

Circadian dysfunction perturbs the female reproductive cycle. In particular, mice lacking the clock gene Bmal1 show severe infertility, implying that BMAL1 plays roles in ovulation and luteinization. Here, we examined temporal changes in clock gene expression in the ovary and oviduct before and during gonadotropin-induced follicular growth, ovulation, and luteinization in sexually immature mice. While the oviduct did not show a drastic change in clock gene expression, Bmal1 expression in the ovary was higher than that in control mice during the period from 4 to 16 hr after human chorionic gonadotropin (hCG) administration. Bmal1 expression reached a maximum at 16 hr after hCG administration, when follicle luteinization occurred. In an interesting manner, administration of hCG to ex vivo-cultured oviduct triggered a shorter circadian period and inevitably resulted in phase advance. Together, our present data suggest that LH surge induces continuous expression of BMAL1 in the mouse ovary and modulates circadian phase in the mouse oviduct., (© 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Recent studies suggest that Day-7 bovine embryo starts to communicate with the uterine epithelium through interferon-tau (IFNT) signaling. However, immune modulatory role of IFNT in the uterus just after the embryo moves from the oviduct is unclear. We aimed to examine the hypothesis that Day-7 bovine embryo secretes IFNT in the uterus, which induces anti-inflammatory response in immune cells. The uterine flush (UF) with multiple embryos was collected from Day-7 donor pregnant cows and peripheral blood mononuclear cells (PBMCs) were then cultured in UF. Transcripts detected in PBMCs revealed that UF from pregnant cows down-regulated pro-inflammatory cytokines (TNFA, IL1B) and up-regulated anti-inflammatory cytokine (IL10) expression, with activation of interferon-stimulated genes (ISGs; ISG15, OAS1) as compared with UF from non-pregnant cows. An addition of specific anti-IFNT antibody to the UF inhibited the effect on PBMCs, indicating that IFNT is a major factor for such immune modulation. The observation that conditioned media from bovine uterine epithelial cells both stimulated with IFNT in vitro and supplemented with fresh IFNT induced similar PBMCs gene expression, confirming that IFNT directly acts on this immune crosstalk. This study shows that IFNT secreted from Day-7 embryo in vivo generates anti-inflammatory response in immune cells, which may provide immunological tolerance to accept the embryo., (Copyright © 2018 Elsevier Inc. All rights reserved.)

SummaryNutrition influences the microenvironment in the proximity of oocyte and affects early embryonic development. Elevated blood urea nitrogen, even in healthy dairy cows, is associated with reduced fertility and there is high correlation between blood urea levels and follicular fluid urea levels. Using a docking calculation (in silico), urea showed a favorable binding activity towards the ZP-N domain of ZP3, that of ZP2, and towards the predicted full-length sperm receptor ZP3. Supplementation of oocyte maturation medium with nutrition-related levels of urea (20 or 40 mg/dl as seen in healthy dairy cows fed on low or high dietary protein, respectively) dose-dependently increased: (i) the proportion of oocytes that remained uncleaved; and (ii) oocyte degeneration; and reduced cleavage, blastocyst and hatching rates. High levels of urea induced shrinkage in oocytes, visualised using scanning electron microscopy. Urea downregulated NANOG while dose-dependently upregulating OCT4, DNMT1, and BCL2 expression. Urea at 20 mg/dl induced BAX expression. Using mathematical modelling, the rate of oocyte degeneration was sensitive to urea levels; while cleavage, blastocyst and hatching rates exhibited negative sensitivity. The present data imply a novel role for urea in reducing oocyte competence and changing gene expression in the resultant embryos.

Recent studies indicate that communication between the bovine embryo and the mother begins in the oviduct. Here, we aimed to investigate the effect of embryos on bovine oviducts for their immune responses using an in vitro model. First, zygotes were cultured with or without bovine oviduct epithelial cells (BOECs) for 4 days, when embryos had reached the 16-cell stage. At that time, we detected interferon-tau (IFNT) in embryos co-cultured with BOECs, but not in embryos cultured alone. Next, peripheral blood mononuclear cells (PBMCs) were incubated either in media from embryo alone cultures or from co-cultures of embryos with BOECs. The medium from embryo alone cultures did not modulate PBMCs gene expression; whereas the embryo-BOEC co-culture medium increased interferon-stimulated genes (ISGs: ISG15, OAS1, MX2), STAT1, PTGES and TGFB1 but suppressed IL17 expression in PBMCs. Both IFNT-treated BOEC culture medium and IFNT-supplemented fresh medium alone without BOEC, modulated PBMCs gene expressions similar to those by the embryo-BOEC co-culture medium. Further, specific antibody to IFNT neutralized the effect of embryo-BOEC co-culture medium on PBMCs gene expression. Our results indicate that BOECs stimulate embryos to produce IFNT, which then acts on immune cells to promote an anti-inflammatory response in the oviduct.

In the cow, cryopreserved semen is inseminated into the uterus, and most of sperm are removed by backflow and phagocytes. Nevertheless, the mechanism responsible for sperm phagocytosis is unclear. Here, we used cultured bovine uterine epithelial cells (BUECs) to investigate the uterine response to sperm and the mechanism that activates polymorphonuclear neutrophils (PMNs). BUEC monolayers were co-cultured with different numbers of washed sperm obtained from cryopreserved semen (10 4 , 10 5 , and 10 6 sperm/ml) for 3 hr. Sperm dose-dependently up-regulated IL8 (Interleukin 8). Sperm at 10 6 /ml increased mRNA expression of TNFA (Tumor necrosis factor alpha), IL1B (Interleukin 1B), NFKB2 (Nuclear factor kappa B2), and C3 (Complement factor 3), as well as PGES (Prostaglandin E synthase) expression and PGE 2 release. Live sperm, but not dead sperm, attached to BUECs, and dead sperm did not induce an acute inflammatory response. Time-dependent effects were evaluated by co-culture of 10 6 /ml washed sperm with BUECs for 0, 1, 3, and 6 hr. The number of detached sperm increased gradually toward 6 hr. Maximum mRNA expression of IL8, TNFA, IL1B, and NFKB2 was induced at 3 hr, while C3 continued to increase toward 6 hr. Sperm did not stimulate mRNA expression of anti-inflammatory cytokines TGFB1 (Transforming growth factor beta 1) or IL10 (Interleukin 10). Medium conditioned by sperm co-incubated with BUECs stimulated PMNs phagocytosis of sperm in vitro. Fresh media supplemented with low levels of IL1B, TNFA, and PGE 2 up-regulated sperm phagocytosis by PMNs as well. In conclusion, our findings strongly suggest that the active sperm attach to BUECs and trigger uterine local innate immunity with induction of a pro-inflammatory response that enhances sperm phagocytosis by PMNs., (© 2018 Wiley Periodicals, Inc.)

Dietary contamination by Zearalenone (ZEN) has a detrimental effect on bovine fertility. Recently, we showed a novel anti-inflammatory response of bovine oviductal epithelial cells (BOEC) to active sperm cells in vitro. The aim of the present study was to investigate the effect of ZEN exposure of BOEC on the immune-related cytokine expression in response to bovine sperm. At concentrations of 100 and 1000ng/mL, ZEN induced the expression of TNF and IL1B (pro-inflammatory cytokines) as well as IL8 (chemokine) in BOEC in a dose-dependent manner. Furthermore, ZEN induced PTGES expression and PGE 2 secretion in BOEC. Sperm co-culture induced an anti-inflammatory response in BOEC with upregulation of TGFB, secretion of PGE 2 and downregulation of TNF. Most importantly, ZEN at 1-1000ng/mL eliminated the response of BOEC to sperm. Estradiol-17β (5ng/mL) treatment did not produce the same effects as ZEN, suggesting that the response of BOEC to ZEN is, at least in part, not mediated by estrogen receptors. Taken together, ZEN can produce inflammatory effects on BOEC by stimulating the expressions of pro-inflammatory cytokines and disrupt the normal interaction between sperm and BOEC at the level of cytokine expressions and PGE 2 production. Thus, exposure of the bovine oviduct to ZEN may negatively affect sperm survival and reduce fertility., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recent observations suggest that the bovine uterus starts to react to the early embryo immediately after its arrival from the oviduct. The present study aimed to investigate the effect of the early developing embryo on the immune-related gene profile in bovine uterine epithelial cells (BUECs) in vitro, and to further examine the impact of conditioned media (CM), either from embryo-BUEC co-culture or embryo culture alone, on gene expression in peripheral blood mononuclear cells (PBMCs). First, BUECs were co-cultured with morulae (n = 10) for D5-D9 (D0 = IVF), and gene expression in BUECs was analyzed. Subsequently, PBMCs were cultured in CM from embryo-BUEC co-culture or D5-D9 embryo culture, and gene expression was evaluated. In BUECs, the embryo induced interferon (IFN)-stimulated genes (ISGs: ISG15, OAS1, and MX2), a key factor for IFN-signaling (STAT1), and type-1 IFN receptors (IFNAR1 and IFNAR2), with suppression of NFkB2, NFkBIA and pro-inflammatory cytokines (TNFA and IL1B). The embryo also stimulated PTGES and PGE2 secretion in BUECs. In PBMCs, both CM from embryo-BUEC co-culture and embryo culture alone induced ISGs, STAT1 and TGFB1, while suppressing TNFA and IL17. Similarly, interferon tau (IFNT) at 100 pg/ml suppressed NFkB2, TNFA and IL1B in BUECs, and also stimulated TGFB1 and suppressed TNFA in PBMCs. Our findings suggest that the bovine embryo, in the first four days in the uterus (D5-D9), starts to induce an anti-inflammatory response in epithelial cells and in immune cells. IFNT is likely to act as one of the intermediators for induction of the anti-inflammatory response in the bovine uterus.

After intercourse/insemination, large numbers of sperm are deposited in the female reproductive tract (FRT), triggering a massive recruitment of neutrophils (PMNs) into the FRT, possibly to eliminate excessive sperm via phagocytosis. Some bovine oviductal fluid components (BOFCs) have been shown to regulate in vitro sperm phagocytosis (spermophagy) by PMNs. The modeling approach-based logistic regression (LR) and autoregressive logistic regression (ALR) can be used to predict the behavior of complex biological systems. We, first, compared the LR and ALR models using in vitro data to find which of them provides a better prediction of in vitro spermophagy in bovine. Then, the best model was used to identify and classify the reciprocal effects of BOFCs in regulating spermophagy. The ALR model was calibrated using an iterative procedure with a dynamical search direction. The superoxide production data were used to illustrate the accuracy in validating logit model-based ALR and LR. The ALR model was more accurate than the LR model. Based on in vitro data, the ALR predicted that the regulation of spermophagy by PMNs in bovine oviduct is more sensitive to alpha-1 acid glycoprotein (AGP), PGE2, bovine serum albumin (BSA), and to the combination of AGP or BSA with other BOFCs.

In dairy cows, inflammatory diseases caused by infection with pathogenic bacteria post calving affect ovarian functions. This study examined the relationship between single-nucleotide polymorphisms (SNPs) of Toll-like receptor 4 (TLR4), reproductive performances [the number of artificial insemination (AI) application and days open], and immune cell functions (apoptosis and migration). Two hundred Holstein cows from the Obihiro University farm were included. The SNPs of TLR4 were genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP) method. Polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood. The number of AI application in the animals with T/C genotype in the TLR4 exon3 was lower than that in animals with C/C genotype (1.6 ± 0.2 and 2.2 ± 0.2, respectively). Among the animals with TLR4 exon3 polymorphisms, the days open was shorter for the T/C cows than that for C/C cows (100.7 ± 6.9 days and 136.6 ± 9.0 days, respectively). The SNPs in the TLR4 intron did not affect the number of AI and days open. The apoptosis percentage of PMNs treated with lipopolysaccharide (LPS; 0.001 and 1 μg/ml) tended to be lower in the T/C genotype compared to that in the C/C genotype. The transmigration rates of PMNs, and IL-1β production in PBMCs were tended to be higher for the animals with the T/C genotype compared to those for animals with the C/C genotype. Taken together, these results suggest that TLR4 polymorphisms offer a meaningful tool to judge the reproductive potential and immune activity in individual cows.

This study examined the relationship between postpartum metritis, reactive oxygen species (ROS) generation, and plasma lipopolysaccharide (LPS) concentration in peripartum dairy cows. Blood was collected twice weekly from 2 weeks prepartum through 6 weeks postpartum. Whole blood chemiluminescence (WBCL) was measured using the luminol-enhanced zymosan-stimulated chemiluminescence assay. Cows were examined for uterine health disorders and classified into two groups, healthy (n = 11) and metritis (n = 5). Metritis had a significant effect on WBCL, with cows with metritis having a higher WBCL. Plasma LPS concentrations in cows with metritis were significantly higher than in healthy cows. To examine the effect of LPS on WBCL, blood was sampled in healthy peripartum cows (1 to 2 weeks prepartum, n = 8; 0 to 3 weeks postpartum, n = 11; and 4 to 8 weeks postpartum, n = 8) and incubated with LPS. At 1 endotoxin units/mL of LPS, similar to the plasma LPS concentration in cows with metritis, the WBCL increased in cows at 0 to 3 weeks postpartum. Results indicate that the increase in ROS generation and plasma LPS concentration are associated with metritis, and LPS may be responsible for enhanced ROS generation in early postpartum dairy cows., (© 2016 Japanese Society of Animal Science.)

The present study investigated over 9 months the changes of fermentative quality of total mixed rations (TMR) containing grass silage (GS) as a major component, associated with changes in the volatile basic nitrogen (VBN) levels in an experimental dairy farm. Effects of VBN levels in TMR on metabolic parameters, reactive oxygen species (ROS) production by blood polymorphonuclear leukocytes (PMNs) and conception rates for dairy cows were analyzed. According to VBN levels in TMR during survey periods, three distinct phases were identified; phase A with low VBN; phase B with high VBN; and phase C with mid-VBN. Metabolic parameters in blood were all within normal range. However, during phases B and C, nitrogen metabolic indices such as blood urea nitrogen and milk urea nitrogen showed higher levels compared to those in phase A, and a simultaneous increase in ROS production by blood PMNs and the load on hepatic function in metabolic parameters was observed in the cows with a lower conception rate. This suggests that feeding TMR with elevated VBN levels due to poor fermented GS results in stimulation of ROS production by PMNs by ammonia, and negatively affects metabolism and reproductive performance in lactating dairy cow., (© 2016 Japanese Society of Animal Science.)