Ovarian sex steroid and epithelial control of immune responses in the uterus and oviduct: human and animal models†.

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.

Heat shock interferes with the amino acid metabolism of bovine cumulus-oocyte complexes in vitro: a multistep analysis.

By affecting the ovarian pool of follicles and their enclosed oocytes, heat stress has an impact on dairy cow fertility. This study aimed to determine how heat shock (HS) during in vitro maturation affected the ability of the bovine cumulus-oocyte complexes (COCs) to develop, as well as their metabolism of amino acids (AAs). In this study, COCs were in vitro matured for 23 h at 38.5 °C (control; n = 322), 39.5 °C (mild HS (MHS); n = 290), or 40.5 °C (severe HS (SHS); n = 245). In comparison to the control group, the MHS and SHS groups significantly decreased the percentage of metaphase-II oocytes, as well as cumulus cell expansion and viability. The SHS decreased the rates of cleavage and blastocyst formation in comparison to the control and MHS. Compared to the control and MHS-COCs, the SHS-COCs produced significantly more phenylalanine, threonine, valine, arginine, alanine, glutamic acid, and citrulline while depleting less leucine, glutamine, and serine. Data showed that SHS-COCs had the highest appearance and turnover of all AAs and essential AAs. Heat shock was positively correlated with the appearance of glutamic acid, glutamine, isoleucine, alanine, serine, valine, phenylalanine, and asparagine. Network analysis identified the relationship between HS and alanine or glutamic acid, as well as the relationship between blastocyst and cleavage rates and ornithine. The findings imply that SHS may have an impact on the quality and metabolism of AAs in COCs. Moreover, the use of a multistep analysis could simply identify the AAs most closely linked to HS and the developmental competence of bovine COCs.

Sex-Specific Variations in the mRNA Levels of Candidate Genes in Peripheral Blood Mononuclear Cells from Patients with Diabetes: A Multistep Study.

BACKGROUND: Type 2 diabetes (T2D) is one of the most prevalent diseases that also show sexual dimorphism in many different aspects. OBJECTIVES: This study aimed to distinguish the mRNA expression of genes in peripheral blood mononuclear cells (PBMCs) in men or women with T2D using a multistep analysis. METHODS: A total of 95 patients with T2D were compared based on their sex in terms of clinical variables and mRNA expression in their PBMCs. RESULTS: Men with T2D had lower LDLC, HDLC, and HbA1c values in their blood, but greater creatinine levels. In men with T2D, TLR4, CCR2, NOX2, and p67phox mRNA expression was greater, but IL6 and NF-κB mRNA expression was lesser in PBMCs. There was a link between fasting plasma glucose (FPG), triglycerides, and hs-CRP, as well as COX1 mRNA in men with T2D. In women with T2D, FPG was associated with the mRNA expression of THBS1 and p67phox, as well as triglycerides and HDLC levels. We found the exclusive effect of FPG on HDLC, HbA1c, as well as p67phox mRNA in PBMCs of women with T2D. Analysis revealed the exclusive effect of FPG on hs-CRP and PAFR mRNA in PBMCs of men with T2D. FPG was shown to be associated with body mass index, hs-CRP, triglycerides, and COX1 mRNA in men with T2D, and with serum triglycerides, THSB1, and p67phox mRNA in women with T2D, according to network analysis. HbA1c was linked with NF-κB mRNA in women with T2D. CONCLUSIONS: Using a multistep analysis, it was shown that network analysis outperformed traditional analytic techniques in identifying sex-specific alterations in mRNA gene expression in PBMCs of T2D patients. The development of sex-specific therapeutic approaches may result from an understanding of these disparities.

Homology Modeling, Molecular Dynamics Simulation, and Prediction of Bovine TLR2 Heterodimerization.

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.

Urea changes oocyte competence and gene expression in resultant bovine embryo in vitro.

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.

Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.

This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E2 (PGE2), which dose-dependently (10(-6), 10(-7), and 10(-8) M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP2 receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE2 or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE2 released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP2 receptors. Thus, the bovine oviduct provides a PGE2-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1.

An acute-phase protein as a regulator of sperm survival in the bovine oviduct: alpha 1-acid-glycoprotein impairs neutrophil phagocytosis of sperm in vitro.

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.

Regulation of innate immune function in bovine oviduct epithelial cells in culture: the homeostatic role of epithelial cells in balancing Th1/Th2 response.

This study aimed to investigate the role of epithelial cells in regulating innate immunity in bovine oviduct epithelial cell (BOEC) culture. We studied the effect of Escherichia coli lipopolysaccharide (LPS) and its interaction with ovarian steroids, estradiol (E2) and progesterone (P4), and luteinizing hormone (LH) at concentrations observed during the preovulatory period on immune responses in BOEC culture. Immunohistochemistry of oviduct tissue showed intensive expression of Toll-like receptor-4 (TLR-4) and TLR-2 in epithelial cells. A dose of 10 ng/ml LPS stimulated TLR-4, cyclooxygenase-2 (COX-2), nuclear factor kappa B inhibitor A (NFKBIA), interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) expression, indicating an early pro-inflammatory response. A dose of 100 ng/ml LPS did not induce expression of these genes but stimulated TLR-2, IL-10,IL-4 and microsomal prostaglandin E synthase-1 (mPGES-1) expression and PGE2 secretion, indicating an anti-inflammatory response. Ovarian steroids and LH completely block LPS (10 ng/ml)-induced TLR-4, IL-1ß and TNF-α expression as well as LPS (100 ng/ml)-induced TLR-2 expression. Taken together, this study suggests the existence of an early signaling system to respond to infection in the BOEC. In addition, ovarian steroids and LH may play a critical role in inducing homeostasis and in controlling hyperactive pro-inflammatory responses detrimental to epithelial cells, sperm and the embryo.

Hyaluronan regulates sperm-induced inflammatory response by enhancing sperm attachment to bovine endometrial epithelial cells via CD44: in-silico and in-vitro approaches.

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 (106/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.

Sperm activate TLR2/TLR1 heterodimerization to induce a weak proinflammatory response in the bovine uterus.

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.

The relationship between bovine blastocyst formation in vitro and follicular fluid amino acids.

Follicular fluid has been found as a possible source of metabolic predictors for oocyte competence, and it is conveniently accessible during ovum pick-up (OPU). We used the OPU procedure to recover oocytes from 41 Holstein heifers for in vitro embryo production in this study. Follicular fluid was collected during OPU in order to establish a link between follicular amino acids and blastocyst formation. Each heifer's oocytes were collected, matured in vitro for 24 h and fertilized separately. The heifers were then divided into two groups based on blastocyst formation: those that produced at least one blastocyst (the blastocyst group, n = 29) and those that did not (the failed group, n = 12). The blastocyst group had higher follicular glutamine concentrations and lower aspartate levels than the failed group. Furthermore, network and Spearman correlation analyses revealed a link between blastocyst formation and aspartate (r = -0.37, p = 0.02) or glutamine (r = 0.38, p = 0.02). The receiver operator characteristic curve revealed that glutamine (AUC = 0.75) was the greatest predictor of blastocyst formation. These findings revealed that follicular amino acid levels in bovines can be used to predict blastocyst development.

Risk of mortality in COVID-19 patients: a meta- and network analysis.

Understanding the most relevant hematological/biochemical characteristics, pre-existing health conditions and complications in survivors and non-survivor will aid in predicting COVID-19 patient mortality, as well as intensive care unit (ICU) referral and death. A literature review was conducted for COVID-19 mortality in PubMed, Scopus, and various preprint servers (bioRxiv, medRxiv and SSRN), with 97 observational studies and preprints, consisting of survivor and non-survivor sub-populations. This meta/network analysis comprised 19,014 COVID-19 patients, consisting of 14,359 survivors and 4655 non-survivors. Meta and network analyses were performed using META-MAR V2.7.0 and PAST software. The study revealed that non-survivors of COVID-19 had elevated levels of gamma-glutamyl transferase and creatinine, as well as a higher number of neutrophils. Non-survivors had fewer lymphocytes and platelets, as well as lower hemoglobin and albumin concentrations. Age, hypertension, and cerebrovascular disease were shown to be the most influential risk factors among non-survivors. The most common complication among non-survivors was heart failure, followed by septic shock and respiratory failure. Platelet counts, creatinine, aspartate aminotransferase, albumin, and blood urea nitrogen levels were all linked to ICU admission. Hemoglobin levels preferred non-ICU patients. Lower levels of hemoglobin, lymphocytes, and albumin were associated with increased mortality in ICU patients. This meta-analysis showed that inexpensive and fast biochemical and hematological tests, as well as pre-existing conditions and complications, can be used to estimate the risk of mortality in COVID-19 patients.

Multi-level analysis reveals the association between diabetes, body mass index, and HbA1c in an Iraqi population.

Type 2 diabetes (T2D) known as a complex metabolic disorder may cause health problems and changes in blood biochemical markers. A growing number of studies have looked into several biomarkers and their connections with T2D risk. However, few have explored the interconnection of these biomarkers, as well as the prospective alterations in the diabetes biomarker correlation network. We conducted a secondary analysis in order to introduce a multi-level approach to establish a relationship between diabetes, pre-diabetes, blood biochemical markers, age, and body mass index (BMI). The dataset was obtained from the Mendeley Data (available at https://data.mendeley.com/datasets/wj9rwkp9c2/1 . In this study, three groups were established: non-diabetic (n = 103), pre-diabetic (n = 53), and diabetic (n = 844). According to the Heatmap analysis, non-diabetic and pre-diabetic individuals had the lowest BMI, age, and HbA1c. Diabetes and pre-diabetes were correlated with BMI (r = 0.58 and - 0.27, respectively), age (r = 0.47 and - 0.28, respectively), and HbA1c (r = 0.55 and - 0.21, respectively) using Pearson analysis. Using multivariate analysis, we found that diabetes, BMI, age, HbA1c, cholesterol, triglyceride, LDL, VLDL, and HDL were all associated. Network analysis revealed a connection between BMI and diabetes at the highest cut-off point. Moreover, receiver operating characteristic (ROC) analysis validated the network findings, revealing that BMI (area under the ROC curve, AUC = 0.95), HbA1c (AUC = 0.94), and age (AUC = 0.84) were the best predictors of diabetes. In conclusion, our multi-step study revealed that identifying significant T2D predictors, such as BMI and HbA1c, required a series of mathematical analyses.

The impact of calcitriol and estradiol on the SARS-CoV-2 biological activity: a molecular modeling approach.

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.

Multistep analysis reveals the relationship between blood indices at the time of ovum pick-up and in vitro embryo production in heifers.

The inflammatory factors of complete blood count (CBC) are associated with a decrease in the in vitro embryo production (IVP) outcome in women. The relation between the blood indices and in vitro fertilization (IVF) outcomes in bovines remains to be elucidated. Using ovum pick-up (OPU), oocytes were retrieved from heifers (n = 60) and inseminated separately with sperm. The blastocyst formation was recorded on day 7 after insemination for each animal and the blood indices were evaluated at the time of OPU. Then, heifers were classified on the basis of (1) blastocyst formation, cleaved vs. failed, or (2) inflammation, low-grade inflammation (lymphocyte counts > 5.6 × 109/L) vs. no inflammation (lymphocyte counts < 5.6 × 109/L). Oocytes derived from heifers with higher lymphocytes, red blood cells (RBC), platelets, hematocrit, red cell distribution width (RDW-SD) and plateletcrit values and lower monocytes, eosinophils, mean corpuscular hemoglobin (MCH) and MCH concentration (MCHC) successfully developed to the blastocyst stage. Heifers with low-grade inflammation numerically had a higher percentage of blastocyst formation than normal heifers. The principle component analysis (PCA) showed that blastocyst formation had the strongest positive association with RDW-cv and RDW-SD, while having a strong negative association with mean corpuscular volume (MCV), hemoglobin, MCHC and MCH. The PCA determined that the number of grade A COCs and the percentage of COCs reached the cleavage stage had a negative association with white blood cells (WBC), lymphocytes, basophils and monocytes, and a positive correlation with platelet to lymphocyte ratio, platelet distribution width (PDW) and plateletcrit. Network mapping detected close similarities between BFR and RDW-SD, MPV, and lymphocytes. The area under the receiver operating characteristic curve (AUC) identified that, eosinophils (AUC 0.80), RDW-SD (AUC 0.76), monocytes (AUC 0.76) and lymphocytes (AUC 0.76) had a good predictive ability to detect heifers with high OPU-IVP outcome (≥60%). In conclusion, these findings suggest that CBC indices at the time of OPU were associated with the IVF outcome and may be incorporated into protocols for the identification of heifers with high potential for blastocyst formation.

Epidermal growth factor alleviates the negative impact of urea on frozen-thawed bovine sperm, but the subsequent developmental competence is compromised.

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.

Day 7 Embryos Change the Proteomics and Exosomal Micro-RNAs Content of Bovine Uterine Fluid: Involvement of Innate Immune Functions.

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.

Effect of pomegranate by-product silage on feed intake, blood metabolites, inflammatory responses, and prostaglandin F2α metabolites in Holstein dairy cows.

Dairy cows often suffer metabolic disorders due to the challenges of the transition period. The aim of study was to determine the effects of feeding pomegranate by-product silage (PBS) on feed intake and some blood parameters, such as non-esterified fatty acids (NEFA) and beta hydroxy butyric acid (BHBA), interleukin-1 (IL-1), serum amyloid-A (SAA), prostaglandin F2α metabolites (PGFM), and progesterone (P4) in fresh Holstein cows. The experimental groups were as follows: CON (0 g PBS of DM) and PBS (120 g PBS of DM). Results showed that replacing corn silage with PBS had no effect on dry matter intake (DMI) throughout the study. The CON group had highest (p < .05) NEFA at 14 d postpartum and serum BHBA was decreased by feeding PBS, especially on days 21 (p < .05) and 28 (p < .05) and the entire study (p < .01). Cows fed PBS had lower (p < .05) serum Malondialdehyde (MDA) than CON cows at different times. Feeding PBS decreased (p < .01) serum IL-1 compared to CON group (216.7 versus. 515.5 Pg/ml) at 28 d postpartum, and also cows fed 120 g PBS had higher (p < .01) serum P4. It was concluded that feeding PBS had no adverse effect on feed intake, milk production, and some reproductive parameters. Moreover, PBS positively affected serum metabolites in fresh dairy cows.

Effect of abortion on milk production, health, and reproductive performance of Holstein dairy cattle.

The objective of this study was to identify the effect of abortion on performance of Iranian Holstein cows that calved between 2005-2014. Abortion records were categorized in two groups: 1) new abortion (NLA), and 2) rebreeding abortion (RA). Production data (305-d milk, fat, and protein yields) were analyzed using PROC MIXED, number of inseminations per lactation period (INS), postpartum health disorders (PPHD), and culling records using PROC GLIMMIX, and calving to conception records using PROC PHREG of SAS software (SAS Institute, 2013). For each separate model, main herd-cow factors and all two-way significant interactions were considered in the analyses with results being reported for each calving status by lactation period. In total, 12.5 % of lactation periods were associated with at least one case of abortion, while 1.9 % of the lactation periods commenced with an abortion. Based on results from the present study, milk production decreased for cows having abortion classified as NLA or RA. Overall, when there was a RA, there was an increase in median days non-pregnant (DNP) by 132 d and INS by 1.6, whereas cows with NLA had median DNP of 15 d less and an INS of 0.17 less than cows with normal parturitions. Compared with cows with normal parturitions, cows with a NLA were 1.24, 1.88, and 2.41 times more likely to have PPHD, and be culled from the herd for involuntary reasons and reasons related to reproduction, respectively. The results of this study indicate abortions were associated with cow health and milk production and reproductive performance, indicating losses due to abortion are not only a direct result of the loss of a pregnancy.

A multilevel analysis identifies the different relationships between amino acids and the competence of oocytes matured individually or in groups.

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.