Characterization of Bacillus pumilus Strains Isolated from Bovine Uteri.

Uterine infections are a major source of economic losses to dairy farmers. The uterine microbiota as well as opportunistic uterine contaminants can contribute to the development of endometritis in dairy cows during the postpartum period. Therefore, it is important to characterize potential pathogens and to further elucidate their role in the disease. In this study, we aimed to characterize Bacillus pumilus field isolates to obtain more details regarding their effect on uterine cells by using an in vitro endometrial epithelial primary cells model. We found that B. pumilus isolates possessed the keratinase genes ker1 and ker2 and therefore may produce keratinases. When primary endometrial epithelial cells were infected with 4 different B. pumilus strains, an effect on cellular viability was observed over the course of 72 h. The effect was dose-dependent and time-dependent. Nevertheless, significant differences between the strains were not observed. All tested strains reduced the viability of the primary cells after 72 h of incubation, indicating that B. pumilus potentially has a pathogenic effect on endometrial epithelial cells.

Endometrial Inflammation at the Time of Insemination and Its Effect on Subsequent Fertility of Dairy Cows.

Our objective was to investigate the level of endometrial immune response at artificial insemination (AI) and to relate it to subsequent fertility. From 71 healthy cows, endometrial cytobrush samples were taken at the first AI for cytological and mRNA analyses. Total RNA isolated from the cytobrushes was used for reverse transcription qPCR for selected transcripts. Animals were grouped into pregnant (PREG; n = 32) and non-pregnant (non-PREG; n = 39) cows following their first AI. The mRNA abundance of the neutrophil-related factor CEACAM1 and the chemokine CXCL5 was 1.2- (p = 0.03) and 2.0-fold (p = 0.04) greater in PREG than in non-PREG cows, respectively. Animals were further subdivided according to the number of inseminations until pregnancy (PREG1, n = 32; PREG2-3, n = 19) and in repeat breeder cows (RBC, n = 13). CEACAM1 and CXCL8 mRNA expression was 1.7- (p = 0.01) and 2.3-fold (p = 0.03) greater in PREG1 than in RBC, respectively. Cox regression showed that cows with PMN ≥ 1% had a 1.8-fold increased chance of pregnancy within 150 days postpartum compared with cows with fewer PMNs. We conclude that a certain level of inflammation before the stimulus of AI might be beneficial for subsequent fertility.

Messenger RNA Expression of Selected Factors at Different Sites of the Bovine Endometrium Associated With Uterine Health.

Recent studies have elucidated the role of several pro-inflammatory factors as mediators of inflammatory processes in the bovine endometrium. Only few studies, however, have analyzed samples collected from different regions of the uterus of the same animal. In this study, we tested the hypothesis that on a molecular level, clinical endometritis is characterized by inflammatory responses spread over the entire endometrium. Furthermore, we assume that subclinical endometritis is described by an inflammation of local regions of the uterus. Therefore, the objective of this study was to assess the mRNA expression of uterus-associated pro-inflammatory factors at five pre-defined endometrial sites, i.e., corpus uteri, left horn base, left horn tip, right horn base, and right horn tip, in cows with clinical and subclinical endometritis and in healthy controls. We analyzed the mRNA expression of interleukin 1 alpha, interleukin 1 beta, C-X-C motif chemokine ligand 8, prostaglandin-endoperoxide synthase 2, protein tyrosine phosphatase receptor type C, carcinoembryonic antigen related cell adhesion molecule 1, and mucin 4 and 16. Based on vaginoscopy and endometrial cytology (≥ 5% polymorphonuclear neutrophils) between 28 to 34 days in milk, 18 Simmental cows were categorized in clinical endometritis group (n = 7), subclinical endometritis group (n = 4), and healthy group (n = 7). In general, the analyses revealed a great variation of mRNA expression between sites and animals. Differences were found between different uterine health statuses, but the variation between the sampling sites within the groups was not significant (P > 0.05). This indicates that inflammatory processes at the end of the postpartum period can be regarded as multi-focal or spread throughout the uterus independent from the uterine health status.

Streptococcus uberis strains originating from bovine uteri provoke upregulation of pro-inflammatory factors mRNA expression of endometrial epithelial cells in vitro.

Streptococcus uberis is an opportunistic pathogen involved in various infections of cattle. It is a well-known etiological agent of bovine mastitis and has recently also been linked to postpartum endometritis in dairy cows. S. uberis is frequently isolated from the uterus of postpartum cows but its actual contribution to host pathophysiology is unknown and information on S. uberis virulence factors potentially involved in the disease is lacking. To gain first insights into the role of S. uberis in the pathology of bovine endometritis, a cell-culture-based infection model was employed to study inflammatory host responses and investigate cytotoxic effects. A comprehensive strain panel, comprising 53 strains previously isolated from bovine uteri, was compiled and screened for known virulence factor genes. Isolates showing distinct virulence gene patterns were used to study their impact on cellular viability and influence on mRNA expression of pro-inflammatory factors in endometrial epithelial cells. Our study revealed that S. uberis negatively impacts the viability of endometrial epithelial cells and provokes an upregulation of specific pro-inflammatory factors, although with certain strains having a greater effect than others. Especially, mRNA expression of IL1A and CXCL8 as well as CXCL1/2 and PTGS2 was found to be stimulated by S. uberis. These results suggest that S. uberis might indeed contribute to the establishment of bovine endometritis.

Formation of Surface Deposits on Steel and Titanium Aviation Fuel Tubes under Real Operating Conditions.

In this study, stainless steel and titanium (Ti) tubes obtained from a turbofan engine after the end of its lifetime were analyzed in order to compare the amount of pyrolytic coke present and its influence on the parent, base material. Various analytical techniques including microhardness and topographical evaluations, optical emission spectrometry (OES), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS) were applied. On steel surfaces, a thick pyrolytic coke deposition layer consisting of carbon and oxygen and also containing elements from the tube material, fuel, and fuel additives was found. The concentration of elements from the pyrolytic coke continuously decreased with distance from the surface of the deposit, while the concentrations of elements from the tube material continuously increased, with the concentrations of elements from the fuel and the fuel additives being relatively constant. With ultrasonic cleaning in distilled water, most of the deposits could be removed. Only carbon-rich patches with a thickness of more than 300 nm remained adhered to the surface and/or had diffused into the original material. On Ti surfaces, the thickness of the C-rich fuel deposit layer was significantly thinner as compared to that on the stainless steel; however, the surface was covered with an ∼3 µm-thick oxide layer, which consisted of elements from the fuel additives. It is believed that the beneficial properties of Ti covered with a thin layer of TiO2, such as low adhesion and/or surface energy, have promoted different deposition mechanisms compared to those of stainless steel and thus prevented pyrolytic coke deposition and the related material deterioration observed on stainless steel.

Detection and characterization of Lactobacillus spp. in the porcine seminal plasma and their influence on boar semen quality.

The presence of pathogenic bacteria in ejaculates has been a topic in boar semen preservation over the last decades. Since little information is available on commensal bacteria in boar semen, the aim of the present study was to identify commensal lactobacilli in fresh cryopreserved boar semen and to examine their influence on boar semen quality. Therefore, 111 boar ejaculates were investigated for the presence of Lactobacillus species. Thirty samples (27%) contained viable Lactobacillus species (e.g. L. amylovorus, L. animalis, L. reuteri and Weisella minor). L. animalis and L. buchneri DSM 32407 (isolated from the bovine uterus) qualified for further examinations based on their growth rate in six antibiotic-free boar semen extenders. After a 120 min short-term incubation with an antibiotic-free BTS-extender, progressive motility was diminished (P = 0.001) upon addition of 105 and 106 colony forming units (CFU/mL) L. animalis. The supplementation with L. buchneri DSM 32407 had no significant (P > 0.05) influence on sperm quality during short-term co-incubation. After 168 h long-term co-incubation, motility analysis revealed a negative (P = 0.026) impact of 105 CFU/mL L. buchneri DSM 32407. A concentration- and storage-dependent effect is particularly obvious (P < 0.001) using 106 CFU/mL L. buchneri DSM 32407. Most notably, the thermo-resistance (TRT) for 106 CFU/mL L. buchneri DSM 32407 (P = 0.001) was inferior to BTS with and without gentamicin after 72 and 168 h of semen co-incubation. The supplementation of 105 CFU/mL L. buchneri DSM 32407 impaired progressive motility to a lesser extent. The percentage of mitochondrially active spermatozoa after 96 h (P = 0.009) and membrane-intact spermatozoa after 168 h (P < 0.001) was lower when 106 CFU/mL L. buchneri DSM 32407 were suspended compared with all other groups. Finally, the addition of L. buchneri DSM 32407 to BTS-extended boar semen had no competitive effect on the total amount of bacteria 48 h after co-incubation. In summary, the present study demonstrated that there are Lactobacillus species present in the porcine seminal plasma, which can be cultivated using standard procedures. However, long-term co-incubation of lactic acid bacteria with spermatozoa had a negative influence on spermatozoa.

In situ tribochemical sulfurization of molybdenum oxide nanotubes.

MoS2 nanoparticles are typically obtained by high temperature sulfurization of organic and inorganic precursors under a S rich atmosphere and have excellent friction reduction properties. We present a novel approach for making the sulfurization unnecessary for MoO3 nanotubes during the synthesis process for friction and wear reduction applications while simultaneously achieving a superb tribological performance. To this end, we report the first in situ sulfurization of MoO3 nanotubes during sliding contact in the presence of sulfur-containing lubricant additives. The sulfurization leads to the tribo-chemical formation of a MoS2-rich low-friction tribofilm as verified using Raman spectroscopy and can be achieved both during sliding contact and under extreme pressure conditions. Under sliding contact conditions, MoO3 nanotubes in synergy with sulfurized olefin polysulfide and pre-formed zinc dialkyl dithiophosphate tribofilms achieve an excellent friction performance. Under these conditions, the tribochemical sulfurization of MoO3 nanotubes leads to a similar coefficient of friction to the one obtained using a model nanolubricant containing MoS2 nanotubes. Under extreme pressure conditions, the in situ sulfurization of MoO3 nanotubes using sulfurized olefin polysulfide results in a superb load carrying capacity capable of outperforming MoS2 nanotubes. The reason is that while MoO3 nanotubes are able to continuously sulfurize during sliding contact conditions, MoS2 nanotubes progressively degrade by oxidation thus losing lubricity.

Bovine Endometrial Epithelial Cells Scale Their Pro-inflammatory Response In vitro to Pathogenic Trueperella pyogenes Isolated from the Bovine Uterus in a Strain-Specific Manner.

Among different bacteria colonizing the bovine uterus, Trueperella pyogenes is found to be associated with clinical endometritis (CE). The ability of cows to defend against T. pyogenes infections depends on the virulence of invading bacteria and on the host's innate immunity. Therefore, to gain insights into bacterial factors contributing to the interplay of this host pathogen, two strains of T. pyogenes were included in this study: one strain (TP2) was isolated from the uterus of a postpartum dairy cow developing CE and a second strain (TP5) was isolated from a uterus of a healthy cow. The two strains were compared in terms of their metabolic fingerprints, growth rate, virulence gene transcription, and effect on bovine endometrial epithelial cells in vitro. In addition, the effect of the presence of peripheral blood mononuclear cells (PBMCs) on the response of endometrial epithelial cells was evaluated. TP2, the strain isolated from the diseased cow, showed a higher growth rate, expressed more virulence factors (cbpA, nanH, fimE, and fimG), and elicited a higher mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, and IL8) in bovine endometrial epithelial cells compared with TP5, the strain isolated from the healthy cow. The presence of PBMCs amplified the mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, IL1A, IL6, and IL8) in bovine endometrial epithelial cells co-cultured with live TP2 compared with untreated cells, especially as early as after 4 h. In conclusion, particular strain characteristics of T. pyogenes were found to be important for the development of CE. Furthermore, immune cells attracted to the site of infection might also play an important role in up-regulation of the pro-inflammatory response in the bovine uterus and thus significantly contribute to the host-pathogen interaction.

A review of the ongoing discussion about definition, diagnosis and pathomechanism of subclinical endometritis in dairy cows.

In the last decade, several new aspects of the inflammation of the bovine endometrium have been investigated and described, including a new definition of subclinical endometritis. This review summarizes the recent discussion about the definition, diagnosis and pathomechanism of subclinical endometritis. Subclinical endometritis also referred to as cytological endometritis is defined by findings of endometrial cytology, which is usually performed with the cytobrush-technique or by low-volume flushing of the uterus. The sampling procedure is minimally invasive and has no negative impact on subsequent conception rate. The suggested threshold value for polymorphonuclear cells (PMN) as diagnostic for subclinical endometritis depends on the time postpartum and varies from 5 to 18%. It has also been shown that a general threshold of 5% PMN is eligible for all cows between 21 and 62 days postpartum. Accuracy and repeatability of counting PMN under the microscope have been evaluated and can be regarded as reliable. The impact of subclinical endometritis on reproductive performance is characterized by decreased conception rates, and prolonged days to first service and days open. In addition, it has been demonstrated that subclinical endometritis has an impact on survival and quality of the embryo. Some studies, however, did not confirm this negative effect of subclinical endometritis on fertility. More detailed analyses of the cytobrush samples revealed higher mRNA expression of several cytokines in cows with subclinical endometritis compared with healthy cows, and contributed to the understanding of detrimental effects of subclinical endometritis on fertility. In contrast to clinical endometritis, there are no predominant bacteria related to subclinical endometritis, but associations between the presence of α-hemolytic streptococci and Trueperella pyogenes and subclinical endometritis have been found. For the treatment of subclinical endometritis, intrauterine infusions with cephapirin as well as the administration of PGF2α have been recommended. Other studies, however, did not confirm the efficiency of these treatments.

mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

BACKGROUND: The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. METHODS: BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. RESULTS: Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. CONCLUSION: This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered.

Increased mRNA expression of selected pro-inflammatory factors in inflamed bovine endometrium in vivo as well as in endometrial epithelial cells exposed to Bacillus pumilus in vitro.

Endometrial epithelium plays a crucial role in the first immune response to invading bacteria by producing cytokines and chemokines. The aim of this study was to investigate the first inflammatory response of the endometrium in vivo and in vitro. Gene expression of several pro-inflammatory factors and Toll-like receptors (TLR2, -4, -6) was determined in endometrial cytobrush samples obtained from healthy cows and cows with clinical or subclinical endometritis. Endometrial epithelial cells were co-cultured with an isolated autochthonous uterine bacterial strain Bacillus pumilus. Total RNA was extracted from in vivo and in vitro samples and subjected to real-time reverse transcription polymerase chain reaction. CXC ligands (CXCL) 1/2 and CXC chemokine receptor (CXCR) 2 mRNA expression was higher in cows with subclinical endometritis and CXCL3 mRNA expression was higher in cows with clinical endometritis compared with healthy cows. B. pumilus induced cell death of epithelial cells within 24h of co-culturing. The presence of B. pumilus resulted in significantly higher mRNA expression of interleukin 1α (IL1A), IL6, IL8, CXCL1-3 and prostaglandin-endoperoxide synthase 2 in co-cultured cells compared with untreated controls. The maximum increase was mainly detected after 2h. These results support the hypothesis that bacterial infection of endometrial cells might induce prompt synthesis of pro-inflammatory cytokines resulting in a local inflammatory reaction.

Detection and characterisation of Lactobacillus spp. in the bovine uterus and their influence on bovine endometrial epithelial cells in vitro.

Bacterial infections and inflammation of the uterus are common in dairy cattle after parturition. In particular, pathogenic bacteria that cause endometritis have been the focus of research in cattle reproduction in the last ten years. The aim of the present study was to identify commensal lactobacilli in the bovine uterus and to examine their influence on the synthesis of pro-inflammatory factors in bovine endometrial epithelial cells in vitro. Lactobacillus species were isolated from healthy bovine uteri and further characterised. Bovine endometrial epithelial cells in the second passage (n = 5 animals) were co-cultured with the autochthonous isolates L. buchneri, L. ruminis and L. amylovorus as well as with a commercially available L. vaginalis in different multiplicities of infection (MOI = 1, 5 and 10, respectively). Endometrial epithelial cells cultured without bacteria served as controls. At distinct points in time (2, 4 and 6 h) total RNA was extracted from co-cultured epithelial cells and subjected to reverse transcription quantitative PCR of pro-inflammatory factors. Furthermore, the release of such factors by co-cultured epithelial cells was measured by ELISA or EIA after 24 and 48 h. L. ruminis and L. amylovorus induced increased interleukin (IL) IL1A, IL6, IL8 and prostaglandin-endoperoxide synthase 2 mRNA levels and the release of IL8 and prostaglandin F(2α) in endometrial epithelial cells compared with control cells. In contrast, L. buchneri did not significantly influence the expression and release of these factors. Toll-like receptors 2 and 6 transcripts were found unchanged in co-cultured and untreated epithelial cells in vitro. However, endometrial epithelial cells of each animal showed individual differences in the response to bacterial load. These results suggest that Lactobacillus species are present in the bovine uterus, revealing immunomodulatory properties.

Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes.

Hot season is a major constraint to production and reproduction in buffaloes. The present work aimed to investigate the effect of season on ovarian function, developmental competence, and the relative abundance of gene expression in buffalo oocytes. Three experiments were conducted. In experiment 1, pairs of buffalo ovaries were collected during cold season (CS, autumn and winter) and hot season (HS, spring and summer), and the number of antral follicles was recorded. Cumulus oocyte complexes (COCs) were aspirated and evaluated according to their morphology into four Grades. In experiment 2, Grade A and B COCs collected during CS and HS were in vitro matured (IVM) for 24 hours under standard conditions at 38.5 °C in a humidified air of 5% CO2. After IVM, cumulus cells were removed and oocytes were fixed, stained with 1% aceto-orcein, and evaluated for nuclear configuration. In vitro matured buffalo oocytes harvested during CS or HS were in vitro fertilized (IVF) using frozen-thawed buffalo semen and cultured in vitro to the blastocyst stage. In experiment 3, buffalo COCs and in vitro matured oocytes were collected during CS and HS, and then snap frozen in liquid nitrogen for gene expression analysis. Total RNA was extracted from COCs and in vitro matured oocytes, and complementary DNA was synthesized; quantitative Reverse Transcription-Polymerase Chain Reaction was performed for eight candidate genes including GAPDH, ACTB, B2M, GDF9, BMP15, HSP70, and SOD2. The results indicated that HS significantly (P < 0.01) decreased the number of antral follicles and the number of COCs recovered per ovary. The number of Grade A, B, and C COCs was lower (P < 0.05) during HS than CS. In vitro maturation of buffalo oocytes during HS significantly (P < 0.01) reduced the number of oocytes reaching the metaphase II stage and increased the percentage of degenerated oocytes compared with CS. Oocytes collected during HS also showed signs of cytoplasmic degeneration. After IVF, cleavage rate was lower (P < 0.01) for oocytes collected during HS, and the percentage of oocytes arrested at the two-cell stage was higher (P < 0.01) than oocytes IVF during CS. Oocytes matured during CS showed a higher (P < 0.01) blastocyst rate than those matured during HS. Also, COCs recovered in HS showed significant (P < 0.05) upregulation of HSP70 mRNA expression compared with those recovered in CS. For in vitro matured oocytes, CS down regulated the transcript abundance of ACTB and upregulated GAPDH and HSP70 mRNA levels compared with HS condition. In conclusion, HS could impair buffalo fertility by reducing the number of antral follicles and oocyte quality. In vitro maturation of buffalo oocytes during HS impairs their nuclear and cytoplasmic maturation, fertilization, and subsequent embryo development to the morula and blastocyst stages. This could be in part because of the altered gene expression found in COCs and in vitro matured oocytes.

Innate immunity and inflammation of the bovine female reproductive tract in health and disease.

Mammalian reproductive physiology and the development of viviparity co-evolved with inflammation and immunity over millennia. Many inflammatory mediators contribute to paracrine and endocrine signalling, and the maintenance of tissue homeostasis in the female reproductive tract. However, inflammation is also a feature of microbial infections of the reproductive tract. Bacteria and viruses commonly cause endometritis, perturb ovarian follicle development and suppress the endocrine activity of the hypothalamus and pituitary in cattle. Innate immunity is an evolutionary ancient system that orchestrates host cell inflammatory responses aimed at eliminating pathogens and repairing damaged tissue. Pattern recognition receptors on host cells bind pathogen-associated molecular patterns and damage-associated molecular patterns, leading to the activation of intracellular MAPK and NFκB signalling pathways and the release of inflammatory mediators. Inflammatory mediators typically include the interleukin cytokines IL1ß and IL6, chemokines such as IL8, interferons and prostaglandins. This review outlines the mechanisms of inflammation and innate immunity in the bovine female reproductive tract during health and disease condition.

Feeding low or pharmacological concentrations of zinc oxide changes the hepatic proteome profiles in weaned piglets.

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs.

Imaging of a tribolayer formed from ionic liquids by laser desorption/ionization-reflectron time-of-flight mass spectrometry.

For the first time, imaging using laser desorption/ionization (LDI) reflectron time-of-flight (RTOF) mass spectrometry (MS) was demonstrated to be a powerful tool for an offline monitoring of tribometrical experiments directly from disc specimen applying selected ammonium-, phosphonium-, and sulfonium-based ionic liquids (IL) with bis(trifluoromethylsulfonyl)imide as counterion for lubrication. The direct measurement of IL tribolayers by LDI-MS allowed the visualization of the lubricants in the form of the distribution of their intact cations and the anion in and outside the wear scar after the tribometrical experiment with a low degree of in-source generated fragmentation. Besides, also, an oxidation product formed during a tribometrical experiment was detected and located exclusively in the wear track. Comparative data of identical wear tracks were obtained by X-ray photoelectron spectroscopy (XPS) imaging not only enabling the determination of elemental distributions of the IL across the area imaged but also corroborating the mass spectrometry imaging (MSI) data, thus generating multimodal images. Merging data from MSI and XPS imaging exhibited that areas, where iron-fluorine bonds were detected in the wear track, are corresponding to data from LDI-MS imaging showing absence of IL cations and anions.

Identification of differentially expressed proteins in ruminal epithelium in response to a concentrate-supplemented diet.

Ruminal epithelium adapts to dietary change with well-coordinated alterations in metabolism, proliferation, and permeability. To further understand the molecular events controlling diet effects, the aim of this study was to evaluate protein expression patterns of ruminal epithelium in response to various feeding regimes. Sheep were fed with a concentrate-supplemented diet for up to 6 wk. The control group received hay only. Proteome analysis with differential in gel electrophoresis technology revealed that, after 2 days, 60 proteins were significantly modulated in ruminal epithelium in a comparison between hay-fed and concentrate-fed sheep (P < 0.05). Forty proteins were upregulated and 20 proteins were downregulated in response to concentrate diet. After 6 wk of this diet, only 14 proteins were differentially expressed. Among these, 11 proteins were upregulated and 3 downregulated. To identify proteins that were modulated by dietary change, two-dimensional electrophoresis was coupled with liquid chromatography electrospray ionization mass spectrometry. The differential expression of selected proteins, such as esterase D, annexin 5, peroxiredoxin 6, carbonic anhydrase I, and actin-related protein 3, was verified by immunoblotting and/or mRNA analysis. The identified proteins were mainly associated with functions related to cellular stress, metabolism, and differentiation. These results suggest new candidate proteins that may contribute to a better understanding of the signaling pathways and mechanisms that mediate rumen epithelial adaptation to high-concentrate diet.

Time-dependent mRNA expression of selected pro-inflammatory factors in the endometrium of primiparous cows postpartum.

BACKGROUND: Inflammatory processes and infections of the uterine wall must be accepted as a physiological event in dairy cows after calving. This might result in clinical or subclinical endometritis which is assumed to impair reproductive performance in the current lactation. Several cytokines and acute phase proteins have been discussed as local and systemic mediators of these inflammatory processes. The aim of the present study was to investigate the endometrial mRNA expression of the chemokine CXC ligand 5 (CXCL5), interleukin 1ß (IL1B), IL6, IL8, tumour necrosis factor alpha (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2) and haptoglobin (HP) in the postpartum period. METHODS: Endometrial samples were obtained from primiparous cows (n = 5) on days 10, 17, 24, 31, 38 and 45 postpartum (pp) using the cytobrush technique. Cytological smears were prepared from cytobrush samples to determine the proportion of polymorphonuclear neutrophils (PMN). Total RNA was extracted from endometrial samples, and real-time RT-PCR was performed. RESULTS: A time-dependent mRNA expression of the investigated factors was found for the course of the postpartum period. In detail, a significantly higher expression of these factors was observed on day 17 pp compared to day 31 pp. Furthermore, the proportion of PMN peaked between days 10-24 pp and decreased thereafter to low percentages (< 5%) on day 31 pp and thereafter. In addition, CXCL5, IL1B, IL8 and HP mRNA expression correlated significantly with the proportion of PMN (P < 0.05). A significantly higher CXCL5, IL1B, IL6, IL8, PTGS2 and TNF mRNA content was observed in samples from cows with an inflamed endometrium compared with samples from cows with a healthy endometrium (P < 0.05). CONCLUSIONS: These results show that inflammatory cytokines and acute phase proteins are expressed in the bovine endometrium in a time-related manner during the postpartum period, with a significant expression peak on day 17 pp as a possible mucosal immune response in the uterus. The evaluation of the expression patterns of such candidate genes may reveal more information than only determining the percentage of PMN to judge the severity of an inflammation.

Prenatal testosterone excess alters Sertoli and germ cell number and testicular FSH receptor expression in rams.

Exposure to excess testosterone (T) during fetal life has a profound impact on the metabolic and reproductive functions in the female's postnatal life. However, less is known about the effects of excess testosterone in males. The aim of the present study was to evaluate the impact (consequences) of an excess of T during fetal development on mature male testis. The testicular evaluation was by histological analysis and by determination of mRNA expression of the FSH receptor (FSH-R), transforming growth factor-ß type I receptor (TßR-I), and two members of the TGF-ß superfamily, transforming growth factor-ß3 (TGFß3) and anti-Müllerian hormone (AMH) in males born to mothers receiving an excess of T during pregnancy. At 42 wk of age, postpubertal males born to mothers treated with 30 mg of T propionate twice weekly from day 30 to 90, followed by 40 mg of T propionate from day 90 to 120 of pregnancy (T males), showed higher concentrations of FSH in response to a GnRH analog, a higher number of Sertoli cells/seminiferous tubule cross-section, and a lower number of germ cells/tubules (P < 0.05) than control males (C males) born to mothers treated with the vehicle. The mRNA expression of FSH-R and of TßR-I was higher in T males compared with C males (P < 0.05). Moreover, in T males, AMH expression level correlated negatively with the expression level of TGFß3. In C males, this latter correlation was not observed. These results suggest that prenatal exposure to an excess of T can negatively modify some histological and molecular characteristics of the mature testis.

Selected pro-inflammatory factor transcripts in bovine endometrial epithelial cells are regulated during the oestrous cycle and elevated in case of subclinical or clinical endometritis.

Endometrial cells take part in embryo-maternal communication, as well as supporting the immune system in defending against invading pathogens. The aim of the present study was to examine the mRNA expression of factors that have been suggested to be involved in both events in the bovine endometrial epithelium, namely bovine granulocyte chemotactic protein 2 (CXCL5), interleukin-1 beta (IL1B), IL6, IL8, tumour necrosis factor (TNF), cyclooxygenase 2 (PTGS2) and haptoglobin (HP). Samples were collected in vivo from cows on Days 21-27 postpartum by the cytobrush method to evaluate the correlation between inflammatory factors and uterine health (cows with signs of clinical or subclinical endometritis and healthy cows). Bovine uteri were collected at the abattoir to investigate oestrous cycle-dependent mRNA expression patterns. Real-time reverse transcription-polymerase chain reaction revealed that the expression of CXCL5, IL1B, IL8 and TNF mRNA was significantly higher in cows with subclinical or clinical endometritis compared with healthy cows. The expression of CXCL5, IL1B and IL8 mRNA was increased around ovulation compared with the luteal phase. There was no indication of either oestrous cycle-dependent expression or a correlation with uterine health for IL6, PTGS2 and HP transcripts. These results suggest that CXCL5, IL1B, IL8 and TNF may represent potential marker genes for the detection of cows with subclinical endometritis and for monitoring new therapeutic approaches.