Mechanism of collagen type IV regulation by focal adhesion kinase during retained fetal membranes in dairy cows.

Retained fetal membranes (RFM) is an important reproductive disease in dairy cows, caused by maternal and fetal placental tissue adhesion. The main collagen in maternal and fetal placenta tissues is collagen type IV (COL-IV) and its breakdown is the key to placental expulsion. Focal adhesion kinase (FAK) has been shown to regulate the hydrolysis of Col-IV by affecting the activity of MMP-2 and MMP-9 activity, but the regulation of the mechanisms involved in placenta expulsion in dairy cows after postpartum are still unclear. The aim of this study was to investigate the pathogenic mechanism of RFM by studying the relationship between the FAK signaling pathway and COL-IV regulation. Maternal placental tissues were collected from six healthy and six cows with RFM of similar age, parity, body condition and milk yield at 12 h postpartum. In vitro experiments were performed on bovine endometrial epithelial cells from three groups including a FAK inhibitor group, a FAK activator group and a control group without FAK inhibitor and activator. The abundance of molecules involved in the FAK signaling pathway and COL-IV was detected by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The immunohistochemical results showed that the key molecules of FAK signaling pathway FAK, Src, MMP-2 and MMP-9 and Col-IV were expressed in placental tissues. The expression level of FAK, Src, MMP-2, and MMP-9 were significantly down-regulated (P < 0.05) and the abundances of COL-IV were significantly up-regulated (P < 0.05) in maternal placental tissues of RFM cows compared with healthy cows. In the FAK inhibitor treatment group, the relative expression levels of FAK and other related proteins were significantly down-regulated (P < 0.05) and the relative expression levels of COL-IV were significantly up-regulated (P < 0.05) with the results of the FAK activation group the opposite. These results indicated that FAK in maternal endometrial epithelial cells could regulate the hydrolysis process of Col-IV through the expression of key factors of signaling pathways and promote collagen hydrolysis, which in turn facilitated the process of postpartum placenta expulsion in dairy cows.

The dependence between glycodelin and selected metalloproteinases concentrations in bovine placenta during early gestation and parturition with and without retained foetal membranes.

Pregnancy course depends on the appropriate connection between the mother and the developing foetus. Pregnancy is completed when the placenta is timely expelled. Placental retention is one of the possible pregnancy complications. Extracellular matrix, including adhesive proteins and enzymes that can break down collagens, seems to be responsible for it. The aim of the present study was to examine the impact of one of the adhesive proteins - glycodelin (Gd) - on selected metalloproteinases degrading collagens (MMP2, MMP3, MMP7). Placental tissues from healthy pregnant cows collected during early-mid pregnancy (2nd month n = 7, 3rd month n = 8, 4th month n = 6) and in cows that properly released placenta (NR; n = 6) and cows with retained foetal membranes (R; n = 6) were experimental material. The concentrations of glycodelin and protein content of selected metalloproteinases were measured by ELISA in the maternal and foetal placental homogenates as well as in the culture of epithelial cells derived from the maternal part of the placenta. The presence of these protein molecules was confirmed by Western Blotting. In the bovine placenta, the concentrations of examined proteins exhibit significant changes during placental formation. Gd, MMP3 and MMP7 concentrations decrease with pregnancy progress (between the 2nd and 4th month), while MMP2 concentrations were on the same level in this period. During parturition, concentrations of Gd and MMP3 were significantly higher in the R group compared to the NR group. In parallel, MMP2 concentrations did not show significant differences between the groups (NR vs R), and MMP7 concentrations decreased significantly in the maternal part of the placenta in cows with retained foetal membranes (R). Obtained results show correlations between the gestational age and proteins' (Gd, MMP3, MMP7) concentration, both in the maternal and foetal part of the placenta.

A pilot study on the relationship between thrombospondin-1 (THBS1) and transforming growth factor beta1 (TGFß1) in the bovine placenta during early mid-pregnancy as well as parturition with normally released and retained placenta.

During pregnancy, it is necessary to create appropriate conditions for the development of the placenta and the fetus. However, during parturition, the placenta must be separated and subsequently removed as soon as possible to not expose the female to the possibility of infection. In this study, the relationship between thrombospondin-1 (THBS1) and transforming growth factor beta1 (TGFß1) concentrations was described during bovine pregnancy (second, fourth, and sixth months; n = 3/each month), at normal parturition (NR) and parturition with fetal membrane retention (R). The presence of THBS1 and TGFß1 was confirmed in bovine placental tissues of both maternal and fetal parts. Enzyme-linked immunosorbent assay showed statistically significant differences (p < 0.05) in THBS1 concentrations (pg/mg protein) between examined parturient samples (maternal part: 5.76 ± 1.61 in R vs. 2.26 ± 1.58 in NR; fetal part: 2.62 ± 1.94 in R vs. 1.70 ± 0.23 in NR). TGFß1 concentrations (pg/mg protein) were significantly lower (p < 0.05) in the retained fetal membranes compared to the released fetal membranes in the maternal part of the placenta (26.22 ± 7.53 in NR vs. 17.80 ± 5.01 in R). The participation of THBS1 in the activation of TGFß1 in parturient bovine placental tissues leading to the normal release of fetal membranes may be suggested.

Profiling the alterations of serum proteome in dairy cows with retained placenta using high-throughput tandem mass tags quantitative approach.

BACKGROUND: Retained placenta (RP), a quite common disorder in dairy cows, shows a high negative impact on their health status and milk production. AIM: To investigate the difference in the serum proteome between the cows with RP and the physiologic puerperium (PP). MATERIAL & METHODS: Analysis of serum samples from nine cows with RP and six with PP using high-resolution liquid chromatography-tandem mass spectrometry approach. The proteins differing in the relative abundance between the PP and RP groups were classified using the Protein Analysis Through Evolutionary Relationship tool. For the pathway enrichment analysis, the REACTOME tool, with the human genome as the background, was employed. The criterion for significance was the false discovery rate corrected P-value less than 0.05. RESULTS: In total 651 proteins were identified with altered relative abundance of ten proteins. Among them, seven had higher, and three showed lower relative abundance in RP than in the PP group. The differently abundant proteins participated in 15 pathways: six related to hemostasis, three involved in lipoprotein metabolism, and the remaining ones associated with for instance redox homeostasis, post-translational modification, and scavenging. Finally, the validation of the proteomic results showed that haptoglobin and lipopolysaccharide-binding protein levels reliably differentiated between the RP and PP groups. CONCLUSION: The pattern of serum proteome alterations in the cows with RP mirrored several interplaying mechanisms underlying the systematic response to the presence of RP, therefore representing a source to mine for predictive or prognostic biomarkers.

Impaired placentomal interferon signaling as the possible cause of retained fetal membrane in parturition-induced cows.

Although hormonal induction of parturition in cattle results in the successful delivery of healthy calves, the risk of retained fetal membrane is significantly increased. In a previous study, a combination of the long-acting glucocorticoid, triamcinolone acetonide, with a high dose of betamethasone partially normalized the placentomal gene expression during parturition; however, the incidence of retained fetal membrane remained high. This study further explored placentomal dysfunction and aimed to elucidate the mechanism of retained fetal membrane in parturition-induced cows. In this study, transcriptome analysis revealed that enhanced glucocorticoid exposure normalized the expression of a substantial fraction of genes in the cotyledons. In contrast, a significant reduction in the multiple signaling pathway activities, including interferon signaling, was found in the caruncles during induced parturition. Real-time PCR showed that the expression of interferon-tau in the caruncles, but not interferon-alpha or interferon-gamma, was significantly lower in induced parturition than spontaneous parturition. Interferon-stimulated gene expression was also significantly decreased in the caruncles during induced parturition. These results indicate that interferon signaling could be important for immunological control in placentomes during parturition. Additionally, this suggests that interferon-tau might be a pivotal ligand for interferon receptors in the caruncles. This study revealed that peripheral blood leukocytes in prepartum cows transcribed interferon-tau. Macrophage infiltration in the placentome is known to participate in the detachment of the fetal membrane from the caruncle. Thus, this study raised the possibility that immune cells migrating into the caruncles at parturition may act as a source of ligands that activate interferon signaling.

Increase in white blood cell counts by pegbovigrastim in primiparous and multiparous grazing dairy cows and the interaction with prepartum body condition score and non-esterified fatty acids concentration.

The objective of this study was to determine if parity affected the effect of pegbovigrastim (PEG) treatment on white blood cell (WBC) counts in grazing dairy cows. Additionally, the association of prepartum body condition score (BCS) and non-esterified fatty acid (Pre-NEFA) concentration with WBC counts was investigated. The effect of early-lactation disease was included in the statistical analysis. A randomized controlled trial on four commercial grazing dairy farms was performed. Holstein primiparous (Control = 87, PEG = 89) and multiparous (Control = 181, PEG = 184) cows were randomly assigned to one of two treatments: first PEG dose 8 ± 5 (mean ± SD) days before the expected calving date and a second dose within 24 h after calving (PEG) compared to untreated controls (Control). Treatment effects were evaluated with mixed linear regression models. Treatment with PEG increased WBC, neutrophil, lymphocyte and monocyte counts at 6 ± 1 (mean ± SD) days in milk. Parity, BCS and their interactions with treatment were not associated with WBC counts. In control cows, Pre-NEFA concentration was associated with reduced WBC, neutrophil and lymphocyte counts and tended to be associated with reduced monocyte counts. Pegbovigrastim treatment reversed the negative association of Pre-NEFA concentration with neutrophil and monocyte counts and tended to reverse the negative association of Pre-NEFA concentration with WBC counts. In the PEG treated group, cows diagnosed with retained placenta or metritis showed lower neutrophil counts when compared to PEG treated cows without these clinical diseases. These data confirm that PEG treatment increases WBC, neutrophil, lymphocyte and monocyte counts in grazing dairy cows and that this effect is independent of parity. Pegbovigrastim treatment reversed the negative association of Pre-NEFA concentration with neutrophil and monocyte counts, and tended to reverse the negative association of Pre-NEFA concentration with WBC counts.

Hormonal and metabolic profiles related to placental retention with emphasis on oxidative stress and serotonin receptors in pluriparous buffaloes.

This study was designed to investigate the hormonal and metabolic factors associated with placental retention in buffaloes with respect to the roles of oxidative stress biomarkers and serotonin receptors. Blood samples were collected at weeks 3, 2 and 1 pre-partum and at calving from 37 buffaloes; thirty normally dropped their placentae (Non-RFM group) and 7 dropped their placentae after 12 hr post-calving (RFM group). Serum progesterone (P4 ), oestradiol, cortisol, non-esterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), antioxidant/oxidant biomarkers and mineral concentrations were assessed. Histopathology and histochemistry were implemented to detect collagen in foetal placental tissues. Immunohistochemistry for serotonin receptors in placental tissues was performed. Significant elevations in P4 , cortisol, NEFA, BHBA and oxidative biomarkers concentrations were observed in the RFM group. However, oestradiol, antioxidants and mineral concentrations were significantly lower in RFM buffaloes than Non-RFM group. Histopathological examination revealed degenerative changes and necrosis in retained placental tissue compared with that in normal placental tissues. Serotonin receptors were significantly expressed with collagen condensation in retained placental tissues. Furthermore, inferior reproductive performance was pronounced in the retained group. In conclusion, retained foetal membranes in buffaloes were associated with hormonal imbalance, metabolic perturbation, oxidative stress, serotonin receptor upregulation and markedly reduced fertility indices.

The preliminary studies on protein profile in retained and not retained foetal membranes in heavy draft mares.

Protein profile of the placenta expresses its function and maintenance. Any alterations can be reflected in qualitative and quantitative changes in this profile. The aim of the present study was the evaluation of protein profile in the placenta of mares suffering from the retention of foetal membranes (FMR) by two separation methods and the comparison with physiologically released tissues. Placentas from 14 healthy, heavy draft mares were collected immediately after the expulsion of newborn. Tissues after homogenization and staining with fluorescent dyes were subjected to electrophoretic as well as chromatographic separation. Electrophoretic gels were statistically analysed for the presence and abundance of examined proteins, while some proteins were identified in chromatographic fractions. Out of 248 spots detected in endometrium, 38 differed significantly between FMR and control animals, while in allantochorion, respective values reached 241 and 27 spots (p < .05). Among identified proteins that expressed higher abundance in endometrium of FMR mares than control animals were prostaglandin reductase, dehydrogenase/reductase SDR family, and placental growth factor. These proteins are involved in regulation of parturition. Additionally, the following proteins responsible for physiological activity of a cell-guanine methyl transferase, aspartyl/asparaginyl beta-hydroxylase and GTP-binding protein, were identified. These proteins expressed higher abundance in allantochorion of FMR mares than in controls. This preliminary study confirmed the disturbances in protein pattern between foetal membranes in FMR and healthy mares. Further qualitative and quantitative experiments are necessary to deepen our knowledge on the mechanisms of the retention of foetal membranes in mares.

Patterns of protein glycosylation in bovine placentomes as a function of gestational age and in retained versus non-retained placenta.

The formation of placenta at the beginning of pregnancy and its separation at parturition require not only deep remodelling of extracellular matrix, which mainly consists of proteins conjugated with sugar moieties, but also the cooperation with cells from both maternal and foetal parts of placenta. The aim of the study was to compare the patterns of selected conjugated proteins with sugar moieties between pregnant and term placenta as well as between released and retained placenta in cows. Placental samples from healthy pregnant cows (3-5 months of pregnancy) were collected at a slaughterhouse (n = 6), and parturient samples were collected during caesarean section at term and retrospectively divided into retained (n = 6) and released (n = 6). The pattern of selected sugar moieties conjugated with proteins was detected by use of lectin blotting with Phaseolus Vulgaris leucoagglutinin, Maackia Amurensis and Sambucus Nigra (Elderberry). The comparison and analysis of obtained band patterns showed differences between their number, molecular weight and abundance related to the intensity of staining. Samples from 3 to 4 months showed similarities, while at the 5th month, clear differences were visible in all 3 lectins, which were used in this study. Samples from retained/released placenta expressed significant differences in PHA-L and SNA pattern in the foetal part. Obtained results indicate that the development of placenta related to extracellular matrix and accompanying cells from both sides of placenta shows dynamic changes during pregnancy. Moreover, in the case of animals with the retention of foetal membranes the patterns of proteins conjugated with sugar moieties are altered, suggesting that the changes in extracellular matrix metabolism can be involved in the attachment and detachment of the placenta in cows.

The comparison of protein map between retained and released bovine placenta.

Placental retention in cows may be the result of altered protein pattern in comparison to physiologically released fetal membranes. Aim of study was to separate and identify proteins from maternal and fetal part of placenta and to compare them between released and retained fetal membranes. Six not retained and 6 retained tissues were obtained from healthy cows during routinely performed caesarian section. Cows were allocated to appropriate groups retrospectively. Samples were homogenized in phosphate buffer and subjected to 2D electrophoresis. After analysis of gels selected spots were excised and proteins were identified by MS. Two-dimensional electrophoresis detected and identified 886 spots in examined tissues. Significant differences (p < .05) were noticed between appropriate parts of retained and released placenta. In maternal part of retained placenta 40 spots showed lower abundance and 47 higher abundance in comparison to healthy samples. While in fetal part of retained placenta respective values were 60 and 125 proteins. Out of 73 identified proteins, 26 were significantly different between respective maternal (19) and fetal (7) part of retained and released placenta. In summary, protein profile of released and retained placenta express the presence and abundance of different proteins. It may suggest that selected proteins could be target molecules in searching for reasons for placental retention. Further identification of spots obtained here may provide with more detailed explanation of mechanisms of placental retention.

The evaluation of superoxide dismutase activity, neutrophil function, and metabolic profile in cows with retained placenta.

The present study aimed to determine the alterations in pre- and postpartum immune function, superoxide dismutase (SOD) activity, and serum metabolite changes in Brown Swiss cows with or without retained placenta (RP). Peripheral blood was sampled at -10 ±â€¯4 days antepartum and 10 ±â€¯4 days postpartum for the assessment of neutrophil phagocytic and oxidative burst activities by using flow cytometry. SOD activity, beta hydroxybutiric acid (BHB), nonesterified fatty acids (NEFA), calcium (Ca), phosphorus, aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), creatinine kinase (CK), gamma-glutamyl transferase (GGT), lactate dehydrogenase, bilirubin, cholesterol, glucose, urea, albumin, total protein, and triglyceride concentrations were also measured from serum samples collected at -10 ±â€¯4, 3 ±â€¯1, 10 ±â€¯4, and 30 ±â€¯4 days relative to parturition. All data were analyzed using the MIXED procedure of SPSS. Neutrophil phagocytic and oxidative burst activities, phagocytic and oxidative fluorescence intensity, and phagocytic and oxidative indexes were similar among the groups (P > 0.05). However, serum SOD activity was 30 ±â€¯4 IU/ml and 17 ±â€¯2 IU/mL for cows that subsequently developed RP and healthy cows at -10 ±â€¯4 days antepartum, respectively (P < 0.05). Body condition scores were similar in both groups (P > 0.05). All investigated serum metabolic parameters varied significantly over time (P < 0.001). Cows affected with RP had subclinical hypocalcemia (8.06 ±â€¯0.3 mg/dl) and had significantly higher BHB levels (1027 ±â€¯137.9 mmoL/l) at 3 ±â€¯1 days postpartum. Furthermore, these cows had greater AST and NEFA concentrations around calving (P < 0.05). In conclusion, retained placenta had no prolonged effect on the measures of neutrophil function during the postpartum period. On the other hand, increased antepartum SOD activity might be a predisposing factor and can be used as a predictive biomarker for retained placenta.

Expression of proinflammatory cytokines IL-1ß, IL-6 and TNFα in the retained placenta of mares.

Retained fetal membranes (RFM) is a prevalent postpartum disease in mares. During parturition, various proinflammatory cytokines are released in the placenta, which further facilitates uterine contractions, expulsion of the fetus and fetal membranes. We hypothesized that an altered inflammatory response in mares results in retained fetal membranes. Samples of the endometrium and the allantochorion were collected from 15 mares with RFM and from 29 control mares within 2 h of foal delivery. Next, the mRNA expression of IL-1ß, IL-6 and TNFα proteins was analyzed by Real Time PCR and Western blot. These cytokines were also localized by immunocytochemistry. In mares with RFM, the mRNA expression of IL-1ß in the endometrium (p < 0.05) and IL-6 in the allantochorion (p < 0.0001) was higher than in mares that expelled fetal membranes physiologically. Higher expression of IL-6 in the allantochorion was confirmed by Western blot. In contrast, no changes in TNFα mRNA and protein expression were observed between the 2 groups of mares. Moreover, TNFα mRNA was barely detectable. The presence of all studied cytokine proteins in the allantochorion was confirmed by immunocytochemistry. The results suggest that the outbreak of inflammation is involved in the mechanism of placenta release in mares. Increased expression of IL-6 in the allantochorion and IL-1ß in the endometrium could reflect a local immune response which leads to the detachment of fetal membranes. The low expression of TNFα mRNA might suggest that this cytokine is not involved in the expulsion of fetal membranes. Further work is needed to determine the exact role and timing of inflammatory activation in fetal membranes during equine parturition.

miRNA-185 regulates the VEGFA signaling pathway in dairy cows with retained fetal membranes.

Retention of fetal membranes (RFM) of cows is an important reproductive disturbance, and is related to miRNAs. Vascular endothelial growth factor (VEGF)A, regulated by miRNA-185, can activate arachidonic acid (ARA) release via the VEGFA signaling pathway, which influences RFM. The aim of this study was to explore the pathogenic mechanism of RFM by investigating the regulatory relationship between miRNA-185 and the VEGFA signaling pathway. Serum samples of healthy Holstein dairy cows (n = 20) and RFM cows (n = 12), with a similar age, parity, weight, and milk yield, were collected to detect VEGFA and ARA concentrations at 6, 12, and 24 h after calving. Caruncle tissues were collected from healthy (n = 6) and RFM cows (n = 6) at 12 h after calving. Quantitative polymerase chain reaction (qPCR) and western blotting (WB) were performed to detect the mRNA and proteins levels, respectively, of genes involved in the VEGFA signaling pathway. Uterine caruncle epithelial (UCE) cells were cultured by the explant culture method, further purified, and subsequently treated with miRNA-185 mimics, miRNA-185 mimics + MEK inhibitor, or left untreated as a control for detection of the mRNA and protein levels of genes involved in the VEGFA signaling pathway. The cellular supernatant was collected for measurement of ARA levels at 12, 24 and 48 h after treatment. Serum levels of VEGFA and ARA from RFM cows were abnormally increased at 12 h after calving, as compared to those in healthy dairy cows. Expression levels of most of the investigated genes (VEGFA, PLC, PRK, RAF, MEK, MAPK, and PLA) were down-regulated in the caruncle tissue of RFM cows. However, P-p44/42 MAPK was up-regulated in the caruncle tissues of cows with RFM (p < .01). In UCE cells treated with the miRNA-185 mimics, expression of VEGFA, PLC, RAF, MEK, MAPK and PLA was significantly down-regulated, while that of P-p44/42 MAPK was significantly up-regulated. Expression of genes involved in the VEGFA signaling pathway was similar to that in the in vivo assay. In UCE cells treated with the miRNA-185 mimics + MEK inhibitors, expression of VEGFA, PLC, RAF, MEK, MAPK and P-p44/42 MAPK was significantly down-regulated, while that of PLA was significantly up-regulated. Meanwhile, the release of ARA was increased (p < .01). These results demonstrate that miRNA-185 can regulate the VEGFA signaling pathway, especially via abnormal expression of P-p44/42 MAPK, which influences the release of the fetal placenta after calving.

Pregnancy-associated glycoproteins in cows with retained fetal membranes.

In cows, retained fetal membranes (RFM) are a major problem in reproduction. The timely detachment of fetal membranes after parturition requires well coordinated maturation processes in the placenta. One feature of placental maturation in cows is a prepartal decline in the number of binucleate trophoblast giant cells (BNC) in the fetal chorion. Pregnancy associated glycoproteins (PAGs) are a group of proteins, produced by trophoblast cells in artiodactyls. We studied aspects of PAG expression in cows with and without RFM. The numerical density of PAG-positive immunostained BNC in placentomal samples, collected from cows with normal expulsion of fetal membranes (n = 20) and cows with RFM (n = 20) was determined. The number of PAG-positive BNCs was significantly higher in cows with RFM, compared to controls. The concentration of PAGs in maternal serum in prepartum, intrapartum, and postpartum cows was measured (RFM n = 20; controls n = 68). No significant differences between RFM and controls were detected. Microarray analysis of placental PAG mRNA expression was done with two types of microarrays: Affymetrix (RFM n = 20; controls n = 20) and Agilent (RFM n = 8; controls n = 8). Both microarrays showed a significantly higher expression of modern PAGs in RFM cases. Our results show that the expression of modern PAGs, which are produced by BNCs and are secreted into the maternal organism, are differentially expressed in RFM. Although the concentration in peripheral maternal blood did not differ between RFM and controls, the local concentration in the placenta is likely to be higher in RFM cases. This suggests the possibility of local regulatory roles of PAG in the release of bovine fetal membranes.

Extracellular matrix proteins in healthy and retained placentas, comparing hemochorial and synepitheliochorial placentas.

The placenta expresses structural and biologically active proteins. Their synthesis is mainly regulated by genomic or nongenomic signals and modulated by hormones. These protein profiles are altered during different stages of pregnancy. The biological properties of extracellular matrix (ECM) proteins were defined and described in a number of tissues including placenta. These properties enable them to be the main players in the processes of attachment or invasion into the endometrium during initial placenta formation and its timely separation after delivery and detachment. In this review, we focused on the role of ECM proteins during attachment of the placenta to the uterine wall, its timely separation, and the implications of this process on retained or pathologically attached placenta. Although the amount of published information in this area is relatively scant, some of the key proteins and processes are well defined. We focused on the available data detailing the ECM protein profiles of human (histologically thin; hemochorial) and bovine (histologically thick; epitheliochorial) placentas and compared the shared and unique ECM proteins that are relevant to placental attachment and separation.

Oxidative stress and inflammation in retained placenta: a pilot study of protein and gene expression of GPX1 and NFκB.

BACKGROUND: Retained placenta is associated with severe postpartum hemorrhage. Its etiology is unknown and its biochemistry has not been studied. We aimed to assess whether levels of the antioxidative enzyme Glutathione Peroxidase 1 (GPX1) and the transcription factor Nuclear Factor κß (NFκß), as markers of oxidative stress and inflammation, were affected in retained placentas compared to spontaneously released placentas from otherwise normal full term pregnancies. METHODS: In a pilot study we assessed concentrations of GPX1 by ELISA and gene (mRNA) expression of GPX1, NFκß and its inhibitor Iκßα, by quantitative real-time-PCR in periumbilical and peripheral samples from retained (n = 29) and non-retained (n = 31) placental tissue. RESULTS: Median periumbilical GPX1 concentrations were 13.32 ng/ml in retained placentas and 17.96 ng/ml in non-retained placentas (p = 0.22), peripheral concentrations were 13.27 ng/ml and 19.09 ng/ml (p = 0.08). Retained placental tissue was more likely to have a low GPX1 protein concentration (OR 3.82, p = 0.02 for periumbilical and OR 3.95, p = 0.02 for peripheral samples). Median periumbilical GPX1 gene expressions were 1.13 for retained placentas and 0.88 for non-retained placentas (p = 0.08), peripheral expression was 1.32 and 1.18 (p = 0.46). Gene expressions of NFκß and Iκßα were not significantly different between retained and non-retained placental tissue. CONCLUSIONS: Women with retained placenta were more likely to have a low level of GPX1 protein concentration in placental tissue compared to women without retained placenta and retained placental tissue showed a tendency of lower median concentrations of GPX1 protein expression. This may indicate decreased antioxidative capacity as a component in this disorder but requires a larger sample to corroborate results.

[Evaluation of sulfur status in dairy cows in Germany]. / Untersuchungen zur Schwefelversorgung von Milchkühen in Deutschland.

OBJECTIVE: Sulfur deficiency and toxicity in livestock is associated with decreased performance and diseases. However, little is known about the diagnostics of sulfur status in dairy herds. The objectives of this study were to describe sulfur concentrations in feed rations in German dairy herds, to assess associations between feed sulfur concentrations and health and milk yield, to determine suitable laboratory diagnostic methods of sulfur status, and to propose reference values for the assessment of sulfur status in different sample media. MATERIALS AND METHODS: Between 2006 and 2014, a total of 569 dairy herds were visited. Blood, urine, and hair samples were obtained from 10 animals per group in the groups -3 to 0, 0 to 1, 3 to 5, and 15 to 18 weeks post partum (p. p.) (farms > 200 animals) or -3 to 0, 0 to 5, and 6 to 20 weeks p. p. (farms ≤ 200 animals). Pooled whole blood, serum, plasma, urine, and hair samples (n = 5663) were analyzed by ICP-OES. The sulfur content of the total mixed ration (TMR) of the group -3 to 0 weeks p. p. was determined in a certified feed laboratory (n = 625). Data analyses were performed using ANOVA, Chi-squared test and Spearman-correlation. Reference values were determined using the 2.5% and 97.5% percentiles. RESULTS: Median sulfur concentration in the feed rations was 2.3 g/kg dry matter (DM). Deficiency (< 1.6 g/kg DM; 31% of farms) and oversupply (> 4.0 g/kg DM; 11% of farms) of sulfur in feed rations were regularly diagnosed. Sulfur deficiency was associated with reduced milk yield (p  <   0.001), retained placenta (OR  =   1.74; p  =  0.037), milk fever (OR  =  2.68; p  <  0.001) and silent heat (OR  = 2.56; p   =  0.014). Positive correlations were found between sulfur concentration in feed rations and sulfur content in urine (r  =  0.50), serum (r  =  0.17), plasma (r  =  0.18) and whole blood (r = 0.16). Reference values for sulfur diagnostics in dairy herds were proposed. CONCLUSION AND CLINICAL RELEVANCE: Diagnostics of sulfur status in dairy herds is relevant for veterinary practitioners and can be performed using TMR analysis and clinical laboratory parameters. Urine can be considered a short-term parameter, serum, plasma, and whole blood as intermediate parameters, and hair as a long-term parameter for the evaluation of the sulfur status.

Changes in endometrial transcription of TLR2, TLR4, and CD14 during the first-week postpartum in dairy cows with retained placenta.

Changes in the endometrial transcription of pattern recognition receptors may increase the susceptibility to postpartum uterine infections in Holstein cows with retained placenta. To test this hypothesis, nine cows with retained placenta and ten cows without retained placenta were submitted to endometrial biopsies at the first and seventh days postpartum. Cows were monitored weekly with clinical and gynecological examinations until 42 days postpartum. Samples of the uterine contents were collected weekly for aerobic bacteria isolation. All cows had endometrial transcription of Toll-like receptors (TLRs) 1/6, 2, 4, 5, and 9; nucleotide-binding oligomerization domain (NOD)-like receptors 1 and 2; and the coreceptors cluster of differentiation 14 (CD14) and myeloid differentiation protein-2 (MD-2), as measured on the first and seventh days postpartum. Escherichia coli was the most common bacterium isolated from the uterine contents of cows with or without retained placenta until 21 days postpartum. Transcription levels of TLR2, TLR4, and CD14 in Holstein cows with retained placenta significantly decreased (P < 0.05) between the first and the seventh day postpartum. Conversely, cows without retained placenta did not have any significant changes in transcription levels between these time points.

Periparturient stress and immune suppression as a potential cause of retained placenta in highly productive dairy cows: examples of prevention.

The immune system during the periparturient period is impaired. At this time the most important factor causing immune-suppression in highly productive cows is metabolic stress resulting from hormonal and metabolic fluctuations, a negative energy balance, shortage of proteins, minerals and vitamins which are required to meet the demands of the fetus as well as the onset of lactation. This stress can activate the hypothalamic-pituitary-adrenocortical axis (HPA), which results in increase plasma corticosteroids. As a result, the cortisol concentration during the periparturient period increases by several folds particularly on the day of calving. Cortisol is a powerful immune-suppressive agent. During stress, this hormone causes depression of the leukocyte proliferation and their functions. Decreased phagocytosis of neutrophils, decreased cytotoxic ability of lymphocytes, as well as depressed activity of their cytokines, make it impossible for the normal, efficient maternal immune recognition and rejection of fetal membranes (as a foreign, allogeneic tissue expressed fetal antigens-MHC class I proteins by trophoblast cells) and finally results in their retention in cows. The metabolic periparturient stress also activates production of catecholamines, especially adrenalin. Adrenalin activates adrenoreceptors of the myometrium and then causes hypotony or atony of the uterus. Thus, cortisol and adrenalin inhibit rejection and expulsion of fetal membranes and cause their retention. These mechanisms of retained placenta (RP) often have a metabolic etiology and occur in herds, where important infectious diseases causing placentitis are absent or prevented. The aim of this article is to show the fundamental mechanisms occurring during periparturient stress and the accompanied immune-suppression in cows, as well as their consequences in relation to RP. The paper also gives examples of the symptomatic prevention of RP in cows caused by metabolic and immune suppressive factors. The prevention of RP was carried out using drugs which inhibit the activity of cortisol or adrenalin in dairy cows during calving.

Usefulness of DIGE for the detection of protein profile in retained and released bovine placental tissues.

INTRODUCTION: Regardless intensive research, the etiology and mechanisms of retention of fetal membranes in cows, still require elucidation. In our research approach, difference in gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization (MALDI) identification were used to obtain first results on protein profile of bovine placental membranes which were properly released or retained for more than 12 h after parturition. METHODS: Placentomes from 6 cows that released placenta and from 6 cows that retained fetal membranes were homogenized, fluorescence labeled and subjected to DIGE. RESULTS: Selected spots that significantly differed between retained and released placenta as well as spots with constant appearance were identified by MALDI. This allowed identification of the following proteins with high statistical reliability: Transforming growth factor beta 2 - high expression in maternal and fetal part of retained fetal membranes, Short transient receptor potential channel 5 -high expression in maternal part of retained and not retained fetal membranes, Rab GDP dissociation inhibitor beta - high expression in fetal part of retained and not retained fetal membranes, Proline dehydrogenase 2 - similar expression in all examined samples, Ras-related protein Rab-7b -high expression only in maternal part of not retained fetal membranes. DISCUSSION: Up to now, these proteins have not been considered as possibly important molecules for the separation/retention of fetal membranes, but their biological roles may suggest it. Further studies are necessary to establish a full profile of bovine placental proteins and define target molecules that may be involved in separation/retention of fetal membranes.