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.

Changes in plasma PLAC-1 concentration and its expression during early-mid pregnancy in bovine placental tissues - a pilot study.

BACKGROUND: Placenta-specific protein 1 (PLAC1) is a small secreted protein considered to be a molecule with a significant role in the development of the placenta and the establishment of the mother-foetus interface. This study aimed to confirm the presence of bovine PLAC1 and to examine its profile in the placenta and plasma in the first six months of pregnancy. The expression pattern of PLAC1 was analysed by RT-qPCR and Western Blotting. Quantitative evaluation was carried out using ELISA. RESULTS: PLAC1 concentrations in the plasma of pregnant cows were significantly higher (p < 0.05) than those obtained from non-pregnant animals. PLAC1 protein concentrations in the placental tissues of the foetal part were significantly (p < 0.05) higher than in the tissues of the maternal part of the placenta. PLAC1 transcripts were detected in both placental tissue samples and epithelial cell cultures. CONCLUSIONS: In conclusion, the results of the present preliminary study suggest that PLAC1 is involved in the development of bovine placenta. The presence of this protein in the plasma of pregnant animals as early as the first month may make it a potential candidate as a pregnancy marker in cows. Further studies on exact mechanisms of action of PLAC1 in bovine placenta are necessary.

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.

Egg Yolk as a New Source of Peptides with Antioxidant and Antimicrobial Properties.

A significant increase in interest in food-derived peptides obtained mostly through enzymatic reactions has been observed in the past few years. One of the best sources of bioactive peptides are defatted egg yolk proteins, which can potentially find application as high-quality nutritional supplements for infants with cow's milk protein intolerance and as natural preservatives. The aim of this study was to obtain peptides from defatted egg yolk protein, to study their antioxidant and antimicrobial properties, and to identify peptides with bioactive properties To control the course of the process, MALDI-TOF/MS (matrix-assisted laser desorption/ionization time-of flight/mass spectrometry) spectra were also examined. The peptide mixture obtained through enzyme digestion was tested for its antioxidant properties by measuring the scavenging activity in 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization (ABTS•+), and ferric reducing activity (FRAP) assays. Antimicrobial activity was also studied. The peptide mixture exhibited significant antioxidant activity: DPPH-1776.66 ± 32.99, ABTS-390.43 ± 8.92, and FRAP-16.45 ± 0.19. The inhibition of bacterial growth by two concentrations of the peptide mixture was examined. The best result was obtained in Bacillus cereus, with an inhibition zone of 20.0 ± 1.0 and 10.7 ± 0.6 mm at the concentrations of 50 and 25 mg/mL, respectively. The results of the study suggest that the mixture of egg yolk peptides may exhibit a number of health-promoting properties.

Effect of Sex Steroids and PGF2α on the Expression of Their Receptors and Decorin in Bovine Caruncular Epithelial Cells in Early-Mid Pregnancy.

Changes in the expression of various genes, including pregnancy-associated hormone receptors and extracellular matrix proteins, have been suggested to play a significant role in bovine placental development. This study aimed to examine the influence of sex steroids and PGF2α on decorin (DCN) expression in the epithelial cells of bovine caruncle in early−mid pregnancy in cows. The expression patterns of DCN, PTGFR, PGR and ESR1 were analyzed by RT-qPCR and Western blotting in primary caruncular epithelial cell cultures (PCECC) and placental tissue homogenates derived from the 2nd and 4th months of pregnancy. PCECC were found to express DCN, PTGFR, PGR and ESR1. The intensity of PGR staining was higher in cells derived from the 4th month of pregnancy (p < 0.05). The 17ß-estradiol, progesterone and PGF2α have not been shown to affect DCN expression. PGF2α decreased PTGFR expression in cells derived from the 4th month of gestation (p < 0.05). In conclusion, the results of the present preliminary study showed that the expression of the PTGFR, ESR1, PGR and DCN in PCECC does not vary throughout early−mid pregnancy. Further studies should be carried out to observe the relationship between hormonal status and cellular adhesion to determine their importance for properly developing placentation and pregnancy in cows.

Comparative Analysis of Saliva and Plasma Proteins Patterns in Pregnant Cows-Preliminary Studies.

Pregnancy is a physiological state that can be described, from a biochemical point of view, using protein patterns. The present study focused on the comparison of protein patterns between the saliva and plasma of pregnant cows to search for possible markers which are present both in plasma and saliva. Saliva and plasma were collected from healthy, pregnant (3-4 months) and non-pregnant (C; n = 4) cows aged between 4 and 8 years (P; n = 8) from the same farm. Biological material was analyzed using 2D electrophoresis and MS identification. Among identified spots, there were those which could be related to pregnancy (e.g., apolipoproteins I and II in all examined matrices or transforming growth factor-beta-induced protein ig-h3 in albumin-free plasma) as well as those which are responsible for regulating of cellular processes (e.g., pyruvate kinase and aspartate aminotransferase in all examined matrices, or lactate dehydrogenase, phosphoglycerate kinase, and NADH dehydrogenase in plasma). Further identification of common spots and those only specific to saliva as well as the comparison between other periods of pregnancy are necessary; it is already clear that saliva can be considered a valuable diagnostic matrix containing potential markers of physiological and pathological status.

Reference gene selection in bovine caruncular epithelial cells under pregnancy-associated hormones exposure.

Examination of transcriptional regulation occurring during pregnancy establishment and maintenance requires the identification of endogenous reference genes characterized by high expression stability. Since the expression of some reference genes may be modulated by pregnancy-associated hormones, the goal of our study was to identify suitable reference genes unaffected by hormonal treatment. In our study bovine caruncular epithelial cells were subjected to progesterone, estrogen and prostaglandin F2α treatment. Ten candidate reference genes (ACTR1A, CNOT11, HDAC1, HPRT1, RPL19, RPS9, SDHA, SUZ12, UXT and ZNF131) were evaluated with the use of four approaches (geNorm, NormFinder, BestKeeper, delta Ct). We found that RPS9 and SUZ12 displayed the highest expression stability in the tested material. Moreover, HPRT1 and SDHA were found inappropriate for RT-qPCR data normalization as they demonstrated the highest expression variability out of all candidates analysed. Hence geNorm calculations shown that the use of just two best-performing genes would be sufficient for obtaining reliable results, we propose that RPS9 and SUZ12 be used as suitable endogenous controls in future studies investigating gene expression in normal and compromised pregnancies.

Antioxidative and Oxidative Profiles in Plasma and Saliva in Dairy Cows during Pregnancy.

Increased metabolism that occurs during pregnancy can result in oxidative stress which is harmful to cells and, consequently, for the proper functioning of the whole organism. Plasma and recently also saliva are important resources for evaluating physiological and pathological conditions in animals. The study aimed to investigate the influence of the metabolic state on the effectiveness of the antioxidant profile of plasma and saliva during the pregnancy of cows. Seventy-six healthy pregnant and twelve non-pregnant control cows were included in the study. Blood and saliva samples were collected each month of the pregnancy course. Examined body fluids were used to evaluate both the total antioxidant capacity (TAC) and the oxidative parameters related to protein and lipid peroxidative processes. TAC, the content of hydroperoxides, and SH groups were determined spectrophotometrically while formylokinurenine and bityrosine contents were measured spectrofluorimetrically. The results showed dynamic changes depending on the period of pregnancy course. The highest antioxidant activity in plasma was mostly noted in early pregnancy and advanced pregnant cows. All tested parameters except SH groups expressed higher values in saliva compared to plasma. Obtained results reveal that the increase in oxidative intensity induced appropriate answers of cells reflected in the increase in antioxidative activity of the organism. Moreover, some examined parameters can indicate the intensity of oxidative stress and therefore could be used in a panel of markers of the physiological course of pregnancy. However, with regards to antioxidant/oxidative parameters, saliva reflects the content of plasma only in part, due to the local metabolism of the salivary gland. Further studies are necessary to establish physiological ranges of antioxidative/oxidative profiles in cows and to define the usefulness of saliva as biological material in oxidative stress diagnostics.

The role of dermatopontin in cell adhesion in bovine placenta during early-mid pregnancy and parturition - Pilot study.

Dermatopontin (DPT) is a small protein molecule thought to have a role in the formation of the extracellular architecture and adhesion. The aim of the study was to confirm the presence of DPT and to examine its role in placental cell adhesion during pregnancy, at parturition and postpartum in cows. Placental tissue samples were obtained at abattoir from healthy pregnant cows (n = 6) while parturient samples were collected during caesarian section and retrospectively divided into released up to 6 h (R; n = 5) and not released up to 6 h (NR; n = 4) foetal membranes. Maternal epithelial cells were isolated from pregnant samples and were used for the examination of the influence of DPT (5, 50 and 100 ng/mL) on cell adhesion. Parturient samples were manually divided into maternal and foetal part and individually homogenized for Western blotting and ELISA analysis. Western blotting confirmed the presence of DPT in examined tissues. ELISA test showed significant (p < 0.05) decrease in DPT concentration within examined pregnancy period with higher concentrations in maternal part (p < 0.05). Moreover, at parturition DPT concentration further decreased in maternal (p < 0.05) but increased (p < 0.05) in fetal part. The examination of not released samples showed opposite relationship in comparison to parturient samples - the increase in maternal (p < 0.05) and the decrease in fetal (p < 0.05) part of placenta. DPT facilitated the adhesion of epithelial cells in examined periods of pregnancy in increasing manner with pregnancy course. The presence of DPT in bovine placenta during pregnancy and parturition was confirmed. This protein may influence cell adhesion during attachment and detachment of placenta. Further studies on mechanisms of action of DPT in bovine placenta are necessary.

The influence of progesterone and prostaglandin F2α on decorin and the adhesion of caruncular epithelial cells of bovine placenta at early-mid pregnancy-Part II.

One of the most important processes determining the proper course of gestation and its physiological termination in cows is the adhesion of epithelial cells allowing for direct contact of maternal and foetal parts of the placenta. Throughout pregnancy, placental cells are under strict hormonal control, which among others regulates the concentration and activity of specific proteins participating in the extracellular matrix remodelling of foetal membranes. The aim of the study was to evaluate the influence of progesterone and prostaglandin F2α on the adhesion of epithelial cells at early-mid pregnancy in cows. Additionally, the impact of selected hormones on anti-adhesive properties of decorin was evaluated. Caruncular epithelial cells were isolated from healthy cows during pregnancy, immediately after slaughter. Primary cell cultures derived from the 2nd and 4th month of gestation were used in the experiments. The viability of cells was assessed by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. The activity of metalloproteinases was confirmed by the metalloproteinase assay. Progesterone (10-5 and 10-7  mol/L) and prostaglandin F2α (10-4 , 10-5 and 10-7  mol/L) increased the viability of bovine caruncular epithelial cells in the 2nd month of pregnancy. The treatment with prostaglandin F2α significantly reduced the number of adherent cells from the 2nd month of gestation at the doses of 10-4 and 10-5  mol/L. Both progesterone and prostaglandin F2α were shown to have an effect of decorin resulting in both a decrease in metalloproteinase activity and an increase in adhesion of cells to fibronectin.

Effect of decorin and selected glycosylation inhibitors on the adhesion of caruncular epithelial cells of pregnant cows-part I.

Adhesion process ensures the formation of the appropriate connection between mother and foetus during placentation and further placental development, which determines physiological pregnancy course. Extracellular matrix of foetal membranes are a rich source of biologically active proteins, the synthesis of which is regulated by hormones. Depending on the stage of pregnancy, the protein profile of the placenta changes, thanks to which its remodelling is possible. The aim of the study was to evaluate the effect of decorin, as well as selected glycosylation inhibitors on the adhesion of caruncular epithelial cells derived from cows during pregnancy. Placental cells were isolated from healthy, pregnant (2nd and 4th month) cows after slaughter, which allowed for the establishment of 4 primary cell cultures without visible cells of fibroblast morphology. The presence of decorin in cell monolayer and cell lysates was determined by the use of immunocytochemistry and Western blotting, respectively. The viability of cells was evaluated by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. Protein N-glycosylation and O-glycosylation have a modulating effect on the adhesion and viability of placental cells during early-mid pregnancy. Decorin and tunicamycin were shown to have anti-adhesive properties with respect to caruncular cells of the pregnant bovine uterus.

The comparison of pro- and antioxidative parameters in plasma and placental tissues during early phase of placental development in cows.

Physiological balance between pro- and antioxidative processes is crucial for placentation and further development of fetus and placenta. Parameters of pro- and antioxidative profile may serve as markers of proper course of pregnancy. The aim of study was to assess whether the balance between pro- and antioxidative parameters during placentation phase in bovine placenta is maintained. Placental and blood samples were collected from healthy, HF, pregnant (2nd-3rd month) cows (n = 8) in slaughterhouse and in farm, respectively. Formylokinurenine and bityrosine content were measured spectrofluorimetrically in blood plasma and tissue homogenates while metabolites of lipid peroxidation, total antioxidant capacity, SH groups and activity of antioxidative enzymes (glutathione peroxidase and superoxide dismutase) were determined in examined tissues by spectrophotometry. Western blotting was used to confirm the presence of enzymatic proteins in placenta. Results: Local profile in tissues was more pronounced than general profile in blood plasma. Activities of antioxidative enzymes were significantly (p < 0.05) higher in 2nd compared to 3rd month of pregnancy in maternal part of placenta while prooxidant parameters showed opposite relationship. Obtained results showed significant differences when compared to data from non-pregnant animals or time of parturition. Further studies are necessary for elucidation of placentation phase in cows.

Activity of selected glycosidases and availability of their substrates in bovine placenta during pregnancy and parturition with and without retained foetal membranes.

The activity of glycosidases is crucial for the function and biological activity of proteins conjugated with sugar moieties, which play an important role in adhesion of cells during attachment and detachment of the foetal membranes. The aim of study was to describe the ability of bovine placental tissues to break down O-glycosidic bonds in different glycoproteins by the determination of activity of ß-galactosidase, α-l-fucosidase, ß-N-acetyl-hexosaminidase and sialidase in early-mid-pregnancy as well as at parturition with released and retained foetal membranes. Moreover, the availability of substrates for these glycosidases in placental homogenates was evaluated. Placental samples were collected from pregnant (2-4 months) cows in slaughterhouse (n = 8) as well as during Caesarean section and divided into released foetal membranes (n = 8) and retained foetal membranes (n = 8). Tissue homogenates were subjected to spectrofluorimetric and spectrophotometric determinations of enzyme activities as well as electrophoretic separations. Enzyme activities expressed changes within examined time with significant (p < .05) differences between pregnancy and physiological parturition in ß-N-acetyl-hexosaminidase and α-l-fucosidase in foetal part of placenta while in maternal part only in the latter one. Decreasing tendency in enzyme activity was noticed in foetal part of retained samples in comparison with released ones with significant (p < .05) differences in α-l-fucosidase activity. The analysis of staining of sugar moieties attached to selected proteins depicted availability of sugar molecules in examined tissues, but their patterns differed between samples. In conclusion, sugar moieties in conjugated proteins express changes in the course of pregnancy which is reflected by the alterations in activities of placental glycosidases.

Bee Pollen Supplementation to Aged Horses Influences Several Blood Parameters.

In humans and animals, aging leads to a decrease in immune function and an increased susceptibility to infection. Decreased immunity and an increase in the incidence of infectious diseases are particularly notable during the autumn. Bee pollen supplementation improves immunity and antioxidant enzyme activity, as well as general performance. The aim of this study was to evaluate the effects of bee pollen supplementation during the autumn on blood parameters in aged horses. The study was performed on 16 warmblood horses aged 15-26 years. Half of this group received 60 g of bee pollen (soaked in water) daily for 30 days during the autumn season. Blood samples were taken from all horses before and after the supplementation period. Numerous hematological and plasma biochemical parameters including indicators of oxidative stress were determined. The data collected after the supplementation were compared with data collected before the experiment using one-way analysis of variance and paired Student's t-test. In the control group, there was a decline in the total number of red blood cells, hemoglobin, and hematocrit and an increase in some lipid parameters, urea, total plasma proteins, and sulfhydryl groups. Supplementation with bee pollen prevented the variation of these parameters, except for low-density lipoprotein cholesterol. We believe that bee pollen supplementation for aged horses during autumn has beneficial effects because it inhibited some of the adverse changes observed in the control horses during this season.