Marta Kankofer

Comparison of antioxidative/oxidative profiles in blood plasma of cows with and without retained fetal placental membranes.

Ante- and postpartum antioxidative/oxidative profiles in blood plasma of cows with and without retained placental membranes (RFM) were investigated. Twenty-two healthy pregnant cows were included in the study. Seven animals out of 22 suffered from RFM. Blood samples were obtained at 4, 3, 2, 1 weeks and 5 days antepartum (a.p.), at parturition and 1, 3, and 5 weeks postpartum. The following antioxidative parameters were measured using spectrophotometric methods: total antioxidant activity (TAC), β-carotene, vitamin A, vitamin C, and ceruloplasmin. The oxidative profile was based on the content of intermediates and end products of lipid and protein peroxidative processes which were measured by spectrophotometric and spectrofluorimetric methods. The examined parameters revealed a dynamic profile within the experimental period. The highest antioxidant and oxidant activity was noted at 2 and 1 week a.p. with a drop towards parturition suggesting the presence of oxidative stress during this time period and an apparent appropriate metabolic response of the macroorganism. Except for TAC and vitamin A, the contents of oxidative and antioxidative blood constituents did not differ between cows with and without RFM. A TAC and vitamin A by time interaction indicated higher antepartal concentrations of TAC and vitamin A in cows without RFM than in cows with RFM suggesting a possible role of antioxidative/oxidative imbalances in the aetiology of RFM.

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.

Profile of Bovine Proteins in Retained and Normally Expelled Placenta in Dairy Cows

Tissue-specific protein profile is determined by its function, structure, intensity of metabolism and usefulness. This profile remains under hormonal control. Any disturbance in the general metabolism may be reflected in changes in both protein quantity and quality. These changes can be of low or high specificity, and some can be used as clinical markers of pathological conditions. The aim of this study was to describe and to compare the protein profile of caruncle and foetal villi of bovine placenta that was either properly released or retained. Placental tissues were collected from healthy cows, divided into releasing and retaining foetal membranes, homogenized and subjected to 1D and 2D electrophoresis. Computer-aided analysis of gel images showed essential qualitative and quantitative alterations in protein profile between tissues that were properly released and retained. Alterations concerned both the number of fractions and spots as well as the intensity of staining. This preliminary study provides a general overview of the differences in the protein profile between released and retained foetal membranes. It may allow for selecting the group of proteins or single molecules, which should be further analysed in detail as possible markers differentiating the retention of foetal membranes in cows from placentas that were released spontaneously. The continuation of the study for the identification of particular spots detected in 2D gels is necessary.

The presence of SOD 1 and GSH-Px in bovine retained and properly released foetal membranes.

The maintenance of antioxidative/oxidative balance is crucial for cellular and extracellular environment. That is why antioxidative enzymes express their activity in different isoforms in different cell compartments and extracellular space. The aim of study was to verify the results of previous experiment on activities of antioxidative enzymes by the determination of their enzymatic proteins in bovine placental tissues by Western blotting technique. Moreover, the presence of particular isoenzymes was detected and differentiated. Homogenates of maternal and foetal part of both properly released and retained bovine placenta were subjected to PAGE electrophoresis in non-reducing and reducing conditions and Western blotting with appropriate antibodies against superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Electrophoresis allowed for the detection of protein bands of molecular weight related to CuZn-SOD as well as cGSH-Px isoenzymes. The reaction with appropriate antibodies confirmed this. Densitometric analysis, although semi-quantitative, allowed for the observation of trends in differences in antioxidative enzyme proteins, which may partly confirm previously described results in cases of retained and released placenta. Local antioxidative enzymatic mechanisms in bovine placental tissues are represented by CuZn-SOD and cGSH-Px, which show the changes in their expression during improper placental release.

Erythrocyte antioxidative enzymes in experimentally induced osteopenia in rats

Abstract The study aimed at the determination of dynamic relationship between mineralisation processes and antioxidative/oxidative status during the development of osteopenia. One hundred and two healthy female Wistar rats at the age of 2 months and initial body weight of 200 g were used in the experiment. The rats were divided into control (CON, n=6), sham operated (SHO, n=48), and ovariectomised (OVX, n=48) groups. Animals from SHO (n=6) and OVX (n=6) groups were sacrificed every week during 8 weeks of the experiment in order to detect dynamic changes in examined parameters. The samples were collected weekly from day 7 to day 56. The femora were examined with the use of DXA (bone mineral density) and pQCT (area, mineral content, volumetric density of trabecular and cortical part of distal femora). The pQCT scans were performed 5 mm from distal end of the tibia. The determination of activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in haemolysates of erythrocytes were performed spectrophotometrically. Obtained data showed wave like changes in both enzyme activities and bone parameters and indicated the importance of the 2nd-3rd and 5th-6th week after surgery as a key moment for bone metabolism and activity of enzymatic antioxidative defence during the development of osteopenia induced by bilateral ovariectomy. The obtained results proved that alterations in activity of GSH-Px and SOD, and pQCT ahead the changes registered by DXA by 7 d.