Poly(ADP-Ribose) Glycohydrolase in Bovine Retained and Not Retained Placenta

Poly(ADP-ribose) glycohydrolase (PARG) is the enzyme which degrades poly(ADP-ribose) polymers synthesized by poly(ADP-ribose) polymerase (PARP). Both enzymes are activated in response to different stimuli like oxidative stress and are involved in DNA repair processes. The retention of bovine foetal membranes (RFM) is supposed to be connected with oxidative stress conditions. The aim of the study was to detect the presence of PARG protein in bovine placenta in order to find the relationship between the process of releasing, retaining placenta and DNA repair. Placentomes, collected alter spontaneous delivery or caesarian section were divided into maternal as well as foetal part of placenta, homogenized and subjected to electrophoresis. Animals were divided into six groups as follows: A--caesarian section before term with RFM; B--caesarian section before term without RFM; C--spontaneous delivery at term with RFM; D--spontaneous delivery at term without RFM; E--caesarian section at term with RFM; F--caesarian section at term without RFM. PARG protein was detected in nitrocellulose membranes using commercially available bovine anti-PARG antibody and Western blotting technique. Single bands referred to bovine PARG standard were observed in all examined tissues as well as in human placenta used as the control of procedure. In addition, the intensity of staining was stronger in retained than properly released term placenta and in foetal than in maternal part of the placenta. These results may suggest the differences in enzyme protein content and careful conclusions can be drawn that the activities of PARG may be altered between compared groups of animals. It may confirm the presence of oxidative stress conditions and their consequences on metabolic pathways, the content of biologically active substances and processes of proper releasing placenta. Further experiments on PARG activity in bovine foetal membranes with respect to proper and improper placental release are necessary.     

Nitric oxide synthase expression in foetal placentas of cows with retained fetal membranes

The objectives of this study were to investigate relationship of retained fetal membranes (RFM) to expression of NOS and NOS mRNA and to analyze pathohistological changes and the distribution of nitric oxide synthase (NOS) in foetal placentas of cows with RFM. Twenty cows were assigned to two groups, a control group (no retained fetal membranes, NRFM, n = 10) and a diseased group (RFM, n = 10). The endpoint method was used to detect the nitric oxide (NO) content and nitric oxide synthase (NOS) activity in foetal placental tissue fluid and the fluorescent quantitation PCR was used to measure the expression of NOS mRNA. Immunohistochemistry and hematoxylin-eosin staining were used to observe pathohistological changes. Tissue from RFM cows showed fibronecrosis of the chorionic villi, and a decreased number of trophoblastic cells. The majority of trophoblastic cells displayed vacuolar degeneration. Interstitium vessels were distended and congested. Expression of induced nitric oxide synthase (iNOS) protein and iNOS mRNA was significantly higher (P < 0.05) in the cytoplasm of placental villus trophoblastic cells in the RFM group. But expression of endothelial nitric oxide synthase (eNOS) protein and eNOS mRNA was significantly lower (P<0.05) in the RFM group. The NO content and NOS activity of cows with RFM were significantly higher (P < 0.05). A high expression of iNOS protein and iNOS mRNA in the cow foetal placenta could produce high content of NO, which might inhibit uterine contraction. So over expression of iNOS protein and iNOS mRNA might be an important agent of retained fetal membranes in cows, and it may be a potential diagnosis biomarker.     

Lecithin cholesterol acyltransferase (LCAT) activity as a predictor for ketosis and parturient haemoglobinuria in Egyptian water buffaloes

Lecithin cholesterol acyltransferase (LCAT) activity was measured in 48 Egyptian water buffaloes four weeks pre-parturient. The activity was significantly low in 37 buffaloes (77.1%). Four weeks post-partum, clinical examination revealed that 23 buffaloes had the clinical signs of ketosis (K) while 14 had the clinical signs of parturient-haemoglobinuria (PHU). Serum samples were collected from 5 buffaloes of each group (K and PHU) besides 5 clinically healthy buffaloes with normal LCAT (control). Glucose level was significantly reduced in K and PHU groups while the phosphorous (P) level was significantly reduced in PHU group compared to control. There were significant reductions in the total cholesterol, free cholesterol, triglycerides, total protein and albumin in K and PHU groups; whereas, significant increases in AST, GGT, non-esterified fatty acids (NEFA) and beta-hydroxybutyric acid (BHBA) in K and PHU groups were detected. Therefore, LCAT could be a predictor for metabolic disorders in Egyptian water buffaloes.     

An association between the level of oxidative stress and the concentrations of NEFA and BHBA in the plasma of ketotic dairy cows

The aim of this study was to evaluate the oxidative status in ketotic cows. We observed changes in the oxidative status and correlations between the oxidative and metabolic status in non‐ketotic (n = 10), subclinical ketotic (n = 10) and ketotic cows (n = 10). Plasma samples were analysed by standard biochemical techniques and ELISA to determine traditional metabolic parameters: triglyceride (TG), phosphonium (P), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), immune globulin (Ig), total cholesterol (TC), high‐density lipoprotein (HDL), very low‐density lipoprotein (VLDL) and lactate dehydrogenase (LDH); energy metabolism indices: glucose, β‐hydroxybutyrate (BHBA) and non‐esterified fatty acids (NEFA); and indices of oxidative status: malondialdehyde (MDA), hydrogen peroxide (H₂O₂), vitamin C, vitamin E, superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), catalase (CAT), xanthine oxidase (XOD) and total antioxidant capacity (TAOC). The results of this study showed that plasma glucose levels were lower in ketotic and subclinical ketotic cows than in non‐ketotic cows; however, the plasma NEFA and BHBA concentrations were higher. In addition, significant decreases in TC, HDL and VLDL and significant increases in AST, ALT and LDH were observed in the plasma of the ketotic cows. The ketotic cows showed decreased plasma SOD, CAT, vitamin C and vitamin E, inhibited hydroxyl radical capacity and increased plasma H₂O₂ and MDA. There were positive correlations between the plasma NEFA and ALT, AST, LDH and MDA and negative correlations between the plasma NEFA and TC, HDL, VLDL, SOD, vitamin C, vitamin E, 1542280 uric acid and inhibited hydroxyl radical capacity. In addition, there were positive correlations between BHBA concentrations and ALT, AST and LDH and negative correlations between plasma BHBA concentrations and TC, HDL, VLDL, vitamin E and inhibited hydroxyl radical capacity. Overall, ketotic dairy cows experience oxidative stress, which is presumably associated with hyperketonemia and higher NEFA.     

Physiology and Treatment of Retained Fetal Membranes in Cattle

Retained fetal membranes (RFM) in cattle have adverse effects on fertility and production. Understanding the pathophysiology and causes of RFM is important for managing this disease. The hormonal processes that lead to normal placental separation are multifactorial and begin before parturition. A variety of risk factors, including early or induced parturition, dystocia, hormonal imbalances, and immunosuppression, can interrupt these normal processes and result in retention of the placenta. Current research does not support the efficacy of many commonly practiced treatments for RFM. Systemic administration of antibiotics can be beneficial for treating metritis after RFM, but antibiotic administration has not been shown to significantly improve future reproduction in cows with RFM. Collagenase injected into the umbilical arteries of retained placentas specifically targets the lack of placentome proteolysis and might enhance placental release. However, such therapy is costly and its benefits in terms of improving subsequent reproductive function have not been evaluated.     

15-ketodihydro-PGF2 alpha, progesterone and cortisol profiles in heifers after induction of parturition by injection of dexamethasone.

In order to study rapid changes in 15-ketodihydro-PGF2 alpha, cortisol and progesterone in the period preceding parturition in cattle, pre-term parturition was induced in 4 late pregnant heifers. Parturitions were induced by 2 intramuscular injections of 20 mg dexamethasone with a 24-h interval. The first injection was made on days 254, 258, 264 and 265 in gestation, respectively. Twenty-four h before the first injection an intravenous polyurethane cannula was inserted. Blood samples were collected at least every hour until 12 h after parturition and during the second stage of labour at least 6 times per hour. Plasma was analysed for 15-ketodihydro-PGF2 alpha and progesterone by radioimmunoassays, and for cortisol by an ELISA. The average time from injection to parturition was 7.7 (6.6-8.9) days (mean (range)). Two of the heifers had retained foetal membranes (RFM). At the start of the experiment the levels of PGF2 alpha metabolite were low (< 300 pmol/L) and increased slowly to levels between 1000 and 2000 pmol/L at one day before parturition. During the last day, however, the levels increased rapidly and the highest levels (> 10,000 pmol/L) were reached at the time of delivery. No pulsatile release was seen. Immediately after foetal expulsion the PG-metabolite levels decreased rapidly in all animals. In the 2 animals with RFM, however, this decline ceased within a few h. The PG-metabolite levels in these animals then started to increase and reached levels as high as during parturition. Luteolysis occurred between 1.6 and 0.4 days before parturition in all animals. The cortisol profile showed a distinct peak at the time of parturition in the RFM heifers. This peak was absent in the non-RFM heifers. This study shows that the PGF2 alpha release at prepartal luteolysis and parturition is not pulsatile in cattle and that cortisol profiles in heifers with retained foetal membranes might differ from the profiles in non-RFM heifers at the time of parturition.     

Effects of nonesterified fatty acids and beta-hydroxybutyrate on functions of mononuclear cells obtained from ewes

OBJECTIVE: To assess the effects of nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) on functions of mononuclear cells obtained from ewes. ANIMALS: 6 Sardinian ewes. PROCEDURE: Mononuclear cells were cultured with concentrations of NEFA (0, 15.6, 31.2, 62.5, 125, 250, 500, 1,000, or 2,000 micromol/L) and BHBA (0, 0.45, 0.9, 1.8, or 3.6 mmol/L). Concentrations of NEFA and BHBA were intended to mimic those of ketotic or healthy ewes, and NEFA and BHBA were tested alone and in combination. Synthesis of DNA was stimulated by use of concanavalin A (Con A) or pokeweed-mitogen (PWM). Secretion of IgM was stimulated by use of PWM. RESULTS: Synthesis of DNA stimulated by Con A and PWM was significantly inhibited by high concentrations of NEFA (> or = 250 micromol/L) or by a combination of high concentrations of NEFA (> or = 250 micromol/L) and all concentrations of BHBA (> or = 0.45 mmol/L). In contrast, DNA synthesis was not inhibited by low concentrations of NEFA (< or = 125 micromol/L) or by a combination of low concentrations of NEFA (< or = 125 micromol/L) and the lowest concentration of BHBA (0.45 mmol/L). Secretion of IgM was significantly inhibited by all concentrations of NEFA and by all combinations of NEFA and BHBA concentrations. When used alone, none of the concentrations of BHBA inhibited DNA synthesis or IgM secretion. CONCLUSIONS AND CLINICAL RELEVANCE: Reduced immunoresponsiveness during ketosis is likely to be associated with an increase in plasma concentration of NEFA and not with an increase in plasma concentration of BH BA.     

Plasma hormones, metabolites, milk production, and cholesterol levels in Murrah buffaloes fed with Asparagus racemosus in transition and postpartum period

Ten dry and pregnant Murrah buffaloes were selected to investigate the effect of Asparagus racemosus feeding on hormones, metabolites, milk yield, and plasma cholesterol levels. The treatment groups of buffaloes were fed with A. racemosus (shatavari) @?150?g/day/animal during prepartum and @?300?g/day/animal during the postpartum period. Blood samples collected on ?6, ?4, ?2-week, day of parturition (0), and +2, +4, and +6-week postpartum were analyzed for plasma total cholesterol, triglycerides, HDL, low-density lipoproteins (LDL), prolactin, cortisol, and blood metabolites. Milk samples collected at weekly intervals (+1, +3, +5, and 7?weeks) were analyzed for total milk fat cholesterol. Prepartum plasma cholesterol concentrations were significantly higher in treatment group over the control (P?<?0.05). Mean plasma triglycerides, LDL cholesterol, HDL cholesterol, glucose, and nonesterified fatty acid (NEFA) levels varied nonsignificantly between groups. Plasma prolactin and cortisol concentrations were significantly (P?<?0.01) more in treatment group than in control group. On day of parturition, plasma prolactin, cortisol, LDL, and plasma total cholesterol were higher (P?<?0.01) in treatment group buffaloes in comparison to control group. A. racemosus feeding significantly (P?<?0.01) increased plasma prolactin, cortisol (P?<?0.01), and milk fat cholesterol (P?<?0.05) without affecting total cholesterol, HDL, LDL, glucose, and NEFA concentrations. The buffaloes of treatment group produced more milk (?@?0.526?kg/animal/day) suggesting thereby that A. racemosus is galactopoietic. It was concluded that feeding of A. racemosus increases plasma prolactin and cortisol and decreased plasma total cholesterol and LDL concentration.     

15-Ketodihydro-PGF2α, Progesterone and Uterine Involution in Primiparous Cows with Induced Retained Placenta and Post-partal Endometritis Treated with Oxytetracycline and Flunixin

Retention of the foetal membranes (RFM) and post‐partal endometritis are common problems in dairy cows. Among other things, the disease is characterized by a bacterial endometritis with aerobic as well as anaerobic bacteria. From an endocrine perspective, cows with RFM have high levels of 15‐ketodihydro‐PGF_2α (PG‐metabolite) immediately after parturition but these levels fall rapidly within 2 weeks post‐partum (early PG‐metabolite elevation). After this decline, the PG‐metabolite levels increase again and the levels (at this time of a lower magnitude) remain elevated during the period of uterine infection (late PG‐metabolite elevation). The aim of this study was to investigate the PG‐metabolite profiles in cows with retained placenta and post‐partal endometritis treated with the prostaglandin synthesis inhibitor flunixin (F), either alone or in combination with oxytetracycline (T). The study was accomplished over 2 years with 12 primiparous cows in each experiment. As a model for RFM, preterm parturition was induced in late‐pregnant heifers by injecting PGF_2α (25 mg i.m) twice with a 24 h interval. In each experiment, the cows were divided into four groups and treated with either T (10 mg/kg b.w. i.m. once daily), F (2.2 mg/kg b.w. p.o. twice per day), a combination of T and F (dosage, as above), or conservatively (0). The treatment periods lasted from day 11 to day 14 post‐partum (pp) in experiment 1 (after placental shedding, groups T1, F1, TF1 and 0) and from day 3 to day 6 pp in experiment 2 (before placental shedding, groups T2, F2, TF2 and 0). Jugular vein blood samples were collected for analyses of PG‐metabolite and flunixin. Uterine biopsies were collected twice weekly for investigation of endometrial microbiology. Rectal palpation and ultrasonographic examinations were performed three times per week for investigations of uterine and cervical involution and ovarian activity. No attempts were made to remove the placentas manually. The experiment lasted until day 56 pp. The induction of parturition was successful in all heifers and 22 of 24 animals had RFM. All RFM cows had bacterial endometritis, based on bacteriological examinations. Flunixin treatment (F1, TF1, F2 and TF2) suppressed PG‐metabolite levels significantly (p=0.006) during the period of treatment in both experiments. However, the early flunixin treatment only suppressed PG synthesis partially. Late oxytetracycline treatment (T1) did not influence the PG‐metabolite levels but oxytetracycline treatment (T2 and TF2) before placental shedding significantly altered the kinetics of the early PG‐metabolite elevation compared with other treatments. Late PG‐metabolite elevation was significantly correlated to duration of uterine infection and cervical involution. In conclusion, flunixin treatment of cows with retained placenta either before or after placental shedding suppresses prostaglandin synthesis. However, early treatment, when the release of prostaglandins is high, might need more intensive treatment in order to prevent the PG synthesis effectively. Oxytetracycline treatment during the period immediately after parturition before placental shedding might influence the PG‐metabolite profile and suggests a bacteriological contribution to the high levels of PG‐metabolite seen during the first 2 weeks pp in cows with retained placenta. In this study, a correlation between prostaglandin release, the final cervical involution and the end of infection was found. This suggests a link between uterine endocrinology, bacteriology and involution in cows with retained placenta.     

In Vitro Effect of the Reproductive Hormones on the Oxidative Burst Activity of Polymorphonuclear Leucocytes from Cows: A Flow Cytometric Study

In this study, the effect of reproductive hormones and substances with hormonal activity on the oxidative burst activity of blood polymorphonuclear leucocytes (PMN) high yielding dairy cows was evaluated. Different concentrations of: progesterone, oestradiol 17β, FSH, LH, GnRH, cortisol and PGF2α were incubated in vitro for 4 h with PMN of seven high milk yielding cows, during the period of anoestrous postpartum. Controls were run in parallel in which each hormone was replaced by its solvent. After incubation with hormones the competence of PMN to generate H₂O₂ was monitored by flow cytometry. A down‐regulation on the oxidative burst activity of PMA‐stimulated PMN was observed when cells were incubated with progesterone. Significant (p ≤ 0.001) differences between control and progesterone incubated cells were observed from 6.56 μg/ml. The same predisposition was observed when PMNs were incubated with cortisol. Besides for all concentrations employed, a decrease in the burst activity was observed, only beyond 0.19 mg/ml, statistical differences between the results obtained by the control and the cortisol incubated cells were obtained. Concerning oestradiol 17β, an increase on H₂O₂‐production was observed when PMN were incubated with 15 pg/ml and 45 pg/ml of this steroid (p ≤ 0.05), followed by a depression of the cell’s activity when unphysiological concentrations were employed. Significant (p ≤ 0.05) differences between the obtained with the control and oestradiol 17β incubated cells were observed only in the highest concentration of oestradiol. No statistical differences were observed in the metabolic burst activity of PMN incubated with FSH, GnRH and LH when compared with the results obtained by the control.     

Vitamin E and Selenium Supplementation Reduces Plasma Cortisol and Oxidative Stress in Dystocia-Affected Buffaloes

The aim of the study was to evaluate oxidative stress around parturition in normally calved and dystocia-affected buffaloes and the clinical efficacy of vitamin E and selenium (Se) in reducing the effects in buffaloes suffering from dystocia. Plasma cortisol concentration, erythrocytic malondialdehyde (MDA) level and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were evaluated in dystocia-affected (supplemented (n = 8) and unsupplemented (n = 5) with vitamin E and Se) and normally calved (unsupplemented; n = 6) buffaloes. The decline in plasma cortisol concentration, MDA level and SOD activity was greater in the group of dystocia-affected buffaloes supplemented with vitamin E and Se (78.62 ± 2.7%, 13.38 ± 9.75% and 28.83 ± 7.72%, respectively) than in the unsupplemented group (54.33 ± 12.62%, 3.48 ± 4.16% and 17.69 ± 12.93%, respectively). These results suggests that supplementation with antioxidants like vitamin E and Se may be beneficial in reducing oxidative stress in dystocia-affected buffaloes in the immediate postpartum period.     

Plasma mineral and energy metabolite concentrations in dairy cows fed an anionic prepartum diet that did or did not have retained fetal membranes after parturition

OBJECTIVE: To compare plasma total calcium, phosphorus, magnesium, nonesterified fatty acids (NEFA), beta hydroxy butyrate (BHB), and glucose concentrations in parturient dairy cows that were fed an anionic prepartum diet between those with and without retained fetal membranes (RFM) at 24 hours after parturition. ANIMALS: 152 Holstein cows that calved during October through December of 1997 PROCEDURE: All cows were fed an anionic prepartum diet. Blood sample was taken within 6 hours after parturition from randomly selected cows. Thirty-nine cows had a diagnosis of RFM at 24 hours after parturition; 113 were not affected with RFM. At calving, body condition score (BCS; 1 [thin] to 5 [obese]), parity, and calving difficulty score were recorded. Plasma calcium, phosphorus, magnesium, NEFA, BHB, and glucose concentrations were compared between cows with or without RFM. RESULTS: Cows with RFM had significantly lower plasma calcium concentration soon after calving, compared with cows without RFM. Cows with a parity of > or = 3 had significantly lower plasma concentrations of calcium and higher concentrations of magnesium, compared with cows with a parity of 1 or 2. Cows with a BCS of > or = 3.25 at calving had significantly higher plasma concentrations of BHB than cows with a BCS of 2.75 to 3.0. Cows with dystocia had significantly higher plasma concentrations of glucose, compared with cows without dystocia. CONCLUSIONS AND CLINICAL RELEVANCE: In parturient cows fed a prepartum anionic diet, those with RFM have lower plasma calcium concentrations than cows without RFM, although this association does not prove a cause-effect relationship.     

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 β-galactosidase, α-l-fucosidase, β-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 β-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.     

Bacterial Isolates Associated With Dystocia And Retained Placenta In Iraqi Buffaloes

The present study was conducted on 50 recently calved Iraqi Buffalo cows. Depending on the kind of parturition, buffalo cows were divided into two main groups, the first group had normal unassisted parturition (NP) (26 animals) and the second group with certain periparturent complications (PPC) (24 animals). After 24 h of parturition, these two groups were further subdivided into two groups as cows expel their foetal membranes in <24 h postpartum and referred as non‐retained placenta (NRP) while cows that did not expel their foetal membrane after 24 h referred as retained placenta (RP). Sampling for bacteriology, uterine discharge for polymorphonuclear cells per cent and blood samples for polymorphonuclear neutrophil (PMN) and the enzyme creatine kinase activity were performed at 6, 24 and 48 h postpartum. In PPC group, the most prevalent bacteria after 6 h of calving were Escherichia coli, β‐haemolytic Streptococci and Lactobacillus acidophilus. Total bacterial isolates in the uterus of buffaloes with RP in PPC group after 24 and 48 h were 129 and 183 respectively. Among the isolates, Archanobacterium pyogenes, Fusobacterium necrophorum, Prevotella melaninogenicus and Staphylococcus aureus were the most prevalent isolates after 48 h of RP buffaloes in PPC group. Polymorphonuclear neutrophil were significantly (p < 0.01) increased in the uterine discharge than in blood in buffaloes with RP in both PPC and NP groups. In conclusion, uterine contamination occurs as a result of postpartum ascending contamination by non‐specific environmental organisms. The presence of Lactobacillus sp. in the uterus indicated a healthy uterus. Peripartum complications followed by retention of foetal membranes with the dominance of E. coli in the uterine lumen might favour the colonization of other bacteria including facultative anaerobic and strictly anaerobic in the uterine wall of buffaloes.     

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.     

Abnormal Expression of TIMP-2, SOD, Vimentin and PAI Proteins in Cloned Bovine Placentae

Cloned mammals suffer from high rates of placental abnormality and foetal loss during pregnancy. We previously used 2-D gel electrophoresis and mass spectrometry for global proteomic analysis of cloned and normal bovine placentae to identify differential protein expression patterns. Here, we used Western blot analysis to confirm the expression levels of several pregnancy-related proteins putatively identified as being differentially expressed in somatic cell nuclear transfer (SCNT) vs normal bovine placentae. The expression levels of tissue inhibitor of metalloproteinase-2 (TIMP-2), its downstream protein, matrix metalloproteinase-2 (MMP-2), superoxide dismutase (SOD), vimentin and plasminogen activator inhibitor-1 (PAI) were analysed in the placentae of SCNT cloned Korean native cattle that died immediately after birth and in normal placentae obtained by AI. Our results revealed that TIMP-2 and SOD were up-regulated in SCNT placenta compared with normal placenta, whereas MMP-2 levels were comparable in cloned and normal placentae, and vimentin and PAI were significantly down-regulated in SCNT compared with normal placentae. Our results suggest that key proteins of placental development are abnormally expressed in SCNT cloned bovine placentae, probably resulting in abnormal placental function and clonal mortality.     

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.     

Effect of cow-specific factors and feeding frequency of concentrate on diurnal variations of blood metabolites in dairy cows

OBJECTIVE: To determine the effect of feeding frequency and cow-specific factors on diurnal variations of blood metabolites concentrations. ANIMALS: 40 dairy cows from 2 herds. PROCEDURE: Each herd was fed concentrate at a specific feeding frequency (automatic vs twice daily). Blood samples were collected 4 times daily. Plasma concentrations of glucose, nonesterified fatty acids (NEFA), and beta-hydroxybutyrate (BHBA) were analyzed, and concentrations of urea, cholesterol, and total protein (TP) were determined in serum samples. A multivariate repeated-measures ANOVA was used to test the effect of time, feeding frequency, production group, parity, days after parturition, and daily milk yield on diurnal variations of metabolites. RESULTS: Concentrations of glucose, BHBA, and urea remained relatively constant in the herd that was fed by use of automatic distribution of concentrate; however, significant diurnal patterns were detected in the herd fed only twice daily. Only slight differences in glucose and urea concentrations were detected between high- and low-producing cows, but concentration of BHBA was significantly influenced by number of days in lactation. In contrast, diurnal variations in NEFA and cholesterol concentrations were similar between the 2 herds but differed with regard to production group. Concentrations of TP did not have relevant diurnal patterns and did not differ between production groups. CONCLUSIONS AND CLINICAL RELEVANCE: Diurnal variations and feeding frequency affect glucose, BHBA, and urea concentrations in cows. In contrast, concentrations of NEFA, cholesterol, and TP appear to be less sensitive to time of sample collection. Feeding frequency, composition of feed, or both, have a major impact on blood metabolites concentrations.     

Macrophages in bovine term placenta: An ultrastructural and molecular study

Retention of foetal membranes (RFM) is a major reproductive disorder in dairy cows. An appropriate immune response is important for a physiological expulsion of the foetal membranes at parturition. Our study aims to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. We used transmission electron microscopy (TEM), immunohistochemistry and semi-quantitative RT-PCR to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. Semi-quantitative RT-PCR was used to define macrophage polarization in foetal and maternal compartments of normal term placenta. Gene expression of factors involved in M1 polarization [interferon regulatory factor-5 (IRF5), interleukin (IL)-12A, IL12B] and in M2 polarization (IL10) were studied. Ultrastructurally, foetal macrophages showed an irregular shape and large vacuoles, whereas the maternal macrophages were spindle shaped. By immunohistochemistry, macrophages were identified by a strong staining with the lysosomal marker Lysosome-associated membrane glycoprotein 1 (LAMP-1), while myofibroblast in the maternal stroma was positive for alpha-smooth muscle actin. We used the LAMP-1 marker to compare the density of foetal stromal macrophages in placentas of cows with RFM and in controls, but no statistically significant difference was observed. RT-PCR showed a higher expression of all studied genes in the maternal compartment of the placenta and generally a higher expression of M1-, compared to M2-associated genes. Our results indicated that at parturition placental macrophages predominantly show the pro-inflammatory M1 polarization. The higher expression of all the target genes in the maternal compartment may denote that maternal macrophages in bovine term placenta are more frequent than foetal macrophages.