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.     

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.     

Videoendoscopic evaluation of the mare's uterus: III. Findings in the pregnant mare.

During a 5-year period 65 Pony and 20 Thoroughbred pregnant mares were subjected to videoendoscopic hysteroscopy from 10 to 266 days of gestation. The aims of these examinations were to 1) observe foetal and placental development in vivo (60 Pony and 10 Thoroughbred mares); 2) eliminate one of unicornuate twin conceptuses (9 Thoroughbred mares); 3) recover embryonic foetal and placental tissues non-surgically for experimental purposes (47 Pony mares); 4) induce focal separation of the placenta in late gestation as an experimental model of placentitis (5 pony mares and 1 Thoroughbred mare). It was possible to view the embryo and all constituent extra-embryonic membranes of the conceptus between Days 10 and 87 of gestation without having to perforate the allantochorion. This allowed study of physiological processes such as the coordinated uterine contractions responsible for conceptus motility between Days 7 and 17, active foetal movements, which began as early as Day 34, and invasion of chorionic girdle cells into the endometrium, which occurred between Days 34 and 38. From Day 90, vision of the foetus was reduced or prevented by the increased thickness of the allantochorion. Transendoscopic recovery of the conceptus was successful in all of 10 mares under 30 days of gestation, whereas only 10 of 18 attempts between Days 30 and 45 produced the conceptus without resorting to uterine lavage after initial rupture of the allantochorion. All 9 attempts to eliminate one of unilateral twin conceptuses were unsuccessful and the technique was abandoned as clinically unsuitable. Nevertheless, 9 of 22 (41%) single conceptuses remained viable after one or more hysteroscopic examinations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retained Fetal Membranes in Heavy Draft Mares Associated with Histological Abnormalities

Retention of fetal membranes is one of the most common life-threatening peripartum problems; however, its etiology is still unknown. Draft mares are more likely to retain fetal membranes than other breeds of mares. Because a strong connection between the fetal membranes and the endometrium has been observed in cases of retention, we studied the changes in the endometrium and the allantochorion of heavy draft mares with this condition. Tissue samples were taken from 90 mares that retained the fetal membranes and from 21 that did not. Retained fetal membranes were associated with adhesion of the allantochorion to the endometrium in 88% of the mares; this adhesion depended on the following factors: occurrence of fibrosis both in the lamina propria of the allantochorial villi and in the allantochorial stromal connective tissue, overdevelopment of the allantochorial stromal connective tissue, oversized allantochorial epithelial cells, and sparser and less-branched allantochorial villi.     

奶牛血浆钙离子浓度及胎儿胎盘eNOS表达的研究

本试验采用终点法检测血浆中Ca2+浓度变化,分别采用免疫组织化学SP法及荧光定量PCR法检测奶牛胎儿胎盘eNOS蛋白及其mRNA的表达。通过对血浆中Ca2+浓度的检测及胎儿胎盘内皮型一氧化氮合酶(eNOS)表达量的研究,探讨奶牛胎衣不下的发生与这些物质变化的关系。结果表明,胎衣不下组奶牛血浆中Ca2+浓度在分娩前后均低于正常组;两组eNOS蛋白均在胎盘绒毛滋养层细胞和内皮细胞胞浆内表达,蛋白的表达量差异显著(P0.05);两组胎儿胎盘eNOS mRNA表达量差异显著(P0.05)。本研究证明分娩前后血浆中Ca2+水平对奶牛胎衣不下的发生有重要的调节作用,低浓度的Ca2+容易导致子宫收缩无力,同时影响eNOS在胎儿胎盘绒毛中的表达。     

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.     

Equine placenta – A clinician's perspective. Part 2: Abnormalities

Any insufficiency of the equine placenta has dramatic effects on the developing equine fetus. Placental abnormalities, such as the separation of the chorioallantois from the maternal endometrium or torsion of the umbilical cord, lead to fetal demise, premature labour or abortion. These conditions are each associated with characteristic lesions on the equine placenta, which can be found during a detailed examination. These findings can be very helpful for diagnosing problems and implementing appropriate treatments for mares and affected newborn foals. Furthermore, the retention of the entire placenta or any small fragment thereof can cause metritis, laminitis and sepsis. The prompt diagnosis and aggressive treatment of this condition is necessary to save the mare from becoming seriously ill. Therefore, a thorough evaluation of the equine placenta is a crucial element of the post partum evaluation of every brood mare.     

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.     

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.     

Proliferation and Apoptosis in Fetal Membranes and Endometrium During Placental Retention in Heavy Draft Mares

Placental retention (retained placenta [RP]) is a serious and common peripartum disease in mares, but the etiology and pathogenesis of RP still remain unclear. The alteration of cell proliferation and apoptosis is considered to be an important factor in RP. Fetal membranes and endometrial biopsies were collected from mares with RP (n = 8) and from control mares (n = 10). The proliferation and apoptosis levels in the chorionic and the endometrial epithelia were assessed by proliferating cell nuclear antigen immunostaining and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, respectively. The study revealed that there was an insignificant decrease in proliferation and a significant increase in apoptosis in the chorionic epithelium from mares with RP. This result excludes a proliferation imbalance from the possible causes of RP. In the area of the nonpregnant horn of the placenta, proliferation was negatively correlated and apoptosis was positively correlated with the degree of fetomaternal anchorage. It was observed that, in all mares with placental retention, the endometrial epithelium (both luminal and glandular) showed decreased proliferation and increased apoptosis, which may indicate a delay in postpartum uterine regeneration.     

The Effect of Manual Removal of Placenta Immediately after Foaling on Subsequent Fertility Parameters in the Mare

Retained placenta is considered to be a common problem in postpartum mares. The incidence varies from 6% to 54% depending on the breed, with higher incidence in heavy draught mares than in light-weight mares. Retained placenta has been linked to lower postpartum oxytocin concentration, impaired uterine involution, and dystocia. The objective of this study was to assess the effect of early manual removal of placenta immediately postpartum on subsequent fertility parameters (development of free intrauterine fluid, inflammatory status of endometrium, and on pregnancy rates) and to compare them with mares with spontaneous expulsion of placenta. A total of 29 mares, mainly Irish draught, were closely monitored during foaling by closed circuit television and allocated to two groups: (1) mares that expelled the placenta spontaneously within 3 hours of foaling; and (2) mares that were cleansed manually immediately after foal delivery. All mares were examined and scanned 5 and 9 days postpartum, and free intrauterine fluid was recorded; endometrial swabs were taken 9 days postpartum for endometrial cytology and culture. None of the fertility parameters analyzed showed statistical difference between groups 1 and 2. Therefore, it can be concluded that early manual removal of placenta has no detrimental effects on subsequent fertility of mares and, therefore, can be recommended when a veterinarian attends a foaling.     

The significance of steroid hormones and prostaglandins for the expulsion of the afterbirth in cattle

This review describes the importance of different hormones (oestrogens, progesterone, prostaglandins and glucocorticoids) for the loosening process of the foetal membranes in cattle. Hormone concentrations, measured ante partum and at the time of parturition are discussed. In the literature conflicting results can be found. Foetal membrane retention seems to coincide with increased plasma progesterone and decreased plasma oestrogen and PGF2 alpha concentrations. Possible effects of particular hormones on loosening of the placenta are discussed. It can be concluded that these hormones do have a considerable influence on the foetal membranes. However, to clarify their definite role for retention of the placenta further investigations are needed.     

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.     

Early foetal death in the mare. Histological, bacteriological and cytological findings in the endometrium.

Fifteen mares which had each lost their pregnancies 2 to 8 times during the preceding 6 years were used in a study comprising 1 or 2 breeding seasons for each mare. During the research period all 15 mares conceived and 10 of the mares had normal pregnancies in the 1st experimental year. Five mares resorbed/aborted once or twice during the 1st year. This was followed by a pregnancy that terminated in a live foal. Histopathological examinations of uterine biopsies showed a wide range of histopathological conditions, from absence of changes excessive for the mare's age and parity to the most severe changes of the endometrium. Most likely, endometritis, acute as well as chronic degenerative endometritis, is the most common cause of early foetal death in the mare. Good breeding management is essential and was often enough to make the mares produce a foal. Additional treatment was needed in some of the mares and was based on the course of the conceptus loss and the condition of the endometrium, as determined by clinical, histological, bacteriological and cytological examinations.     

Observations and procedures at foaling on a thoroughbred stud

The behaviour and management of Thoroughbred mares prior to, and at foaling time, is detailed. There is a discussion of the losses that occur at birth, traumatic injuries to the mares, and the subsequent treatment of these. The postnatal behavioural patterns of the mare emphasise that she must be isolated at foaling. The normal feeding of the foal and the consequences of this not occurring, the lack of normal bowel function, and the retention of the foetal membranes by the mare are postnatal conditions likely to cause most losses.     

Enzymatic activity in the placenta in cows in relation to the course of parturition

The activity of aminopeptidases and cathepsins was determined in placentoma homogenates; placentomas of cows were extirpated immediately after parturition and in four and eight hours. In cows with afterbirth retention (a. r.) following induced parturition, the activity of these enzymes was always higher than in cows without a. r., no matter if after induced and spontaneous parturitions; it was at a similar or slightly higher level than in the eighth month of pregnancy. The content of total proteins in placentomas of cows with a. r. was also higher. These findings point to the insufficient ripening, or aging of placental tissue, which is related to a release of lysosomal enzymes. The enzymes are active through hydrolysis in the separation of foetal placenta from the maternal one and in the modification of proteins participating in intercellular linkages.     

Could treatment of pregnant mares prevent abortions due to equine piroplasmosis?

Treatment of pregnant mares to prevent abortions due to equine piroplasmosis is a novel idea practised empirically at some Thoroughbred studs in South Africa. This paper presents the results of an investigation to ascertain whether imidocarb dipropionate crosses the equine placenta. Three pregnant mares were injected intramuscularly with imidocarb and their foetuses were mechanically aborted at varying time intervals thereafter. Imidocarb was found in foetal blood at a level similar to that in the dam's blood, suggesting that imidocarb administered to the dam would be available for anti-parasitic activity in the foetal circulation. Uncertainty concerning the time of treatment to achieve the desired effect currently makes this a questionable exercise.     

Qualitative analysis and functional classification of the uterine proteome of mares in oestrus and dioestrus

Quantitative analysis of the uterine flush fluid proteome of mares in oestrus and dioestrus has been previously reported. The objectives of this study were to: a) evaluate qualitative differences in the uterine flush fluid proteome between mares in oestrus and mares in dioestrus and b) perform a functional classification of proteins either unique to each stage or common between the two stages. Uterine flush fluid samples were collected from 8 light breed mares in either oestrus (n = 5) or dioestrus (n = 3). Proteomic analysis of the samples was conducted using liquid chromatography–tandem mass spectrometry. Proteins exclusively detected in oestrus or dioestrus and those common to both stages were identified using the Scaffold software (version 4.4.8, Proteome Software Inc., Portland, OR). The identified proteins were classified into gene ontology (GO) categories (cellular component [CC], molecular function [MF] and biological process [BP]) using the PANTHER ( www.pantherdb.org ) classification system version 14.0. Of 172 proteins identified, 51 and 28 were exclusively detected in mares in oestrus and dioestrus, respectively, and 93 proteins were common to both stages. The most represented terms in various GO categories were similar among the three subsets of proteins. The most represented CC terms were extracellular region and cell, the most represented MF terms were catalytic activity and binding, and the most represented BP terms were metabolic process and cellular process. In conclusion, proteomic analysis of the uterine flush fluid enabled the identification of subsets of proteins unique to oestrus or dioestrus, or common to both stages. The results of this study can serve as a baseline for future research focused on finding stage-specific protein markers or evaluating differences in the uterine flush fluid proteome between normal mares and those with uterine disease.     

Serial investigations of early pregnancy in pony mares using real time ultrasound scanning

The uteri of 13 pony mares were examined daily by ultrasound during the first two months of gestation. The conceptus was first identified between 12 and 16 days after ovulation and the embryo was seen on the ventral surface of the conceptus after Day 21. The foetal heart could be visualised after Day 22 and spontaneous movement of the foetus occurred after Day 39. No consistent pattern was seen in the development of the foetal membranes, although attachment of the umbilical cord to the allantochorion was always on the dorsal aspect of the conceptus. Daily measurements were made of the diameter of the conceptus and the length of the embryo and foetus.     

Abortion of virologically negative foetuses following experimental challenge of pregnant pony mares with equid herpesvirus 1.

From 1988 to 1991, 51 pregnant pony mares were challenged intranasally or by aerosol with an isolate of EHV-1 (AB4) originally recovered from a quadriplegic mare. This resulted in 32 abortions, occurring from 9 to 29 days after infection. In 14 of the early abortions (Days 9-14), EHV-1 was not demonstrated in the foetal tissues by virus isolation or immunostaining despite no other non-viral cause for the abortion being evident. Application of the polymerase chain reaction to foetal tissues from 9 of these cases also proved negative. One of the 14 mares was destroyed immediately after abortion, and post-mortem examination revealed severe and widespread vasculitis, thrombosis and secondary ischaemic damage in the endometrium with replication of EHV-1 in endothelial cells. These findings suggest that EHV-1 abortion can occur due to endometrial damage without the establishment of a foetal infection.