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

Contents Poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks using NAD⁺ as a substrate. It leads to consequences for metabolism not only on a cellular level, but also on a tissue level, among others: NAD⁺ and ATP depletion. Retention of foetal membranes (RF) in cows is supposed to be connected with the imbalance between production and neutralization of reactive oxygen species, leading to oxidative damage to DNA, lipids and proteins. The aim of this preliminary study was to detect the presence of PARP in bovine placenta and to describe the enzyme with respect to type of placental tissue, time and mode of delivery. Placentomes, collected after spontaneous delivery or caesarian section, were divided into maternal and foetal parts of placenta, homogenized, and subjected to electrophoresis. Cows were divided into six groups as follows: (A) caesarian section before term with RF, (B) caesarian section before term without RF, (C) spontaneous delivery at term with RF, (D) spontaneous delivery at term without RF, (E) caesarian section at term with RF, (F) caesarian section at term without RF. PARP was detected by Western blotting using commercially available bovine anti PARP antibody. Bands referred to as bovine PARP standard were present in all examined tissues as well as the products of its cleavage. However, the patterns of bands were different with respect to type of tissue, time, and mode of delivery. Further experiments on detailed relationship between PARP activity and the process of releasing and retaining of bovine placenta are necessary.     

Non-enzymatic Antioxidative Defence Mechanisms Against Reactive Oxygen Species in Bovine-retained and not-retained Placenta: Vitamin C and Glutathione

Vitamin C and glutathione (GSH) are water-soluble antioxidants which take part in defence mechanisms against reactive oxygen species (ROS). They may also be involved in processes of releasing/retaining bovine fetal membranes. Hence vitamin C, reduced (GSH) and oxidised (GSSG) glutathione levels were determined in retained and not-retained bovine fetal membranes in order to describe the non-enzymatic antioxidative status. Placental samples were collected immediately after spontaneous delivery or during caesarean section before term and at term, and 6 groups were formed as follows: (A) pre-term caesarean section without retained placenta; (B) pre-term caesarean section with retained placenta; (C) term caesarean section without retained placenta; (D) term caesarean section with retained placenta; (E) spontaneous delivery without retained placenta and (F) spontaneous delivery with retained placenta. Homogenates of maternal and fetal placental tissues were prepared, and vitamin C, GSH and GSSG were measured spectrophotometrically. Vitamin C levels were significantly higher in the maternal part than in the fetal part of the placenta in all groups examined. In retained placenta cases the levels were significantly lower than in control cows, except in pre-term groups. GSH concentrations were significantly higher in placentas without retention than with retention. GSSG levels showed the opposite relationship and were significantly higher in samples with retention of fetal membranes than in controls. Further experiments on antioxidative as well as oxidative status in bovine placenta are necessary.     

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.     

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.     

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.     

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.     

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

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

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.     

Bovine foetal sex determination—Different DNA extraction and amplification approaches for efficient livestock production

Foetal sex determination using polymerase chain reaction (PCR) in mammals is based on the amplification of gender‐specific foetal DNA sequences circulating in maternal blood. The bovine synepitheliochorial placenta does not allow a direct contact between the trophoblast and the maternal blood, resulting in difficult passage of foetal DNA and, consequently, its very small amounts in maternal bloodstream. Circulating cell‐free foetal DNA (ccffDNA) encompasses short nucleotide fragments (300–600 bp) in maternal circulation. The aim of this study was to assess this non‐invasive method in accurate prenatal sexing in early and late gestational periods in comparison with ultrasound diagnostics. As various DNA isolation and amplification methods were tested, their success in obtaining reliable results was evaluated. Two groups were tested, each consisting of 20 pregnant cows. Blood of a bull and a non‐pregnant heifer was the controls. Extraction of foetal DNA was accomplished by three different methods: using tubes with silicone membranes, a single‐tube extraction without silicone membranes and phenol–chloroform extraction. Following each extraction method, foetal DNA was amplified using PCR and real‐time PCR with both bAML and TSPY primers in a separate reaction. Positive results were obtained only after amplification of foetal DNA extracted with a single‐tube extraction kit. In comparison with ultrasound examination results and foetal gender recorded at birth, the sensitivity of the PCR test was 90% in Group I, but the technique failed to detect male foetuses in Group II. The real‐time PCR test sensitivity in Group I was 90% and in Group II 91.6%.     

Term placenta shows methylation independent down regulation of Cyp19 gene in animals with retained fetal membranes

Retention of fetal membranes (RFM) is the major post-partum disorder in dairy cattle. Cyp19 gene encodes the aromatase enzyme responsible for catalyzing the rate limiting step in estrogen biosynthesis, an important hormone for placental maturation and expulsion. The present study was aimed for comparative analysis of Cyp19 gene expression and its epigenetic regulation in placental cotyledons of animals with and without RFM. Significantly lower expression of Cyp19 gene was found in placental samples of RFM affected animals in comparison to normal animals. Methylation analysis of 5 CpG dinucleotides of placenta specific Cyp19 gene promoter I.1 and proximal promoter, PII showed hypo-methylation of both PI.1 and PII in term placenta of normal and diseased animals. In conclusion, a mechanism other than promoter methylation is responsible for decreased aromatase expression in placental cotyledons of animals suffering from RFM.     

Retained fetal membranes in the mare: A retrospective study

A retrospective study of 3456 deliveries was conducted from the records of four Standardbred broodmare farms where mares were bred by artificial insemination and maintained under close veterinary supervision. Retained fetal membranes (RFM) were observed in 10.6% of the deliveries. Retained fetal membranes occurred more frequently (p < 0.05) after dystocia and in mares which had RFM the previous year. Retained fetal membranes after normal foaling had no significant effect on the reproductive performance (pregnancy rate, pregnancy loss rate, or foaling rate), nor on the general health of the mares, regardless of the duration of RFM (3 to 144 hours). Postfoaling laminitis was not observed. Oxytocin therapy of mares with RFM starting at two hours postpartum significantly reduced the incidence of RFM ≥ 8 hours. Mares with RFM which had received intrauterine antimicrobials between foaling and first breeding had a foaling rate similar to mares with RFM which had not received intrauterine therapy.     

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

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

The Intrauterine Treatment of the Retained Foetal Membrane in Dairy Goats by Ozone: Novel Alternative to Antibiotic Therapy

One of the major post‐parturient complications in dairy goats is the retention of foetal membrane (RFM), which negatively influences their health, reproductive efficacy and welfare. The aim of this study was to compare the efficiency of intrauterine either ozone (OZ) or antibiotic (AB) treatments to establish the use of OZ as a novel and potential alternative to AB therapy in does with the RFM. The study was performed on 7 herds of dairy goats (n = 563) kept in the farms in Croatia. The conception rate was 563 of 641 total matings or 87.83%. The does from selected farms were observed during early puerperium and were divided into animals without the RFM (n = 522) and with the RFM (n = 41), treated either with foam spray OZ (n = 21) or with foaming AB oxytetracycline tablets (n = 20). The does with the RFM were mated successfully and became pregnant next kidding season, regardless of the treatment applied. Treatment with OZ attained similar results to the standard AB therapy, indicating that it could be novel potential alternative therapy of the RFM in dairy goats.     

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.     

Analysis and Validation of the Differentially Expressed ENO1 in the Maternal Placenta of Cow with Retained Foetal Membrane

The paper aimed to analyze and validate the function of differentially expressed ENO1 in the cow with retained foetal membrane.In our research,we chose three healthy Holstein dairy cows and three Holstein dairy cows with retained foetal membrane of similar age,foetal times,weight and milk yield and divided them into two groups.The total protein of maternal placenta was extracted in the control and retained foetal membrane of cow.The differential expression of proteins were found out by the 2-DIGE,and the differential expression of ENO1 was validated by the Western blotting and Real-time quantitative PCR.The results showed that ENO1 was significant expression and large multiple in the two groups,and it was significant increased expression in the retained foetal membrane of cow after Western blotting and Real-time quantitative PCR (P=0.015<0.05;P=0.001<0.01).The ENO1 participated in the energy homeostasis,immune and fibrinolysis process which related to the retained foetal membrane of cow.It suggested that ENO1 was likely connected with the retained foetal membrane.     

胎衣不下奶牛母体胎盘α-烯醇化酶异常表达分析及验证

试验通过对胎衣不下奶牛母体胎盘组织中差异α-烯醇化酶(enolase,ENO1)的分析验证,探讨ENO1在胎衣不下中的作用。本研究选取了年龄、胎次、体重和泌乳量均相近的产后胎衣不下和产后胎衣正常排出奶牛各3头,分为两组,提取了胎衣不下组与胎衣正常排出组母体胎盘组织中的总蛋白,采用双向凝胶电泳的方法筛选出差异蛋白,并利用Western blotting及实时荧光定量PCR的方法对其中差异表达量大的ENO1进行验证。结果发现,胎衣不下组和胎衣正常组母体胎盘组织中ENO1差异表达量大且倍数较大,Western blotting及实时荧光定量PCR验证发现ENO1在胎衣不下奶牛母体胎盘中的表达量升高,t检验结果分别为P=0.015<0.05,差异显著;P=0.001<0.01,差异极显著。该基因参与机体的能量调节、免疫和纤溶等过程,而这些过程与奶牛胎衣不下密切相关,提示ENO1可能参与该病的发生发展。     

Is poly(ADP-ribose) polymerase involved in bovine placental retention?

Poly(ADP-ribose) polymerase (PARP) is the enzyme which utilises NAD to synthesise poly(ADP-ribose) polymers. This process appears in response to DNA lesions. Oxidative stress, which might be involved in bovine placental retention, is the reason for oxidative DNA injury. In this mini-review, the relationship between PARP activity and bovine placental retention is discussed. The results of our experiments on PARP activity in placental tissues showed that the enzyme of 113 kDa and its cleavage products were present in retained as well as released fetal membranes. Western blotting technique showed different intensities in the staining of bands which might suggest different activities of the enzyme.     

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.     

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.