奶牛血浆钙离子浓度及胎儿胎盘eNOS表达的研究

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。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。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)。说明分娩前后血浆中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。     

浅谈奶牛胎衣不下症

胎衣不下为奶牛常见的一种疾病,是指成年母牛分娩后经过一定时间胎衣仍然滞留于子宫内.正常情况下,奶牛分娩后8～12 h仍不排出胎衣,就属于胎衣不下.奶牛胎衣不下的发生率因季节、营养状况、胎次及分娩正常与否而有所不同,其也与牛的胎盘组织构造有关.牛的胎盘属于上皮绒毛膜与结缔组织绒毛膜混合型构造,胎儿胎盘与母体胎盘联系比较紧...     

胎衣不下奶牛胎儿胎盘中IL-2含量的测定

通过对胎盘组织中IL-2的含量检测,探讨奶牛发生胎衣不下的免疫学机制。将20头奶牛分为健康对照组和胎衣不下组,采集健康奶牛和胎衣不下奶牛的胎儿胎盘,制备胎盘组织匀浆液,用酶联免疫吸附试验(ELISA)检测其中的IL-2的含量。结果表明,胎衣不下组奶牛胎盘组织中IL-2的含量极显著高于健康组(P0.01),说明胎盘组织中IL-2含量与奶牛发生胎衣不下有密切关系。     

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。     

胎衣不下奶牛血浆中矿物质及维生素含量变化

选取胎衣不下组和胎衣正常排出组奶牛各6头,通过终点法对两组奶牛血浆中矿物质指标Ca、P、Fe、Zn的含量检测,同时采用高效液相色谱法测量两组奶牛血浆中维生素指标VA、VD、VE的含量。结果表明,胎衣不下组奶牛血浆中矿物质Ca、Zn、Fe的含量较胎衣排出组正常奶牛低,而血浆中P的含量较正常奶牛的高;胎衣不下组奶牛血浆中维生素VD和VE的含量低,而血浆VA的含量与胎衣正常排出组奶牛无差异性。     

胎衣不下奶牛血清和胎盘中激素含量变化的研究

本试验在产犊后立即无菌采集32头奶牛血液,其中17头为胎衣正常排出奶牛,15头为胎衣不下奶牛。胎衣正常排出奶牛在胎衣排出后立即采集胎盘,胎衣不下奶牛在确诊后再次静脉采血并采集其胎盘;用酶联免疫吸附测定法对胎衣不下奶牛和胎衣正常排出奶牛血清和胎盘中的激素含量进行检测,并对检测结果进行分析。结果表明,胎衣不下奶牛血清和胎盘中孕酮、纤维蛋白溶酶原和前列腺素(PGF_(2α))的含量与胎衣正常排出组相比差异显著降低(P0.05),而雌二醇含量则显著升高(P0.05)。本试验通过对胎衣不下和胎衣正常排出奶牛血清和胎盘中激素的含量进行检测并比较,结果发现奶牛胎衣不下的发生与血清和胎盘组织中的孕酮、雌二醇、纤维蛋白溶酶原和前列腺素(PGF_(2α))的含量变化有密切相关的联系,表明奶牛胎衣不下的发生可能与机体内激素分泌紊乱具有很大的关联。     

胎衣不下奶牛分娩前后血浆PGE2水平变化

采用放射性免疫测定法,探讨了胎衣不下奶牛分娩前后血浆中前列腺素PGE2水平的变化.结果发现正常分娩的奶牛PGE2的量从分娩前4d开始上升直到分娩前2d达最高,分娩前1d又降到最低,分娩时与分娩后略有回升.而胎衣不下奶牛PGE2的量从分娩前4d开始下降,到分娩前2d降到最低,分娩前1d突然升达最高值,分娩时和分娩后与正常分娩的奶牛PGE2的量持平.这揭示正常分娩和胎衣不下奶牛的PGE2量和时相差异可引起免疫失调,造成奶牛胎衣不下.     

胎衣不下奶牛分娩前后血浆PGE2水平变化

采用放射性免疫测定法，探讨了胎衣不下奶牛分娩前后血浆中前列腺素PGE2水平的变化。结果发现正常分娩的奶牛PGE2的量从分娩前4d开始上升直到分娩前2d达最高，分娩前1d又降到最低，分娩时与分娩后略有回升，而胎衣不下奶牛PGE2的量分分娩前4d开始下降，到分娩前2d降到最低，分娩前1d突然升达最高值，分娩时和分娩后与正常分娩的奶牛PGE2的量持平，这揭示正常分娩和胎衣不下奶牛的PGE2量和时相差异可引起免疫失调，造成奶牛胎衣不下。     

奶牛胎衣不下的防治措施

奶牛胎衣不下是分娩第三期(胎膜排出期)胎衣不能在8—12小时内自然排出,滞留在子宫内的一种常见奶牛产后期疾病。其特征主要是胎儿胎盘不能从母体胎盘腺窝中分离开来。子宫肌收缩无力或弛缓,仅见一少部分胎膜悬垂于阴门之外。胎衣不下的奶牛外周血浆孕酮含量增高,17β-雌二醇减少,催产素降低,使分娩第二期(胎儿排出期)与第三期的内分     

奶牛胎衣不下的诊断及防治

<正>胎衣不下是指怀孕母畜正常分娩后,胎衣在正常时限内没有完全排出子宫而引起的产科疾病,也叫胎衣滞留。奶牛胎衣正常排出时间为8h左右,在18h后仍然没有排出视为胎衣不下。胎衣为胎膜的俗称,也就是胎盘。奶牛胎盘属于上皮绒毛膜与结缔组织绒毛膜混合型,胎儿胎盘与母体胎盘联系比较紧密,这是胎衣不下发生较多的原因。胎衣不下     

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.     

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.     

Peripartal changes in serum alkaline phosphatase activity and lactate dehydrogenase activity in dairy cows.

Peripartal serum alkaline phosphatase activity and lactate dehydrogenase activity were measured in 30 dairy cows in order to examine the association between retained fetal membranes and enzyme activity. Daily blood samples were obtained from pregnant cows, starting 15 days before the expected day of calving until eight days after parturition. Sera from 15 cows which retained fetal membranes longer than 24 hours and 15 cows which shed fetal membranes within six hours after parturition were analyzed for alkaline phosphatase and lactate dehydrogenase enzyme activities. Mean alkaline phosphatase enzyme activities ranged from 15.93 to 32.6 U/L in retained and nonretained placenta cows. There was a trend towards higher serum alkaline phosphatase activities in retained placenta cows but the differences were not significant among the groups (P greater than 0.05). Mean lactate dehydrogenase activities ranged from 307.2 to 438.86 U/L in nonretained and retained placenta cows. Lactate dehydrogenase enzyme activities in nonretained and retained placenta cows were similar (P greater than 0.05). The alkaline phosphatase and lactate dehydrogenase enzyme activities peaked at the time of parturition in both groups. However, the differences in alkaline phosphatase and lactate dehydrogenase activities on different days within non-retained and retained placenta cows were significant (P less than 0.05). Results indicate that prepartal changes in alkaline phosphatase and lactate dehydrogenase enzyme activities are not predictive of placental retention postpartum.     

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.     

Histologic and histochemical study of placentomes in cows after induced labor

The placentomes were extirpated from 16 cows after parturition induced with 750 micrograms cloprostenol or 20 mg dexamethasone on the 277th day of gravidity, on an average, from 9 cows after spontaneous parturition, and from 7 cows after hysterectomy in the eighth month of gravidity. In the cows with induced calving the foetal placenta was not expelled within 12 hours after calving in 68.7% of the cases whereas in the spontaneous parturitions this proportion was only 22.2% of cases. The placentomes obtained immediately after calf expulsion, and then after four and eight hours, were subjected to histological and histochemical examination. In the terminal crypts of the placentome in cross sections obtained from cows which expelled the placenta in time after natural and induced parturitions, the number of binuclear cells of the fetal syncytium and of cells of the dam epithelium (P less than 0.001) was found to be significantly lower than in the cases of afterbirth retention (1.2 and 3.9; 6.4 and 18.5). The cells of the cow's epithelium of the expelled placentae had a higher activity of acid phosphatase and lipids and the foetal syncytium had a higher activity of non-specific esterase. Increased alkaline phosphatase activity was characteristic of the cow's epithelium in the cases of subsequent retention of afterbirth. These findings should be taken into account in efforts for developing new methods of the induction of parturition if the undesired occurrence of afterbirth retention is to be reduced.     

Messenger RNA expression of angiotensin-converting enzyme, endothelin, cyclooxygenase-2 and prostaglandin synthases in bovine placentomes during gestation and the postpartum period

The bovine placenta contains local vasoactive-related systems, including angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), ET-A receptor (ETAR) and ET-B receptor (ETBR), as well as arachidonic acid (AA) cascade-related enzymes, such as cyclooxygenase-2 (Cox-2), prostaglandin E-synthase (PGES) and prostaglandin F-synthase (PGFS). The mRNA expression of these molecules was examined in bovine placentomes (caruncles and cotyledons) collected immediately (0 h) and 6h after spontaneous parturition from 15 cows with early (fetal membranes released within 6 h of parturition) or late (fetal membranes released 6-12 h after parturition) detachment, as well as from 15 pregnant cows at a slaughterhouse. Significant differences were observed in expression of ET-1, ETAR and ETBR mRNAs between gestation and the postpartum period in both caruncles and cotyledons. Significant differences were also found between 0 and 6 h postpartum in the expression of ETBR mRNA in the early detachment group and PGES mRNA in the early and late detachment groups. Compared to PGFS, both Cox-2 and PGES exhibited opposite mRNA expression patterns during gestation and the postpartum period. The vasoactive-related peptide systems and AA cascade-related enzymes may mediate placental development and fetal membrane detachment after parturition in cattle.     

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.     

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.