Biochemical Profile of Amniotic and Allantoic Fluid During Different Gestational Phases in Mares

Fetal fluids have different vital functions that sustain both pregnancy and normal parturition. The biochemical composition of amniotic fluid during gestation is not well established; thus the purpose of the present study was to determine the biochemical profile of both amniotic and allantoic fluids from mares during initial, mid, and latter third phases of pregnancy. Samples were collected after slaughter, using allantocentesis and amniocentesis. Sixty samples of fetal fluids were analyzed. Alkaline phosphatase (AP), glucose, total protein (TP), urea, creatinine, Ca, chloride (Cl), Na, and K concentrations were measured using commercially available kits. The AP concentration in amniotic fluid was higher than that in allantoic fluid during the three gestational phases (P < .05). There were no differences between glucose mean values of allantoic and those of amniotic fluids (P < .05). However, glucose values were higher in the allantoic fluid in the last trimester of pregnancy. TP was higher in the amniotic fluid than in allantoic fluid (P < .05). Urea values varied among the phases; however, there were no differences between the amniotic and allantoic fluid values (P > .05). Creatinine values were higher in allantoic fluid (P < .05). Na and Cl concentrations were higher in amniotic fluid (P < .05). However, Ca and K concentrations were higher in the allantoic fluid.     

Metabolomic Profile of Allantoic and Amniotic Fluid in Late-term Gestational Mares Characterized by 1H-nuclear Magnetic Resonance Spectroscopy

The amniotic and allantoic fluid compartments in the mare serve essential roles throughout pregnancy and parturition. Although the global metabolomic profile of amniotic fluid in women has been extensively characterized, current data for equine fetal fluids are limited. Therefore, the goal of this study was to characterize the global metabolomic profile of equine allantoic and amniotic fluid through nuclear magnetic resonance spectroscopy. Fetal fluids were collected between 270 and 295 days of gestation from 12 pregnancies through ultrasound-guided transabdominal puncture. A total of 24 samples (n = 10 allantoic fluid; n = 9 amniotic fluid; n = 5 admixed fluid) were analyzed by one-dimensional proton (¹H) and two-dimensional (¹H-¹³ C) nuclear magnetic resonance spectroscopy. Metabolites were integrated and compared between fluid types using a Kruskal–Wallis test at P < .05 significance. A total of 28 distinct metabolites were found in allantoic and admixed fluid, whereas 23 metabolites were identified in amniotic fluid. Allantoic fluid contained significant elevations (P < .05) in the metabolites betaine, creatine, creatinine, citrate, histidine, nitrophenol, tryptophan, π-methylhistidine, and unknown metabolite #1 compared with amniotic fluid, whereas amniotic fluid contained statistically increased concentrations of the metabolite lactate compared with allantoic fluid (P = .003).     

鸡胚发育过程中胚胎液中几种游离氨基酸的动态变化

本实验旨在研究鸡胚发育过程中尿囊液和羊水中天冬氨酸、谷氨酸、精氨酸和亮氨酸浓度的动态变化。选用72枚6胚龄的三黄鸡鸡胚,按重量相近原则分为6组,每组12个重复,分别在孵化第7、8、11、13、14、18天采集尿囊液和羊水,并利用高效液相色谱法分析其氨基酸浓度。结果表明:尿囊液中,除8胚龄外,其他各胚龄精氨酸浓度均高于另外3种氨基酸(P<0.05),13胚龄精氨酸浓度显著低于其他胚龄(P<0.05),而18胚龄显则著高于其他胚龄(P<0.05)。羊水中,各胚龄精氨酸浓度均高于其他3种氨基酸;除11胚龄外,13胚龄精氨酸浓度显著高于其他胚龄(P<0.05);7胚龄天门冬氨酸浓度显著高于13、14、18胚龄(P<0.05);但18胚龄谷氨酸及亮氨酸浓度则显著升高(P<0.05)。以上结果说明,在尿囊液及羊水中,所测4种氨基酸浓度变化不尽相同,但总体精氨酸含量最丰富,这可能与鸡胚发育的营养与代谢有关。     

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.     

Experimental Neospora Caninum Infection in Pregnant Dairy Heifers Raises Concentrations of Pregnancy‐Associated Glycoproteins 1 and 2 in Foetal Fluids

Plasma concentrations of PAG‐1 are used for pregnancy diagnosis and as a marker of placental/foetal well‐being, while those of PAG‐2 may be an indicator of abortion risk in Neospora caninum‐infected cows. Studies have shown that N. caninum infection modifies PAG‐1 and PAG‐2 patterns in maternal blood plasma. However, no prior work has examined the effects of N. caninum infection on concentrations of PAGs in foetal fluids. In this study, PAG‐1, PAG‐2 and pH levels were determined in the amniotic and allantoic fluids of foetuses collected at 152 days of gestation from control uninfected dams and from dams experimentally infected with N. caninum on Day 110 of gestation. Foetal fluids from infected foetuses had significantly higher PAG‐2 concentrations (p = 0.026) and pH values (p = 0.02) than fluids from non‐infected foetuses. In infected foetuses, significantly higher concentrations of PAG‐1 (p < 0.001) and PAG‐2 (p < 0.001) were detected in fluid samples showing antibodies against N. caninum than those without antibodies. Moreover, pH values were significantly higher (p = 0.011) in foetal fluid samples with antibodies than in samples from non‐infected foetuses. In conclusion, this is the first report on the effect of N. caninum infection on PAG levels in foetal fluids. Our results indicate that following the experimental infection of dams with N. caninum on Day 110 of gestation, foetal fluids collected from the infected foetuses of these dams featured higher PAG‐1 and PAG‐2 levels and pH values than fluids from non‐infected controls, provided that the samples tested showed the presence of antibodies. The clinical implications of these findings are that following infection with N. caninum, most cows will experience some level of placental damage and that this injury correlates with foetal fluid PAG levels and pH.     

Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes

Toxoplasma gondii, an intracellular protozoan parasite, is one of the major causes of infectious abortion in sheep. To further understand the pathogenesis of toxoplasmosis, serum, amniotic and allantoic fluids and foetal stomach contents were collected from experimentally infected pregnant ewes to determine pathogen numbers and other markers of infection. Fifteen pregnant ewes (90 days of gestation) were each orally inoculated with 3000 sporulated oocysts of T. gondii. Serum samples were collected weekly following challenge. Amniotic and allantoic fluids and foetal stomach contents were collected at 21, 25, 28, 33 and 35 days post-infection. Characteristic placental lesions were detected in 1 of 4 challenged ewes at day 25, 3 of 4 challenged ewes at day 28 and in all challenged ewes at days 33 and 35 post-infection. T. gondii was detected only sporadically in amniotic and allantoic fluids before 35 days of infection, by real-time PCR, and only in ewes with placental lesions. At 35 days post-infection, high numbers of parasite were detected in both amniotic and allantoic fluids. An increase in the number of fluids from challenged animals with IgM and IgG was detected over time, except for IgG in allantoic fluid, which was detected in all samples from day 21 post-infection. IgG in amniotic and allantoic fluids was shown to be specific for T. gondii, and reacted with antigens with an apparent molecular mass of approximately 22 kDa and 30 kDa. Results suggest a maternal source of immunoglobulin in the allantoic fluid and a foetal source of immunoglobulin in the amniotic fluid early in infection but that both sources may contribute immunoglobulin to both fluids at a later stage.     

The protein composition of sheep foetal fluids

Amniotic fluid contains several proteins including alpha-foetoprotein derived from the foetal plasma, but lacks the higher molecular weight alpha2- and gamma-globulins. The protein composition of the fluid, like that of the serum, remains unchanged after the first month of gestation, and is buffered from the influence of the allantoic fluid by the amnion. Allantoic fluid contains a significantly higher concentration and a greater variety of proteins; it is not a simple transudate of either foetal or maternal plasma, and though containing proteins derived from both sources, it is deficient in albumin but relatively abundant in gamma-globulins. The latter comprises gamma-foetoprotein and mature type IgG. It is believed that maternal IgG may reach the allantoic sac in appreciable amount, but its transmission to the amniotic sac and thence to the foetus may be prevented by the impermeability of the amnion.     

Cytology of bovine foetal fluids.

Cell contents of foetal fluids obtained separately from amniotic and allantoic cavities of live cows by transvaginal amniocentesis and by aspiration from pregnant uteri immediately after slaughter were studied using Papanicolaou's staining procedure. The samples obtained belonged to cows which were between ten to 24 weeks of gestation. Cell scrapings from foetal and membranous surfaces normally in contact with the foetal fluids were also stained simultaneously with foetal fluid smears by Papanicolaou's method to determine the probable origin of the cells detected in the amniotic and allantoic cavities. The cell types were classified according to morphology and tinctorial patterns and their origin was deduced on the basis of similarity to cell scrapings from the surface layers of foetus and foetal cavities. The evaluation of foetal fluid cells revealed that a majority of cells are of foetal origin and that the cytological features of these cells may be useful in assessing the condition of the foetus.     

日粮中添加NCG对妊娠前期鄂尔多斯细毛羊血液、尿囊液和羊水中氨基酸浓度的影响

为了研究日粮中添加NCG(N-氨甲酰谷氨酸)对妊娠前期鄂尔多斯细毛羊血液、尿囊液和羊水中氨基酸浓度的影响,试验选择年龄、胎次和体况相近、平均体重为50.62±0.52 kg的受孕鄂尔多斯细毛羊母羊40只,随机分为3组(NCG1组饲喂基础日粮+0.30 g NCG/d(n=13),NCG2组饲喂基础日粮+0.40 g N...     

Anatomy, physiology and pathology of the amniotic and allantoic compartments in the sheep and cow

In both cows and sheep the allantoic fluid is confined to 2 sacs connected by a flattened isthmus, one in the non-pregnant horn and one in the upper part of the pregnant horn. The chorion encloses the amniotic and allantoic compartments, forming the amniochorion and chorioallantois, respectively. In the last third of gestation the compositions of both amniotic and allantoic fluids differ substantially from each other and from those of foetal urine, and maternal and foetal plasma. There is less variation in composition than in volume for a given gestational age. Abnormalities of volume are more common in cows than sheep, and hydrallantois is more common than hydramnois. Data obtained from both physiological experiments and pathological cases suggest that the foetal membranes play an important role in the regulation of composition and volume of foetal fluids. Evidence is presented that the permeability of the membranes to various solutes, as well as their capacity to produce and respond to a number of hormones, can affect the foetal fluid composition and/or volume. Progesterone, oestrogens and prolactin are some of the hormones known to affect foetal fluids. Foetal adrenal insufficiency has been associated with hydramnios implying a lack of hormones from this gland in this disease. The changes in allantoic fluid composition from normal to that closely resembling maternal/foetal extracellular fluid, in hydrallantois, suggests an alteration of membrane function as an aetiology and the continued production of fluid, after removal of the foetus in some cases, favours this hypothesis.     

Morphological Characteristics of the Placenta and Umbilical Cord of Arabian Mares Foaling in the United Arab Emirates

A total of 127 normal placentas from Arabian mares resident in the United Arab Emirates were examined. The mean linear dimensions of the placenta were, on average, 84% of those previously recorded for the placentas of the Thoroughbred. Significant differences in the size of the allantochorion between primigravid and multiparous mares were seen only in the linear dimensions of the body portion. The pregnant horn was more commonly on the right than left side of the uterus (P = .01; 74/127; 58%). Cord attachment was primarily at the base of the two placental horns (112/127; 88%), with the remainder showing anomalies from this position. The mean (±SEM) total length of the umbilical cords was 62.2 ± 1.2 cm, being composed of the allantoic portion (29.7 ± 0.9 cm) and amniotic portion (32.5 ± 0.6 cm), which averaged 53 ± 0.01% of the total length. The amniotic portion was usually, but not always, longer than the allantoic portion (79/127; 62%). Longer cords were associated with a greater proportion of allantoic length. An enlarged yolk sac remnant (YSR) was present in 16/127 (13%) placentas. Cords with YSRs displayed a significantly longer allantoic portion than those without (P = .02). The total cord length was not correlated with the weight or area of the allantochorion or amnion, any linear measurement of the allantochorion, gestation length, the month of foaling, parity of the dam, or birthweight or sex of the foal. The purpose for which the mare was bred (i.e., racing or showing) influenced the cord length, those of show mares being significantly longer.     

Abnormalities of foetal fluids in sheep: two case reports

SUMMARY Two cases of abnormal accumulation of foetal fluids in sheep are described. In the first case, a ewe, at 64 days of pregnancy, had 2 sacs of fluid, one at each of the tips of the pregnant and non-pregnant horns. Neither sac contained a foetus. The composition of the fluids differed from those of the amniotic and allantoic fluids associated with a single foetus. The composition resembled that of foetal plasma. We conclude that the 2 sacs were amniotic sacs from conceptuses that had been resorbed, and that at this early (0.4) stage of pregnancy, amniotic fluid can accumulate in the absence of a viable foetus.
The second case was a rare case of hydrallantois in a ewe at 100 days of pregnancy. The volume of allantoic fluid (1800 mL) was more than 10 times normal. The composition was normal, except for high chloride and calcium concentrations and a very low creatinine concentration. The foetus and the volume and composition of the amniotic fluid were normal. The hydrallantois might have occurred as a result of some alteration in function of the allantoic membrane, perhaps as a result of altered hormonal status of the ewe.     

Immunoglobulin concentrations and bovine enterovirus inhibitors in fetal bovine fluids.

Fluids from 53 bovine fetuses ranging in age from 90 to 240 days were examined for immunoglobulin G (IgG) and immunoglobulin M (IgM) and neutralizing activity to ten bovine viruses. Non-specific inhibitors to bovine enteroviruses were found in serum, allantoic, and amniotic fluids of most samples tested. In most cases, serum IgG were within normal values. Neither IgG nor IgM was detected in amniotic fluids, whereas 2 samples of allantoic fluid contained traces of IgG.     

Clinical evaluation of ketone bodies in body fluids of cows in late pregancy and their fetuses

Ketone body concentrations were determined in six body fluids of cows in late pregnancy and their fetuses (blood plasma and urine of cows, amniotic and allantoic fetal fluids, and fetal blood serum and urine) using the head-space gas chromatographic method. Physiological ratios 0/T% of oxidized (acetone and acetoacetic acid) to total (acetone, isopropanol, acetoacetic and 3-hydroxybutyric acids) ketone bodies were below 10% in cow blood plasma and urine, fetal serum and urine, and allantoic fluid, and below 20% in amniotic fluid. A scheme for the evaluation of the degree and duration of ketosis based on the 0/T% ratio is proposed. The existence of a renal regulatory mechanism for excretion of ketone bodies is suggested by analogy with the renal acidobasic regulation.     

Clinical evaluation of ketone bodies in body fluids of cows in late pregnancy and their fetuses

Ketone body concentrations were determined in six body fluids of cows in late pregnancy and their fetuses (blood plasma and urine of cows, amniotic and allantoic fetal fluids, and fetal blood serum and urine) using the head-space gas chromatographic method. Physiological ratios O/T% of oxidized (acetone and acetoacetic acid) to total (acetone, isopropanol, acetoacetic and 3-hydroxybutyric acid) ketone bodies were below 10% in cow blood plasma and urine, fetal serum and urine, and allantoic fluid, and below 20% in amniotic fluid. A scheme for the evaluation of the degree and duration of ketosis based on the O/T% ratio is proposed. The existence of a renal regulatory mechanism for excretion of ketone bodies is suggested by analogy with the renal acidobasic regulation.     

Efficiency of uterine fluid cytology in the diagnosis of subclinical endometritis in the water buffalo (Bubalus bubalis)

This study compared endometrial cytology vis‐a‐vis uterine fluid cytology for assessment of uterine health in clinically normal and subclinical endometritis (SE)‐affected buffaloes. Uterine fluid samples and endometrial samples were collected from the buffaloes (n = 38) at oestrus using blue sheath and cytobrush, respectively. The smears were stained with Field stain for 3 minutes, and a minimum of 400 cells were counted in each smear for determination of the percentage of polymorphonuclear (PMN) leucocyte. The incidence of subclinical endometritis, based on the cytobrush cytology, was 23.08%. The correlation between cytobrush cytology with uterine fluid cytology was positive and significant (r = .37; p = .02). The ratio of PMN leucocyte in cytobrush cytology to uterine fluid cytology was 1:2.4. ROC analysis revealed that the threshold value of 6.16% PMN leucocyte in uterine fluid cytology showed a diagnostic sensitivity and specificity of 100% in differentiating normal from SE‐affected buffaloes. In conclusion, collection of uterine fluid was easier compared to collection of endometrial samples using cytobrush and the percentage of PMN leucocyte in uterine fluid cytology can be used as a tool for diagnosis of subclinical endometritis in buffaloes.     

Pharmacokinetics of ceftiofur sodium in equine pregnancy

Eleven pregnant pony mares (D270‐326) were administered ceftiofur sodium intramuscularly at 2.2 mg/kg (n = 6) or 4.4 mg/kg (n = 5), once daily. Plasma was obtained prior to ceftiofur administration and at 0.5, 1, 2, 4, 8, 12, and 24 hr after administration. Eight pony mares were re‐enrolled in the study at least 3 days from expected foaling to ensure steady‐state concentrations of drug at the time of foaling. Mares were administered ceftiofur sodium (4.4 mg/kg, IM) daily until foaling. Parturition was induced using oxytocin 1 hr after ceftiofur sodium administration. Allantoic and amniotic fluid, plasma, and colostrum samples were collected at time of foaling. Serial foal plasma samples were obtained. Placental tissues were collected. Desfuroylceftiofur acetamide (DCA) concentrations were measured in samples by high‐performance liquid chromatography (HPLC). Mean (±SD) peak serum concentrations of DCA were 3.97 ± 0.50 μg/ml (low dose) and 7.45 ± 1.05 μg/ml (high dose). Terminal half‐life was significantly (p = .014) shorter after administration of the low dose (2.91 ± 0.59 hr) than after administration of the high dose (4.10 ± 0.72 hr). The mean serum concentration of DCA from mares at time of foaling was 7.96 ± 1.39 μg/ml. The mean DCA concentration in colostrum was 1.39 ± 0.70 μg/ml. DCA concentrations in allantoic fluid, amniotic fluid, placental tissues, and foal plasma were below the limit of quantification (<0.1 μg/ml) and below the minimum inhibitory concentration of ceftiofur against relevant pathogens. These results infer incomplete passage of DCA across fetal membranes after administration of ceftiofur sodium to normal pony mares.     

Biochemical analysis of uterine fluid for identification of indicators for subclinical endometritis in the water buffalo (Bubalus bubalis)

Alterations in biochemical constituents of uterine fluid have been suggested for diagnosis of subclinical uterine infection in the bovine. This study was undertaken to investigate whether uterine fluid biomolecules could act as tool for diagnosis of subclinical endometritis in the buffalo. Uterine fluid samples from normal (n = 22) and subclinical endometritis (n = 18; diagnosed based on uterine cytology)‐affected buffaloes were subjected to biochemical analysis. Among the different biochemical constituents estimated, urea, urea N, cholesterol, total bilirubin and alkaline phosphatase (ALP) concentrations were significantly (p < 0.05) higher in uterine fluid obtained from subclinical endometritis‐affected buffaloes. The extent of difference between normal and subclinical endometritis‐affected buffaloes was highest in ALP (69%) followed by cholesterol (55%), bilirubin (48%), urea (30%) and urea N (30%) concentrations. Receiver operating characteristic (ROC) analysis indicated that the likelihood ratio (LR) was 3.63 for urea, indicating that buffaloes having less than the threshold concentration (47.5 mg/dl) of urea in their uterine fluid were at 3.6 times more risk to be affected with SE. The LRs for urea N, cholesterol, ALP and bilirubin were 2.33, 2.54, 2.12 and 1.65, respectively. It was concluded that ALP, urea, urea N and cholesterol concentrations in uterine fluid may serve as an aid for diagnosing subclinical endometritis in the buffalo.     

A comparison of the uterine proteome of mares in oestrus and dioestrus

Proteomic analysis of mare uterine flush fluid provides a minimally invasive technique for studying protein changes associated with the oestrous cycle. The aim of this study was to identify differentially abundant proteins in the uterine flush fluid of mares in oestrus and dioestrus. In this study, uterine flush fluid samples were collected from eight reproductively healthy mares in either oestrus (n = 5) or dioestrus (n = 3). Proteomic analysis was performed using liquid chromatography‐tandem mass spectrometry. Of 172 proteins identified, six proteins (immunoglobulin lambda‐like polypeptide 1, haemoglobin subunit alpha, alpha‐1B‐glycoprotein, serotransferrin, apolipoprotein A‐1, and haemoglobin subunit beta) were significantly more abundant in oestrus. These proteins may contribute to the endometrial defence system through roles in inflammation, immunity or antimicrobial activity. In other species, some of these proteins have been described as immunoglobulins, negative acute phase proteins or defence agents against micro‐organisms. During dioestrus, immunoglobulin alpha‐1 chain C region‐related, complement factor I, CD 109 antigen and uterocalin, were significantly more abundant. Research in other species suggests that these four proteins contribute to the immune response through proposed immunoregulatory characteristics, complement system involvement or roles in B cell–T cell interactions. In conclusion, ten differentially abundant proteins were identified in the uterine flush fluid of mares in oestrus and dioestrus. Targeted studies on these proteins could elucidate their role in uterine defence mechanisms during the oestrous cycle in the mare.     

Role of uteroferrin in placental iron transport in swine: relationship between uteroferrin levels and iron deposition in the conceptus during gestation

This study was to examine the relationship between uteroferrin and Fe, and Fe and Cu in the fetal pig. In Exp. 1, conceptus tissues and fluids were obtained on d 30, 45, 60, 75 and 90 of gestation for Fe and Cu analyses. In fetus minus liver, total Fe and Cu increased constantly between d 30 and 90, but Fe and Cu concentrations (microgram/g dry tissue) both decreased between d 30 and 45 and then remained relatively constant to d 90. For fetal liver, total Fe increased from d 30 (27 micrograms) to d 90 (3,222 micrograms), as did total Cu (14 micrograms on d 30 to 960 micrograms on d 90). Fetal liver Fe concentration (microgram/g dry tissue) decreased from d 30 (1,021) to d 60 (472) and then increased to d 90 (1,082), whereas Cu concentration increased between d 30 (537) and 60 (830) and then decreased between d 60 (471) and 90 (329). In allantoic fluid, both total Fe and Cu increased between d 30 and 60 and then decreased to d 90. Data from this study indicated a close temporal relationship between Fe and Cu in the fetal tissues and fluids examined. In Exp. 2, the relationship between Fe and uteroferrin in fetal tissues and fluids was studied. Uteroferrin, measured indirectly by acid phosphatase activity and Fe in fetal tissues and fluid underwent closely related temporal changes between d 30 and 112 of gestation. Changes in total Fe and Fe concentration during gestation were similar to those described for Exp. 1 in fetus minus liver, fetal liver and allantoic fluid. In addition, total Fe and Fe concentration in placental and endometrial tissues were analyzed. It was concluded that uteroferrin provides a major source of Fe in endometrial secretion and that it may be stored in placental and endometrial tissues. The relationship between Fe and Cu in conceptus tissues and erythropoiesis also is discussed.