Proteins in the amniotic and allantoic fluids of fetal pigs

Seven proteins were identified in the amniotic and allantoic fluids of fetal pigs (Sus scrofa domesticus) using crossed immunoelectrophoresis: albumin, fetuin, transferrin, alpha-fetoprotein (AFP), alpha 1-acid glycoprotein, alpha 1-antitrypsin and alpha 2-macroglobulin. The total protein concentrations were determined by the method of Bradford and individual protein concentrations by radial immunodiffusion or rocket immunoelectrophoresis. Transferrin and fetuin were the major proteins in amniotic fluid during the second trimester of gestation and together with AFP and albumin accounted for the majority of the total protein in amniotic, but not allantoic fluid.     

Distribution of alpha-fetoprotein in fetal plasma and in amniotic and allantoic fluids of the pig

The concentration of AFP in the plasma of fetal pigs was highest in early gestation (Days 15-30) and declined with advancing gestation. This pattern was comparable to those observed in other mammals with similar lengths of gestation. In contrast, allantoic and amniotic fluid AFP concentrations were highest during the middle third of gestation but these concentrations were much lower than those in fetal plasma throughout gestation. The allantois appears to be a significant distribution space for AFP in early gestation.     

Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid.

Intestinal lactase activity (with its associated cellobiase, 4-methylumbelliferyl-beta-galactosidase and -beta-glucosidase activities) was used as a specific intestinal marker enzyme to study the release of protein and enzymes of intestinal origin in sheep amniotic fluid during gestation. In amniotic fluid, intestinal lactase activity peaked at 66--85 days of gestation and then decreased with gestation. This enzyme activity was very low or absent in allantoic fluid throughout gestation suggesting that there is no important transfer of amniotic fluid lactase towards the allantoic cavity. Maltase and 4-methylumbelliferyl-alpha-glucosidase showed no statistically significant variation with gestation in both amniotic and allantoic fluid whereas alpha-galactosidase and N-acetyl-beta-hexosaminidase which were first higher in allantoic than in amniotic fluid increased in amniotic fluid to reach allantoic fluid levels near term. Such patterns are consistent with the suggestion that the fetal urine is a source of alpha-galactosidase and N-acety-beta-hexosaminidase activities and that sheep urine is first accumulated in the allantoic sac via the urachus up to 86--90 days of gestation and thereafter passes more and more into the amniotic sac.     

Prostaglandins in fetal tracheal and amniotic fluid from late pregnant sheep

Concentrations of prostaglandin E (PGE), prostaglandin F (PGF) and 13,14-dihydro-15-keto-prostaglandin F (PGFM) have been measured in fetal tracheal and amniotic fluid from chronically catheterized sheep during late pregnancy. Amniotic fluid contained significantly greater concentrations of these prostaglandins than tracheal fluid (p less than 0.01); there was no correlation between the level of prostaglandins found in each fluid. In tracheal fluid concentrations of PGE and PGFM exceeded those of PGF (P less than 0.01) whereas no significant differences were found in amniotic fluid. The levels of prostaglandins in these fluids were similar in ewes bearing hypophysectomized fetuses.     

Bovine amniotic and allantoic fluids for the culture of murine embryos

This study evaluated bovine amniotic and allantoic fluids as culture media for two-cell murine embryos to the hatched blastocyst stage. Amniotic and allantoic fluids were collected from four 70-d periods of pregnancy and pooled from at least five different animals. In Experiment 1 (n = 470) the fluids were frozen twice. Treatments consisted of twice frozen amniotic or allantoic fluid from each pregnancy period, Whitten's medium and fetal calf serum. The later two media were controls. Twice-frozen amniotic fluid <70 d pregnancy period, fetal calf serum and Whitten's medium supported the development of embryos to the hatched blastocyst stage. Whitten's medium was superior to twice-frozen amniotic fluid <70 d pregnancy period or fetal calf serum (P<0.01). Biochemical analysis showed lower glucose in amniotic and allantoic fluids than in Whitten's medium. Experiment 2 (n = 425) was performed to evaluate the effect of glucose supplementation to amniotic fluid. No benefit of glucose supplementation of the amniotic fluid was observed. In Experiment 3 (n = 432), the fluids were transported nonfrozen on ice. Treatments consisted of nonfrozen amniotic fluid <70 d pregnancy period; nonfrozen amniotic fluid <70 d pregnancy period + glucose), nonfrozen allantoic fluid <70 d pregnancy period; and Whitten's medium. The percentages of embryos developing to hatched blastocyst stage were 66.6, 56.5, 57.4 and 63.9% respectively, for each of the four treatments. No differences were found between any two treatments (P<0.05). In Experiment 4 (n = 231) the fluids were stored at -20 degrees C for 15 d. Whitten's medium was superior to amniotic or allantoic fluid <70 d pregnancy period in sustaining embryo development (P<0.05). In conclusion, these data indicate that nonfrozen bovine amniotic or allantoic fluid <70 d pregnancy period can support the development of murine embryos to the hatched blastocyst stage comparable to culture in Whitten's medium. Glucose supplementation of the amniotic fluid offered no advantage, and freezing of fluids had an adverse effect on in vitro embryo development.     

Ovine fetal adaptations to chronically reduced urine flow: preservation of amniotic fluid volume

Mann, Stephanie E., Mark J. M. Nijland, and Michael G. Ross.Ovine fetal adaptations to chronically reduced urine flow: preservation of amniotic fluid volume. J. Appl.Physiol. 81(6): 2588-2594, 1996.Adequateamniotic fluid (AF) volume is maintained by a balance of fetal fluidproduction (lung liquid and urine) and resorption (swallowing andintramembranous flow). Because fetal urine is the principle source ofAF, alterations in urine flow and composition directly impact AFdynamics. Intra-amniotic 1-desamino-8-D-argininevasopressin (DDAVP) is rapidly absorbed into fetal plasma and induces amarked fetal urinary antidiuresis. To examine the effect ofintra-amniotic- DDAVP-induced fetal urinary responses on AF volume andcomposition, six chronically prepared ewes with singleton fetuses(gestation 128 ± 2 days) were studied for 72 h after a singleintra-amniotic DDAVP (50-µg) injection. After DDAVP, fetal urineosmolality significantly increased at 2 h (157 ± 13 to 253 ± 21 mosmol/kg) and remained elevated at 72 h (400 ± 13 mosmol/kg). Urinary sodium (33.0 ± 4.5 to 117.2 ± 9.7 meq/l)and chloride (26.0 ± 2.8 to 92.4 ± 8.1 meq/l) concentrations similarly increased. AF osmolality increased (285 ± 3 to 299 ± 4 mosmol/kgH₂O), although there was no change in fetalplasma osmolality (294 ± 2 mosmol/kg). Despite a 50% reductionin fetal urine flow (0.12 ± 0.03 to 0.05 ± 0.02 ml ^· kg^{1 ·} min¹at 2 h and 0.06 ± 0.01 ml ^· kg^{1 ·} min¹after 72 h), AF volume did not change (693 ± 226 to 679 ± 214 ml). There were no changes in fetal arterial blood pressures, pH,PCO₂, orPO₂ after DDAVP. We conclude the following. 1)Intra-amniotic DDAVP injection induces a prolonged decrease in fetalurine flow and increases in urine and AF osmolalities. 2) Despite decreased urine flow, AFvolume does not change. We speculate that, in response to DDAVP-inducedfetal oliguria, reversed intramembranous flow (from isotonic fetalplasma to hypertonic AF) preserves AF volume.

     

Biochemical characteristics of amniotic and allantoic fluid in late gestational mares

The purpose of this study was to determine certain clinically important parameters of amniotic and allantoic fluid. Amniocentesis was performed on 10 normal mares in late gestation (323.8±10.2 d) and fluid was collected from both amniotic and allantoic cavities. Compared to amniotic fluid, allantoic fluid had significantly higher values of specific gravity, sorbitol dehydrogenase, total bilirubin, gamma glutamyltransferase, creatinine, phosphorus, total protein, and globulin. However, allantoic fluid had significantly lower values for sodium, chloride and alkaline phosphatase than amniotic fluid.     

Distribution of alpha 1-fetoprotein in fetal plasma, allantoic fluid, amniotic fluid and maternal plasma of cows.

Maximal concentrations of AFP, measured by RIA, were obtained in fetal plasma and amniotic and allantoic fluid between the 3rd and 4th month of gestation, with levels declining thereafter until term. AFP values in maternal plasma were unchanged. Throughout gestation, AFP values were higher in allantoic than in amniotic fluid and the ratio of allantoic fluid/amniotic fluid AFP was significantly correlated with gestational age.     

Suppression of lymphocyte reactivity in vitro by porcine allantoic and amniotic fluids

Porcine allantoic and amniotic fluids from early, mid and late pregnancy were analyzed for protein constituents and ability to suppress phytohemagglutinin (PHA)-induced lymphocyte transformation. All fetal fluids contained alpha-fetoprotein as evaluated by polyacrylamide gel electrophoresis (PAGE), with the highest concentrations appearing in mid-pregnancy amniotic fluids. In addition, allantoic fluids from mid-pregnancy contained proteins assumed to be secreted by the uterus. Both allantoic and amniotic fluids from mid-pregnancy were suppressive to PHA-induced lymphocyte transformation (P<.01). Thus it was concluded that substances, potentially able to suppress the immune response locally, exist within porcine fetal fluids, but it was not determined whether this material is of solely fetal or maternal origin, or a combination.     

Modification of respiratory center output in the unanesthetized fetal sheep "in utero".

Diaphragmatic electromyographic activity, tracheal and amniotic fluid pressures, lung liquid flow, and carotid and jugular venous pressures were measured on eight fetal lambs who survived for periods of 9-43 days postoperatively. The fetal gestational age ranged from 98 to 113 days at operation. Respiratory center output of the fetus as indicated by electromyographic activity was modified by the following stimuli. It was suppressed by anesthesia and fetal hypoxia (Pao2 = 12 mmHg), tonically reduced by lung inflation, and stimulated by cyanide injections (150-600 mug) into the fetal jugular vein. Neuromuscular transmission to the diaphragm was blocked with d-tubocurarine (0.2-0.6 mg). These experiments indicate that central and motor pathways to the diaphragm are sufficiently mature by 101 days in the fetal sheep to permit their output to be modified by chemical and mechanical stimuli.     

Water exchange between the pregnant ewe,the foetus and its amniotic and allantoic fluids

Water exchange between the ewe and its foetus was measured in two Merino ewes maintained with continuous feeding under thermoneutral conditions from about day 90 of gestation to term. Catheters were established in the maternal pulmonary artery (MPA), the foetal dorsal aorta (FDA) and the amniotic and allantoic sacs. Doses of tritiated water were given into either the MPA or the FDA on five occasions, three for one ewe and two for the other, at least 6 days apart and samples were taken from all catheters for 31 h following the dose. An open, eight-compartment restricted model was developed which simultaneously fitted the tracer data and the ewes water balance, determined by measurement and calculation. The ewes delivered live lambs at term. Water was exchanged between ewe and foetus at 16–43 l h^–1 whereas net flow to the foetus averaged 82 ml day^–1. Turnover times were 20–39 min in the ewes body water, 2–7 min in foetal body water, 10–58 min in amniotic water and 3–22 min in allantoic water; the whole-body half-times were 4.5–5.7 days. The data suggest that intramembranous exchange was the major contributor to water exchange in amniotic and allantoic fluids. The mean residence time of water in the rumen (39–52 min) was shorter than in non-pregnant sheep, suggesting that blood flow to the rumen increased during mid to late gestation.     

Urethral plugs and urine retention in male mice

Because MM male mice suffer from a high incidence of urinary tract infection, an assessment was made as to whether the urethral plugs, which occur in male rodents, might be involved in its aetiology. When killed, males more commonly retained urine in their bladders than females but there was no significant difference between strains or method of killing. Males also voided urine more often during stressful handling followed by abdominal pressure, but also retained some urine more often than females. The study of sexually immature mice demonstrated no sex differences and no urine was retained in any bladder. It was concluded that the high frequency of urine retention in mature males is attributable to the presence of urethral plugs but these could not be implicated in the cause of MM urinary tract infection because of comparable findings in the controls. However, the possibility was considered that the plugs might facilitate infection of the kidneys once a bladder infection had become established.     

Regulatory response to washout of amniotic fluid in sheep

To test the hypothesis that a substance present in the amniotic fluid could serve as a regulator of amniotic fluid volume, we drained and discarded amniotic fluid while replacing it with lactated Ringer solution that was isotonic to amniotic fluid. Seven ewes with singleton fetuses at 119 +/- 1 days of gestation (mean +/- SE) were instrumented with multiple indwelling catheters in the pedal artery, pedal vein, and amniotic cavity. During the exchange periods, an average of 3,019 +/- 171 ml/day of lactated Ringer solution was infused into the amniotic cavity while an equal amount of amniotic fluid was pumped out and discarded. During the control period, amniotic fluid composition and volume were not altered. Exchange and control periods started with the same amniotic fluid volume, lasted 3 or 4 days, and were randomized with regard to order. Amniotic fluid volume measured by vacuum drainage was 556 +/- 98 ml at the end of the control period and 986 +/- 209 ml (P = 0.03) at the end of the exchange period. Fetal arterial blood gases, hemodynamic parameters and the osmolality gradient between fetal plasma and amniotic fluid were not altered by the exchange process. A linear relationship between the control amniotic fluid volume and the volume at the end of the exchange period (P = 0.003) suggests that the animals with larger control volumes responded to isovolumic dilution with a larger volume increase. We conclude that amniotic fluid may contain a substance that regulates amniotic volume.     

Distribution of bovine fetuin and albumin in plasma, allantoic and amniotic fluids during development

A radioimmunoassay for bovine fetuin was developed and its specificity and validity established. Albumin was measured by radial-immunodiffusion assay. Fetuin levels in fetal plasma increased from 10 to 15 mg/ml between 4 and 8 months of gestation; albumin levels remained higher than fetuin. Neonatal plasma fetuin levels rapidly declined during the first 14 days post partum, coincident with a marked reciprocal increase in albumin levels. In allantoic fluid fetuin and albumin concentrations reached a peak at 7 months but fetuin values were always higher. In amniotic fluid both proteins peaked at 8 months; albumin levels were similar to those in allantoic fluid but fetuin values remained consistently lower than those in allantoic fluid throughout gestation. Fetuin levels in maternal plasma declined from 0.7 to 0.4 mg/ml between 1 month and term. We conclude that (1) at term there is an abrupt change from fetuin synthesis to increased albumin synthesis by the neonatal liver; (2) fetuin appears to be preferentially accumulated in the allantois whereas albumin is equally concentrated in the allantois and amnion.     

Metabolic and hormonal responses to cooling the fetal sheep in utero

The metabolic and hormonal effects of cooling 10 fetal sheep in utero (115-142 days of gestation) for 2h were studied. The fetal core temperature fell by 2.81 +/- 0.14 degrees C while the maternal temperature fell 0.86 +/- 0.15 degrees C. This hypothermia caused a significant rise in the fetal and maternal plasma glucose concentrations (P less than 0.001) and a fall in the fetal insulin concentrations (P less than 0.01). The fetal plasma lactate and cortisol concentrations rose rapidly (P less than 0.01) while the growth hormone fell (P less than 0.01) and remained low until cooling ceased when a rapid rebound occurred. There was no significant change in any of the fetal iodothyronines and no elevation of nonesterified free fatty acid concentrations, in contrast to the rapid rise (P less than 0.01) which occurred when newborn lambs were cooled. These observations demonstrate that appropriate glucose, insulin, lactate and cortisol responses to hypothermia have differentiated by 120 days of gestation. However, neither a thyroid hormone response nor an elevation in free fatty acid levels was observed. Thus not all components of the thermogenic response to hypothermia can be demonstrated in the late gestation fetail sheep in utero.