Proteomic analysis of amniotic and allantoic fluid from buffaloes during foetal development

The objective of this study was to describe the dynamic changes in protein composition and protein abundance in amniotic and allantoic fluids from buffaloes during gestation. Amniotic and allantoic fluids were collected during the first, second and third trimesters of gestation. The foetuses were measured and weighed. Fluid samples were centrifuged at 800 g for 10 min and then at 10,000 g for 60 min at 4°C. The supernatant was collected to determine the total protein concentration. Based on total protein concentration, an aliquot (50 μg) was used for in‐solution tryptic digestion, and mass spectrometry analysis (nano‐LC‐MS/MS) was performed. A multivariate statistical analysis of the proteomic data was conducted. Across the different stages of buffalo gestation, fifty‐one proteins were found in the amniotic fluid, and twenty‐one were found in the allantoic fluid. A total of twelve proteins were common among the stages, and four presented significant differences (VIP score α > 1). Fibronectin and alpha‐1‐antiproteinase were more abundant in the amniotic fluid than in the allantoic fluid. Alpha‐2‐macroglobulin and alpha‐2‐HS‐glycoprotein were more abundant in the allantoic fluid than in the amniotic fluid. Alpha‐2‐macroglobulin participates in remodelling and growth of the uterus at beginning of the gestation (first trimester), and these findings indicate that can serve as a potential tool for the early diagnosis of pregnancy in buffaloes.     

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.     

Ascorbic acid concentration in plasma, in amniotic and allantoic fluids, in the placenta and in 13 tissues of sheep fetuses and newborn lambs]

At each 8 on an average 92 (body weight 615 +/- 49 g) and 120 days (b.w. 1.8 +/- 0.40 kg) old fetuses as well as at 6 newborn lambs (b.w. 2.8 +/- 0.65 kg) the concentration of ascorbic acid in the plasma, in the amniotic- and allantois-fluid, in the fetal and maternal part of the placenta and in 13 tissues was analyzed. The highest concentration in the plasma (73 +/- 11 micrograms/ml) was determined in the newborns. The concentration in the amniotic-fluid decreased and that in the allantoic-fluid, the adrenals and the fetal part of the placenta increased in the course of growth. In the cerebrum, the cerebellum, the brain stem, the lungs, the heart, the kidneys, the M. longissimus and the M. semimembranosus the concentration diminished during the fetal growth. In the liver, the spleen and the pancreas the concentration was similar. The function of the ascorbic acid during the fetal development is discussed. The results are compared with those at human, pig and calf fetuses. In analyzing the ascorbic concentration in fetal body fluids of ungulates the high concentration of fructose as an influence-factor in the usual applied methods has to be considered.     

The activity and properties of adenosine triphosphatase in various swine organs (liver, cerebral and kidney cortex, small intestinal mucosa)]

Studies into the activity of adenosine triphosphatase (ATPase) in homogenates of liver, cerebral cortex, renal cortex, and mucosa of small intestine of swine have shown differentiated activity patterns, with peak activity developing in the liver. This has been related to a particularly high metabolism performance of the liver in fattening pigs. No difference was found to exist between magnesium activation of ATPase of swine tissue homogenates and that in tissue obtained from ruminants. ATPase which could be activated by sodium and potassium ions and inhibited by ouabain was detectable from cerebral and renal cortex. Sodium and potassium ATPases accounts from some 25 per cent of the total activity. ATPase that could be stimulated by calcium ions was recorded only from liver homogenate. The optimum pH values of ATPase were between 7.5 and 8 in the liver, 9 in mucosa of small intestine, and 9.5 in cerebral and renal cortex.     

Histochemical localization of adenosine triphosphatase activity in bovine ruminal epithelium.

A fine-structural histochemical technique was used to localize magnesium-dependent adenosine triphosphatase (Mg-ATPase) activity in ruminal mucosa. Precipitate appeared on the cytoplasmic surface of the plasmalemma in cells of the upper stratum spinosum, the stratum granulosum, and the deepest layer of the stratum corneum. This ATPase activity was sensitive to glutaraldehyde fixation and possibly to ouabain, but was unaffected by sodium and potassium. The preponderance of Mg-ATPase activity in bovine ruminal epithelium may make it impossible to detect sodium-potassium-activated adenosine triphosphatase ((Na + K)-ATPase) activity histochemically. A Mg-ATPase activity also occurred in mitochondria of the stratum spinosum and stratum granulosum. None of the ruminal sections hydrolyzed adenosine diphosphate, inosine triphosphate, or beta-glycerophosphate when these compounds were used as substitute substrates for adenosine triphosphate. When adenosine-5'-monophosphate was the available substrate, a reaction product appeared in the same layers as Mg-ATPase activity, but the reaction product was confined to the intercellular space.     

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.     

Serum adenosine deaminase activity in bovine liver diseases

A total of 60 cattle were examined for the presence of pathological liver lesions. The liver lesions were classified as glycogen degeneration, liver abscess, sawdust liver and fatty degeneration. The value of serum adenosine deaminase (ADA) activity was investigated as a pilot study for diagnosing liver diseases in cattle. Serum ADA activity was significantly higher in cases with glycogen degeneration (9.8 +/- 3.8 U/l) , liver abscess (10.4 +/- 3.2 U/l), sawdust liver (11.5 +/- 7.3 U/l) and fatty degeneration (20.8 +/- 7.7 U/l) than in the controls. The results indicate that ADA activity increases with the degree of hepatocellular damage. We concluded that serum ADA activity may be of value in bovine liver disease diagnosis.     

Amniotic and allantoic fluid levels of oestrogens at different stages of development in the goat

During our studies on certain aspects of reproduction in the goat (Lyngset 1971) we collected several samples of foetal fluid which were stored in the frozen state for further investigation. We have now processed this material with respect to contents of oestrogens.     

Activity and properties of adenosine triphosphatase in erythrocytes and various tissues (brain, liver, kidney, small intestine mucosa) of cattle]

In 15 samples of haemolysate of bovine erythrocytes, the splitting of phosphate from adenosine triphosphate average 158 +/- 63 X 10(-3) muMol/min/g haemolysate haemoglobin. Estimation of total adenosine triphosphatase in homogenates of various organs from cattle showed that spleen, liver, kidney and brain possessed high activity, while the activity was moderate in lung, myocardium and skeletal muscle, and low in endometrial mucosa and spinal cord. There was a relatively large proportion of Na-K-adenosine triphosphatase in brain and kidney. In various organs the activity of the enzyme was dependent upon the concentrations of Mg, Na, K and Ca. The inhibition of adenosine triphosphatase in various tissues by ouabain was studied. The optimum pH for the enzyme lay in the weakly alkaline region.     

Copper, zinc and molybdenum in goat liver.

The concentrations of copper, zinc and molybdenum were measured in the liver of goats from western and south-eastern Norway. The mean copper concentrations in the liver of goats from these two districts were 23±19 µg Cu/g and 59±31 µg Cu/g wet weight, respectively. As for zinc and molybdenum, no difference was found between the two groups of animals. No correlations were detected between copper and zinc, zinc and molybdenum, or copper and molybdenum. The copper levels in Norwegian goat liver are considerably lower than in sheep liver, and the ranges are significantly more narrow. The concentrations of molybdenum in goat liver are at the same levels as in sheep and swine, while the levels of zinc are somewhat lower.     

Characterization of myofibrillar adenosine triphosphatase activity and liberation of actin from myofibrils upon heating chicken breast meat

Denaturation of actin and myosin in myofibrils induced by heating at 50°C was investigated to reveal the mechanism of irreversible liberation of actin from myofibrils on heating at lower temperatures than conventional cooking. Denaturation of these proteins was determined by Mg²⁺‐ATPase (adenosine triphosphatase) and Ca²⁺‐ATPase activities. When minced meat was heated for 20 min, actin was liberated accompanying denaturation of 80% of actin and 50% of myosin. Heating of the myofibrillar fraction (MFF) isolated from meat homogenate induced much slower denaturation of actin than myosin. When MFF was heated with sarcoplasmic fractions, denaturation of actin was facilitated, suggesting that sarcoplasmic fractions contain factors to facilitate actin denaturation. Inosine‐5′‐monophosphate, a component of sarcoplasmic fractions, was shown to have no effect on actin and myosin denaturation. These results suggest that heating meat at 50°C dissociates binding (‘Bond A’) between actin and myosin participating in ATPase activities, resulting in denaturation of both proteins under influence of sarcoplasmic components. Although denaturation of actin and myosin disrupted Bond A, actin was not liberated simultaneously, suggesting the presence of another bond (‘Bond B’, more heat‐stable than Bond A) between both proteins and necessity of disruption of Bond B for actin release from myofibrils.     

Studies on reproduction in the goat. VII. Pregnancy and the development of the foetus and the foetal accessories of the goat

1. A total of 18 goats were ovariohysterectomized at different stages — 26 days to 145 days — of pregnancy.
2. The amniotic fluid increases from about 3.5 ml at 30 days to 600–1,000 ml per foetus at birth.
3. At birth the amount of allantoic fluid is approximately 600–1,100 ml and is to a small extent dependent on the number of foetuses.
4. The length of the foetus is about 1.4 cm at 30 days and 43 cm at 145 days, the lengthwise growth curve from the 30th day and onward being almost linear.
5. Hair follicles are visible around the 60th day and the eyes open between the 130th and 140th day.
6. The average length and weight of the goat kids at birth are 42.06 cm and 2.81 kg respectively.
7. The birth weight and length tend to increase with the increasing age of the dam.
8. The male does not appear to have any influence on the birth weight and length of the offspring.