Ovine Plasma Research Articles

A combined radioimmunoassay for the measurement of unconjugated and sulfoconjugated pregnenolone, 17 hydroxypregnenolone, dehydroepiandrosterone, and estrone applied to fetal and maternal ovine plasma

This report describes a radioimmunoassay (RIA) method for the combined measurement of four steroid sulfoconjugates and their four unconjugated counterparts in maternal and fetal ovine plasma: pregnenolone (Δ 5P), 17-hydroxypregnenolone (17Δ 5P), dehydroepiandrosterone (DHEA), and estrone (E 1). In the procedure a preliminary ether extraction is utilized to isolate the unconjugated steroids followed by salting out, ethyl acetate extraction, and mineral acid solvolysis of the remaining sulfated steroids. The hydrolyzed sulfoconjugates are then separated chromatographically and measured in a manner identical to their unconjugated counterparts. The combined measurement of these eight steroids in single samples of fetal and maternal ovine plasma has not been reported previously, and plasma concentrations of these steroids were heretofore unknown. Since no previous data was available for comparison, rigorous specificity evaluation of this RIA system was required prior to its use for physiologic studies and the reporting of concentrations in this species.

Changing patterns of steroid production in the fetus and placenta and their effects on development.

A variety of radioimmunoassays specifically characterized for use with fetal and maternal ovine plasma have been used to measure steroid hormone concentrations in small plasma samples drawn simultaneously from mother and fetus. By repeated sampling from the same animal preparation the changes over time of plasma concentrations of cortisol, pregnenolone and its sulphate, and oestrone and oestrone sulphate have been systematically studied. In animals delivering spontaneously at term the fetal plasma cortisol concentration rises between 120 and 130 days gestation and plays an important part in the maturation of several vital physiological systems, including the fetal thyroid axis. In normal term deliveries and premature deliveries induced by synthetic adrenocorticotropin (ACTH(1-24), 1 microgram h-1), the concentration of oestrone sulphate in maternal plasma increases before the fetal plasma concentration. Fetal and maternal oestrogens are probably important in the control of the low-grade tonic myometrial activity that occurs throughout gestation, in the initiation of labour and in the control of uterine blood flow. Low-grade tonic myometrial activity affects fetal oxygenation, fetal breathing and the fetal sleep state and may constitute a pathway through which the mother influences fetal development, with important physiological and possibly pathological consequences.

Time-trend analysis of plasma cortisol concentrations in the fetal sheep in relation to parturition.

Using a cortisol RIA carefully validated for specificity in fetal ovine plasma, we studied plasma cortisol time-trend changes in eight chronically catheterized sheep fetuses in the last 22 days of gestation before the spontaneous onset of labor. Best fit exponential type curves were drawn for each individual fetus to define the time before the onset of labor at which fetal plasma cortisol was rising at different rates. Fetal plasma cortisol increased at a rate of 0.5 ng ml-1 day-1 as early as 17.1 days before labor in one fetus, whereas the mean value for all eight fetuses occurred 11.8 +/- 1.0 (SEM) days before labor. The rate of increase doubled to 1.0 ng cortisol ml-1 day-1 as early as 14.4 days before labor in one fetus, whereas the mean value for all eight fetuses occurred 9.3 +/- 0.9 days before labor. These findings suggest that the regulatory mechanisms responsible for the prelabor increase in fetal cortisol production are activated earlier in gestation than we previously thought.

Effects of season, lactation and plane of nutrition on prolactin concentrations in ovine plasma and the role of prolactin in the control of ewe fertility.

Plasma prolactin concentrations during the first 2 months after lambing, at oestrus, and during early pregnancy were investigated in 2 experiments in which Finn x Dorset Horn ewes were mated at an induced oestrus approximately 9 weeks after lambing. Mean prolactin concentrations between lambing and mating were dependent on seasons, being greater than 260 ng/ml plasma in lactating ewes mated in July and less than 150 ng/ml in those mated in October. Within 8 days of weaning of the lambs at 50 days post partum values declined to 122 and 30 ng/ml respectively. Plane of nutrition had little effect on prolactin levels. Higher prolactin values were recorded during oestrus in ewes mated in March or July, the normal period of anoestrus, than in December, the normal breeding season, mean values being approximately 200 ng/ml and 35 ng/ml respectively. The mean increases in the concentrations of prolactin during oestrus were smaller in lactating than non-lactating ewes. It is suggested that these differences in prolactin levels may be responsible for the effects of season and lactation on ewe fertility.

Circulating plasma protein concentrations in the fetal and neonatal sheep.

Fetal and neonatal ovine plasma protein and lactate dehydrogenase concentrations have been measured between 124 and 146 days gestation in th chronically catheterized fetal sheep and in the newborn lamb. Fetal plasma protein concentration ranged from 24.7 to 77.0 mg . ml-1 and increased at a rate of 1.65 mg . ml-1 . day-1. In contrast, in the first 10 days of neonatal life plasma protein concentration fell steadilyu at a rate of 2.64 mg . ml-1 . day-1. Fetal plasma lactate dehydrogenase concentration increased at a rate of 0.014 IU . ml-1 . day-1 during the period of study. The increase correlated significantly with the rise of fetal plasma protein concentration. In contrast, in the newborn lamb no significant change in plasma lactate dehydrogenase concentration was observed during ther first 10 days after birth.

Comparison of two rapid cortisol radioimmunoassays for use in the fetal sheep

Cortisol radioimmunoassays (RIA's) utilizing highly specific antisera combined with a simple ethanol protein precipitation procedure (ETOH-PPT) are widely utilized to measure cortisol in human plasma. This same type of RIA has been assumed specific for measurement of cortisol in the plasma of several different species of experimental animals. In order to test this assumption as applied to fetal ovine plasma, we compared an ETOH-PPT cortisol RIA with another rapid cortisol assay which utilizes a dichloromethane extraction (DM-E) step. The DM-E assay in turn was compared with a chromatographic assay previously shown to be highly specific for measurement of fetal plasma cortisol in this species. Fetal ovine plasma cortisol concentrations determined by the DM-E method were nearly identical to the concentrations obtained by the specific chromatographic RIA procedure. On the other hand, the ETOH-PPT RIA grossly overestimated cortisol concentrations when compared with the DM-E RIA. While the rapid DM-E RIA appears to be suitable for use in fetal ovine plasma, the widely used ETOH-PPT RIA yields spuriously high and unpredictable values and must be considered unreliable. These comparisons demonstrate the need for careful reassessment of steroid assays prior to their application in experimental animals even though they have been previously documented as specific in human plasma.

Validation of a specific radioimmunoassay to measure plasma cortisol in the fetal sheep

A procedure utilizing co-chromatography and complementary antiserum comparisons was employed to assess the specificity of a cortisol radioimmunoassay for use in the chronically catheterized fetal sheep preparation. Complementary antiserum comparisons is a technique by which two different cortisol antisera, prepared from conjugates attached at opposite ends of the cortisol molecule, were used to determine cortisol concentrations in the same ovine fetal plasma specimens. Results were not significantly different between the two groups, each measured by a different antiserum. This procedure may be used to assess assay specificity in any species in which steroid radioimmunoassays are being adapted.

Stimulation of ovine placental lactogen secretion by arginine infusion.

Arginine has been demonstrated to be a potent stimulus to GH and PRL secretion. To determine the effect of arginine on plasma ovine placental lactogen (oPL) concentrations, arginine (50 g in 350 ml distilled water, pH 7.4) or hypertonic saline of identical volume, osmolality, and pH was infused iv over a 30-min period into nine pregnant ewes, and blood samples from chronic indwelling venous catheters were obtained at frequent intervals before and for 8 h after the infusions. After the infusion of hypertonic saline, plasma oPL concentrations (measured by homologous RIA) decreased 20--50% over 1--2 h and then returned to baseline concentrations. After the infusion of arginine, plasma oPL concentrations also decreased by 20--50% for 1--2 h. However, 2--3 h after the infusion, plasma oPL concentrations in creased 79--115% (delta = 204--700 ng/ml) over preinfusion concentrations in seven ewes and 454% (2930 ng/ml) and 1142% (2042 ng/ml) in two ewes and remained elevated for the remainder of the 8-h interval. When the amount of arginine infused was reduced from 50 to 25 g, an increase in plasma oPL concentrations occurred in only one of five ewes. Plasma oPL concentrations increased by 8--58% after infusions of 50 g alanine or glycine but did not increase after 50 g glutamic acid. The delayed oPL response to arginine suggests that the increase in plasma oPL concentrations is not caused directly by arginine but rather by changes in the synthesis, secretion, and/or degradation of oPL induced indirectly by arginine.

Gas chromatographic determinant of indole and 3-methylindole in ovine plasma and rumen fluid using a nitrogen-phosphorous selective detector

Gas chromatographic determinant of indole and 3-methylindole in ovine plasma and rumen fluid using a nitrogen-phosphorous selective detector

Dexamethasone concentrations in ovine plasma during its intravenous infusion, its relation to the production of some endogenous hormones, and some of the effects on wool growth.

Plasma concentrations of dexamethasone have been measured in sheep during an 8-day infusion of dexamethasone-21 phosphate. The dexamethasone concentration profiles generally revealed a reproducible pattern with three phases--a peak during the first 48 h infusion which was followed by falling concentrations during the next 5 days, and a small increase in dexamethasone concentration during the final 24 h infusion was not uncommon. The pattern of dexamethasone concentrations was retained when dosage was arranged in such a way as to infuse increasing quantities of hormonal analogue as infusion progressed. Aspects of the metabolism of the analogue are discussed. Endogenous thyroxine and cortisol were significantly depressed during infusion. In these experiments wool was completely shed in three out of four animals dosed at a rate of 8.5 mg dexamethasone/kg0.75. The recovery of wool growth to its pretreatment values occurred by about one month after infusion. The consumption of food and body weight increases were satisfactory during the post-infusion period.

A Maternal-fetal comparison of ovine plasma esterases

The esterases of maternal and fetal ovine plasma were characterized and compared early in the third trimester of gestation. Various techniques were used including statistical evaluation of kinetic parameters, gel-filtration and sensitivity to various activators and inhibitors. Maternal esterase activity, expressed in terms of p-nitrophenyl acetate hydrolysis, was found to be 8-fold higher than fetal activity and although both arylesterase and aliesterase appeared to be present in fetal plasma, their contribution to the total activity was relatively small.

Changes in relaxin and prolactin immunoactivities in ovine plasma following suckling.

Department of Anatomy & Reproductive Biology, School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, U.S.A., and Reproductive Research Section, University of Melbourne, S.S. Cameron Laboratory, Werribee 3030, Australia

Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F during pregnancy in sheep

A specific and sensitive radioimmunoassay is described for 13,14-dihydro-15-keto-prostaglandin F in ovine plasma. Using this assay it has been shown that, in sheep, jugular venous 13,14-dihydro-15-keto-prostaglandin F concentrations increase at parturition and correlate well with concentrations of prostaglandin F in the utero-ovarian vein. It is suggested that uterine prostaglandin F production under these conditions may be assessed by measuring peripheral venous 13,14-dihydro-15-keto-prostaglandin F, thereby avoiding the need for chronic utero-ovarian venous catheters.

Characterization of maternal and fetal ovine plasma cholinesterase

Cholinesterase of maternal and fetal ovine plasma, obtained at 112–115 days of gestation, has been characterized and compared by using various techniques including statistical evaluation of kinetic parameters when using acetyl-, propionyl-, and butyrylthiocholine, gel filtration, polyacrylamide-gel electrophoresis, and sensitivity to various inhibitors. Hydrolysis of acetylthiocholine, the optimum substrate, was found to be two to three times faster in fetal plasma than in maternal plasma, although the Michaelis constants did not differ. In both maternal and fetal plasma, gel filtration yielded a major and a minor peak of cholinesterase activity, whereas electrophoresis yielded six bands of activity, including three major bands. These findings suggested quantitative rather than qualitative differences between the maternal and fetal activity. Despite the quantitative differences, there was no difference in the maternal and fetal cholinesterase sensitivity to inhibition by eserine, diisopropylfluorophosphate, and dichlorvos.

Plasma Esterase Development in the Perinatal Lamb

Ovine plasma cholinesterase and nonspecific esterase activities were determined during the last 45 days of fetal development and first 48 h of extrauterine life and compared to adult activities. Marked differences in the development pattern of these enzymes were found. Fetal plasma cholinesterase activity increased between 102 and 112 days gestation to approximately twice adult values and thereafter progressively decreased to adult levels at 48 h postpartum. In contrast, fetal nonspecific esterase activity at 102 days gestation was only about 8% of the adult activity; a steady increase in this activity was found from 102 days gestation to term. From term to 48 h postpartum there was a marked increase of nonspecific esterase activity but the activity at 48 h was still only about 25% of the adult value.

The first James Russel Goding Memorial Lecture of the Australian Society for Reproductive Biology. The ovine and bovine oestrous cycle. An appreciation of J. R. Goding's contribution to the understanding of the oestrous cycle.

The First James Russell Goding Memorial Lecture of the Australian Society for Reproductive Biology AN APPRECIATION OF J. R. GODING'S CONTRIBUTION TO THE UNDERSTANDING OF THE OESTROUS CYCLE Department of Agriculture, Victoria, S.S. Cameron Laboratory, State Research Farm, Werribee, Victoria 3030, Australia (Received 1st October 1974) Today I honour the memory of James Russell Goding, my past colleague and mentor. I worked closely with Jim Goding throughout a period in his life when he contributed much to our understanding of reproductive physiology. I first met him in the mid 1960s when he and Dr W. Hansel visited the State Research Farm, Werribee. From that day, our friendship quickly developed and continued to grow as we undertook joint research programmes. To us both, it felt as if we were moving from one exciting discovery to the next—as if we were continually 'riding high'. The excitement of measuring LH in ovine plasma,

A radioimmunoassay for ovine calcitonin: an evaluation of calcitonin secretion during gestation, lactation and foetal life.

SUMMARY A radioimmunoassay capable of measuring ovine calcitonin in unextracted ovine plasma has been developed. Guinea-pig antisera to partially purified porcine calcitonin were highly specific for calcitonin and were capable of detecting 5 pg porcine calcitonin. One of these antisera cross-reacted significantly with pure ovine calcitonin and with foetal ovine plasma. The mean basal calcitonin secretion rate for the left thyroid lobe found in sheep foetuses 10 days before term was 3·36 ng/min/kg body weight. An infusion of calcium into the foetus was followed by an increase in the calcitonin secretion rate. The mean fasting calcitonin concentration in samples from the jugular vein of five pregnant ewes 10 days before term was 5·28 ng/ml. An infusion of calcium into the mother induced a sevenfold increase in plasma calcitonin concentration whereas the calcitonin concentration was unchanged in the foetal thyroid venous plasma. At parturition the plasma calcitonin concentration fell in the mother (1·85 ng/ml), and a sharp increase was observed during lactation (11·60 ng/ml). In the lamb from birth to 6 weeks post partum the calcitonin concentration was very high (8·10–10·40 ng/ml).

Proceedings: Radioimmunoassay for gonadotrophin-releasing hormone (GnRH): its application to the measurement of GnRH in ovine plasma.

Proceedings: Radioimmunoassay for gonadotrophin-releasing hormone (GnRH): its application to the measurement of GnRH in ovine plasma.

Assay of cortisol, corticosterone, 11-deoxycortisol and cortisone in ovine peripheral and adrenal venous plasma.

SUMMARY An assay was devised, using Sephadex LH-20 chromatography and competitive protein binding, to measure cortisol, corticosterone, 11-deoxycortisol and cortisone in a single extract of 4 ml peripheral plasma or 50–300 μl adrenal venous plasma in sheep. The method is especially suited for measuring the low concentrations of these steroids in ovine plasma. Mean values (μg/100 ml) in dexamethasone-suppressed plasma were cortisol, 0·042; corticosterone, 0·004; 11-deoxycortisol, 0·004; and cortisone, 0·021. Mean values (μg/100 ml) in basal plasma were cortisol, 1·11; corticosterone, 0·044; 11-deoxycortisol, 0·020; and cortisone, 0·24. Peripheral concentrations of all four steroids increased in response to corticotrophin, but none increased after angiotensin. Metyrapone administration produced a marked rise in deoxycortisol. The method provided direct evidence that cortisone and deoxycortisol were secreted by the adrenal gland in sheep.

The binding of oestrogens to ovine serum albumin.

A major factor in determining the metabolic clearance rate (MCRB) (Tait & Burstein, 1964) of many steroids is the extent to which these may be metabolized by the liver, and those steroids which are present in plasma largely as the unbound or albumin-bound moiety have been shown to have a high splanchnic extraction (Baird, Horton, Longcope & Tait, 1969). In conscious sheep MCRB values for oestradiol-17β and oestrone (Challis, Harrison & Heap, 1973 and in preparation) are similar to or greater than hepatic blood flow (Katz & Bergman, 1969; Paterson & Harrison, 1972), and it seemed probable that in plasma these steroids were largely associated with albumin rather than bound by high affinity proteins. The binding of oestrogens to ovine serum albumin (OSA) and ovine plasma has therefore been studied using an equilibrium dialysis technique (Paterson & Hills, 1967). Tritiated oestrogens (oestradiol-17β, E2β; oestrone, E1