bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal function in the chronically cannulated fetal llama: comparison with studies in the ovine fetus
Authors: Wintour EM , Riquelme R , Gaete C , Rabasa C , Sanhueza E , Silver M , Towstoless M , Llanos A
Published: October 2005

Samples of maternal and fetal plasma, fetal urine, and amniotic fluid were collected from 8 chronically cannulated pregnant llamas, in the last third of gestation. The samples were obtained for up to 18 days post-surgery. Osmolality, sodium (Na), potassium (K), chloride (Cl), and urea were measured on 40 samples collected on days 1, 2, 3, 4-5, 6-7, 8-9, and 10-19. The osmolalities of maternal and fetal plasma, fetal urine and amniotic fluid, averaged over these 7 time periods, were, respectively, 312 +/- 2, 311 +/- 1, 484 +/- 14, and 317 +/- 1 mosmol kg-1. Values are given as mean +/- s.e. The major differences from fetal fluid values in the ovine fetus (from previously published values) were the higher osmolality and urea concentration of llama fetal urine. Urine flow rate measured in 6 fetuses, 4.5-6.5 kg body weight, was 5.8 +/- 0.4 mliter h-1; urea clearance rate was 55.5 +/- 11.8 mliter h-1. Glomerular filtration rate (GFR), measured with 51Cr-EDTA in 5 fetuses on 1-4 occasions, was 111.4 +/- 23.3 mliter h-1. Fractional reabsorptions (FR) of Na, K and Cl were 97.9 +/- 1, 75.9 +/- 13.5 and 97.7 +/- 0.4% respectively. The GFR (25 mliter kg-1 h-1) and urine flow rate (1 mL kg-1 h-1) were less than half and about one-tenth the respective values in ovine fetuses. As Na reabsorption is the major oxygen-consuming activity of the kidney, the llama fetal kidney requires only half the oxygen needed by the ovine fetal kidney to reabsorb the filtered sodium load. The reason for the formation of hypertonic, rather than hypotonic, urine in the fetal llama may be due to both greater morphological maturity of the kidney and the excretion of as yet unidentified osmotically active organic substances.
Ontogeny of 11 beta-hydroxysteroid dehydrogenase in ovine fetal kidney and lung
Authors: Wood CE , Srun R
Published: October 2005

Eleven-beta-hydroxysteroid dehydrogenase (11 beta-HSD) is an enzyme which degrades 11-hydroxycorticosteroids to biologically inactive 11-oxocorticosteroids (cortisone and 11-dehydrocorticosterone). In some tissues, the activity of this enzyme prevents binding of cortisol to mineralocorticoid receptors. The present experiments were designed to test the hypothesis that the fetal kidney contains 11 beta-HSD, that the activity of 11 beta-HSD in fetal kidney increases near term, and that the fetal lung does not contain significant 11 beta-HSD activity. In kidney and lung tissue from 23 fetal sheep ranging in age between 86 and 145 days' gestation, we measured 11 beta-HSD activity. We found significant activity in fetal kidney (14-85% conversion from cortisol to cortisone) but no measurable activity in fetal lung (0-9%). The activity of 11 beta-HSD was significantly related to fetal gestational age (r = 0.76, n = 14). We conclude that 11 beta-HSD activity in the fetal kidney develops as a function of fetal gestational age, and that activity cannot be demonstrated in fetal lung. We speculate 11 beta-HSD in the fetus might function to alter the sensitivity of target organs to glucocorticoids, as well as to mineralocorticoids, and that the absence of activity in the lung allows a high sensitivity of pulmonary tissue to cortisol at the end of gestation.
Pre- versus post-ventilatory surfactant treatment in surfactant-deficient preterm lambs
Authors: Cummings JJ , Holm BA , Nickerson PA , Ferguson WH , Egan EA
Published: October 2005

Twenty lambs at 127 days' gestation (term is 145 days) were randomly assigned to receive Infasurf (Calf Lung Surfactant Extract, ONY Inc., Amherst, NY) as an intratracheal bolus (3 mliter kg-1) either into a fluid-filled lung before ventilation (n = 10), or after ventilation for 5 min (n = 10). All lambs were surfactant-deficient by analysis of lung liquid obtained before surfactant administration. Lambs were then mechanically ventilated for 4 h. Oxygenation for the lambs given surfactant before ventilation did not change during the experiment; a/A pO2 was 0.50 +/- 0.13 at 1 h and 0.52 +/- 0.17 at 4 h. For the lambs given surfactant after initial ventilation, oxygenation decreased over time; a/A pO2 decreased from 0.48 +/- 0.23 at 1 h to 0.37 +/- 0.22 at 4 h (P < 0.05). Compliance, as calculated from the Ventilator Efficiency Index (VEI), improved over time in both groups, but was always significantly higher for lambs given surfactant before ventilation (P = 0.03). Histologic examination of the lungs revealed no differences between the groups; no evidence of epithelial denudation or hyaline membrane formation was seen in either group. Thus, ventilation of surfactant-deficient newborn lambs for 5 min before surfactant administration results in significantly decreased lung function when compared with surfactant administration before ventilation. These differences in lung function are not dependent on a histopathologic injury to the lung. It is possible that unevenness of deposition of the surfactant in an air-filled lung, compared to more uniform deposition in a fluid-filled unventilated lung, produces these differences.
Effect of exposure in vitro to ethanol and hypoxia on gamma-aminobutyric acid efflux in the hippocampus of the fetal and adult guinea-pig
Authors: Catlin MC , Penning DH , Brien JF
Published: October 2005

The objective of this study was to determine the effects of acute direct exposure to ethanol, hypoxia or ethanol plus hypoxia on K+-stimulated gamma-aminobutyric acid (GABA) efflux (neuronal release minus uptake) in the hippocampus of the near-term fetal and adult guinea-pig. Transverse hippocampal slices were studied in a static-interface system. Exposure in vitro to ethanol or hypoxia involved 10-min incubation with 50 mM ethanol or 10-min incubation in a 95% N2/5% CO2 environment. GABA was quantitated by HPLC. Ethanol did not alter K+-stimulated GABA efflux; hypoxia augmented K+-stimulated GABA efflux three-fold in the near-term fetus and seven-fold in the adult; concurrent exposure to ethanol did not alter the effect of hypoxia. The data demonstrate that, for acute direct exposure to hypoxia and/or ethanol, only hypoxia increases K+-stimulated GABA efflux, the magnitude of which is dependent on the extent of development of the GABA system.
The developmental switch in ventricular myosin expression in vivo is not triggered by an increase in cyclic AMP
Authors: Schachat F , Seidler FJ , Slotkin TA
Published: October 2005

Ventricular myosin heavy chain (HC) expression undergoes a rapid change from the beta to the alpha isoform shortly after birth. Thyroid hormone is required for this transition to occur, but the time course of developmental changes in circulating thyroid hormone levels suggests that it cannot be the trigger for this event. In this study, the possibility was examined that cyclic AMP (cAMP), either acting separately or as a mediator of the permissive actions of thyroid hormone, triggers the developmental transition in ventricular myosin HC expression. One-day-old euthyroid or propylthiouracil-hypothyroid rat pups were treated with a membrane-permeable cAMP analogue, 8-bromo-cAMP (8-Br-cAMP) or triiodothyronine (T3). Two and four hours after acute treatment, the relative synthetic rates of alpha and beta myosin HC were measured using an in vivo pulse labelling technique. Myosin HC isoforms were separated electrophoretically and then quantitated by densitometry. Acute treatment in vivo with 8-Br-cAMP did not alter the relative rate of alpha myosin HC synthesis in euthyroid animals at either 2 or 4 h. Hypothyroidism significantly reduced the relative rate of alpha HC synthesis and obturated the transition from beta to alpha HC. The effect on synthesis was rapidly reversed by acute treatment with T3, but not with 8-Br-cAMP. Thus, an increase in cAMP does not appear to trigger the developmental change in myosin isoform expression, nor does it appear to mediate the stimulatory effect of thyroid hormone on alpha myosin HC synthesis. Instead, thyroid hormone is likely to be acting directly on the alpha HC gene by binding to the thyroid response element. The identity of stimuli that mediate the increased responsiveness to thyroid hormone during development remains to be determined, but it is not due to an increase in the levels of cAMP in perinatal cardiocytes in vivo.
Effects of hypercarbia on autoregulation of brain blood flow and cerebral metabolism in newborn piglets
Authors: Stonestreet BS , Barefield ES , Piva D , Goldstein M
Published: October 2005

We tested the hypothesis that, in newborn piglets, hypercarbia impairs autoregulation of total and regional brain blood flow at the lower limb of the autoregulatory curve. Cerebral oxygen metabolism was measured in the same piglets to relate changes in metabolism to blood flow. Instrumented hypercarbic (n = 9) and normocarbic (n = 8) newborn piglets exposed to phlebotomy were studied during normotension and graded hypotension with mean arterial blood pressures of 55-41, 40-31 and < 30 mmHg. In the hypercarbic piglets, total brain blood flow decreased (P < 0.01) from the hypercarbic-normotensive value of 187 +/- 15 mliter min-1 100 g-1 to 139 +/- 18, 66 +/- 11 and 34 +/- 6 at mean arterial blood pressures of 55-41, 40-31 and < 30 mmHg, respectively; in the normocarbic piglets, total brain blood flow did not change from the normotensive value (70 +/- 11 mliter min-1 100 g-1) until the mean arterial blood pressure was < 30 mmHg, when brain blood flow had decreased (P < 0.01) to 49 +/- 8 mliter min-1 100 g-1. In the hypercarbic piglets, all brain regions (cerebrum, caudate nucleus, cerebellum, brainstem and medulla) demonstrated similar response patterns to that of total brain blood flow during hypotension. Thus, during hypercarbia, none of the brain regions demonstrated autoregulation. In the normocarbic piglets, cerebral blood flow decreased (P < 0.01) from the normocarbic-normotensive value of 74 +/- 6 mloter min-1 100 g-1 to 51 +/- 8 and 37 +/- 7 at mean arterial blood pressures of 40-31 and < 30 mmHg, respectively, and blood flow to the caudate nucleus, cerebellum and brainstem did not decrease significantly, and in fact increased (P < 0.01) to the medulla during hypotension. Although cerebral oxygen metabolism was compromised in the hypercarbic and normocarbic piglets, the relationship between metabolism and blood flow was altered such that the cerebral metabolic rate of oxygen per unit of blood flow was lower in the hypercarbic than the normocarbic piglets. We conclude that hypercarbia impairs total and regional brain blood flow autoregulation in newborn piglets.
Current studies on human implantation: a brief overview
Authors: Rogers PA
Published: October 2005

Embryo implantation involves a series of complex interactions between the developing embryo and the maternal endometrium. Results of studies with animal models suggest that the uterus must undergo a series of morphological and biochemical changes, mediated primarily by oestrogen and progesterone, before it becomes receptive for successful implantation. At present there is little understanding of the endometrial changes required to achieve endometrial receptivity for implantation in the human. It appears that control of receptivity is not as stringent in the human as in some other species, with IVF data suggesting that the duration of receptivity is at least 4 days, and that successful implantation can occur under a relatively wide range of morphological and ultrastructural conditions. Research on the later stages of implantation, including embryo positioning within the uterus, attachment and invasion, has been almost non-existent in the human. Further studies are critical for a better understanding of this complex process, although human studies will always be limited by ethical constraints.
Role of epidermal growth factor (EGF) and its receptor in the development of the human placenta
Authors: Maruo T , Matsuo H , Otani T , Mochizuki M
Published: October 2005

To elucidate the role of EGF in human placental development, effects of EGF on the proliferation and differentiation of trophoblasts were investigated. Explants of trophoblastic tissues obtained from 4-5 week or 6-12 week placentas were, respectively, cultured with or without EGF, in the presence or absence of triiodo-L-thyronine (T3) in a serum-free condition. The proliferative activity was examined by immunocytochemical staining with an antibody Ki-67, and the differentiated function was assessed by the ability to secrete human chorionic gonadotrophin (hCG) and human placental lactogen (hPL). In 4-5 week placentas, EGF and EGF receptor were localized in cytotrophoblast (C-cell), and EGF augmented the proliferation of C-cell without affecting the ability to secrete hCG and hPL. In contrast, in 6-12 week placentas, EGF and EGF receptor were localized in syncytiotrophoblast (S-cell), and EGF stimulated the secretion of hCG and hPL without affecting the proliferation of C-cell. In situ hybridization with c-erb B probe revealed that c-erb B mRNA is expressed in the S-cell after 6 weeks' gestation. Column chromatography of the serum-free media obtained by 5-day culture of early placental tissues resulted in the elution of immunoreactive EGF. The addition of T3 (10(-8) mol L(-1)) resulted in increased secretion of immunoreactive EGF by placental explants. These findings suggest that EGF acts as an autocrine factor in regulating early placental growth and function in synergy with thyroid hormone.
Secretory type II phospholipase A2 and the generation of intrauterine signals
Authors: Rice GE
Published: October 2005

Recent advances in the understanding of the metabolism of cell membrane glycerophospholipids and the subsequent generation of biologically active metabolites have provided new insight into their role in normal spontaneous-onset labour and delivery at term, and in preterm labour. In particular, multiple isozymes of the lipolytic enzyme phospholipase A2 (PLA2) have been identified in human gestational tissue during late pregnancy and at the time of labour. Specific isozymes have been identified that are active intracellularly (e.g. cytosolic PLA2) or extracellularly (e.g. Type II and Type IV secretory PLA2). The relative contribution of these isozymes to total PLA2 activity is tissue specific and affected by labour status. The development of isozyme-specific inhibitors of PLA2 may afford the opportunity to better regulate the formation of biologically active phospholipid metabolites within intrauterine compartments. With the identification of secretory PLA2 isozymes in human gestational tissues, a new pathway for materno-fetal and/or intrauterine communication has been established--a pathway in which secretory PLA2 isozymes released by intrauterine tissues act in an autocrine, paracrine or even endocrine fashion to affect glycerophospholipid metabolism, cell membrane fluidity and the generation of biological mediators.
Drug delivery during pregnancy: evaluation in vitro of new drugs
Authors: Dancis J , Liebes L
Published: October 2005

Indications for the treatment of the pregnant woman fall into three general categories: mother and infant require treatment, only the mother should be treated, or only the infant. Directing therapy towards the affected subject is an important aspect of good care, although it is not the only one. It is argued that a rational selection of the appropriate drug can be made without endangering mother or infant, with the aid of select laboratory investigations, including placental perfusion, and a knowledge of placental physiology. Examples are presented to support this contention. The intra-amniotic administration of drugs is briefly discussed. A plea is made to develop drugs that are designed specifically for use during pregnancy.
Transport and metabolism of adenosine in diabetic human placenta
Authors: Osses N , Sobrevia L , Cordova C , Jarvis SM , Yudilevich DL
Published: October 2005

Pregnancy complicated by diabetes is a relatively frequent event and may result in fetal embriopathy. However, little is known regarding whether placental transport functions are altered. In this study, we have compared the activity of the nitrobenzylthioinosine (NBMPR)-sensitive adenosine transporter and adenosine metabolism in human placental brush-border- and basal-membrane vesicles from placentas of normal and diabetic pregnancies. Neither [3H]NBMPR binding, a marker of the facilitative-diffusion nucleoside transporter in the human placenta, nor adenosine metabolism exhibited a significant difference in either the brush-border- or the basal-membrane vesicles between the normal and diabetic group, except for an increased affinity in [3H]NBMPR binding at the basal side in diabetic placenta. This result contrasts with an earlier finding using the same group of patients that adenosine transport is downregulated in umbilical vein endothelial cells from diabetic pregnancies. It is concluded that adenosine transport is modulated selectively in different tissues in diabetic pregnancies.
Human placental and fetal membrane nitric oxide synthase activity before, during and after labour at term
Authors: Di Iulio JL , Gude NM , King RG , Brennecke SP
Published: October 2005

The aim of this study was to determine whether any labour-associated changes in nitric oxide synthase (NOS) activity occur in human placenta and fetal membranes. NOS activity in amnion, choriodecidua, and placenta obtained from women before (at Caesarean section, not in labour), during (at Caesarean section, in labour) and after (spontaneous onset labour, normal vaginal delivery) labour was assessed by measuring conversion of radio-labelled L-arginine to L-citrulline. NOS activity, as judged by its inhibition by the specific NOS inhibitor N omega-nitro-L-arginine, was present in placental and amnionic tissues, but not in choriodecidual tissue specimens. Activity detected in choriodecidua was significantly blocked during incubation with a high concentration of valine, suggesting that L-arginine was being consumed by reactions other than NOS under the experimental conditions in that tissue. There were no significant differences among the labour groups in either amnion or placental NOS activities measured in the presence of 1 microM L-arginine. Amnion NOS activity was significantly less than that in placenta. Placental V(max) and Km values (determined after removal of endogenous L-arginine) did not differ significantly among the different labour groups.
Human placental cytochrome P450 and quinone reductase enzyme induction in relation to maternal smoking
Authors: Boden AG , Bush PG , Burke MD , Abramovich DR , Aggett P , Mayhew TM , Page KR
Published: October 2005

Components of cigarette smoke such as cadmium and polycyclic aromatic hydrocarbons have been shown to induce quinone reductase (QR) activity in placental explants. This study examines the relationship of maternal smoking habit and maternal plasma cotinine concentration with the activities in vitro of both QR and the cytochrome P450 (CYP1A) marker ethoxyresorufin O-deethylase (EROD) in placental tissue. Maternal plasma samples were taken at Week 34 of gestation, and placental tissues were obtained at term. Plasma cotinine concentrations were determined by high-performance liquid chromatography. Trophoblast cytosolic QR and microsomal EROD activities were measured by resazurin reduction and ethoxyresorufin O-dealkylation respectively. QR activity was inhibited 70% by a mixture of dicoumarol (1 microM) and rutin (20 microM). Plasma cotinine concentrations correlated significantly (P < 0.001) with both declared smoking rate (r = 0.67, N = 37) and placental EROD activity (r = 0.63, N = 36), but not with QR activity, whether measured as total QR activity or specifically as either DT-diaphorase or carbonyl reductase. It is concluded that smoking up to 40 cigarettes per day induces EROD but does not affect QR activity in the placenta at term.
Effect of asymmetric dimethyl arginine on nitric oxide synthase activity in normal and pre-eclamptic placentae
Authors: King RG , Di Iulio JL , Gude NM , Brennecke SP
Published: October 2005

An endogenous inhibitor of nitric oxide synthase (NOS), NG,NG dimethylarginine (asymmetric dimethylarginine, ADMA), which is present in human plasma and urine, has been reported to be elevated in the plasma of women with pre-eclampsia. As ADMA inhibition may contribute to reduced placental NOS activity observed in pre-eclampsia, the aim of this study was to compare the effects of ADMA on placental NOS activity from pre-eclamptic and normal pregnancies (gestational ages 38.4 +/- 0.9 and 38.3 +/- 0.3 weeks respectively). NOS activity was determined by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in homogenates of normal and pre-eclamptic placentae in the absence and presence of increasing concentrations of ADMA (1-100 microM). The IC50 for ADMA for the pre-eclamptic placentae (22.1 +/- 2.1 microM, n = 6) was not significantly different from that for the normal placentae (18.8 +/- 1.4 microM, n = 6). When ADMA and L-arginine in homogenates was removed by ion exchange chromatography and exogenous L-arginine replaced (32 microM), the IC50 for the pre-eclamptic placentae (19.5 +/- 1.8 microM, n = 6) was not significantly different than that for the normal placentae (20.9 +/- 1.0 microM, n = 6), and NOS activity in the absence of endogenous and exogenous ADMA was still reduced in pre-eclamptic placentae. These results provide no evidence that the sensitivity of placental NOS to ADMA is affected by pre-eclampsia, or that placental ADMA contributes to the reduction of placental NOS in pre-eclampsia.
Optical imaging of the ventral medullary surface of developing kittens during ventilatory challenges
Authors: Gozal D , Dong XW , Rector DM , Harper RK , Harper RM
Published: October 2005

We used large-array optical recording procedures to examine maturation of regional neural activity within the ventral medullary surface (VMS) of anaesthetized kittens during graded hypercapnic and hypoxic challenges. The VMS was exposed through a ventral surgical approach in 10, 20, 30, and 45-day-old kittens and in adult cats under sodium pentobarbital anaesthesia. Arterial pressure, costal diaphragmatic EMG, and ECG were continuously monitored. A coherent image conduit with 12 mu fibre resolution was attached to a charge-coupled-device camera and positioned over the VMS. Reflected 660 nm light was digitized continuously at 2-s intervals during a baseline period, hyperoxic hypercapnia, (3, 5, and 10% CO2 in O2), and poikylocapnic hypoxia (6%, 9%, and 12% O2 in N2), and recovery. Sixty to seventy-five images within each epoch were averaged, and subtracted from baseline. Regional differences within the image were determined by ANOVA procedures (alpha = 0.05). During hypercapnia, an overall decrease in neural activity (increase in scattered light) occurred, which was marginally age-dependent. By 30 days, regional bidirectional reflectance changes in response to CO2 emerged in a small proportion of animals, and were similar to adult responses. Hypoxia induced a dose- and age-dependent decrease in overall scattered light. Transient 'on' and 'off' responses were common under both ventilatory stimuli. In 20-30-day kittens, marked rebound responses in reflectance accompanied cessation of hypoxic stimuli; such patterns were absent at other ages. At 30 days, a caudal-rostral bidirectionality in response to mild hypoxia (12% O2) began to emerge in a subset of animals. We conclude that dose-dependent response to ventilatory stimuli occur in the VMS at all post-natal ages of the kitten; however, in hypoxia, the magnitude of the overall reflectance changes is diminished relative to adult patterns. Rebound responses to hypoxia are present at particular ages, and older kittens begin to show a topographical organization of neural activation.
Changes in M-CSF-like activity during chicken embryonic development
Authors: Shao X , Kikuchi K , Watari E , Norose Y , Araki T , Yokomuro K
Published: October 2005

We have established a method for separation of chicken bone-marrow cells using Percoll density gradient centrifugation, and have developed a new method for determining chicken M-CSF-like activity employing a liquid culture. Using this method, we determined M-CSF-like activities in egg yolk, chorioallantoic fluid (CAF) and amniotic fluid (AmF), and studied the effects of M-CSF on development of chicken embryos. M-CSF-like activity in egg yolk was at a high level before the incubation of the egg; it began to decrease on the third day of incubation and rapidly decreased on the fourth day, and no significant activity was detected after the tenth day of incubation. M-CSF-like activity in CAF was very low, and it exhibited almost no change during development. No M-CSF-like activity was detected in AmF throughout the experimental period.
Changes in thoracic dimensions induced by breathing movements in fetal sheep
Authors: Harding R , Liggins GC
Published: October 2005

The dimensions of the three major axes of the thorax (longitudinal, transverse and antero-posterior) were monitored in utero from the ultrasonic transit-time between pairs of piezo-electric transducers chronically implanted at opposite sides of the thorax in five fetal sheep at 119-122 days of gestation; tracheal and vascular catheters and diaphragmatic EMG electrodes were also implanted. To measure thoracic length, ultrasound transducers were implanted on the diaphragm and over the upper sternum and manubrium sterni. A pair was implanted on opposite sides of the chest to measure thoracic width, and another pair was implanted over the lower thoracic spine and lower sternum (antero-posterior dimension). The width of the thorax either decreased (mean 0.6 +/- 0.2 mm) or increased (mean 0.7 +/- 0.1 mm) during fetal breathing movements (FBM). The depth of the thorax (antero-posterior dimension) usually decreased (mean 0.9 +/- 0.1 mm) during FBM. The distance between the upper sternum and the diaphragm decreased by 1.0 +/- 0.1 mm (left side) and 1.6 +/- 0.3 mm (right side) during inspiratory efforts. The distance between the upper thorax and a fixed point on the lower thoracic spine decreased by a similar amount. Overall, fetal thoracic dimensions changed by 1-3%. The largest changes measured, and the most surprising, were reductions, rather than increases, in the separation between the dome of the diaphragm and the upper thorax; this suggests that, during inspiratory efforts, the upper thorax moves caudally by a greater distance than the diaphragm. FBM induce complex and variable changes in thoracic dimensions; these are likely to induce small alterations in the shape of the lungs that may act as a stimulus to lung growth.
Cerebral, tympanic and colonic thermometry in the piglet
Authors: Haaland K , Steen PA , Thoresen M
Published: October 2005

The temperature of the brain is crucial for the outcome of hypoxic/ischaemic brain damage. In clinical medicine and in animal experiments involving survival after hypoxia/ischaemia, non-invasive measurement of cerebral temperature is needed. We have therefore compared tympanic and colonic temperature with cerebral temperature in the newborn piglet during hypothermia. Ten piglets aged 12-60 h were cooled to 35 degrees C (mild hypothermia) for 150 min and rewarmed. Thereafter, four of the piglets were again cooled to approximately 29 degrees C for less than one hour (moderate hypothermia). During stable mild hypothermia and normothermia the cerebro-tympanic temperature difference in individual piglets was less than +/- 0.4 degrees C (95% confidence intervals < or = 0.18 degrees C) and the cerebro-colonic temperature difference was -0.7 to 0.4 (95% confidence interval < or = 0.28 degrees C). The differences were larger during moderate than during mild hypothermia and largest during rapid changes in body temperature. Then the tympanic temperature correlated with the cerebral temperature significantly better than did the colonic temperature (95% confidence interval -0.3 to 0.3 versus -0.6 to 1.4 for the ten minutes with the least good correlation).
Brown adipose tissue and liver development during early postnatal life in hand-reared and ewe-reared lambs
Authors: Darby CJ , Clarke L , Lomax MA , Symonds ME
Published: October 2005

This study examined the effects of modest changes in ambient temperature in hand-reared lambs (experiment one) and in ewe-reared lambs (experiment two). Lambs were killed at either 8 or 31 days of age and perirenal adipose tissue was identified as being brown adipose tissue (BAT) from measurements of thermogenic activity (i.e. GDP binding to uncoupling protein in isolated mitochondria) or thermogenic capacity (i.e. detection of uncoupling protein by immunoblotting). In addition, type I and II iodothyronine 5' monodeiodonase (5'MDI) activities were assayed in perirenal adipose tissue, plus type I 5'MDI activity in liver. Plasma samples were also taken for measurements of glucose, lactate, insulin, triiodothyronine (T3) and thyroxine (T4) concentrations. In experiment one, lambs were hand-reared at either warm (WR; 25 degrees C) or cool (CR; 10-15 degrees C) ambient temperatures. Mean growth rate over the first 8 days of life in CR lambs was 88 g/day and increased to 128 g/day over the first month of life. Growth rate in WR lambs was constant at 141 g/day. Thermogenic activity of BAT was significantly higher in CR than WR lambs, but total weight and tissue lipid content of perirenal adipose tissue were significantly lower in the CR group. In both WR and CR lambs, the thermogenic activity of BAT fell by an average of 71% between 8 and 31 days. At 31 days of age, uncoupling protein in mitochondria could be detected only by immunoblotting in adipose tissue sampled from CR lambs. There was no effect of ambient temperature on type I or type II 5'MDI activity in BAT or liver; it decreased in adipose but not liver tissue between 8 and 31 days of age. The plasma concentrations of glucose, insulin and T3 tended to decline with age in CR but not in WR lambs. In ewe-reared lambs perirenal adipose tissue weight and tissue lipid content more than doubled between 8 and 31 days of age, but the level of GDP binding decreased from 85 to 5 pmol/mg mitochondrial protein over this period. Liver weight increased by 55% between 8 and 31 days of age, but hepatic 5'MDI activity remained unchanged. The plasma concentrations of T3, T4 and lactate, but not glucose or insulin, increased between 8 and 31 days of age. It is concluded that hand-rearing lambs at a cool ambient temperature significantly delays postnatal development, to the extent that BAT characteristics are retained. Ewe-rearing lambs enhances the rate at which BAT adopts the characteristics of white adipose tissue, and it prevents the postnatal decline in plasma concentrations of thyroid hormones.
Fetal insulin-like growth factor (IGF)-I and IGF-II are regulated differently by glucose or insulin in the sheep fetus
Authors: Oliver MH , Harding JE , Breier BH , Gluckman PD
Published: October 2005

We investigated the effect of restoration of normoglycaemia or normoinsulinaemia in fetuses of starved ewes on plasma IGF-I and IGF-II concentrations. Paired maternal and fetal blood samples were taken during an initial 2-day control period, after 48 h of maternal starvation, during 24 h fetal infusion of glucose (n = 6) or insulin (n = 4) while maintaining maternal starvation and after 48 h maternal refeeding. After 48 h starvation maternal and fetal plasma IGF-I, insulin and blood glucose fell (maternal IGF-I 38.9 +/- 3.6 to 16.4 +/- 1.8 nM and fetal IGF-I 13.2 +/- 0.8 to 7.1 +/- 0.7 nM, both P < 0.05). Fetal plasma IGF-II also fell (147.8 +/- 9.1 to 112.2 +/- 3.8 nM, P < 0.05), but maternal plasma IGF-II rose (71.8 +/- 6.3 to 88.8 +/- 9.2 nM, P = 0.10). Fetal glucose replacement raised fetal plasma IGF-I (11.4 +/- 1.2 nM), IGF-II (149.7 +/- 6.5 nM), insulin and blood glucose to near control values (all P < 0.05). Fetal insulin replacement raised fetal plasma IGF-I (9.0 +/- 0.6 nM) and insulin (all P < 0.05) while IGF-II (105.2 +/- 8.4 nM) and blood glucose remained depressed. Neither fetal infusion had any significant effect on maternal plasma IGF-I (13.1 +/- 1.6 nM), IGF-II (77.5 +/- 8.7 nM), insulin or blood glucose. After 48 h maternal refeeding fetal IGF-I (12.4 +/- 0.4 nM), fetal IGF-II (158.4 +/- 8.9 nM), maternal IGF-II (67.1 +/- 3.0 nM), maternal and fetal insulin and glucose had returned to near control values in both groups. Maternal IGF-I remained below control values (24.7 +/- 2.5 nM, P < 0.05). The data suggest that fetal IGF-I and IGF-II are independently regulated in the fetal circulation. While glucose plays an important role in the regulation of both IGF-I and IGF-II, the influence of glucose on fetal IGF-I is likely to be mediated by insulin, whereas for IGF-II the effect of glucose is insulin-independent.
ACTH-like bioactivity and immunoactivity in fetal lamb pituitaries at 0.65 and 0.95 gestation
Authors: Fora MA , Valego NK , Lively MO , Castro MI , Rose JC
Published: October 2005

We wished to determine if the concentration of bioactive ACTH-like activity increased during development and if there was heterogeneity in ovine fetal anterior pituitary ACTH activity as measured by bioassay and radioimmunoassay (RIA). We obtained anterior pituitaries from eight sheep fetuses (four at 0.65 and four at 0.95 gestation; term 145 +/- 5 days) and extracted and homogenized them in ice-cold 5N acetic acid, 0.3% phenylmethanesulfonyl fluoride (PMSF) and 0.2% BSA. Fractionation of each pituitary extract was performed by size-exclusion chromatography using Sepadex G-50. The ACTH-like immunoactivity (ALI) profile for each pituitary showed two well-defined peaks. One eluted with human ACTH1-39 and the other eluted with the high molecular weight fraction in the void volume. Four fractions from the first peak representing the high molecular weight forms of ACTH activity and four fractions from the second peak representing the low molecular weight forms of ACTH activity were pooled separately. These two pools were subjected to reverse-phase chromatography (RPC) on a C-8 column using a linear gradient of 70% acetonitrile in 0.8% trifluoroacetic acid over a 60 min period. Based upon the RIA, the high molecular weight forms of ACTH from the G-50 column were resolved into three main fractions, one eluting similar to the standard ACTH1-39 and the remaining two eluting after that. The low molecular weight forms of ACTH from the G-50 column were resolved into three peaks, before, with, and after the standard. We used collagenase-dispersed rat adrenal cells to test the ACTH-like bioactivity (ALB) of the crude extracts and of the different fractions obtained from the RPC of the high and low molecular weight material. The concentration of ACTH-like bioactivity in the crude extracts was similar at the two stages of gestation. However, there was a trend for the low molecular weight peak to have more peptide eluting with human ACTH1-39 and higher ratios of ALB/ALI than did the high molecular weight peak. These results suggest that multiple ACTH molecular forms with different ALB/ALI ratios are present in the ovine fetal pituitary and that there is no selective increase in ACTH1-39 concentration in the fetal pituitary in late gestation.
Optimization of a continuous real-time computerized semen analysis system for ram sperm motility assessment, and evaluation of four methods of semen preparation
Authors: Holt WV , Palomo MJ
Published: October 2005

Sampling conditions that affect the biological validity of computer-assisted analysis of ram sperm motion were examined using a continuous real-time computerized semen analysis system (Hobson Sperm Tracker). Search radius (SR, 10 settings) and minimum track point (MTP, 10 settings) were varied factorially to evaluate their effects on the inclusion of sperm subpopulations within derived datasets. Low SR (< 12 microns) or high MTP values (> 26 frames) precluded measurements of rapidly moving cells, whereas high SR (> 17 microns) and low MTP settings (< 22 frames) led to erroneous tracking and poor data quality. Suitable settings for these set-up parameters were derived and tested for their biological consistency with four methods of preparing ram semen for computerized analysis. The preparation techniques tested were: centrifugation through sucrose-based Ficoll and Percoll media, a swim-up technique and simple dilution in Tyrode's media. The 'selective' Percoll and swim-up methods generated sperm populations with significantly higher linear velocities and a lower tendency to deviate from linear trajectories than from either the Ficoll method or dilution technique. Deleterious effects of centrifugation were evident, particularly on sperm survival in vitro over several hours. It is concluded that computer-assisted semen analysis provides useful information about the behaviour of ram spermatozoa in vitro, but the measurement conditions must be defined carefully.
The relationship between chromosomal abnormality in the human preimplantation embryo and development in vitro
Authors: Almeida PA , Bolton VN
Published: October 2005

The relationship between the survival of the human preimplantation embryo in vitro and chromosomal abnormality was investigated by cytogenetic analysis of a total of 250 embryos of varying morphology between the pronucleate stage and the 8-cell stage. The overall incidence of chromosomal abnormality among these embryos was 49%. At the pronucleate stage (n = 46) the incidence was 65.2%, at the 2-4-cell stage (n = 126) it was 54.6%, and at the 5-8-cell stage (n = 78) it was 27.4%. Cleavage-stage embryos with poor morphology (irregular shaped blastomeres with severe extracellular fragmentation) showed a higher incidence of chromosomal abnormality (62%; 54 of 87 analysed) than those with good morphology (22.2%; 26 of 117 analysed). This study demonstrates: (i) that there is progressive loss of chromosomally-abnormal embryos during preimplantation development; and (ii) that there is an association between chromosomal abnormality and embryo morphology.
Intracellular calcium in cultured rabbit oviduct epithelial cells
Authors: Dickens CJ , Cox CI , Leese HJ
Published: October 2005

Oviduct fluid is the medium in which fertilization and early embryonic development occur but little is known about the ionic basis of fluid secretion or its control. Since calcium ions (Ca2+) are involved in the mechanism of secretion in other epithelia, the intracellular calcium concentration ([Ca2+]i) was measured in single, rabbit oviduct epithelial cells in primary culture using the fluorescent dye Fura-2. The resting [Ca2+]i was constant (115 nM) in cells cultured for 2-7 days. Ion substitution experiments demonstrated the presence of a Na+/Ca(2+)-exchange system in the plasma membrane, whereas influx through channels was found to have only a minor role maintaining the resting [Ca2+]i. The addition of dibutyryl cAMP (db cAMP) induced two types of response: the first was an increase in [Ca2+]i, dependent on the presence of extracellular Ca2+, and the second was a zero response. Extracellular ATP induced a transient increase in [Ca2+]i owing to the release of Ca2+ from intracellular stores and Ca2+ entering the cell across the plasma membrane. It is proposed that these effects may be due to the presence of two types of cell in culture-the ciliated and non-ciliated (secretory type) oviduct epithelial cells.
Bovine granulosa cells express extracellular matrix proteins and their regulators during luteinization in culture
Authors: Zhao Y , Luck MR
Published: October 2005

This study investigated the ability of bovine granulosa cells to express and secrete collagen, metalloproteinase (MMP) activity and a tissue inhibitor of metalloproteinase (TIMP-1) during luteinization in vitro. Cells from mature (1-2 mL fluid volume) bovine follicles were cultured over 4 days in serum-free medium. Their luteinization during culture was confirmed by a 10-fold increase in progesterone secretion. Samples of cell extracts, culture media and follicular fluid were subjected to Western blotting to identify secreted proteins and to gelatin zymography to detect enzyme activity. Poly A+ RNA, isolated from cells before and after culture, was probed to detect expression of collagen alpha 1(I), collagen alpha 3(IV) and TIMP-1. The results revealed that: (1) the collagen alpha 1(I) subunit gene was expressed in cells before culture but with greater intensity by Day 4 culture; collagen I protein, on the other hand, was not detectable in culture medium; (2) the collagen alpha 3(IV) subunit gene was expressed at a low level in uncultured cells and could be detected on Day 4 of culture; low amounts of the protein were detected in medium; (3) a 92-kDa band of gelatinase activity (presumed MMP-9) was present in all medium samples, together with bands of unidentified activity; and (5) the TIMP-1 gene was expressed in uncultured cells but its expression increased markedly up to Day 4 of culture. These results show that granulosa luteinization is associated with an increase in the expression of collagen, collagen-degrading enzymes and TIMP-1. Collagen protein, however, may be only poorly synthesized in this culture model. The results suggest that granulosa-derived cells are a likely source of components of the extracellular matrix during post-ovulatory remodelling of early luteal tissue.
Cryopreservation of germ cells from bovine fetal ovaries
Authors: Lavoir MC , Rumph N , Betteridge KJ , Leibo SP
Published: October 2005

Bovine fetuses at stages required for studies of female germ cells (primordial germ cells and oogonia) become available from the abattoir at unpredictable times. To alleviate this logistical problem, a procedure to cryopreserve these ovarian germ cells has been devised. Fetal ovarian cells were dispersed and suspended in 1.5 M dimethyl sulfoxide (DMSO) prepared in modified TCM 199 medium. The suspensions were aspirated into plastic semen straws, cooled, seeded to induce ice formation at -7 degrees C, and then cooled at 1 degree C min-1 to -70 degrees C before being plunged into liquid nitrogen at -196 degrees C for storage. The straws were thawed at a moderate rate of approximately 250 degrees C min-1, the DMSO was diluted 28-fold with culture medium, and then the cells were cultured for > 2 h before their viability was tested or they were used for nuclear transfer. No statistically significant difference in viability before and after cryopreservation was detected by vital staining with fluorescein diacetate (P > 0.05). When frozen-thawed germ cells were fused to cytoplasts, the cleavage rate of the resultant reconstructed embryos 44 h after fusion was 31%, although none developed into blastocysts. It is concluded that cryopreservation of bovine fetal ovarian germ cells is feasible and can play a major role in facilitating future experimentation.
Relationships between follicle-stimulating hormone, follicle growth and ovulation rate in sheep
Authors: Fry RC , Driancourt MA
Published: October 2005

The changes in follicle-stimulating hormone (FSH) concentration required to affect follicle growth and ovulation rate within individual ewes were examined. Relationships between peripheral FSH concentrations during the late-luteal and follicular phase and subsequent ovulation rates were investigated in 22 ewes from 4 breeds over 3 successive cycles (Experiment 1). Ewes were grouped as follows: Group 1 (n = 6), ewes exhibiting the same ovulation rate at each oestrous cycle: Group 2 (n = 5), ewes with three different ovulation rates at each oestrous cycle; and Group 3 (n = 11), ewes with the same ovulation rate at two oestrous cycles and a different ovulation rate on one occasion. Data from ewes in Group 1 and 3 provided estimates on the variation in FSH concentrations between cycles which were not large enough to alter ovulation rate (range, 0-67% variation in FSH concentration). In Group-2 ewes, there was no consistent association between increases in ovulation rate and the proportional increases in FSH concentrations. Differences in FSH concentrations were often less than those that did not alter ovulation rate in Group-I ewes. Furthermore, only 3 of 11 Group-3 ewes demonstrated high FSH concentrations associated with high ovulation rate (or low FSH concentrations and low ovulation rate) when compared with the concentrations found at the two cycles in which ovulation rate was similar. Hence, there was little evidence that FSH concentrations during the late-luteal and follicular phase are associated with changes in ovulation rate within individual ewes. In Experiment 2, follicles of similar size obtained from the same ewe (FecBFec+ and Romanov) showed markedly different responses in vitro to graded doses of FSH as measured by aromatase activity. It is concluded that, within a ewe, the large variability between gonadotrophin-dependent follicles in their requirement for FSH prevented the expression of any thresholds of ovarian response to FSH.
Fetal gastrointestinal composition: implications for water and electrolyte absorption
Authors: Sherman DJ , Ervin MG , Ross MG
Published: October 2005

In the late-gestation human or ovine fetus, daily swallowed volumes (100-300 ml/kg body weight) are markedly greater than adult values (40-60 ml/kg). This volume of amniotic fluid water, electrolytes and cellular debris is almost completely resorbed within the fetal gastrointestinal (GI) tract, resulting in the formation of meconium within the distal fetal colon. Despite this capacity for fetal GI water resorption, little is known regarding the sites or processes by which absorption occurs. We examined the composition of GI contents of 13 ovine fetuses (121-148 days). Fetuses were operatively delivered and killed and the Gi tract (rumen to sigmoid colon) was ligated in sections. Luminal samples were obtained and analysed for water and organic content and osmolality and electrolyte composition. The water content significantly decreased (98.7% to 70.1%) while the organic content significantly increased (55.0% to 95.3% of dry weight) from the rumen to colon. Osmolality significantly increased from the rumen to the distal small intestine (303 +/- 3 to 596 +/- 50 mOsm/kg) and returned to near isotonic values in the sigmoid colon. These results indicate significant water absorption within the fetal GI tract, with a pattern similar to that of the adult. However, fetal colonic sodium absorption is either suppresses or immature near term.