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Animal studies
A low protein diet throughout pregnancy in rats results in increased blood pressure (Langley and Jackson, 1994) and impaired glucose tolerance (Dahri et al., 1991) in the offspring. A 35% overall reduction in intake in rats in late pregnancy causes fetal ß cell hypertrophy and increased insulin response to glucose in vitro, but not if the undernutrition occurred in early pregnancy (Alvarez et al., 1997). In guinea pigs, a two day fast in late gestation changes fetal hypothalamic GR expression (Lingas et al., 1999), and moderate undernutrition throughout pregnancy results in insulin resistance (Kind et al., 2003). In contrast to studies in small mammals with short gestations, studies in sheep allow investigation of discrete episodes of undernutrition in early, mid or late gestation. In addition to decreasing birthweight in sheep (Luther et al., 2005), late-gestation maternal undernutrition alters fetal somatotrophic axis regulation (Bauer et al., 1995) and pituitary and adrenal responses to hypoglycaemia (Edwards et al., 2001).
Postmortem
On completion of the studies the ewes were killed with an overdose of pentobarbitone (25 mL of 300 mg.mL-1 ). The uterus was removed and placed in a container that had been tared on the scales with two incontinence sheets. For singleton pregnancies, the fetus and uterus were dried with the sheets, which were weighed with the amniotic fluid. The following measurements were taken of the fetus: weight, forelimb and hindlimb lengths, chest girth, abdominal girth, crown-rump length, and biparietal diameter. In addition, the lengths of growth catheters from sternum attachment to spine were measured. The fetal organs were then rapidly dissected out and weighed, with tissues collected for histology, molecular biology and in situ studies.
Insulin
Plasma insulin concentrations were measured by specific radioimmunoassay (RIA) established and validated for maternal and fetal sheep plasma. The first antibody, guinea pig anti-ovine insulin (GC4), was diluted in phosphate buffered saline (PBS) (pH 6.2) to a concentration of 1:80000, and combined with the samples, controls, and standards (ovine insulin, Sigma Chemical, St. Louis, MO, batch # I9254). After 24 h incubation, 100 µL of tracer (125I-rhIns, Eli Lilly, Indianapolis, IN, USA) was added and incubated for a further 24 h. The second antibody-PEG complex was then added (0.5% sheep anti-guinea pig gamma globulin, 0.1% normal guinea pig serum, and 8% polyethylene glycol (PEG) 6000 in 0.01 mol.L-1 PBS), and after 3 h further incubation at room temperature, the tubes were centrifuged at 3300 rpm at 4°C for 45 min.
ACTH
ACTH was measured with a commercial RIA (Diasorin, Stillwater, MO, USA). Samples were thawed on ice and 100 µL of samples, standards or controls added to 200 µL of ACTH antiserum in all tubes except total count tubes and non-specific binding tubes. After addition of 200 µL of 125I-ACTH to all tubes and incubating for 24 h, 500 µL of the precipitating complex was added to all except the total count tubes. After further incubation for 25 min, the tubes were centrifuged at 300 rpm for 20 min at 20°C. The supernatant was aspirated and the pellet counted, with the results calculated from the standard curve. The assay had inter- and intra-assay CV values of 16.1% and 15.2% respectively.
CNP and NTproCNP
CNP and NTproCNP analyses were performed by Dr Tim Prickett at the Department of Medicine, Christchurch School of Medicine, Christchurch, New Zealand. Plasma CNP was assayed by RIA as previously described (Yandle et al., 1993) using a commercial antiserum (Phoenix Pharmaceuticals Inc., Belmont, CA, USA). Antiserum to CNP-22 was diluted 1:104 with assay buffer prior to assay. The sample, standards and control were combined with 100 µL each of antiserum and radiolabelled CNP, incubated for 22 h at 4°C, and bound and free label separated by a solid-phase second antibody method. Intra- and inter-assay CVs were 3.6% and 8.3% respectively.
Table of Contents :
- ABSTRACT
- ACKNOWLEDGEMENTS
- TABLE OF CONTENTS
- LIST OF TABLES
- LIST OF FIGURES
- ABBREVIATIONS
- 1. INTRODUCTION
- 1.1 Normal fetal growth and development
- 1.1.1 Fetal nutrition
- 1.1.2 Regulation of fetal growth by maternal nutrition
- 1.1.3 Placental regulation of fetal growth
- 1.1.4 Genetic and epigenetic determinants of fetal growth
- 1.1.5 Endocrine regulation of fetal growth and development
- 1.2 Altered fetal growth and development – causes, adaptations and consequences
- 1.2.1 The Developmental Origins of Health and Disease hypothesis
- 1.2.2 Maternal nutrition and the developmental origins of health and disease
- 1.2.3 Twinning and the developmental origins of health and disease
- 1.3 Aims of this thesis
- 1.1 Normal fetal growth and development
- 2. MATERIALS AND METHODS
- 2.1 Animals
- 2.2 Nutrition
- 2.3 Surgery
- 2.3.1 Catheters
- 2.3.2 Animal preparation and anaesthesia
- 2.3.3 Catheterisation
- 2.3.4 Closure
- 2.3.5 Postoperative care
- 2.4 Experimental Procedures
- 2.4.1 Sample collection
- 2.4.2 Uterine blood flow measurement
- 2.4.3 Glucose and arginine challenges
- 2.4.4 Maternal fast and refeed
- 2.4.5 CRH/AVP challenge
- 2.4.6 Metyrapone challenge
- 2.4.7 Postmortem
- 2.5 Development of a method for estimation of blood volume parameters in pregnant sheep
- 2.5.1 Introduction
- 2.5.2 Methods
- 2.5.3 Results
- 2.5.4 Discussion
- 2.6 Biochemical Assays
- 2.6.1 Metabolite assays
- 2.6.2 Hormone Assays
- 2.7 Data Analysis
- 3. EFFECTS OF PERICONCEPTIONAL UNDERNUTRITION ON MATERNAL CARDIOVASCULAR ADAPTATION TO PREGNANCY
- 3.1 Introduction
- 3.2 Methods
- 3.2.1 Experimental design
- 3.2.2 Data analysis
- 3.3 Results
- 3.3.1 Maternal Weight
- 3.3.2 Blood volume
- 3.3.3 Uterine Blood Flow
- 3.3.4 Morphometric data
- 3.4 Discussion
- 4. EFFECTS OF PERICONCEPTIONAL UNDERNUTRITION ON OVINE SINGLETON PREGNANCY
- 4.1 Introduction
- 4.2 Methods
- 4.2.1 Experimental design
- 4.2.2 Data analysis
- 4.3 Results
- 4.3.1 Maternal weight
- 4.3.2 Fetal growth
- 4.3.3 Maternal metabolism
- 4.3.4 Fetal metabolism
- 4.3.5 Fetal glucose and arginine challenges
- 4.3.6 Maternal HPA axis
- 4.3.7 Fetal HPA axis
- 4.4 Discussion
- 4.4.1 Preconception undernutrition
- 4.4.2 Postconception undernutrition
- 4.4.3 Pre- and postconception undernutrition
- 4.4.4 Conclusions
- 5. EFFECTS OF TWINNING AND PERICONCEPTIONAL UNDERNUTRITION ON MATERNAL METABOLISM, FETAL GROWTH AND GLUCOSE-INSULIN AXIS FUNCTION IN OVINE PREGNANCY
- 6. EFFECTS OF TWINNING AND PERICONCEPTIONAL UNDERNUTRITION ON HYPOTHALAMIC-PITUITARYADRENAL AXIS FUNCTION IN OVINE PREGNANCY
- 7. C-TYPE NATRIURETIC PEPTIDE FORMS IN THE OVINE FETAL AND MATERNAL CIRCULATIONS
- 8. CONCLUSIONS
- 9. APPENDICES
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Effects of periconceptional undernutrition and twinning on ovine pregnancy