Effects of birth weight and postnatal nutrition on neonatal sheep: III. Regulation of energy metabolism.

This study investigated effects of birth weight and postnatal nutrition on regulation of energy metabolism in the neonatal lamb. Low (mean +/- SD 2.289 +/- 0.341 kg, n = 28) and high (4.840 +/- 0.446 kg, n = 20) birth weight male Suffolk x (Finnsheep x Dorset) lambs were individually reared on a liquid diet to grow rapidly (ad libitum fed, ADG = 337 g, n = 20) or slowly (ADG = 150 g, n = 20) from birth to live weights (LW) up to approximately 20 kg. At birth, small newborns had higher plasma concentrations of urea nitrogen (mean +/- SEM 8.31 +/- 0.25 vs 6.39 +/- 0.32 mM, P = 0.002) and somatotropin (ST, 49.1 +/- 17.0 vs 10.8 +/- 4.3 ng/mL, P = .045) and lower IGF-I (36.1 +/- 6.8 vs 157.7 +/- 21.8 ng/mL, P < 0.001) than large newborns. Plasma glucose (1.42 +/- 0.23 vs 2.63 +/- 0.95 mM, P = 0.147) and insulin (0.09 +/- 0.02 vs 0.13 +/- 0.06 ng/mL, P = 0.264) concentrations did not differ. Urea nitrogen concentration in plasma peaked and then declined rapidly in all lambs during the first week postpartum, and plasma ST declined on a body-weight-related basis from birth. During rearing to 20 kg LW, plasma insulin was higher in low- vs high-birth-weight lambs. Lambs fed ad libitum had greater plasma concentrations of glucose, urea nitrogen, insulin, and IGF-I compared to those fed a restricted diet (ADG = 150 g). The results suggest that during the early postpartum period, newborn lambs exhibit the fetal characteristic of high rates of amino acid oxidation. The results also support the notion that, at birth, low-birth-weight lambs are less mature than high-birth-weight lambs in aspects of metabolic and endocrine development, which may enhance their capacity to utilize amino acids for energy production and to support gluconeogenesis during the immediate postpartum period. Being small at birth also resulted in elevated plasma insulin concentrations when adequate nutriment to support moderate or rapid growth was provided postpartum, although it remains to be elucidated whether this more chronic effect persists in the longer term.

Maternal leptin is elevated during pregnancy in sheep.

Maternal plasma leptin is elevated during pregnancy in several species, but it is unclear to what extent this elevation reflects changes in adiposity or energy balance. Therefore, Karakul ewes (n = 8) were fed to minimize changes in maternal energy status over the pregnancy-lactation cycle. They were studied 20-40 d before breeding and during mid pregnancy (d 50-60 post coitus [PC]), late pregnancy (d 125-135 PC) and early lactation (d 15-22 post partum). Consistent with the maintenance of near energy equilibrium in nongravid maternal tissues, maternal body weight was increased only during late pregnancy when the weight of the conceptus became significant and plasma concentrations of insulin, NEFA and glucose did not vary with physiological state. In contrast, maternal plasma leptin concentration rose from 5.3 to 9.5 ng/mL between prebreeding and mid pregnancy and then declined progressively through late pregnancy and early lactation. Leptin gene expression increased 2.3 fold in maternal white adipose tissue (WAT) from prebreeding to mid pregnancy and declined to prebreeding levels during early lactation. To determine whether tissue response to insulin was involved in this effect, insulin tolerance tests were performed. The maternal plasma glucose response declined from prebreeding to early lactation, but was not correlated with either plasma leptin concentration or WAT leptin mRNA abundance. In conclusion, pregnancy causes an increase in the synthesis of leptin in sheep. This stimulation does not require increases in adiposity or energy balance and is unrelated to the ability of insulin to promote glucose utilization.

Development of a specific radioimmunoassay to measure physiological changes of circulating leptin in cattle and sheep.

Studies of leptin in large domestic ruminants have been limited to measurements of gene expression because methods to measure circulating levels are not available. To develop a bovine leptin radioimmunoassay, we produced recombinant bovine leptin and used it to immunize rabbits, and to prepare bovine leptin tracer and standards. A single antiserum with sufficient affinity and titer was identified. Using this antiserum, logit-transformed binding of (125)I-labeled bovine leptin was linearly related (R(2)= 0.99) to the log of added bovine or ovine leptin between 0.1 to 2.0 ng. Serial dilution of bovine and ovine plasma, chicken serum and bovine milk gave displacement curves that were parallel to those of bovine or ovine leptin. Recoveries of external addition of bovine leptin in ewe and cow plasma ranged between 94 and 104%. Plasma leptin concentration measured by this assay was directly related to the plane of! nutrition in growing calves and lambs. At 11-14 weeks of age, ewe lambs had a higher circulating leptin concentration than ram lambs. Finally, plasma leptin concentration was linearly related to the fat content of the empty carcass in growing cattle and to body condition score in lactating dairy cows. We conclude that circulating leptin in sheep and cattle is increased by fatness and plane of nutrition, consistent with results in humans and rodents. This assay provides an important tool to investigate mechanisms that regulate plasma leptin in cattle and sheep.

Influences on fetal and placental weights during mid to late gestation in prolific ewes well nourished throughout pregnancy.

This study investigated associations between fetal and placental weights from 85 to 130 days gestation in 49 fetuses from 21 ewes of a prolific genotype used as an experimental model of intrauterine growth retardation. The proportion of variation in fetal weight explained by placental weight increased from zero at 85 days to 91% (residual standard deviation (RSD) = 260 g) at 130 days. Overall, stage of pregnancy plus placental weight accounted for 96% of fetal weight variation (RSD = 212 g). Litter size and number of fetuses per uterine horn also influenced individual fetal weights. Gestational age, litter size, placental weight per ewe, and liveweight and condition score of ewes during early to mid gestation (initial LW and CS) explained 99.5% of the variation in fetal weight per ewe (RSD = 236 g). Most variation (86%) in placental weight was explained by stage of pregnancy, litter size, number of placentomes, and initial LW and CS (RSD = 53 g). Placental weight per ewe was influenced by stage of pregnancy, litter size and initial ewe LW and CS (R2 = 0.97; RSD = 89 g). The association of fetal and placental weights with initial ewe LW was positive, and with initial CS was negative. The results show that in the absence of overt nutritional restriction of pregnant ewes, fetal and placental weights are tightly coupled during late gestation and ewe fatness during early pregnancy is inversely related to placental and fetal weights. They demonstrate that placental weight explains most of the variation in fetal weight in the present intrauterine growth retardation model.

Intrauterine growth retardation is associated with reduced cell cycle activity, but not myofibre number, in ovine fetal muscle.

Cellular development of muscle was studied in sheep fetuses at 85 days of gestation. Large and small fetuses were compared at 100, 115 and 130 days, and an additional group of large 130-day fetuses were studied following 7 days of maternal undernutrition. Myogenesis in the peroneus longus muscle was completed between 100 and 115 days of gestation, and myofibre number did not differ between small and large fetuses. The proportion of myofibre-related nuclei identified as entering S-phase of the cell cycle was 1.7% per hour in 85-day fetuses. In large fetuses, subsequent rates were relatively constant (approximately 1.5% h(-1)), whereas in small fetuses cell cycle activity declined with age from 1.3 to 0.9% h(-1), and was 0.5% h(-1) in 130-day fetuses of restricted ewes. The constant rate of cell cycle activity in large fetuses was associated with an increasing estimated rate of muscle growth (peroneus longus (mg) = 0.831 x 10(0.024 x age [d]), r2 = 0.98), which contrasted with slow and relatively constant muscle accretion in small fetuses (8.4 mg day(-1)), and slower muscle accretion at 130 days in large fetuses from restricted ewes. Differences in DNA and RNA content in the semimembranosus muscle increased with age, large fetuses having 70% more muscle DNA, 108% more muscle RNA and 104% larger muscles than small fetuses at 130 days (all P<0.001). The results demonstrate that myonuclei accumulation, but not myofibre number, is associated with fetal growth in sheep and, therefore, with fetal nutrition during mid to late gestation.

An ultrasound-guided procedure to administer a label of DNA synthesis into fetal sheep.

A novel technique was developed to deliver a bolus dose of a DNA label into the peritoneal cavity of fetal sheep at 85-130 days gestation. Use of markers to identify the site of injection in fetuses from litters up to quadruplets, and immunohistochemistry to detect the DNA label, 5-bromo-2'-deoxyuridine (BrdU), confirmed the procedure was successful in 85% of cases. Duration of the procedure was (mean +/- SD) 44 +/- 16 min, and recovery from anaesthesia was rapid and uneventful in all cases. Fetal weight was estimated with a high degree of accuracy (residual standard deviation (RSD) = 297 g and r2 = 0.93, P<0.001) and the dose of label administered (110 +/- 33 mg BrdU/kg fetal weight) was adequate in all cases. BrdU detected in fetal nuclei following injection into amniotic fluid highlights the need for positive identification of the injection site in timed, short-term studies, and suggests potential to further develop the technique to investigate cellular events in fetal sheep younger than 85 days of gestation. The results demonstrate that the procedure can be used to determine in vivo whether or not nuclei have entered the S-phase of the cell cycle.