Raymond P. Aitken

Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors

In the first of two experiments investigating the effect of dietary urea on the survival and metabolism of ovine embryos, 30 Border Leicester x Scottish Blackface ewes received a maintenance diet (milled hay, molasses, minerals, vitamins) with no urea (control, C; n = 10) or with added urea at 15 g (low urea, LU; n = 10) or 30 g (high urea, HU; n = 10) kg-1 feed for a 12 week period. The degraded nitrogen (N) status relative to estimated rumen microbial N requirements was -2, +9 and +20 g per day, respectively. One week after allocation to diets, progesterone priming (12 days) commenced. Ewes received 800 IU of equine chorionic gonadotrophin at progesterone withdrawal, were inseminated 52 h later (Day 0) and embryos were collected from five ewes per group at Day 4 and from five ewes at Day 11. If available, one embryo was returned to each ewe; the rest were cultured in vitro. There was no effect of treatment on progesterone, luteinizing hormone (LH), or time of oestrus onset C, LU and HU plasma urea (P < 0.001) and ammonia levels (C vs. HU, P < 0.01; LU vs. HU, P < 0.05) differed. Day 4 HU embryos were retarded relative to C and LU embryos. After 3 days of culture, 70%, 66% and 0% of C, LU and HU embryos, respectively, were viable. Mid-term pregnancy rates following transfer were 63%, 43% and 33%. Only one HU lamb (male) was born following embryo transfer, its birthweight (10.1 kg) exceeded that of its C (n = 3; 7.0, 7.0, 7.5 kg) and LU (n = 2; 7.3, 8.2 kg) counterparts (P < 0.025). In the second experiment, C2 (2.5 g urea kg-1; n = 5) and HU2 (30 g kg-1; n = 7) diets which provided similar intakes of degraded N relative to microbial requirements as those for C and HU ewes in Experiment 1 were fed to Border Leicester x Scottish Blackface ewes superovulated with 16 mg of porcine follicle-stimulating hormone. Urea and ammonia levels in utero-oviductal samples were elevated in HU2 ewes (P < 0.05). At collection (Day 3), HU2 embryos used more glucose (P < 0.01) and, following culture, some exhibited up to a 2.8-fold increase in metabolism. In conclusion, excess rumen degradable N in ewe diets elevates urea and ammonia in plasma and in utero, with an associated increase in embryo mortality. Nevertheless, metabolism appears to be up-regulated in some embryos and, among those that survive, fetal growth appears to be enhanced.

Nutritionally Mediated Placental Growth Restriction in the Growing Adolescent: Consequences for the Fetus

Abstract Human adolescent pregnancy is characterized by poor pregnancy outcome; the risks of spontaneous miscarriage, prematurity, and low birth weight are particularly acute in girls who are still growing at the time of conception. Studies using a highly controlled sheep paradigm demonstrate that, in growing adolescents who are overnourished throughout pregnancy, growth of the placenta is impaired, resulting in a decrease in lamb birth weight relative to control-fed adolescents of equivalent age. Rapid maternal growth is also associated with increased spontaneous abortion rates in late gestation and a reduction in gestation length. Nutritionally sensitive hormones of the maternal somatotrophic axis may orchestrate nutrient partitioning in this paradigm and the particular role of growth hormone is discussed. At midgestation, the placentae of rapidly growing dams exhibit less proliferation in the fetal trophectoderm and reduced placental mRNA expression of a range of angiogenic factors. These changes occur before differences in placental size are apparent but may impact on subsequent vascularity. By late pregnancy, placental mass in the rapidly growing versus the control dams is reduced by approximately 45%; the fetuses display asymmetric growth restriction and are hypoxic and hypoglycemic. These growth-restricted pregnancies are associated with major reductions in absolute uterine and umbilical blood flows, leading to attenuated fetal oxygen, glucose, and amino acid uptakes. Placental glucose transport capacity is markedly reduced in the rapidly growing dams but is normal when expressed on a weight-specific placental basis. Thus, it is the small size of the placenta per se rather than alterations in its nutrient metabolism or transfer capacity that is the major limitation to fetal growth in the growing adolescent sheep. Information obtained from this highly controlled paradigm is clearly relevant to the clinical management of human adolescent pregnancies. In addition, the paradigm provides a robust model of placental growth restriction that replicates many of the key features of human intrauterine growth restriction per se.

Influence of Maternal Nutrition on Messenger RNA Expression of Placental Angiogenic Factors and Their Receptors at Midgestation in Adolescent Sheep

Abstract Previous studies have shown that placental growth and pregnancy outcome are severely compromised in adolescent ewes overnourished to promote rapid maternal growth. Using this paradigm, the aim of the present study was to investigate expression of the major angiogenic factors and their receptors in the placenta at the onset of the most rapid phase of fetal growth. Singleton pregnancies to a single sire were established by embryo transfer, and thereafter, adolescent dams were offered a high or moderate nutrient intake predicted to induce compromised or normal fetoplacental size at term, respectively. Ovine-specific oligonucleotide probe and primer sets for several angiogenic factors and their receptors were developed for quantitative real-time reverse transcription-polymerase chain reaction determination of placentome mRNA expression at Day 81 of gestation. Total placentome weight and fetal weight were equivalent in high- compared with moderate-intake groups at this stage of gestation. Placentome expression of the angiogenic factors, vascular endothelial growth factor, angiopoietins 1 and 2, and nitric oxide synthase 3, were reduced in overfed ewes. Similarly, level of expression of vascular endothelial growth factor/vascular permeability factor receptor (FLT1) was less in overfed ewes. Thus, in the adolescent, maternal overnutrition has a negative impact on midgestation placental angiogenic factor/ receptor expression. This may impact placental vascularity and explain why uteroplacental mass, blood flow, and nutrient uptake are compromised in late pregnancy, resulting in low-birth-weight offspring.

Dietary-induced suppression of pre-ovulatory progesterone concentrations in superovulated ewes impairs the subsequent in vivo and in vitro development of their ova

In the first of three experiments, eight ovariectomised Greyface ewes primed with exogenous progesterone were used to provide quantitative data on the effects of two contrasting feeding levels (0.3 vs. 1.4 × maintenance) on plasma progesterone concentrations. Over the 9 day study period, mean (± SEM) daily progesterone concentrations were 4.3 ± 0.13 and 3.3 ± 0.17 μg l−1 for the low and high feeding regimens, respectively (P = 0.06), indicating that high feed intake suppressed circulating progesterone levels. The second experiment examined the effect in superovulated Finn-Dorset ewes of a diet supplying either 0.6 (Group L, n = 8) or 2.3 (Group H, n = 8) times their daily energy needs for maintenance, from 1 day before introduction of exogenous progesterone to the time of insemination, on plasma progesterone concentrations and the viability of ova recovered 4 days after insemination. Mean (± SEM) plasma progesterone concentrations were 4.5 ± 0.17 μg l−1 and 2.8 ± 0.16 μg l−1 for L and H ewes, respectively, during the 12 day priming period (P 0.10) at ovum collection. Intervals (mean ± SEM) to oestrous onset (14.5 ± 0.38 h) and the luteinising hormone (LH) surge (27.1 ± 0.98 h) were unaffected by feed intake. Mean (± SEM) ovulation rates (8.1 ± 1.57 vs. 7.8 ± 1.10) and numbers of ova recovered (5.0 ± 1.39 vs. 4.8 ± 1.11) were also similar for each group. However, the proportions of ova considered viable (over 32 cells) at recovery were 0.53 and 0.22 for L and H groups, respectively (P < 0.005). Following 72 h culture (Tissue Culture Medium-199 (M199) + 10% foetal calf serum (FCS)), 0.55 and 0.27, respectively, had developed to blastocysts (P < 0.025). Of ova assessed as viable at recovery, similar proportions (0.86 vs. 0.75) from L and H treatments developed to blastocysts, with corresponding nuclei counts (mean ± SEM) of 55 ± 5.2 and 55 ± 13.2. The third experiment used 12 superovulated Greyface ewes, each offered a different feed level within the range 0.6–2.5 × maintenance, to determine the nature of the relationship between feeding level, pre-ovulatory progesterone concentrations and ovum development at Day 2 following insemination and subsequently during 7 day co-culture (M199 + FCS). Increases in feeding level were accompanied by linear decreases in plasma progesterone (r2 = 0.79, P < 0.001), the interval to oestrous onset (r2 = 0.52, P < 0.01) and timing of the LH surge (r2 = 0.32, P < 0.06). Although undetectable at ovum collection, and somewhat equivocal after 4 day culture, high feeding levels prior to ovulation reduced the proportion of ova (0.16 vs. 0.58) developing to or beyond the expanding blastocyst stage after 7 day culture. Quantitative indices of cell division and protein synthesis confirmed this. In conclusion, excessive feeding during follicular recruitment and oocyte maturation in superovulated ewes imparts a legacy of embryonic loss and developmental retardation.

Placental glucose transport in growth‐restricted pregnancies induced by overnourishing adolescent sheep

Glucose clamp procedures were used to determine whether the slowing of fetal growth during the final third of gestation in overnourished adolescent ewes is due to a reduction in placental glucose transport capacity. Singleton pregnancies to a single sire were established by embryo transfer and thereafter adolescent dams were offered a high (n= 11) or moderate (n= 7) nutrient intake. Studies were conducted at 130 ± 0.5 days gestation. Uterine and umbilical blood flows were studied by the steady‐state transplacental diffusion technique and glucose fluxes quantified by the Fick principle. To determine the relationship between the transplacental glucose gradient and umbilical (fetal) glucose uptake, studies were conducted with maternal arterial glucose clamped at 5 µmol ml−1 and fetal glucose at spontaneously occurring and two additional higher levels. Maternal body weight gain during gestation averaged 282 and 57 g day−1 for high‐ and moderate‐intake dams, respectively. Total placentome weight (209 ± 23 vs. 386 ± 34 g) and fetal weight (3072 ± 266 vs. 4670 ± 196 g) were lower (P < 0.001) in high‐ than in moderate‐intake groups. The growth‐restricted pregnancies in the high‐intake dams were associated with reduced uterine (P < 0.05) and umbilical (P < 0.02) blood flows and, in the non‐perturbed state, the fetuses were relatively hypoxic (2.1 vs. 3.0 µmol ml−1, P < 0.05) and hypoglycaemic (0.90 vs. 1.31 µmol ml−1, P < 0.002). Linear regression analysis of umbilical glucose uptake at three steady‐state uterine‐umbilical arterial transplacental plasma glucose concentration gradients revealed that absolute placental glucose transport capacity was lower in high‐ than in moderate‐intake dams (mean slope, 0.8 vs. 1.5 dl min−1, P < 0.05; and mean intercept, 1.84 vs. 3.40 µmol ml−1). However, glucose transfer capacity was not different between the two groups when expressed on a placental weight‐specific basis. This confirms that the small size of the placenta per se is the major limitation to placental glucose transfer in the overnourished adolescent pregnant sheep.

Repeated recoveries of embryos from ewes by laparoscopy

Abstract Eight ewes were synchronised in oestrus and induced to superovulate on three occasions over a period of two months. Eggs a were recovered on Day 7 (Day 0 = oestrus) of each cycle by a laparoscopic technique which avoids the adhesion formation normally associated with the conventional surgical procedure. Egg recovery rates were 35% ( 22 62 ), 76% ( 13 17 ) and 66% ( 21 32 ) for the three consecutive recovery sessions. This technique allows ewes to be used repeatedly as embryo donors and should be valuable to research workers and those involved with commercial embryo transfer in sheep.

The evaluation of a laparoscopic insemination technique in ewes

Following synchronisation of oestrus using FGA and PMSG, ewes were inseminated by either the conventional cervical (CC) method or directly into the uterus by laparoscopy (LI). The CC method was carried out either at 48 and 60 hours following progestagen withdrawal with 480 x 10(6) spermatozoa per inseminate or once only at 56 hours with 600 x 10(6) spermatozoa. The laparoscopic method was performed at 52 hr using 48 x 10(6) spermatozoa per ewe. In the first two trials eggs were recovered at laparotomy. The egg recovery rate was significantly lower (P<0.05) for those ewes which had been inseminated by the LI method (74%) compared with those inseminated by the CC method (85%); fertilization rates were not significantly different (92% and 89% respectively). In the third trial 20 ewes were bled to determine their periovulatory LH concentrations and the timing of peak LH concentrations correlated with the outcome of each insemination. Ewes inseminated using laparoscopy did not conceive when their LH surge occurred >58 hr after progestagen withdrawal. In this and in the final experiment, the combined pregnancy rates and litter sizes (assessed radiographically) were 67% (n = 51) and 2.21 (n = 34) for the CC method and 75% (n = 48) and 1.97 (n = 36) for the LI method (P>0.05).

Maternal and Fetal Growth, Body Composition, Endocrinology, and Metabolic Status in Undernourished Adolescent Sheep

Abstract The influence of relative maternal undernutrition on growth, endocrinology, and metabolic status in the adolescent ewe and her fetus were investigated at Days 90 and 130 of gestation. Singleton pregnancies to a single sire were established, and thereafter ewes were offered an optimal control (C; n = 14) or low (L [0.7 × C]; n = 21) dietary intake. Seven ewes receiving the L intake were switched to the C intake on Day 90 of gestation (L-C). At Day 90, live weight and adiposity score were reduced (P < 0.001) in L versus C dams. Plasma insulin and IGF1 concentrations were decreased (P < 0.02), whereas glucose concentrations were preserved in L relative to C intake dams. Fetal and placental mass was independent of maternal nutrition at this stage. By Day 130 of gestation, when compared to C and L-C dams, maternal adiposity was further depleted in L intake dams; concentrations of insulin, IGF1, and glucose were reduced; and nonesterified fatty acids increased. At Day 130, placental mass remained independent of maternal nutrition, but body weight was reduced (P < 0.01) in L compared with C fetuses (3555 g vs. 4273 g). Body weight was intermediate (3836 g) in L-C fetuses. Plasma glucose (P < 0.03), insulin (P < 0.07), and total liver glycogen content (P < 0.04) were attenuated in L fetuses. Fetal carcass analyses revealed absolute reductions (P < 0.05) in dry matter, crude protein, and fat, and a relative (g/kg) increase in carcass ash (P < 0.01) in L compared with C fetuses. Thus, limiting maternal intake during adolescent pregnancy gradually depleted maternal body reserves, impaired fetal nutrient supply, and slowed fetal soft tissue growth.

The effect of overnourishing singleton-bearing adult ewes on nutrient partitioning to the gravid uterus.

Overnourishing the singleton-bearing adolescent sheep throughout pregnancy promotes maternal tissue synthesis at the expense of the nutrient requirements of the gravid uterus. Consequently, the growth of the placenta is impaired and results in the premature delivery of low-birth-weight lambs relative to moderately fed adolescents of equivalent age. To establish if this phenomenon is unique to the growing animal, singleton pregnancies to a single sire were established by embryo transfer into primiparous adult ewes who had attained the normal mature body size for their genotype. Thereafter ewes were offered a maintenance or a high level of a complete diet throughout gestation. High maternal intakes resulted in elevated maternal insulin, no significant change in growth hormone or glucose, and attenuated progesterone and NEFA concentrations. Live weight gain during the first 93 d of gestation was 48 and 244 g/d, and adiposity score at term was 2.4 and 3.7 in the maintenance and high groups, respectively (P<0.001). In spite of achieving levels of adiposity similar to overnourished adolescents, placental (477 (sem 30) v. 518 (sem 41) g) and fetal (5190 (sem 320) v. 5420 (sem 250) g) weights were equivalent in maintenance and high groups. Gestation length was shorter (P<0.01) and colostrum yield at parturition lower (P<0.05) in high v. maintenance dams. Thus, adult sheep appear to be relatively insensitive to the oversupply of nutrients during pregnancy and have the ability to meet the nutrient requirements for normal conceptus growth in spite of their increased adiposity.

Fetoplacental growth and vascular development in overnourished adolescent sheep at day 50, 90 and 130 of gestation

To establish the basis for altered placental development and function previously observed at late gestation, fetoplacental growth and placental vascular development were measured at three stages of gestation in a nutritional paradigm of compromised pregnancy. Singleton pregnancies to a single sire were established and thereafter adolescent ewes were offered an optimal control (C) or a high (H) dietary intake. At day 50, the H group had elevated maternal insulin and amniotic glucose, whereas mass of the fetus and placenta were unaltered. At day 90, the H group exhibited elevated maternal insulin, IGF1 and glucose; fetal weight and glucose concentrations in H were increased relative to C, but placental weight was independent of nutrition. By day 130, total placentome weight in the H group was reduced by 46% and was associated with lower fetal glucose and a 20% reduction in fetal weight. As pregnancy progressed from day 50 to 130, the parameters of vascular development in the maternal and fetal components of the placenta increased. In the fetal cotyledon, high dietary intakes were associated with impaired vascular development at day 50 and an increase in capillary number at day 90. At day 130, all vascular indices were independent of nutrition. Thus, high dietary intakes to promote rapid maternal growth influence capillary development in the fetal portion of the placenta during early to mid-pregnancy and may underlie the subsequent reduction in placental mass and hence fetal nutrient supply observed during the final third of gestation.