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a Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The number of uterine caruncles occupied by the developing trophoblast is generally considered to be fixed by Day 50 of gestation [10–12], whereas the growth of the placenta has variously been reported to reach an apex in placental wet weight between Days 75 and 90 of gestation [10, 13, 14]. The profound decrease in placental weight recorded at term in adolescent sheep overnourished throughout pregnancy reflects a significant reduction in both the number of fetal cotyledons per placenta and mean fetal cotyledon weight [8, 9], implying that nutrition during both the first and second trimesters may potentially have an impact on placental growth and lamb birth weight in this experimental paradigm.
The aim of the present study was to investigate whether placental growth and hence pregnancy outcome at term could be altered by switching adolescent dams from a high to a moderate nutrient intake at the end of the first trimester (Day 50) and vice-versa. In addition, to determine maternal body composition in relation to growth of the products of conception, a number of ewes were killed at Day 104 of gestation, by which time placental growth has ceased but the fetus is considered to have achieved only 30% of its final birth weight [15].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All procedures were licensed under the UK Animals (Scientific Procedures) Act of 1986.
Adult Border Leicester x Scottish Blackface ewes (Greyface) were superovulated and used as donors of embryos, which were transferred into recipient ewe lambs (Suffolk or Dorset Horn x Greyface) on seven separate days during the mid-breeding season. All animals were housed in individual pens under natural lighting conditions at the Rowett Research Institute (57°N, 2°W). At the time of embryo transfer, the recipient ewe lambs were peripubertal and 190 ± 1.5 days old, with a mean live weight of 43.7 ± 0.27 kg (means ± SEM). For donors and recipients, synchronization of estrus was achieved by withdrawing progestagen-impregnated vaginal pessaries (40 mg flurogestone acetate [Chronogest]; Intervet, Cambridge, UK) 12 days after their insertion. In donor ewes, multiple ovulations were stimulated by i.m. injection of 1500 IU eCG (Intervet) 28 h before pessary withdrawal and a further (i.m) injection of GnRH (0.008 mg buserilin [Receptal]; Hoechst UK Ltd., Milton Keynes, UK) given 20–24 h after pessary withdrawal. For recipient ewe lambs, vaginal pessaries were withdrawn 4 h in advance of pessary withdrawal in the donors, and ovulation was stimulated by i.m. administration of 700 IU eCG given at pessary withdrawal. The onset of estrus was assessed by presenting females to vasectomized rams three times daily to ensure good synchrony between donor and recipient animals. Fifty hours after pessary withdrawal, donor ewes were inseminated directly into the uterus under laparoscopic visualization as described by McKelvey [16], using fresh semen collected by an artificial vagina from a single sire of proven fertility (Dorset Horn). Oocytes were recovered from donor ewes at laparotomy on Day 4 of the estrous cycle (Day 0 is day of insemination) using a standard technique of retrograde flushing of each oviduct [17]. After assessment of developmental stage using a stereomicroscope, the embryos were held at 33°C in ovum culture media (ICN Biomedicals Inc., Aurora, OH) until transferred to recipients within 4 h of recovery. Sixty-five embryos of transferable quality (loose or compact morula) were recovered from 11 donor ewes and were synchronously transferred in singleton, into the uteri of the recipient ewes using a laparoscope-aided embryo transfer procedure [18]. Ovulation rate was recorded, and the embryo was transferred into the tip of the uterine horn ipsilateral to the ovary bearing the greater number of corpora lutea. Pregnancy rate was initially determined by measurement of plasma progesterone concentrations at Day 18 of the estrous cycle.
Immediately after embryo transfer, recipient ewe lambs were allocated to one of two dietary treatments on the basis of live weight, body condition score, and ovulation rate at the time of transfer. When possible, care was also taken to randomize for embryo source. The recipients were individually offered either a high (H, n = 33) or moderate (M, n = 32) level of a diet calculated to promote rapid (~300 g/day) or moderate (~55 g/day) maternal growth rates. At Day 50 of gestation, half the ewes in each group had their dietary intakes switched, yielding 4 treatment groups: HH, MM, HM, and MH. The diet supplied 10.2 MJ metabolizable energy and 137 g crude protein per kg dry matter and was offered in two equal feeds at 0800 and 1600 h daily. Animals on moderate intakes were offered their entire ration immediately after embryo transfer; those on high intakes had the level of feed gradually increased over a 2-wk period until the level of daily feed refusal was approximately 15% of the total offered (which was equivalent to ad libitum intakes). The level of feed offered was reviewed three times weekly and adjusted on an individual basis if required, on the basis of changes in body weight (recorded weekly) and level of feed refused (recorded daily). After Day 100 of gestation, the feed intake of the moderate-intake groups (MM and HM) were adjusted weekly to maintain body condition score between 2 and 2.5 score units [19] and meet the increasing nutrient demands of the developing fetus during the final trimester. Body condition score is assessed on a five-point scale (1 = emaciated, 5 = obese).
Measurements
On Day 104 of gestation, a subset of ewes (n = 4 or 5 per group) received their morning feed, were weighed and scored for body condition, and were then killed by i.v. injection of an overdose of sodium pentobarbitone (20 ml Euthesate; 200 mg pentobarbitone/ml; Willows Francis Veterinary, Crawley, UK) and exsanguination (by severing the main vessels of the neck). The gravid uterus was removed and opened, and a fetal blood sample was collected by cardiac puncture immediately before intracardiac administration of sodium pentobarbitone (2 ml Euthesate) to kill the fetus. After clamping the umbilical cord, the fetus was removed, dried, and weighed. The crown rump length and girth at the umbilicus were measured, and the major fetal organs were dissected and weighed. Eight representative placentomes, from the entire length of the gravid uterine horn, with the fetal cotyledon and maternal caruncle intact, were removed, and their total weight was recorded. These placentomes were completely representative: mean placentome weight for the 8 placentomes sampled was not different from the mean combined fetal and maternal caruncle weights for the remaining placentomes. For the remaining placental tissue, the number of placentomes was recorded, and fetal cotyledons were separated from the maternal caruncles by gentle traction. The fetal cotyledons were then dissected from the chorioallantoic membranes, and the maternal caruncles were dissected from the endometrium. This allowed the relative weights of the fetal and the maternal component of the placentome to be determined. The 8 placentomes removed intact represented between 5% and 10% of the total placentomes dissected (mean = 7.6%). Thus, the majority of placentomes were dissected into their fetal and maternal components, and the estimated component weights of the 8 entire placentomes did not have a major effect on the data presented below.
The maternal body was dissected exactly as described previously [20]. For chemical analyses, the maternal body was divided into carcass and noncarcass components, which were minced separately in a Wolfking (Slagelse, Denmark) mincer [21]. The noncarcass included the head, feet, pelt (with fleece), blood, empty alimentary tract, omental and mesenteric fat, and the internal organs, excluding the gravid uterus. After mixing, three samples of each homogenate were freeze-dried, ground in a laboratory mincer, and subsampled for analyses. The residual moisture content of the carcass and noncarcass samples remaining after freeze-drying was obtained by drying to constant weight at 100°C. The fat content was determined by the chloroform-methanol technique [22], and the nitrogen by the Kjeldahl method [23]. The ash content of the samples was determined by ashing at 600°C to constant weight.
For the remaining ewes, pregnancy outcome was determined after spontaneous vaginal delivery at term. All ewes were supervised throughout the delivery period. Immediately after parturition, the lamb was dried, and its weight, abdominal girth and crown rump length were recorded. After the placenta was delivered, it was washed, blotted, and weighed. Fetal cotyledons were dissected and counted, and their total weight was recorded.
Blood Sampling and RIAs
Blood samples were collected from all ewes by jugular venepuncture three times weekly (Monday, Wednesday, and Friday) throughout gestation. All samples were analyzed for insulin, and samples collected on Monday and Friday were also analyzed for insulin-like growth factor-I (IGF-I). On Days 49 and 99 of gestation, samples were collected from 6 ewes in each of the final four nutritional treatments at 3-h intervals for 26 h, commencing at 0800 h. These samples were analyzed using established procedures for nonesterified free fatty acids (NEFA) [24] and glucose [25]. For these ewes, which delivered spontaneously at term, blood samples collected three times weekly throughout gestation were also analyzed for glucose. Fetal blood samples collected at slaughter were analyzed for urea [26] and IGF-I. Fetal plasma glucose levels were below the reliable detection limit. Insulin and IGF-I concentrations were measured in duplicate by RIAs described previously [27, 28]. The sensitivities of the assays were 4 µU insulin/ml and 6 pmol IGF-I/ml. The intra- and interassay coefficients of variation were 3.5% and 10.7% for insulin and 6.2% and 12.9% for IGF-I.
Data Analysis
The physical and endocrine data were analyzed by ANOVA (General Linear Models, Minitab 9.0; Minitab Inc., State College, PA) for the main effects of nutrition during the first and second trimester of pregnancy, and the interaction between these periods. For all endocrine data, the individual mean hormone concentrations were calculated for three discrete periods spanning the first, second, and third trimesters, where appropriate, and these individual means were analyzed using ANOVA as described above. In addition, and within nutritional treatments only, the overall group means were compared by Student's t-test. Glucose and NEFA concentrations during frequent blood sampling on Days 49 and 99 of gestation were also initially analyzed by ANOVA. These metabolic variables were strongly dependent on current nutritional treatment. Thus, in order to assess the effect of current nutritional status throughout the frequent sampling period, the data were combined and analyzed by an antedependence model [29]. An order 3 model (implying that observations at a given time point, given the previous 3 observations, are independent of earlier observations) was indicated for the glucose (Days 49 and 99) data, and an order 6 model for the NEFA data. Correlation analysis was by Pearson's product-moment test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pregnancy rates after embryo transfer were equivalent in high (79%) compared with moderate (78%)-dietary-intake groups. One ewe in the HH group spontaneously aborted an autolyzed fetus weighing 200 g on Day 103 of gestation, and one MM ewe had a stillborn fetus, weighing 3650 g, on Day 140 of gestation. The latter fetus had been dead for at least 48 h before parturition and had brain lesions consistent with infection by Toxoplasma gondii. A further HH ewe was euthanized due to an acute abdominal episode on Day 95 of gestation. Her fetus was alive at the point of euthanasia and weighed 498 g. However, as total placental weight was 82 g, with only 26 cotyledons weighing a total of 34 g, it is highly unlikely that this fetus would have survived to term. None of the data pertaining to these three pregnancies were included in any of the following statistical analyses. All remaining pregnancies continued until the ewes were either killed on Day 104 of gestation or delivered live young at term.
The dietary intakes and live weight changes for those ewes delivering live young at term are presented in Figure 1. From an equivalent starting weight at the time of embryo transfer, the mean live weight gain during the first trimester was 52, 51, 251, and 250 g/day for the MM, MH, HH, and HM groups, respectively. After the nutritional switch at Day 50 of gestation, mean live weight gain during the second trimester was 62, 342, 310, and 49 g/day for the MM, MH, HH, and HM groups, respectively. Body condition score at Day 100 of gestation was significantly influenced by the plane of nutrition experienced during both the first (p < 0.009) and second (p < 0.001) trimester (1.9, 2.4, 2.6, and 2.2 score units for the MM, MH, HH, and HM groups, respectively). Body condition score was maintained during the third trimester in moderate-intake groups, and condition score immediately before parturition was 2.0, 2.8, 2.9, and 2.3 score units for MM, MH, HH, and HM groups, respectively. Within all four nutritional treatment groups, the dietary intake, live weight changes, and body condition score data of the adolescent dams killed on Day 104 of gestation were identical to those delivering live young at term.
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Weekly plasma glucose concentrations measured approximately 2.5 h after the morning feed were highly dependent on the current maternal dietary intake. Glucose concentrations were lower in moderate- compared with high-intake groups during the first (p < 0.004), second (p < 0.001), and third (p < 0.04) trimesters (Fig. 2). During the frequent sampling periods on Days 49 and 99 of gestation, glucose concentrations were significantly (p < 0.001) lower in current moderate (MM and MH) versus current high (HH, HM) intake groups (Fig. 3). Conversely, NEFA concentrations were significantly (p < 0.001) elevated in current moderate- versus high-intake groups during both Days 49 and 99 of gestation (Fig. 3). NEFA concentrations in current moderate-intake groups at Day 99 of gestation were within the acceptable range for ewes fed to meet maternal requirements at this stage of pregnancy [30].
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Maternal Body Composition and Pregnancy Outcome at Day 104 of Gestation
A summary of maternal body composition for ewes killed at Day 104 of gestation is presented in Table 1. Ewe live weight, condition score, carcass and noncarcass weights, carcass and noncarcass dry matter, and fat contents were significantly influenced by maternal dietary intake during both the first and second trimesters. In contrast, the crude protein content of the carcass and noncarcass components were influenced by maternal dietary intake during the second trimester only. The increase in weight of both the carcass and noncarcass components of the maternal body in the HH- compared with the MM-intake group was largely due to a 2- to 3-fold increase in fat content. As expected, the carcass and noncarcass weights in the MH and HM groups were intermediate between the MM and HH groups, and also predominantly reflected an increase in fat content. Irrespective of treatment group, our subjective assessment of body fat, i.e., body condition score, was highly "predictive" of the fat content of the carcass (r = 0.838, n = 18, p < 0.001) and noncarcass components (r = 0.822, p < 0.001), and of the internal fat depots per se (r = 0.800, p < 0.001) at this stage of gestation.
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The placental and fetal parameters for ewes killed at Day 104 of gestation are presented in Table 2. At Day 104 of gestation, total fetal cotyledon weight was significantly lower (p < 0.002) in ewes offered a high compared with a moderate dietary intake during the second trimester. A similar trend was evident in the maternal caruncle weight data but did not reach statistical significance. Fetal weight, umbilical girth, crown-rump length, and the weights of all individual fetal organs except the spleen were not significantly influenced by dietary intake during the first or second trimester. Fetal spleen weight was significantly (p < 0.03) lower in ewes receiving a high intake during the second trimester. Irrespective of treatment, total cotyledon weight was significantly correlated with fetal weight (r = 0.650, n = 18, p < 0.01) and the weight of the fetal liver (r = 0.760, p < 0.001), spleen (r = 0.784, p < 0.001), kidneys (r = 0.521, p < 0.05), and gut (r = 0.599, p < 0.01). Fetal plasma IGF-I concentrations immediately before killing at Day 104 of gestation were 7.8 ± 0.89 and 9.5 ± 0.84 pmol/ml for fetuses derived from ewes on a high compared with a moderate nutrient intake during the second trimester. Irrespective of treatment group, these fetal IGF-I concentrations were positively correlated with fetal weight (r = 0.763, n = 18, p < 0.001). Plasma urea concentrations were elevated (p < 0.02) in fetuses derived from ewes on a high compared with a moderate nutrient intake during the second trimester (6.9 ± 0.46 vs. 5.3 ± 0.42 mM, respectively) and, irrespective of maternal nutritional treatment, fetal urea concentrations were negatively correlated with total cotyledon weight (r = -0.489, n = 18, p < 0.05).
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Pregnancy Outcome Following Vaginal Delivery at Term
Pregnancy outcome data for adolescent dams spontaneously delivering live young at term are presented in Table 3. The duration of gestation was shorter (p < 0.009) in ewes receiving a high compared with a moderate plane of nutrition from Day 50 of gestation onwards. Similarly, a high compared with a moderate plane of nutrition from the end of the first trimester was associated with significant reductions in total placental weight (p < 0.002), total fetal cotyledon weight (p < 0.001), and mean fetal cotyledon weight per placenta (p < 0.001). In contrast, fetal cotyledon number was dependent on the plane of nutrition during the first trimester only and was lower (p < 0.007) in ewes offered a high versus a moderate dietary intake at this time. Although fetal cotyledon number at term was 18% higher in the HM compared with the HH group, no statistical interaction was observed. The inhibition of fetal cotyledon growth during the second trimester in the HH and MH groups was associated with a major decrease (p < 0.001) in lamb birth weight at term relative to the MM and HM groups. Total placental weight was positively correlated with lamb birth weight in all nutritional treatment groups (r = 0.989, 0.929, 0.817, and 0.735 in MM, HH, MH, and HM groups, respectively; p < 0.05 to p < 0.01). A similar relationship existed between total fetal cotyledon weight and birth weight (r = 0.962, 0.798, 0.835, and 0.529 for the MM, HH, MH, and HM groups, respectively), but lamb birth weight was completely independent of fetal cotyledon number. The fetal:placental weight ratio at term was variable, and significantly (p < 0.02) influenced by the plane of nutrition during the first trimester, being greater in ewes receiving a high nutrient intake at this time.
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Maternal Endocrine Status throughout Gestation
Maternal plasma insulin concentrations throughout gestation were highly dependent on the dietary intake at the time of sampling (Fig. 4). Plasma insulin concentrations diverged rapidly after the application of the dietary treatments immediately after embryo transfer (equivalent to Day 5 of gestation), and mean insulin concentrations during the first trimester were elevated (p < 0.001) in high- compared with moderate-intake groups. After the nutritional switch at Day 50 of gestation, plasma insulin concentrations in the HM group decreased on average by 40%, while insulin concentrations in the MH group increased by 55%. Consequently, plasma insulin concentrations during the second trimester were significantly greater (p < 0.001) in current high- versus current moderate-intake groups. Within both current high-intake groups, mean insulin concentrations increased significantly between the second and third trimester (p < 0.05 and p < 0.01 for HH and MH groups, respectively). Mean insulin concentrations were equivalent during the first and second trimesters in the MM group and increased in response to elevated nutrient intakes during the final trimester in both MM and HM groups (p < 0.05 and p < 0.01, respectively). Irrespective of treatment group, mean maternal insulin concentrations during both the second and third trimesters were negatively correlated with both total placental weight (r = -0.623 and -0.626, respectively, n = 29, p < 0.001) and lamb birth weight (r = -0.699 and -0.711, respectively, n = 30, p < 0.001). There was no relationship between insulin concentrations during the first trimester and pregnancy outcome.
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Peripheral maternal plasma IGF-I concentrations throughout gestation are illustrated in Figure 5 and were similar to those presented for insulin. During the first trimester, overall mean IGF-I concentrations were greater (p < 0.006) in high- than in moderate-intake groups. After the switch in nutritional intake at Day 50 of gestation, mean plasma IGF-I concentrations in the HM group remained the same while those in the MH group increased by 38% (p < 0.001). Plasma IGF-I concentrations during the second and third trimesters were significantly elevated (p < 0.001) in current high- compared with current moderate-intake groups. Irrespective of treatment group, IGF-I concentrations during the first, second, or third trimesters were not significantly correlated with placental weight at term. However, maternal plasma IGF-I concentrations during the second and third trimesters were negatively correlated with lamb birth weight (r = -0.551 and -0.450, n = 30, p < 0.01).
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There was no significant interaction between first- and second-trimester nutritional treatments and plasma insulin or IGF-I concentrations at any stage of gestation. Maternal plasma insulin and IGF-I concentrations during the first and second trimester in ewes killed at Day 104 of gestation were identical to those presented for ewes delivering live young at term (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this and our previous studies reported to date [8, 9], the reduction in fetal cotyledon number recorded at term in adolescent ewes overnourished throughout pregnancy varies from 18% to 24%. The design of this study precludes precise quantification of the influence of such a modest but significant reduction in cotyledon number on lamb birth weight in that cotyledon number fixed during the first trimester cannot be completely separated from proliferative growth during both the first and second trimesters. No interaction was observed between birth weight and nutritional intake during the first compared with the second trimester and, irrespective of treatment group, cotyledon number was not correlated with birth weight. However, it is interesting to note that in other reports, ablation of only 20% of cotyledons associated with one of a pair of fetuses at Day 80 of gestation resulted in a significant reduction in the placental and fetal weights of that fetus (21% and 11%, respectively) relative to the control twin [31]. Moreover, this latter study suggests that, in twin pregnancies at least, the functional reserve capacity of the ovine placenta after Day 80 of gestation is less than 20%.
Thus, in this study, increasing maternal dietary intake at the end of the first trimester resulted in a major restriction in individual fetal cotyledon growth as determined at Day 104 of gestation and after spontaneous delivery of the placenta at term. Furthermore, total placental weight was low and was associated with a major restriction in lamb birth weight equivalent to that of ewes on a high dietary intake throughout gestation. In contrast, abruptly decreasing maternal dietary intake at the end of the first trimester resulted in a major stimulation of individual fetal cotyledon growth, resulting in normal placental mass and lamb birth weights equal to those of ewes on moderate intakes throughout pregnancy.
It has been previously shown that in carunclectomy studies, surgical reduction of potential implantation sites can result in overgrowth or structural remodeling of the remaining cotyledons to such an extent that placental transport and normal fetal growth can be maintained in a proportion of animals [3, 32]. Similarly, experimentally induced, unilateral pregnancy results in compensatory placental growth by Day 60 of gestation, suggesting that the compensatory growth response can occur early in gestation [33]. In this study, the reduction in cotyledon number in ewes on a high dietary intake during the first trimester was not associated with a compensation in placental growth in ewes maintained on a high intake thereafter for the second and third trimesters, in that mean fetal cotyledon weight per placenta remained low. In contrast, the increase in mean fetal cotyledon weight in ewes switched from a high to a moderate intake can be interpreted as a direct stimulation of placental growth induced by the major decrease in maternal dietary intake. The mechanisms underlying nutritionally mediated alterations in placental growth during mid-pregnancy are unknown. Vastly different dietary intakes, as achieved in this study, have previously been associated with major shifts in blood flow in the portal vein [34], and experimental restriction of placental growth is associated with attenuated uteroplacental blood flow in late pregnancy [35, 36]. The extent to which uterine blood flow can limit growth of the placenta per se during mid-pregnancy is unknown, but it is possible that when nutrient intakes are high, blood flow to support maternal tissue growth is maintained at the expense of uteroplacental blood flow and hence ultimately placental growth.
Analysis of placental growth at 10-day intervals between Days 40 and 100 of gestation reveals that maximum proliferative growth of the ovine placenta is reached between Days 50 and 60 of gestation, with an abrupt cessation of DNA and mass accumulation occurring between Days 75 and 80 of gestation [14]. In this study, high maternal dietary intakes from Day 50 of gestation onwards severely compromised placental growth, irrespective of maternal nutrition during the first trimester. This strongly suggests that maternal nutrition during the second trimester is vital in determining final placental size and function. In adult ewes, the response to a high plane of nutrition during mid-pregnancy is less clear. In nine of sixteen studies reviewed by Kelly [7], a high compared with a low plane of nutrition between Days 40 and 100 of gestation increased placental weight. Two studies reported no effect, and in three studies, ewes on a low plane of nutrition had significantly heavier placentas. A recent study [37] reinforces these latter observations and further suggests that it is the fetal component of the placenta that is primarily enhanced after mild mid-pregnancy nutrient restriction.
There is evidence that the variable response to manipulating maternal nutrition during mid-pregnancy may depend on maternal live weight and body condition score at mating. In ewes that were heavy and in good body condition at mating, feed restriction between Days 30 and 98 of gestation increased lamb birth weight by 17%, while in ewes that were light and in poor body condition at mating, a low nutrient intake was associated with a 13% decrease in birth weight [38]. Similar effects have been documented by De Barro [39] and appear to be mediated by alterations in both placental growth and morphology. However, in this study, the adolescent dams were of equivalent weight and body condition score at the time of embryo transfer. By Day 50 of gestation, the adolescents in the MH compared with the HH groups had a significantly lower live weight and body condition score, yet the response to a high dietary intake thereafter, with respect to placental weight at Day 104 of gestation and at term, was equivalent between groups. Similarly, a high versus a low live weight and body condition score in the HM compared with the MM groups at Day 50 of gestation did not influence the placental response to a moderate dietary intake thereafter. Interestingly, a recent study suggests that when placental weight and feed intakes are controlled in fat versus lean adult ewes, ewe fatness per se has no effect on lamb birth weight; this leads the authors to conclude that placental size is the main factor controlling lamb birth weight when ewes are fed close to their requirements [40].
Our current adolescent ewe model strictly controls many of the known variables suggested by Kelly [7] to confound comparisons of the effects of maternal nutrition on placental growth. In the present study, the adolescent recipients were of equivalent age, weight, and body condition score at the start of the study and were housed in individual pens to facilitate precise nutritional management throughout pregnancy. In addition, the donor ewes were inseminated by a single sire, and embryo transfer was used to establish singleton pregnancies. It remains to be established whether using adult rather than adolescent animals in these strictly controlled studies would result in similar perturbations in placental and fetal growth. Moreover, it is important to note that in the present study the plasma concentrations of free fatty acids and glucose in the moderately fed adolescent ewes was in fact characteristic of well-fed adults during mid-late gestation [1, 30].
As expected, mean total fetal cotyledon weight decreased by approximately 40% in all groups during the final trimester because of tissue dehydration and structural remodeling [41]. More importantly, the relative reduction in total fetal cotyledon weight in ewes consuming a high versus a moderate intake during the second trimester and killed at Day 104 of gestation was equal to that observed at term. This suggests that continuing the high dietary intakes throughout the final trimester (HH and MH groups) did not further influence placental function and that the placental limits to lamb birth weight are established before the end of the second trimester. Indeed, although the fetus weighs less than a third of its potential birth weight, at Day 104 of gestation a positive relationship between placental size and fetal growth was evident and suggests that placental nutrient transfer was already compromised in the high-intake groups. Total cotyledon weight was positively correlated with fetal weight and the weight of the liver, spleen, kidneys, and intestines. Similarly, early studies of fetal growth in undernourished ewes reveal that the liver and spleen are particularly sensitive to fetal nutrient restriction mediated by impaired placental growth [11]. In chronically undernourished ewes, amino acids are key energy substrates for fetal metabolism during late gestation as fetal glucose concentrations decline [42]. While fetal glucose concentrations were too low to measure accurately at Day 104 of gestation in this study, it is intriguing to observe that plasma urea levels were higher in fetuses derived from ewes on a high compared with a moderate maternal intake at Day 104 and with growth-restricted versus normal placentae.
In this study, maternal insulin and IGF-I concentrations during gestation closely mirrored current maternal dietary intake and were similar to those reported and discussed previously for ewes on high versus moderate nutrient intakes throughout their entire pregnancy [9]. Assessment of maternal body composition at Day 104 of gestation revealed that the increase in maternal live weight in response to high versus moderate nutrient intakes during the first and second trimester was primarily due to a major increase in body fat deposition, with a much less dramatic effect on body protein. Insulin, acting via its receptor on the adipocyte, is generally accepted as the major stimulator of lipogenesis in the subcutaneous and omental fat of pregnant sheep [43, 44], and the markedly elevated insulin levels in the high-intake group in this study are commensurate with this role. IGF-I is not an acute regulator of lipolysis in sheep [45], but together with insulin is known to alter the protein economy of the growing sheep in favor of protein deposition [46]. The adolescent sheep used in this study were appropriately grown for their age but had only obtained approximately 60% of their adult weight at the time of embryo transfer. During the second trimester, it is probable that the elevated concentrations of insulin and IGF-I in high-intake ewes provided a sustained anabolic stimulus to maternal tissue deposition at the expense of placental growth. The mechanism(s) underpinning this inappropriate nutrient partitioning between the maternal and uterine compartments is as yet unknown.
In conclusion, this study has clearly demonstrated that nutritionally mediated alterations in placental growth during the second trimester can profoundly influence lamb birth weight in adolescent sheep. In this nutritionally sensitive paradigm, reducing maternal dietary intake from a high to a moderate level at the end of the first trimester stimulates placental growth and enhances pregnancy outcome, and increasing maternal dietary intake at this time has a deleterious effect on placental development and fetal growth.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 44 1224 716622; jwra{at}rri.sari.ac.uk
Accepted: February 19, 1999.
Received: November 2, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J.W. Ward, A.J. Forhead, F.B.P. Wooding, and A.L. Fowden Functional Significance and Cortisol Dependence of the Gross Morphology of Ovine Placentomes During Late Gestation Biol Reprod, January 1, 2006; 74(1): 137 - 145. [Abstract] [Full Text] [PDF]
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L. P. Reynolds, P. P. Borowicz, K. A. Vonnahme, M. L. Johnson, A. T. Grazul-Bilska, D. A. Redmer, and J. S. Caton Placental angiogenesis in sheep models of compromised pregnancy J. Physiol., May 15, 2005; 565(1): 43 - 58. [Abstract] [Full Text] [PDF]
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J. M. Wallace, T. R. H. Regnault, S. W. Limesand, W. W. Hay Jr, and R. V. Anthony Investigating the causes of low birth weight in contrasting ovine paradigms J. Physiol., May 15, 2005; 565(1): 19 - 26. [Abstract] [Full Text] [PDF]
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 I. C. Mcmillen and J. S. Robinson Developmental Origins of the Metabolic Syndrome: Prediction, Plasticity, and Programming Physiol Rev, April 1, 2005; 85(2): 571 - 633. [Abstract] [Full Text] [PDF]
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B. A. Costine, E. K. Inskeep, and M. E. Wilson Growth hormone at breeding modifies conceptus development and postnatal growth in sheep J Anim Sci, April 1, 2005; 83(4): 810 - 815. [Abstract] [Full Text] [PDF]
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J. M. Wallace, R. P. Aitken, J. S. Milne, and W. W. Hay Jr. Nutritionally Mediated Placental Growth Restriction in the Growing Adolescent: Consequences for the Fetus Biol Reprod, October 1, 2004; 71(4): 1055 - 1062. [Abstract] [Full Text] [PDF]
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J. M. Wallace, J. S. Milne, and R. P. Aitken Maternal Growth Hormone Treatment from Day 35 to 80 of Gestation Alters Nutrient Partitioning in Favor of Uteroplacental Growth in the Overnourished Adolescent Sheep Biol Reprod, May 1, 2004; 70(5): 1277 - 1285. [Abstract] [Full Text] [PDF]
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J. M Wallace, D. A Bourke, R. P Aitken, J. S Milne, and W. W Hay Jr Placental glucose transport in growth-restricted pregnancies induced by overnourishing adolescent sheep J. Physiol., February 15, 2003; 547(1): 85 - 94. [Abstract] [Full Text] [PDF]
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B.S. Muhlhausler, C.T. Roberts, J.R. McFarlane, K.G. Kauter, and I.C. McMillen Fetal Leptin Is a Signal of Fat Mass Independent of Maternal Nutrition in Ewes Fed at or above Maintenance Energy Requirements Biol Reprod, August 1, 2002; 67(2): 493 - 499. [Abstract] [Full Text] [PDF]
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 J. M. Wallace, D. A. Bourke, R. P. Aitken, N. Leitch, and W. W. Hay Jr. Blood flows and nutrient uptakes in growth-restricted pregnancies induced by overnourishing adolescent sheep Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2002; 282(4): R1027 - R1036. [Abstract] [Full Text] [PDF]
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