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Journal of Animal Science - 2011 and 2012 Early Careers Achievement Awards

2011 AND 2012 EARLY CAREERS ACHIEVEMENT AWARDS: Placental programming: How the maternal environment can impact placental function12


This article in JAS

  1. Vol. 91 No. 6, p. 2467-2480
    Received: Oct 02, 2012
    Accepted: Dec 27, 2012
    Published: November 25, 2014

    3 Corresponding author(s):

  1. K. A. Vonnahme 3,
  2. C. O. Lemley,
  3. P. Shukla and
  4. S. T. O’Rourke
  1. Department of Animal Sciences
    Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State 39762
    Department of Pharmaceutical Sciences, North Dakota State University, Fargo 58108


Proper establishment of the placenta is important for fetal survival; however, placental adaptations to inadequate maternal nutrition or other stressors are imperative for fetal growth to be optimal. The effects of maternal nutritional status and activity level on placental vascular function and uteroplacental blood flows are important to understand as improper placental function leads to reduced growth of the fetus. In environments where fetal growth can be compromised, potential therapeutics may augment placental function and delivery of nutrients to improve offspring performance during postnatal life. Factors that could enhance placental function include supplementation of specific nutrients, such as protein, hormone supplements, such as indolamines, and increased activity levels of the dam. To understand the mechanism of how the maternal environment can impact uterine or umbilical blood flows, assessment of placental vascular reactivity has been studied in several large animal models. As we begin to understand how the maternal environment impacts uterine and umbilical blood flows and other uteroplacental hemodynamic parameters, development of management methods and therapeutics for proper fetal growth can be achieved.


Livestock producers are interested in using nutrients in the most efficient way to optimize growth. Often, one tends to focus on the growth that an animal achieves after birth; however, the majority of mammalian livestock (i.e., swine, sheep, and cattle) spend 35 to 40% of their life (i.e., from conception to consumption) within the uterus being nourished solely by the placenta. Therefore, it is especially relevant to understand the influences of the maternal environment on placental growth and development because this directly impacts fetal growth. The trajectory of prenatal growth is sensitive to direct and indirect effects of maternal environment, particularly during early stages of embryonic life (Robinson et al., 1995), the time when placental growth is exponential. Moreover, it is recognized that the maternal system can be influenced by many different extrinsic factors, including nutritional status and level of activity, which can program nutrient partitioning and ultimately growth, development, and function of the major fetal organ systems (Wallace, 1948a,b; Wallace et al., 1999; Godfrey and Barker, 2000; Wu et al., 2006).

Stress on the dam can result in preterm delivery and/or fetal growth restriction, which in turn are associated with greater risk of neonatal mortality and morbidity in livestock. Offspring born at an above average BW have an increased chance of survival compared with those born at a below average BW in all domestic livestock species, including cattle, sheep, and swine. Complications of low birth weight reported in livestock include increased neonatal morbidities and mortalities (Hammer et al., 2011), intestinal and respiratory dysfunctions, and slow postnatal growth (reviewed in Wu et al., 2006). However, it should be noted that oftentimes there is a lack of difference in birth weight, yet there are reported differences in postnatal performance (reviewed in Funston et al., 2012). So, whereas early postnatal health may be better predicted by birth weight, it appears that many of the phenotypes that are economically important to producers (i.e., reproductive function, milking ability, or carcass quality) may not be readily predicted by birth weight alone. Perhaps the trajectory of growth, including prenatal growth that is dependent on placental function, could be a better predictor of postnatal performance in our livestock. The continual desire to enhance management methods to produce healthy livestock has led to increased research in the area of developmental programming of our livestock species. By understanding how the maternal system can or cannot adapt to differing stressors during normal pregnancies, we can develop interventions or therapeutics to augment placental development or function and enhance uterine and/or umbilical blood flows and nutrient delivery to produce optimally developed offspring.

Several reviews (Regnault et al., 2002; Reynolds et al., 2005, 2006; Myatt, 2006; Fowden et al., 2006, 2008; Jansson and Powell, 2007; Morrison, 2008; Belkacemi et al., 2010) of animal models of compromised pregnancy have shown that fetal growth restriction is highly associated with many placental characteristics. These characteristics include aberrations in uterine blood flow, decreased placental vascularity and/or placental size, inadequate placental nutrient-transfer capacity, nutrient transporter abundance, nutrient synthesis and metabolism, and altered hormone synthesis and metabolism. The purpose of this review is to focus on how the uteroplacenta adapts to various stressors to provide nutrients to the developing fetus, with emphasis on placental arterial vascular function and uterine and umbilical blood flow in cattle, sheep, and swine. Although the intent is to focus on livestock species, data and information from other species will be discussed when appropriate.


Livestock are unique compared with other mammals, as they have a noninvasive placenta. Gross morphology of the ruminant placenta is termed cotyledonary, and pigs and horses have a diffuse type of placentation. Microscopically, livestock species have epitheliochorial placentation, with 6 cellular layers separating maternal and fetal blood. Some can argue that ruminant placentas are better classified as syndesmochorial due to their formation of binucleate cells by chorionic and uterine epithelia (Björkman, 1965). In swine, the diffuse placenta has chorionic villi attachment distributed over the entire surface of the chorion. The presence of primary and secondary rugae increases the relative surface area of attachment between the endometrium and the fetal membranes (Dantzer, 1984). Within the large white breeds of domestic pigs, placental area of attachment continues to increase as gestation advances (Knight et al., 1977; Vonnahme et al., 2001). Vascular development of the porcine placenta, as measured by the density of larger blood vessels (i.e., arterioles), increases approximately 200% in the fetal portion of the placenta (Vonnahme et al., 2001) with maternal vascular density remaining similar (Vonnahme et al., 2001; Vonnahme and Ford, 2004) from mid to late gestation. In ruminants, the fetal placenta attaches to discrete sites on the uterine wall called caruncles. These caruncles are aglandular sites that appear as knobs along the uterine luminal surface of nonpregnant animals and are arranged in 2 dorsal and 2 ventral rows throughout the length of the uterine horns (Ford, 1999). The placental membranes attach at these sites via chorionic villi in areas termed cotyledons. The caruncular-cotyledonary unit is called a placentome and is the primary functional area of physiological exchanges between mother and fetus. In the ewe, the growth of the cotyledonary mass is exponential during the first 70 to 80 d of pregnancy, thereafter slowing markedly until term (Stegeman, 1974). In contrast, the placental growth in the cow progressively increases throughout gestation (Reynolds et al., 1990; Vonnahme et al., 2007). Perhaps these alterations in growth patterns in the sheep and cow placenta help explain the change of capillary area density (i.e., a blood flow related measure; Borowicz et al., 2007) that exist from mid to late gestation (Funston et al., 2010). Whereas the sheep placenta remains relatively similar in weight from mid to late gestation, caruncular and cotyledonary capillary area density increase approximately 200 and 400%, respectively (Borowicz et al., 2007; Funston et al., 2010). Bovine placentas exhibit relatively modest changes in capillary area density from mid to late gestation compared with sheep, with capillary area density in caruncular tissue decreasing approximately 30% and cotyledonary tissue increasing approximately 190% and with caruncular and cotyledonary tissue weights increasing approximately 530% and approximately 650%, respectively (Vonnahme et al., 2007).


During pregnancy, dramatic changes occur in the maternal cardiovascular system, including dramatic growth and development of the uteroplacental vascular bed. If the vascular beds are not properly developed, intrauterine growth restriction or fetal death may occur, which total up to 15% of the pregnancies and lead to perinatal and maternal morbidity and mortality (Vandenbosche and Kirchner, 1998). In normal pregnancies, the vasculature of the placenta offers little resistance to blood flow, enabling an adequate supply of blood for nutrient and gas exchange between fetal and maternal circulation. In human pregnancies associated with intrauterine growth restriction and/or preeclampsia, the placental vascular resistance may rise, resulting in poor uteroplacental blood flow. Control of placental blood flow may be regulated, in part, by locally produced factors that regulate vascular smooth muscle and endothelial cells. Endothelial dysfunction is associated with hypertension, type 2 diabetes, obesity, intrauterine growth restriction, and other diseases (Feletou and Vanhoutte, 2006). The mechanisms underlying impaired endothelium-dependent responses in various tissue beds during different physiological states (e.g., pregnancy, inadequate nutrition during pregnancy) are still not fully understood.

Placental nutrient transport efficiency is directly related to uteroplacental blood flow (Reynolds and Redmer, 1995). Uteroplacental blood flow increases dramatically to support the nutritional demands of the rapidly growing fetus. To keep up with this demand, the maternal system increases its plasma volume by 30 to 40% as well as its cardiac output [i.e., 35% increase in stroke volume and 15% in heart rate (Rosenfeld, 1984)]. The fractional distribution of cardiac output to the uterus increases from 0.5% in the nonpregnant ewe to over 16% in the late pregnant ewe (Rosenfeld, 1984). Of the overall increase in blood flow to the gravid uterus by late gestation, more than 85% is directed toward the caruncular vascular beds, which transfer oxygen and nutrients to the placenta and fetus through the associated cotyledonary vasculature (Rosenfeld and Fixler, 1977). The expansion in blood volume in the ewe allows for this increased uteroplacental demand while delivering similar volumes of blood to other maternal tissues (Rosenfeld, 1984). Similar results have been reported for rats, where the percentage of the cardiac output distributed to the reproductive organs was increased compared with nonpregnant controls (Ahokas et al., 1984). These authors also report that rats nutrient restricted 50% from d 5 to 21 (i.e., to term) had >30% decrease in blood flow to the uterus and placenta (both absolute and on a percentage of cardiac output), a reduced total cardiac output, and increased arterial blood pressure, despite other organ systems receiving similar amounts of blood flow, both absolute and as a percentage of cardiac output (Ahokas et al., 1984). Therefore, the maternal metabolic system has developed ways to distribute cardiac output in a nutrient-restricted animal.

Factors Controlling Uterine Hemodynamics

Changes in gravid uterine hemodynamics occur in the face of increasing concentrations of catecholamines and angiotensin II in systemic blood (Rosenfeld, 2001). Both of these circulating agents are potent vasoconstrictors that are believed to modulate systemic and uterine blood flow and vascular reactivity. With regard to vasodilation, bradykinin (BK) could perhaps play a role in how the maternal vascular system responds. A summary of how catecholamines, angiotensin II, and BK influence the uteroplacental is discussed subsequently.

Adrenergic Receptors and Calcium Channels

Uterine blood flow is controlled by 2 distinct phenomena: phasic contractility and tonic contractility (Ford, 1995). Phasic contractility mediates short-term contractions (i.e., 5 to 10 min in duration) of uterine arterial smooth muscle, primarily in response to maternal stress. In contrast, tonic contraction of the uterine arterial vasculature represents a prolonged contractile state, which resets arterial diameter and alters the baseline rate of flow. In ewes and women, adrenergic control of uterine blood flow during gestation is mediated predominantly through catecholamine stimulation of the α1-adrenergic receptor (AR) whereas the activity of postsynaptic α2-AR is decreased, resulting in reduced uterine vascular tone (Shnider et al., 1979; Ribeiro and Macedo, 1986; Isla and Dyer, 1990; Ford, 1995). Consistent with this observation, the ability of the gravid uterine vasculature to exhibit powerful phasic contractions (which depend primarily on α1-AR), in which blood flow decreases can exceed 90% in response to circulating catecholamines, remains intact throughout pregnancy. This response occurs even though the uterine arterial diameter and uterine blood flows are increasing (Shnider et al., 1979). These phasic contractions of the uterine arterial bed are necessary in acute stress situations requiring the shunting of blood flow away from the viscera, including the uterus, toward the skeletal muscle (e.g., in the fight or flight response). Because these contractions are of such short duration, the fetus remains viable until baseline flow returns.

These data are also consistent with the concept that reduced sensitivity of the caruncular arterial vasculature to α2-AR stimulation as gestation advances may be important to allow for the decrease in the tone of these vessels, with resulting increases in caruncular blood flow. This suggestion is supported by the observation that phasic contractions of uterine arterial smooth muscle require only α1-AR stimulation whereas a change in arterial tone (i.e., vessel diameter) requires the combined and sustained stimulation of α1- and α2-AR (Ford, 1995). In agreement with these data, Sauer et al. (1989), using an in vitro perfused bovine placentome model, reported that a decrease in caruncular arterial tone appeared to result from a decreasing sensitivity of the caruncular artery to α2-AR-induced constriction as gestation advanced.

Stimulation of α2-AR is thought to stimulate calcium entry through voltage-gated channels (VGC; also called voltage-sensitive or potential-sensitive channels) on the uterine arterial smooth muscle plasma membrane, thereby increasing arterial tone, and as a consequence decreasing caruncular arterial diameter (Ford and Stice, 1985; Ford, 1995). Because Sauer et al. (1989) found no decreases in caruncular arterial α2-AR populations with advancing gestation, they suggested that the decrease in VGC activity accounted for the declining α2-AR-mediated contractile response. To our knowledge, there is limited information on the role of catecholamines in placentas from nutrient-restricted dams, be it sheep, cattle or swine. However, Jansson (1988) reported that if norepinephrine is infused into guinea pigs carrying normal and growth-restricted fetuses, there was no difference in adrenergic responsiveness of the placental vessels. Preliminary data from our laboratory indicates that nutrient restriction in beef cows has little effect on the placental response to catecholamines (K. A. Vonnahme, unpublished data). However, on realimentation, cows that were previously nutrient restricted are less responsive to phenylephrine than placentas from cows that were never nutrient restricted (K. A. Vonnahme, unpublished data).

Angiotensin II

As stated previously, the progressive increase in blood flow to the gravid uterus throughout gestation in the ewe occurs in the face of increasing blood concentrations of angiotensin II, a potent constrictor of the uterine arterial bed (Mackanjee et al., 1991; Rosenfeld, 2001). On vascular smooth muscle cell membranes, angiotensin II binds to two receptor subtypes, termed angiotensin II receptor type 1 (AT1) and angiotensin II receptor type 2 (AT2). The AT1 is expressed in virtually all adult vascular beds (Bottari et al., 1993; Cox et al., 1993) and mediates angiotensin II-induced vasoconstrictor activity largely through interaction with and stimulation of calcium entry via the VGC on the vascular smooth muscle cell membrane (Ma et al., 2001). In contrast, the AT2 is the product of a different gene (Inagami et al., 1994) and does not mediate vasoconstriction (Dudley et al., 1990; Bottari et al., 1993; Cox et al., 1993). In fact, AT2 have been shown to inhibit AT1-induced vascular contractility (McMullen et al., 1999). Interestingly, Walther et al. (2003) provided evidence that activation of AT2 by angiotensin II may actually stimulate angiogenesis. In addition, the AT2 has been reported to be the predominant angiotensin II receptor subtype in the uterine arterial bed of the pregnant ewe (Burrell and Lumbers, 1997). These are exciting observations because the increase in blood concentrations of angiotensin II may account, at least in part, for the dramatic uteroplacental vascular growth that occurs throughout gestation (Reynolds and Redmer, 1995; Borowicz et al., 2007). The expression and relative proportion of AT1 and AT2 subtypes varies markedly among different tissues and organs within the same species (Nishimura, 2001). More recently, it has been reported that early in gestation (i.e., d 41 to 51) in the ewe, angiotensin receptors are located in maternal stromal cells rather than the placental blood vessels (Koukoulas et al., 2002). Receptor ligand binding studies demonstrated that greater levels of angiotensin receptors are observed early in gestation compared with late, with the predominant receptor being AT1 (Koukoulas et al., 2002). In the cow, however, the predominant receptor subtype is AT2, being localized primarily in the fetal tissue (i.e., mesenchyme cells near the trophoblast and surrounding arterioles), and again with increased abundance during early pregnancy compared with late (Schauser et al., 1998). In pigs, AT2 abundance is much greater than AT1 throughout all of pregnancy (Nielsen et al., 1996).

Analogous to the situation in the maternal systemic and uterine arterial vasculature, angiotensin II causes dose-dependent increases in ovine fetal mean arterial pressure and umbilical vascular resistance (Adamson et al., 1989; Clark et al., 1990; Yoshimura et al., 1990). Furthermore, Rosenfeld et al. (1995) reported that the umbilical circulation demonstrated greater sensitivity to infused angiotensin II than the fetal systemic vasculature, confirming observations by Cox and Rosenfeld (1999) who reported that AT2 was the predominant receptor subtype expressed in several fetal systemic vessels whereas AT1 was the major receptor expressed in the umbilical artery. Our laboratory has demonstrated that the responsiveness to angiotensin II within the caruncular vascular bed in the ewe is greater in larger (i.e., ∼40 to 45 g) vs. smaller (i.e., ∼5 to 10 g) placentomes, regardless of morphology (Vonnahme et al., 2008), and therefore, comparison of similar sized placentomes is needed when performing vascular reactivity studies. In cattle, Sauer (1987) simultaneously perfused angiotensin II into the caruncular and cotyledonary arterial beds of in vitro perfused late gestation bovine placentomes and observed dose-dependent increases in contractility in both vascular beds. Our laboratory has recently investigated the role of angiotensin II in placental arteries from beef cows that were nutrient restricted for various durations throughout pregnancy. Placental arterial vasoreactivity in response to angiotensin II was variable, despite the high densities of angiotensin receptors reported in the bovine placenta (Schauser et al., 1998). Further analyses of receptor populations in our model are underway.

Angiotensin II is part of a larger family of mediators than the catecholamines (Fig. 1). Most of the contributors to the shared molecules in renin-angiotensin system (RAS) and kallikrein-kinin system (KKS) can be grouped into 3 different areas: 1) angiotensin converting enzyme (ACE), 2) kinin receptors, and 3) enzymes involved in generating, inactivating, or modulating kinin activity. Angiotensin converting enzyme inactivates BK in the KKS pathway whereas ACE increases angiotensin activity by converting angiotensin I to angiotensin II. As mentioned previously, binding of angiotensin II to AT1 results in vasoconstriction. However, although not completely understood, when angiotensin II binds to AT2 it antagonizes many of the actions of AT1 activation (McMullen et al., 1999); these effects may be mediated, in part, by BK, a potent vasodilator (see subsequent discussion). Angiotensin-(1-7) has been shown to have opposite effects of angiotensin II. Angiotensin-(1-7), a derivative of both angiotensin I and angiotensin II, increases regional blood flow to the kidney, mesentery, brain, and skin as well as causing an increase in cardiac index and reduction in total peripheral resistance (Sampaio et al., 2003). Angiotensin-(1-7) can also work through enhancing BK binding to BK2 receptors to release nitric oxide (NO) or prostacyclin for vasodilatation (Ferrario and Iyer, 1998). The effect of AT1 antagonists may be mediated, in part, by increased endogenous BK as well as by enhanced endogenous angiotensin II binding to AT2 that accompany AT1 antagonism (Wiemer et al., 1993).

Figure 1.
Figure 1.

The angiotensin-bradykinin pathways. Angiotensin-converting enzyme (ACE) plays are role in the regulation of the bradykinin and angiotensin pathways. Bradykinin can elicit vasodilation by increasing endothelial derived hyperpolarizing factors (EDHF), prostacyclin (PGI2), or nitric oxide (NO). Angiotensin II will cause vasoconstriction. However, if angiotensin II is further converted to angiotensin 1-7, vasodilation could occur via NO and PGI2.


The KKS includes the potent vasodilator, BK, which is produced when plasma kallikrein cleaves high molecular weight kininogen (Bhoola et al., 1992). Bradykinin interacts with G protein-coupled receptors, BK receptor 1 (BKR1) and BK receptor 2 (BKR2), to elicit its vasoreactive effects. In endothelial cells, BK stimulates endothelial nitric oxide synthase (eNOS), leading to increased production of NO and resulting in vasorelaxation. Bradykinin can also stimulate NO release through upregulation of eNOS gene transcription (Feron and Balligand, 2006). Additionally, BK can induce vasodilation by stimulating prostacyclin formation (Ignarro et al., 1987), which increases cyclic adenosine monophosphate (cAMP) production in vascular smooth muscle cells, and by activating NO-independent mechanisms that may be accounted for by one or more endothelium-dependent hyperpolarizing factors (reviewed by Madeddu et al., 2007). Bradykinin receptor 2-deficient animals have endothelial dysfunction and display markedly reduced flow-dependent vasodilation in the carotid artery, indicating an important role for the KKS in arterial function (Bergaya et al., 2001).

In our models of placental programming, we have initiated investigations of the role that maternal nutrition has on placental vascular reactivity to BK. In our beef cattle model, we set out to test the hypothesis that nutrient restriction in early to mid pregnancy programs the placenta to be more sensitive to vasodilators, particularly BK. Using tissue baths and myography, cotyledonary (i.e., terminating at the placentome) and caruncular (i.e., third branch from the main uterine artery) arteries were treated with increasing BK concentrations in the presence or absence of different downstream inhibitors, including n-(nitro)-l-arginine (i.e., a nitric oxide synthase inhibitor), indomethacin (i.e., an inhibitor of prostacyclin synthesis), and iberiotoxin (i.e., a selective blockers of large-conductance, calcium-activated K channels, which were used to assess the potential role of endothelial derived hyperpolarizing factor). Our preliminary analyses indicate that nutrient restriction does indeed enhance the ability of the placental arteries to respond to increasing doses of BK, with the cotyledonary response being more dynamic (Reyaz et al., 2012). In both arteries, it appears that BK-induced vasodilation is mediated via NO (K. A. Vonnahme, S. T. O’Rourke, and A. Reyaz, North Dakota State University, Fargo; unpublished data).

We have also investigated if the protein content of the diet may impact vascular reactivity. In sheep fed isocaloric diets but with 60, 100, or 140% of their predicted metabolizable protein requirements during the last third of pregnancy, caruncular and cotyledonary arteries were collected and analyzed similarly to that described above (Lekatz et al., 2012). Caruncular and cotyledonary arteries responded differently to in vitro treatments. In the caruncular arteries, maternal protein concentration did not affect the response to BK. The maternal vasodilation induced by BK appears to be a result of NO or endothelial derived hyperpolarizing factor and not prostacyclin because pretreatment with indomethacin did not block vasodilation. However, cotyledonary arteries from ewes fed high or low protein were more sensitive to BK-induced vasodilation compared with control ewes. The mechanism is still being elucidated because it appears there may be a nonclassical response to BK in the fetal portion of the placenta (K. A. Vonnahme, S. T. O’Rourke, and L. A. Lekatz, North Dakota State University, Fargo; unpublished data). In a previous study, we demonstrated that there was no difference among low protein and control treatments in sensitivity to sodium nitroprusside, a NO donor (Lekatz et al., 2010). This indicates that changes in BK-induced vasodilation in fetal placental arteries due to maternal protein intake cannot solely be explained by a change in sensitivity of the smooth muscle to NO.

Recent reports have shown that maternal melatonin supplementation may alter fetal growth and/or birth weights (Richter et al., 2009; Lemley et al., 2012). The observed alteration in fetal size after melatonin supplementation may be mediated through specific arterial melatonergic receptors. These pathways are relevant in the ovine fetus, where the addition of physiological concentrations of melatonin prevented the vasoconstriction caused by norepinephrine in late term fetal sheep cerebral arteries (Torres-Farfan et al., 2008). It is interesting to note that fetal cardiac output and blood flow distribution may be altered via arterial melatonergic receptor pathways within the developing fetus, which could lead to disproportionate fetal growth. Using a mid to late gestation ovine maternal nutrient restriction model of intrauterine growth restriction, we examined caruncular artery and cotyledonary artery vascular reactivity during melatonin supplementation in a 2 × 2 factorial arrangement of treatments. Ewes were supplemented with 5 mg of melatonin or no melatonin and were allocated to receive 100 or 60% of nutrient requirements from d 50 until d 130 of gestation. At d 130 of gestation, caruncular and cotyledonary arteries were sensitive to vasoconstriction induced by norepinephrine and angiotensin II, respectively; however, no differences were observed between treatment groups (K. A. Vonnahme, C. O. Lemley, P. Shukla, and S. T. O’Rourke, unpublished data). Maternal nutrient restriction did not alter sodium nitroprusside-induced or BK-induced relaxation in caruncular or cotyledonary arteries. In contrast, maternal melatonin supplementation increased sensitivity of cotyledonary artery to the BK induced relaxation, with no effect in the caruncular arteries. Although we are currently examining the potential mechanisms by which melatonin supplementation leads to increased sensitivity of cotyledonary arteries to BK, a portion of these responses may be melatonergic receptor dependent. In addition, melatonin receptors are located on both the fetal and maternal portions of the ovine placenta at Day 130 of gestation (Lemley et al., 2011).


Understanding how placental vascular function is altered due to the maternal diet allows for the development of methods to prevent intrauterine growth restriction (e.g., simple management changes, introduction of a therapeutic). However, it is also important to understand if the results that we obtained in vitro can be or are mirrored within the animal itself. Our laboratories have recently focused on estimating uterine and umbilical blood flows and other uteroplacental indices of arterial pulsatility and resistance with Doppler ultrasonography. In all models of placental insufficiency in the sheep, uterine and umbilical blood flows were reduced (Reynolds et al., 2006). Our recent studies have investigated how a therapeutic or simply realimentation may alter the trajectory of uterine or umbilical blood flow and other indices of blood flow.

Maternal Nutrition

At North Dakota State University, we have been using a primiparous ewe model carrying a singleton fetus that is nutrient restricted 40% compared with controls from d 50 of gestation (Table 1). We have individually fed these ewes and, in studies where lambs were born, they were immediately separated from their dam to prevent confounding our postnatal assessments, as we have reported colostrum and milking performance of the dam to be altered due to gestational treatment (Swanson et al., 2008; Meyer et al., 2010). Therefore, we have confidence that our postnatal responses are due to gestational treatments. In normal pregnancies, resistance of the uteroplacental arteries have been documented to decrease as gestation advances. Our laboratory has reported that when pregnant ewe lambs are nutrient restricted, lamb birth weight is reduced compared with control fed ewes (Swanson et al., 2008; Meyer et al., 2010). Moreover, we have demonstrated that when ewes are restricted, there is approximately 33% and approximately 22% decrease in eNOS mRNA abundance on d 130 of gestation in the maternal and fetal portions of the placenta, respectively, compared with control-fed animals (Lekatz et al., 2011a). We hypothesized that this reduction in birth weight was due to a greater placental vascular resistance in restricted ewes compared with control ewes. Vascular resistance is often presented as pulsatility index {PI; PI = [peak systolic velocity (cm/s) – end diastolic velocity (cm/s)]/mean velocity (cm/s)} and resistance index {RI; RI = [peak systolic velocity (cm/s) – end diastolic velocity (cm/s)]/peak systolic velocity (cm/s)}. Indeed, when PI and RI were assessed, restricted ewes had increased PI and RI in the umbilical artery compared with control ewes (Lekatz et al., 2013; Table 1). These data demonstrate a greater resistance in umbilical artery, which is consistent with reduced flow. In another model of intrauterine growth restriction, adolescent ewes overfed throughout pregnancy have reported decreased uterine blood flow (Wallace et al., 2002). Similar to our nutrient restriction model, Carr et al. (2012) report increased PI and RI in overfed ewes compared with controls during mid to late gestation. In our maternal nutrient restriction model, we showed a decrease in gravid uterine artery blood flow [i.e., blood flow (mL/min) = mean velocity (cm/s) × cross-sectional area of the vessel (cm2) × 60 s] with increasing gravid uterine arterial resistance index and, moreover, this was irrespective of nutritional treatment (Fig. 2). Interestingly, gravid uterine arterial resistance index decreased with increasing total placentome weight but only in the nutrient-restricted dams (Fig. 3). Therefore, during a compromised pregnancy such as maternal nutrient restriction, those animals with smaller placentas show an increased gravid uterine arterial vascular resistance, and this suggests both a reduced maternal placental blood flow and potentially reflects a smaller placental vascular bed.

View Full Table | Close Full ViewTable 1.

Summary of data generated from a North Dakota State University ewe nutrient restriction model1

Item Response in RES compared with CON
Measured during gestation
    Plasma concentrations d 130 of gestation2
        Maternal glucose Decreased 12%
        Fetal glucose Decreased 16%
        Maternal NEFA Increased 54%
        Fetal NEFA Increased 6% (NS)3
        Maternal BUN Decreased 10%
        Fetus BUN Decreased 12%
    Placental wt2,4 Similar
    Fetal wt2,4 Decreased 15 to 20%
    Umbilical blood flow (d 50 to 110)4 Decreased approximately 20%
    Umbilical vascular resistance5 Increased 15 to 25%
    Cot eNOS mRNA expression6 Decreased approximately 33%
    Car eNOS mRNA expression6 Decreased approximately 22%
Measured at term7
    Birth weight Decreased 13%
    Crown rump length Decreased 4%
    Abdominal girth Decreased 5%
    Placental wt Similar
    Organ characteristics4
        Heart wt Decreased 8%
        RV binucleated cell area Increased 36%
        LV binucleated cell area Increased 25%
        Ovarian follicle proliferation8 Decreased 40%
    Plasma IgG 24 h old9 Increased 33%
    Organ weights at 21 d10
        Brain Similar
        Heart Decreased 16%
        Total gastrointestinal Decreased 11%
        Liver Decreased 16%
        Visceral adiposity Decreased 23%
        Adrenal Decreased 11%
Offspring from 3 to 6 mo7,11
    Live weight, 6 mo Similar
    Glucose tolerance, 3 and 5 mo Decreased 40%
    Carcass weight Similar
    Internal fat mass12 Decreased 23% (NS)
1CON = 100% requirements; RES = 60% nutrient restriction from d 50 to d 130 or to term; BUN = blood urea nitrogen; Cot = cotyledonary; eNOS = endothelial nitric oxide synthase; Car = caruncle.
3NS = not statistically different at P < 0.05.
12K. Vonnahme and J. Caton, unpublished data.
Figure 2.
Figure 2.

Linear regression analysis of gravid uterine artery blood flow with gravid uterine artery resistance index at d 130 of gestation in ewes (n = 31). Data are from Lemley et al. (2013).

Figure 3.
Figure 3.

Linear regression analysis of gravid uterine artery resistance index with total placentome weight at d 130 of gestation. Linear regression analysis was only significant in nutrient-restricted ewes (n = 15; data shown) although no significant effects were observed in adequate fed ewes (data not shown). Data are from Lemley et al. (2013).


Therapeutic supplements thought to target placental blood flow and nutrient delivery to the fetus have been shown to increase fetal growth in animal models of intrauterine growth restriction (Vosatka et al., 1998; Richter et al., 2009; Satterfield et al., 2010); however, few studies have addressed uteroplacental hemodynamics in models of improved fetal growth. As mentioned previously, melatonin supplementation negated the decreased birth weight in nutrient-restricted rats (Richter et al., 2009). Our hypothesis was that dietary melatonin treatment during a compromised pregnancy would improve fetal growth and placental nutrient transfer capacity by increasing uterine and umbilical blood flows. The uteroplacental hemodynamics and fetal growth were determined in ewes that received a dietary supplementation with 0 or 5 mg melatonin in adequately fed [i.e., 100% of NRC recommendations (NRC, 2007)] or nutrient-restricted [i.e., 60% of NRC recommendations (NRC, 2007)] ewes. Dietary treatments were initiated on d 50 of gestation and umbilical blood flow as well as fetal growth (measured by abdominal and biparietal distances) were determined every 10 d from d 50 to d 110 of gestation. By d 110 of gestation, fetuses from restricted ewes had a 9% reduction in abdominal diameter compared with fetuses from adequately nourished ewes whereas fetuses from melatonin-supplemented ewes tended to have a 9% increase in biparietal diameter (Lemley et al., 2012). When ewes are restricted, umbilical blood flow was decreased 30 d after the nutrient restriction was initiated although melatonin supplementation increased umbilical blood flow just 10 d after supplementation. On d 90 of gestation, restricted ewes receiving melatonin had similar umbilical blood flows compared with adequately fed ewes not receiving melatonin (Fig. 4; Lemley et al., 2012).

Figure 4.
Figure 4.

Umbilical artery blood flow (BF) throughout gestation in ewes. Blood flow was evaluated at least 1h postfeeding and before lights off (5 h postfeeding). Baseline measurements were taken on d 48 of gestation and treatments commenced on d 50 of gestation. Treatment groups consisted of no melatonin (CON), 5 mg of melatonin (MEL), 100% of NRC recommendations (ADQ), or 60% of NRC recommendations (RES). Figure 3A depicts individual treatment groups. The 3-way interaction of melatonin treatment by nutritional plane by day was not significant (Panel A; P = 0.15). Significant interactions were observed for melatonin treatment by day (Panel B; P < 0.001) and nutritional plane by day (Panel C; P < 0.0001). Asterisks (*) represent differences (P < 0.05) among means within the same time point. From Lemley et al. (2013); published with permission.


In the same study, umbilical blood flow at d 130 of gestation increased with increasing fetal weight at d 130 of gestation in dams not supplemented with melatonin but not in dams receiving supplemental melatonin (Fig. 5). The lack of association between umbilical blood flow and fetal weight in dams supplemented with melatonin may be due to the fact that melatonin increased umbilical blood flow such that it was similar across both nutritional planes (data not shown). From this study we also examined amino acid use across the uterus by measuring total α-AA in maternal arterial circulation and the gravid uterine vein. This technique allows researchers to determine uterine arterial-venous concentration differences as well as the uterine flux of amino acids. Melatonin supplementation did not alter uterine arterial-venous differences in total α-AA; however, maternal nutrient restriction increased uterine arterial-venous differences in total α-AA by 96% compared with ewes receiving 100% of NRC recommendations (Lemley and Vonnahme, 2013). It is interesting to note that this increase in AA concentration difference in nutrient-restricted dams does not reflect an increase in uterine consumption of amino acids because uterine artery blood flow is decreased by approximately 20% in nutrient-restricted dams (Lemley et al., 2012). This relationship highlights the importance of both sufficient AA uptake and sufficient uterine blood perfusion during late gestation. While we are continuing our investigations into the impacts of melatonin supplementation in at-risk pregnancies, we feel that melatonin treatment may be useful in negating the consequences of intrauterine growth restriction that occur due to specific abnormalities in umbilical blood flow.

Figure 5.
Figure 5.

Linear regression analysis of umbilical blood flow with fetal weight at d 130 of gestation in ewes. Linear regression analysis was only significant in no melatonin supplemented ewes (n = 15; data shown) although no significant effects were observed in ewes receiving 5 mg of melatonin per day (data not shown). Data are from Lemley et al. (2013).


Interesting data has been provided in a series of beef cattle papers where protein is supplemented to dams gestated on low quality range (i.e., ∼6% CP) during the last third of gestation (Stalker et al., 2006; Martin et al., 2007; Larson et al., 2009). Although calf BW is similar at birth, postnatal growth is accelerated in offspring from supplemented cows (Stalker et al., 2006; Martin et al., 2007; Larson et al., 2009). Moreover, pregnancy rates in heifer calves born from protein-supplemented cows were enhanced compared with heifer calves from unsupplemented cows (93 vs. 80%; Martin et al., 2007). It was our hypothesis that the increased fertility and growth rate of the calves from protein-supplemented dams may be due to enhanced uteroplacental blood flow and/or placental nutrient transfer. To more fully understand the impacts of maternal dietary protein on uteroplacental blood flow and placental vascular development, we are currently using a pregnant ewe model in which the diets are isocaloric but with differing levels of protein in the diet. In this model, singleton fetuses from ewes consuming the high-protein diet were heavier on d 130 of gestation compared with fetuses from ewes consuming the low-protein diet, with no differences in placental weight (Camacho et al., 2010). When uterine blood flow was obtained from a single time point (d 130 of gestation), ewes consuming the low-protein diet had increased uterine blood flow but also reduced fetal weight, with no differences in placental weight, and higher blood pressure (Camacho et al., 2010). Moreover, BK-induced vasodilation of placental arteries was decreased in ewes fed a high-protein diet compared with control and low-protein fed ewes (Lekatz et al., 2010). Therefore, in isocaloric diets protein levels may be an important component in modulating uteroplacental hemodynamics.

Reports of changes in placental vascularity in response to realimentation of nutrient-restricted ewes and cows are very limited. McMullen et al. (2005) demonstrated that a short duration (7 d) of fasting during mid pregnancy in the ewe resulted in a decrease in the mRNA abundance of the angiogenic factor, vascular endothelial growth factor (VEGF), and placental weights on d 90. Although differences in VEGF mRNA were not evaluated at term, placental weights were similar at lambing in nutrient-restricted and control ewes. In the cow, there was a decrease in total placentome weight on d 125 in nutrient-restricted vs. control cows that remained suppressed even after realimentation on d 250 (Vonnahme et al., 2007; Zhu et al., 2007). Looking more closely at placentome weight in the cow, both the cotyledonary and caruncular portions were decreased in nutrient-restricted versus control cows at the end of the nutrient restriction (d 125); however, only the weight of the cotyledonary tissue remained suppressed at d 250. In contrast, in several sheep models that experienced nutrient restriction from early to mid pregnancy and were then realimented, significant compensatory growth of the entire placentome was found to occur (Foote et al., 1958; Robinson et al., 1995; Heasman et al., 1998; McMullen et al., 2005).

The differences in the impacts of nutrient restriction and realimentation in the cow (Vonnahme et al., 2007) and the sheep models described above may result from inherent species differences in placental development between sheep and cattle or may result from the type of diet that these cows received on realimentation. Our laboratory has been studying the impacts of realimentation in the beef cow after different durations of nutrient restriction (i.e., 55 or 110 d of restriction). In cows that experienced a short duration (d 30 to 85) of restriction followed by a realimentation, blood flow was augmented during late gestation compared with adequately fed control cows and those that experienced a longer duration of restriction (K. A. Vonnahme, C. O. Lemley, and L. E. Camacho, North Dakota State University, Fargo, unpublished data; Fig. 6). These data parallel our in vitro studies where it appears the placental arteries from previously restricted cows are more sensitive to the vasodilator, BK, than control fed cows (K. A. Vonnahme, S. T. O’Rourke, and A. Reyaz , unpublished data). Further studies are underway to determine the proper time of intervention/supplementation in beef cattle to restore proper conceptus development.

Figure 6.
Figure 6.

Uterine artery blood flow during late gestation in pregnant beef cows that were fed 100% of NRC requirements (circles) restricted from d 30 to 85 and then realimented to control levels (triangles) or restricted from d 30 to d 140 and then realimented to control levels (squares).


Maternal Activity

As gestational housing of swine in the United States appears to be shifting from housing in individual stalls to group housing, there is limited information on how fetal development is being altered. Although the impact of group housing on litter size is inconclusive, Lammers et al. (2007) hypothesized the increase in litter size and decrease in stillborn fetuses from sows in their study that were housed in groups during gestation could be attributed to the ability of the female to move about during gestation. Exercise during gestation has been studied in several animal species including rats (Garris et al., 1985; Houghton et al., 2000) and sheep (Lotgering et al., 1983a,b; Chandler et al., 1985) with the duration and intensity of exercise impacting both umbilical and uterine blood flows (Lotgering et al., 1983b; McMurray et al., 1993) as well as birth weight (Garris et al., 1985). Our laboratory hypothesized that umbilical blood flow to the fetus would increase in female swine that were given the ability to increase their activity during gestation. For 2 parities, pregnant female swine were individually housed, and beginning on d 40 of a 114-d gestation, a subset of females were selected to increase their activity levels. Whereas control females remained in their gestation stall for the duration of pregnancy, females selected for exercise were individually walked for 30 min, 3 times per week, at the pace of each individual. All animals received the same diet and were housed in the same room. Beginning on d 39 and approximately every 14 d until approximately d 90 of gestation, umbilical blood flows were determined from 2 independent fetuses per litter by Doppler ultrasonography. On d 70 and 84 of gestation, umbilical blood flows were increased approximately 25% in females that were allowed to exercise compared with control females (Harris et al., 2013). Gestation length, obstetrical interventions, length of parturition, average birth weight, and placental weight did not differ. Upon harvest of offspring at 6 mo of age, it was determined that HCW was not different between groups, but pigs from the exercised gilts had increased carcass quality as measured by muscle color [Minolta (L*)], muscle pH at 45 min, and water content of the muscle (Vonnahme, 2012), indicating that carcass quality may be improved in pigs from active gestating females. Studies are underway to determine if maternal activity alters nutrient transport across the placenta and if this impacts the muscular development of the fetus.


The goal of our laboratory is to improve approaches to management of livestock during pregnancy, which enhances not only the reproductive success of the dam but the growth potential and performance of her offspring later in life. Although the future applications of this research may be used to develop therapeutics or alter management methods for pregnant dams that are nutritionally or environmentally at risk, further work is needed to determine if altered placental function and/or uteroplacental blood flows equate to proper offspring development in our domestic livestock. If therapeutics can be used on farm, producers would have the ability to increase animal health while also reducing costs of animal production. Whereas each species is unique in its placental development and vascularity, comparative studies may ultimately assist researchers in understanding how the maternal environmental impacts placental and thus fetal development.




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