Sildenafil citrate treatment enhances amino acid availability in the conceptus and fetal growth in an ovine model of intrauterine growth restriction.

Abstract

Adequate placental blood flow is essential for the optimal delivery of nutrients from mother to fetus for conceptus growth. Restricted fetal development results from pathophysiological and environmental factors that alter utero-placental blood flow, placental function, and, therefore, nutrient availability in the fetus. To test this hypothesis, 0, 75, or 150 mg/d sildenafil citrate (Viagra) was administered subcutaneously from d 28 to 115 of gestation to either nutrient-restricted [50% of NRC requirements) or adequately-fed ewes (100% of NRC requirements). On d 115, maternal, fetal, and placental tissues and fluids were collected. Concentrations of total amino acids and polyamines in uterine venous and arterial sera, amniotic and allantoic fluids, and fetal umbilical venous serum were lower (P < 0.05) in nutrient-restricted ewes than in adequately fed ewes, as were the ratios of total amino acids in fetal umbilical venous serum to uterine arterial serum. Sildenafil citrate dose-dependently increased (P < 0.05) total amino acids and polyamines in amniotic fluid, allantoic fluid, and fetal serum without affecting values in maternal serum. Fetal weight was lower (P < 0.05) in nutrient-restricted ewes on d 115. Sildenafil citrate treatment dose-dependently increased (P < 0.05) fetal weight in both nutrient-restricted and adequately fed ewes. This study supports the hypothesis that long-term sildenafil citrate treatment enhances fetal growth, at least in part, by increasing the availability of amino acids in the conceptus. These findings may lead to the clinical use of sildenafil citrate in human pregnancies suspected to be at risk for intrauterine fetal growth retardation.

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The Journal of Nutrition
Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
Sildenafil Citrate Treatment Enhances Amino
Acid Availability in the Conceptus and
Fetal Growth in an Ovine Model of
Intrauterine Growth Restriction1–3
M. Carey Satterfield, Fuller W. Bazer, Thomas E. Spencer, and Guoyao Wu*
Department of Animal Science, Texas A&M University, College Station, TX 77843-2471
Abstract
Adequate placental blood flow is essential for the optimal delivery of nutrients from mother to fetus for conceptus growth.
Restricted fetal development results from pathophysiological and environmental factors that alter utero-placental blood
flow, placental function, and, therefore, nutrient availability in the fetus. To test this hypothesis, 0, 75, or 150 mg/d
sildenafil citrate (Viagra) was administered subcutaneously from d 28 to 115 of gestation to either nutrient-restricted [50%
of NRC requirements) or adequately-fed ewes (100% of NRC requirements). On d 115, maternal, fetal, and placental
tissues and fluids were collected. Concentrations of total amino acids and polyamines in uterine venous and arterial sera,
amniotic and allantoic fluids, and fetal umbilical venous serum were lower (P , 0.05) in nutrient-restricted ewes than in
adequately fed ewes, as were the ratios of total amino acids in fetal umbilical venous serum to uterine arterial serum.
Sildenafil citrate dose-dependently increased (P , 0.05) total amino acids and polyamines in amniotic fluid, allantoic fluid,
and fetal serum without affecting values in maternal serum. Fetal weight was lower (P , 0.05) in nutrient-restricted ewes
on d 115. Sildenafil citrate treatment dose-dependently increased (P , 0.05) fetal weight in both nutrient-restricted and
adequatelyfedewes.Thisstudysupportsthehypothesis thatlong-termsildenafilcitratetreatmentenhancesfetalgrowth,
at least in part, by increasing the availability of amino acids in the conceptus. These findings may lead to the clinical use of
sildenafil citrate in human pregnancies suspected to be at risk for intrauterine fetal growth retardation.J. Nutr. 140: 251–
258, 2010.
Introduction
The programmed link between fetal environment and adult
metabolic functions is well accepted, yet therapeutic means for
increasing nutrient transport from pregnant mothers to the
conceptus to prevent fetal growth retardation have not been
developed (1). Recent evidence indicates that 7–15% of preg-
nancies in women are compromised by fetal intrauterine growth
restriction (IUGR) (2,3). During pregnancy, transport of nutri-
ents from the mother to fetus(es) is predominantly dependent on
utero-placental blood flow, which increases considerably during
the second and third trimesters of gestation (4,5).
Several new vasodilatory drugs have recently been approved
for treatment of erectile dysfunction (6–8). Researchers have
explored other potential therapeutic applications for these drugs
to augment blood flow to tissues, including enhancement of
uteroplacental blood flow. One such drug, sildenafil citrate
(Viagra), induces vasodilation through inhibition of type 5
phosphodiesterase (PDE5) (7). PDE5 is responsible for the
degradation of cGMP to guanosine monophosphate. Therefore,
inhibiting PDE5 delays the breakdown of cGMP and increases
vasodilation (6). A recent report suggested that sildenafil citrate
stimulates vasodilation in myometrial biopsies collected from
IUGR pregnancies at the time of Cesarean section (7). These
findings were preceded by observations that sildenafil citrate
rapidly reduces mean arterial pressure and cardiac output while
simultaneously increasing heart rate andblood flow to the uterus
in an ovine model of surgically induced menopause (8).
Collectively, available data indicate that sildenafil citrate may
be a viable clinical treatment to reduce the incidence and/or
severity of IUGR, but direct evidence is lacking.
Although glucose is an important energy source for the
developing fetus, amino acids play a vital role in development
of the conceptus (embryo/fetus and associated placental mem-
branes) (9). In addition to serving as building blocks for tissue
protein synthesis, amino acids function as antioxidants, regula-
tors of hormone secretion, major fuels for fetal growth, and
cell signaling molecules (9–11). Furthermore, amino acids are
1Supported by the Viagra Research Grants Program-2003, grant no. 594 from
Pfizer.
2Author disclosures: M. C. Satterfield, F. W. Bazer, T. E. Spencer, and G. Wu, no
conflicts of interest.
3Supplemental Tables 1–5 are available with the online posting of this paper at
jn.nutrition.org.
* To whom correspondence should be addressed. E-mail: g-wu@tamu.edu.
0022-3166/08 $8.00 ã 2010 American Society for Nutrition.
Manuscript received August 18, 2009. Initial review completed September 22, 2009. Revision accepted November 11, 2009.
First published online December 16, 2009; doi:10.3945/jn.109.114678.
251
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essential precursors for the synthesis of nonprotein substances
with biological importance, including nitric oxide, polyamines,
neurotransmitters, amino sugars, purine and pyrimidine nucle-
otides, creatine, carnitine, porphyrins, melatonin, melanin, and
sphingolipids (11,12). Nitric oxide, a product of arginine
catabolism, plays a crucial role in regulating placental angio-
genesis and fetal-placental blood flow during gestation (13–15).
Polyamines (polycationic molecules) regulate gene expression,
signal transduction, ion channel function, and DNA and protein
synthesis, as well as cell proliferation, differentiation, and
function (10) and are pivotally linked to placental angiogenesis
(16). Importantly, a number of studies have shown that placental
transport of amino acids is dysregulated in clinical conditions
that often result in IUGR (17).
In this study, we tested the hypothesis that sildenafil citrate
would ameliorate fetal growth restriction in an ovine model of
IUGR induced by maternal nutrient restriction. The objectives
were to assess the effect of 2 doses of sildenafil citrate treatment
on maternal, fetal, and placental indices of growth and devel-
opment in the IUGR model. Results indicated that administra-
tion of sildenafil citrate to sheep with IUGR pregnancies
enhanced fetal growth and should be considered as a potential
therapeutic treatment.
Materials and Methods
Animals. Multiparous Suffolkewes were maintainedat the Texas A&M
University Sheep Center and fed an alfalfa-based diet (18). A total of 60
ewes were mated to Suffolk rams when detected in estrus (d 0 of
gestation). All ewes received 100% of NRC requirements of all nutrients
and energy from d 0 to 28 (18). Starting on d 21 of pregnancy, ewes were
individually housed. On d 28 postmating, ewes were assigned randomly
to 1 of 6 groups: 100% NRC + 0 mg/d sildenafil citrate (vehicle, 10%
dimethylsulfoxide); 100% NRC + 75 mg/d sildenafil citrate; 100%
NRC + 150 mg/d sildenafil citrate; 50% NRC + 0 mg/d sildenafil citrate;
50% NRC + 75 mg/d sildenafil citrate; and 50% NRC + 150 mg/d
sildenafil citrate. Nutrient restriction (50% NRC) was achieved by
feedingone-halfofthetotalcompletediet calculatedto meet100%NRC
requirements. Between d 28 and 115 of gestation, ewes received a daily
dose of 0, 75, or 150 mg/d sildenafil citrate (Pfizer) in vehicle according
to the experimental design. Vehicle or sildenafil citrate (1 mL solution)
was administeredsubcutaneously to ewes thrice dailyat 0700,1500, and
2300. Throughout the study, body weight and body condition score (5 =
the best, 1 = the worst) were determined every 7 d and rations were
adjusted based on changes in body weight. On d 115, conceptus (fetal-
placental unit) development was determined at necropsy. For necropsy,
ewes were stunned via captive bolt gun, followed immediately by
exsanguination. All experimental and surgical procedures were in
compliance with the Guide for the Care and Use of Agricultural Animals
in Research and Teaching and approvedby the Institutional Animal Care
and Use Committee of Texas A&M University.
Tissue collection and handling following necropsy. Immediately
upon exsanguination, blood samples were collected from a uterine artery
and a uterine ovarian vein, processed, and stored at 2808C until
analyzed. Amniotic and allantoic fluids were obtained through the
amniochorion and chorioallantoic membranes, respectively, and stored
at 2808C until analyzed (18). Following collection of the fetal fluids, the
fetus was removed, weighed, and the crown-rump length measured.
Fetal umbilical venous blood samples were collected and processed prior
to fetal dissection. The placenta was further dissected to isolate all
placentomes for the assessment of placentome number, gross morphol-
ogy, and weight. Simultaneously, the maternal organs were dissected and
weighed. To avoid a confounding effect of litter size on fetal growth and
other measured factors, we used only ewes with singleton pregnancies
for data analysis, resulting in a variable number of ewes among
treatment groups.
Biochemical analyses of maternal and fetal serum and placental
fluids. Amino acids and polyamines were quantified using fluorometric
HPLC methods after a derivatization reaction with o-phthaldialdehyde
(19–21). The integration of chromatographic peaks was performed using
Millenium-32 software (Waters). Ammonia, glucose, and lactate were
measured using enzymatic methods as previously described (22). Urea
was analyzed using a colorimetric method involving urease, phenol, and
hypochlorite (23). FFA were measured using a commercial assay kit
(catalog no. 994–75409; Wako Chemicals) according to the manufac-
turer’s recommendations (23).
Statistical analyses. This study represents a 2 3 3 factorial design.
Values are reported as least squares means and pooled SE. Fetal organ
weights were analyzed by 2-way ANOVA using fetal weight as a
covariate. All other data were analyzed by 2-way ANOVA. For body and
organ weights of ewes and fetuses as well as biochemical metabolites,
differences among means were determined by the Tukey’s multiple
comparison test. All analyses were performed using the General Linear
Model procedures of the SAS (SAS Institute). P-values # 0.05 were
considered significant.
Results
Maternal and placental variables. Changes in maternal body
weight from d 28 to necropsy (d 115) and body condition score
at necropsy were affected by diet (P , 0.01) but not by sildenafil
citrate (Table 1). Utero-placental, gastrocnemius muscle, and
liver weights were lower (P , 0.01) in nutrient-restricted ewes
compared with adequately fed ewes but were not affected by
sildenafil citrate, or diet and sildenafil citrate interaction (Table
1). A diet 3 sildenafil citrate interaction was detected (P , 0.05)
for maternal heart weight, which was reduced (P , 0.05) in
nutrient-restricted ewes receiving 75 mg/d sildenafil citrate
compared with nutrient-restricted ewes receiving 150 mg/d
sildenafil citrate (Table 1). Weights of the maternal left ventricle,
right ventricle, septum, intestine, pancreas, and adrenal glands
were not affected by diet, sildenafil citrate, or their interaction
(Supplemental Table 1). Further, placentome weight and num-
ber, as well as amniotic and allantoic fluid volumes, were not
affected by diet, sildenafil citrate, or their interaction (Supple-
mental Table 1).
TABLE 1
Effects of maternal nutrient restriction and sildenafil
citrate on maternal weight change, body condition
score, and maternal organ weight in sheep1,2
Diet
Sildenafil
citraten
Maternal
weight
changeBCSUPWGM LiverHeart
mg/dkg/87 d
29.7b
29.9b
210.0b
11.7a
9.9a
8.8a
0.6
AU
2.0b
1.9b
2.2b
2.9a
3.1a
3.3a
0.1
kg
5.1a
4.5b
5.1a
5.5a
5.5a
5.4a
0.2
g
50% NRC05
6
6
6
4
3
435bc
355c
441b
484ab
562a
480ab
15
536c
579bc
631bc
677ab
744a
673ab
19
300ab
260b
343a
341a
331a
288ab
9
75
150
100% NRC0
75
150
SE
P-value
Diet
SC
Diet 3 SC
,0.01
0.51
0.63
,0.01
0.62
0.86
,0.01
0.29
0.31
,0.01
0.99
0.07
,0.01
0.46
0.45
0.33
0.51
,0.05
1Values are means and pooled SE. Means in a column without a common letter differ,
P , 0.05.
2AU, arbitrary unit; BCS, body condition score; GM, gastrocnemius muscle; SC,
sildenafil citrate; UPW, uteroplacental weight.
252Satterfield et al.
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Fetal variables. On d 115 of gestation, fetuses from ewes
receiving 50% of NRC nutrient requirements weighed less (P ,
0.01) than fetuses from ewes fed 100% of NRC requirements
(Table 2). Sildenafil citrate treatment enhanced (P , 0.01)
weights of fetuses from both nutrient-restricted and adequately
fed ewes in a dose-dependent manner (Table 2). When individual
organ weights were calculated as a proportion of fetal weight,
only weights of the fetal left ventricle, pancreas, and spleen were
affected (P , 0.05; Table 2). The fetal left ventricle was heavier
(P , 0.05) in fetuses from nutrient-restricted ewes receiving
150 mg/d sildenafil citrate. The fetal pancreas was heavier (P ,
0.05) in fetuses from adequately fed ewes receiving 150 mg/d
sildenafilcitrate.Incontrast,thefetalspleenwaslighter(P,0.05)
in fetuses from adequately fed ewes receiving 150 mg/d sildenafil
citrate. Sildenafil citrate treatment did not affect the weights of
other fetal organs (Supplemental Table 2).
Amino acids in maternal, fetal, and placental fluids. Total
amino acids in uterine arterial serum were affected (P , 0.01) by
diet, but not sildenafil citrate or diet and sildenafil citrate
interaction (Table 3). Concentrations of total amino acids and
most of individual amino acids in uterine arterial serum were
lower (P , 0.01) in nutrient-restricted ewes than in adequately
fed ewes. In contrast, concentrations of aspartate, glutamate,
glycine, and b-alanine did not differ between maternal diets.
Notably, concentrations of only citrulline in maternal serum
were reduced (P , 0.05) by sildenafil citrate treatment in ewes.
Total amino acids in amniotic fluid were affected (P , 0.01)
by maternal diet, sildenafil citrate, and their interaction (Sup-
plemental Table 3). Total amino acids were lower (P , 0.05) in
nutrient-restricted ewes than in adequately fed ewes. Within
nutrient-restricted ewes, sildenafil citrate had a dose-dependent
TABLE 2
Effects of nutrient restriction and sildenafil citrate on
fetal organ weights in sheep1
DietSC2
n
Fetal
weight
Left
ventricle PancreasSpleen
mg/dkg
2.16c
2.21c
2.47b
2.35b
2.70a
2.64a
0.03
g
50% NRC05
6
6
6
4
3
4.64b
4.68b
5.98a
4.68b
4.67b
4.95b
0.12
3.27ab
2.80b
2.92b
2.85b
3.54ab
3.73a
0.11
4.11ab
3.86ab
3.51bc
4.52a
4.04ab
2.91c
0.12
75
150
100% NRC0
75
150
SE
P-value
Diet
SC
Diet 3 SC
,0.01
,0.01
0.10
0.29 0.21
0.74
0.99
,0.05
0.15
,0.05
0.27
,0.05
1Values are means and pooled SE. Means in a column without a common letter differ,
P , 0.05.
2SC, Sildenafil citrate.
TABLE 3
Effects of maternal nutrient restriction and sildenafil citrate on concentrations of amino acids
in maternal uterine arterial serum of sheep1
Amino acid
50% NRC 100% NRC
SE
P-value
0 mg/d SC,2
n = 5
75 mg/d SC,
n = 6
150 mg/d SC,
n = 6
0 mg/d SC,
n = 6
75 mg/d SC,
n = 4
150 mg/d SC,
n = 3DietSCDiet 3 SC
mmol/L
Ala
Arg
Asn
Asp
b-Ala
Cit
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Orn
Phe
Pro
Ser
Tau
Thr
Trp
Tyr
Val
Total
171bc
157b
26b
13
24
55b
96b
226c
80
607
30b
68b
81b
72b
19c
58b
34b
72b
75
64b
67bc
23bc
41bc
118bc
2277b
164c
160b
29b
13
24
50b
92b
239c
78
607
29b
73ab
79b
70b
19c
54b
36b
75b
76
66b
65c
24bc
40bc
113bc
2274b
174bc
156b
31ab
13
22
46b
89b
247bc
83
588
28b
74ab
76b
71b
20bc
52b
34b
72b
74
60b
63c
21c
37c
111c
2241b
212a
202a
36a
13
26
106a
177a
268ab
80
660
43a
82a
105a
116a
27a
73a
46a
132a
80
85a
78a
32a
52a
154a
2883a
192ab
217a
30b
13
24
101a
199a
290a
86
573
40a
79ab
114a
103a
26a
75a
43a
113a
80
83a
81a
30a
49ab
152a
2793a
203a
233a
31ab
12
26
96a
178a
287ab
86
587
41a
78ab
105a
107a
24ab
70a
41a
120a
79
75a
75ab
29ab
48ab
136ab
2766a
3.5
4.4
0.7
0.3
0.6
1.4
3.3
5.5
1.0
9.5
0.8
1.1
2.0
1.9
0.5
1.3
0.8
3.6
1.1
1.5
1.3
0.8
1.1
3.2
18
,0.01
,0.01
,0.05
0.78
0.14
,0.01 ,0.05
,0.01
,0.01
0.09
0.77
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.05
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
0.26
0.42
0.66
0.66
0.78
0.74
0.39
,0.05
0.63
0.52
0.99
0.27
0.92
0.31
0.18
0.84
0.27
0.52
0.44
0.32
0.59
0.35
,0.05
0.89
0.74
0.60
0.83
0.97
0.70
0.54
0.33
0.30
0.34
0.10
0.48
0.92
0.46
0.32
0.65
0.46
0.57
0.11
0.84
0.17
0.42
0.33
0.37
0.34
0.24
1Values are means with pooled SE. Means in a row without a common letter differ, P , 0.05.
2SC, Sildenafil citrate.
Viagra, amino acids, and fetal growth253
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effect (P , 0.05) to increase total amino acids in amniotic fluid;
however, in adequately fed sheep, sildenafil citrate did not affect
total amino acids. Concentrations of most amino acids (aspar-
tate, glutamate, glutamine, histidine, citrulline, arginine, ala-
nine, methionine, valine, isoleucine, ornithine, lysine, proline,
and cysteine) were elevated (P , 0.05) by sildenafil citrate
treatment. A diet 3 sildenafil citrate interaction was detected
(P , 0.05) for serine, glycine, threonine, alanine, tyrosine,
tryptophan, valine, isoleucine, and lysine. In response to
administration of sildenafil citrate, amniotic fluid concentrations
of serine and glycine were enhanced (P , 0.05) in nutrient-
restricted ewes but lowered (P , 0.05) in adequately fed ewes.
Amniotic fluid concentrations of threonine, alanine, tyrosine,
tryptophan, valine, isoleucine, and lysine were elevated (P ,
0.05) by sildenafil citrate in nutrient-restricted ewes but not in
adequately fed ewes.
In allantoic fluid, total amino acids were affected (P , 0.01)
by both diet and sildenafil citrate, but not by diet 3 sildenafil
citrate interaction (Supplemental Table 4). Concentrations of
total amino acids were lower (P , 0.05) in nutrient-restricted
ewes than in adequately fed ewes and there was a dose-
dependent effect of sildenafil citrate to increase (P , 0.05) total
amino acids in allantoic fluid regardless of diet. In allantoic fluid,
glycine was the only amino acid not affected by maternal diet or
sildenafil citrate. Concentrations of most amino acids were
greater (P , 0.05) in ewes treated with sildenafil citrate than in
ewes receiving administration of vehicle. A diet 3 sildenafil
citrate interaction was detected (P , 0.05) for serine, glycine,
threonine, and arginine in allantoic fluid. The concentration of
glycine in allantoic fluid was greater (P , 0.01) in adequately fed
ewes receiving 150 mg/d sildenafil citrate than in ewes receiving
administration of vehicle.
Concentrations of amino acids in fetal umbilical vein serum
were affected by diet (P , 0.01), sildenafil citrate (P , 0.01),
and their interaction (P , 0.05) (Table 4). Total amino acids
were reduced (P , 0.05) in fetal serum from nutrient-restricted
ewes compared with adequately fed ewes. Sildenafil citrate dose-
dependently enhanced (P , 0.05) total amino acids in fetal
serum from nutrient-restricted ewes and, to a lesser extent, in
adequately fed ewes. Individually, in fetal serum, only aspartate
and alanine were not affected by maternal diet. Further,
sildenafil citrate had a dose-dependent effect to increase
(P , 0.05) concentrations of most amino acids in fetal serum.
A diet 3 sildenafil citrate interaction was detected (P , 0.05) for
asparagine and serine in allantoic fluid. Notably, concentrations
of serine in fetal serum were markedly elevated (P , 0.01) due to
increasing doses of sildenafil citrate in nutrient-restricted ewes
but were only modestly affected (P , 0.05) in adequately fed
ewes receiving 150 mg/d sildenafil citrate.
The ratio of total amino acids in serum from fetal umbilical
vein to maternal uterine artery was affected by diet (P , 0.001),
sildenafil citrate (P , 0.01), and their interaction (P , 0.05)
(Table 5). The ratio of total amino acids in fetal to maternal
serum was lower (P , 0.05) in nutrient-restricted ewes
compared with adequately fed ewes. Sildenafil citrate dose-
dependently enhanced (P , 0.05) the ratio of total amino acids
TABLE 4
Effects of maternal nutrient restriction and sildenafil citrate on concentrations of amino acids
in fetal umbilical venous serum of sheep1
Amino acid
50% NRC 100% NRC
SE
P-value
0 mg/d SC,2
n = 5
75 mg/d SC,
n = 6
150 mg/d SC,
n = 6
0 mg/d SC,
n = 6
75 mg/d SC,
n = 4
150 mg/d SC,
n = 3 DietSCDiet 3 SC
mmol/L
Ala
Arg
Asn
Asp
b-Ala
Cit
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Orn
Phe
Pro
Ser
Tau
Thr
Trp
Tyr
Val
Total
295b
252e
68e
16
200bc
220d
123b
541e
35c
590e
58d
74c
142c
154b
40d
78d
79c
181d
533d
107d
221c
43d
85c
211d
4346f
340b
304d
81d
17
210bc
252cd
135b
645d
40bc
681d
67c
86bc
185b
190ab
51c
88cd
89bc
214cd
663c
123cd
243c
49cd
102c
239cd
5091e
344b
343bc
94c
18
231ab
310ab
175a
715c
44ab
774c
75b
98b
220b
211a
58bc
106bc
109ab
249bc
719bc
141c
282b
58ac
131b
278c
5779d
432a
329cd
94c
17
206bc
278bc
173a
778bc
48a
868b
74b
121a
206b
211a
64b
98bc
112a
259b
801a
170b
290b
55bc
145ab
352ab
6181c
489a
371b
123b
17
196c
316a
196a
819ab
47a
933ab
82ab
132a
212b
224a
74a
122ab
122a
281ab
789ab
176b
304b
64ab
148ab
341b
6577b
477a
446a
143a
20
247a
334a
198a
891a
51a
1,004a
85a
131a
261a
220a
80a
147a
129a
318a
861a
214a
363a
66a
156a
402a
7242a
8.2
4.9
1.8
0.5
4.7
5.1
4.6
10
1.0
12
1.2
3.3
5.8
5.4
1.2
3.6
3.3
6.2
11
2.9
5.0
1.4
3.1
7.5
30
,0.01
,0.01
,0.01
0.49
0.79
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.05
,0.01
,0.01
0.17
,0.01
,0.01
,0.01
,0.01
0.09
,0.01
,0.01
0.12
,0.01
0.06
,0.01
,0.01
,0.05
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.05
,0.01
0.93
0.34
,0.05
0.71
0.44
0.27
0.27
0.37
0.29
0.74
0.53
0.66
0.43
0.24
0.94
0.49
0.67
0.93
,0.05
0.40
0.73
0.59
0.10
0.59
,0.05
1Values are means with pooled SE. Means in a row without a common letter differ, P , 0.05.
2SC, Sildenafil citrate.
254Satterfield et al.
by guest on October 13, 2011
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Page 5

in fetal to maternal serum in nutrient-restricted ewes and, to a
lesser extent, in adequately fed ewes. In adequately fed ewes,
fetal:maternal ratios for only 5 amino acids (asparagine, glycine,
methionine, taurine, and valine) in serum were affected (P ,
0.05) by sildenafil citrate treatment. Individually, the ratios of
most amino acids in serum from fetal umbilical vein to maternal
uterine artery were lower (P , 0.05) in underfed ewes than in
adequately fed ewes. In contrast, the ratios of histidine,
citrulline, lysine, proline, and cysteine in serum from fetal
umbilical vein to maternal uterine artery were lower (P , 0.05)
in underfed ewes than in adequately fed ewes. There was a dose-
dependent effect of sildenafil citrate (P , 0.05) on enhancing the
fetal:maternal ratios for most amino acids in serum.
Polyamines in maternal, fetal, and placental fluids. Total
polyamines in serum from uterine artery and fetal umbilical
vein, as well as amniotic and allantoic fluid, were affected (P ,
0.01) by both diet and sildenafil citrate (Table 6). Concentra-
tions of total polyamines in serum from uterine artery and fetal
umbilical vein, as well as amniotic and allantoic fluids, were
lower (P , 0.01) in nutrient-restricted than in adequately fed
ewes. Sildenafil citrate enhanced (P , 0.01) concentrations of
total polyamines in serum from uterine artery and fetal umbilical
vein, as well as amniotic and allantoic fluids, regardless of
maternal diet.
Metabolites in maternal, fetal, and placental fluids. Con-
centrations of FFA and lactate in uterine arterial serum were
lowered (P , 0.01) by maternal nutrient restriction (Supple-
mental Table 5). Concentrations of ammonia, urea, and glucose
in maternal serum were not affected by diet, sildenafil citrate, or
their interaction. Concentrations of ammonia in amniotic fluid
were affected (P , 0.05) by maternal diet, sildenafil citrate, and
their interaction. Within nutrient-restricted ewes, sildenafil
citrate enhanced (P , 0.05) concentrations of ammonia in
amniotic fluid; however, within adequately fed ewes, sildenafil
citrate lowered (P , 0.05) concentrations of ammonia in
amniotic fluid. Concentrations of lactate in amniotic fluid were
reduced (P , 0.01) by nutrient restriction but were dose-
dependently increased (P , 0.01) by sildenafil citrate, irrespec-
tive of diet. Concentrations of urea, glucose, and FFA in
amniotic fluid were not affected by diet, sildenafil citrate, or
their interaction. Concentrations of lactate in allantoic fluid
were lower (P , 0.01) in nutrient-restricted ewes than in
adequately fed ewes but were greater (P , 0.01) in ewes
receiving sildenafil citrate than in ewes without sildenafil citrate
treatment, irrespective of diet. Concentrations of urea in
allantoic fluid were greater (P , 0.05) in sildenafil citrate-
treated ewes, irrespective of maternal diet. Concentrations of
ammonia in allantoic fluid were affected (P , 0.05) by sildenafil
citrate and diet 3 sildenafil citrate interaction. Concentrations
of glucose and FFA in allantoic fluid were not affected by diet,
sildenafil citrate, or their interaction. Concentrations of lactate
in fetal umbilical vein serum were lower (P , 0.01) in nutrient-
restricted ewes than in adequately fed ewes, but were greater
(P , 0.01) in ewes receiving sildenafil citrate than in ewes
TABLE 5
Effects of maternal nutrient restriction and sildenafil citrate on the ratios for concentrations of
amino acids in fetal umbilical venous serum to maternal uterine arterial serum of sheep1
Amino acid
50% NRC100% NRC
SE
P-value
0 mg/d SC,2
n = 5
75 mg/d SC,
n = 6
150 mg/d SC,
n = 6
0 mg/d SC,
n = 6
75 mg/d SC,
n = 4
150 mg/d SC,
n = 3 DietSC Diet x SC
mmol/L
Ala
Arg
Asn
Asp
b-Ala
Cit
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Orn
Phe
Pro
Ser
Tau
Thr
Trp
Tyr
Val
Total
1.7c
1.6b
2.7b
1.3
8.6b
4.1c
1.3bc
2.4b
0.4b
1.0c
1.9bc
1.1c
1.8c
2.2bc
2.1d
1.4b
2.3c
2.5bc
7.1c
1.7d
3.3c
1.9b
2.1d
1.8d
1.9d
2.1bc
1.9ab
2.8b
1.4
8.9b
5.1b
1.5b
2.7ab
0.5b
1.1c
2.3b
1.2bc
2.4ab
2.7ab
2.7bc
1.6ab
2.5bc
2.9ab
8.7b
1.9cd
3.8bc
2.1b
2.6cd
2.1cd
2.2c
2.0bc
2.2a
3.1b
1.4
10.6a
6.8a
2.0a
2.9a
0.5b
1.3b
2.7a
1.3bc
3.0a
3.0a
2.9ab
2.0a
3.2a
3.5a
9.9ab
2.3b
4.5ab
2.8a
3.5a
2.6ab
2.6a
2.1bc
1.7b
2.7b
1.3
8.0b
2.7d
1.0c
2.9a
0.6a
1.3b
1.7c
1.5ab
2.0bc
1.9c
2.4cd
1.4b
2.5bc
2.0c
10.0ab
2.0bc
3.8bc
1.7b
2.8bc
2.3bc
2.1c
2.6a
1.7b
4.3a
1.4
8.2b
3.1d
1.0c
2.9a
0.6a
1.6a
2.0bc
1.7a
1.9bc
2.2bc
3.0ab
1.6ab
2.9ab
2.5bc
10.0ab
2.2bc
3.7bc
2.1b
3.1abc
2.3bc
2.4b
2.4ab
1.9ab
4.7a
1.7
9.8ab
3.5cd
1.1c
3.1a
0.6a
1.7a
2.1bc
1.7a
2.5ab
2.1bc
3.4a
2.1a
3.2a
2.7b
10.9a
2.8a
4.9a
2.3ab
3.3ab
2.9a
2.6a
0.06
0.05
0.09
0.06
0.26
0.12
0.06
0.06
0.01
0.02
0.06
0.04
0.09
0.08
0.08
0.06
0.10
0.09
0.2
0.05
0.11
0.08
0.09
0.07
0.01
,0.01
0.20
,0.01
0.63
0.19
,0.01
,0.01
,0.05
,0.01
,0.01
,0.01
,0.01
0.22
,0.01
,0.05
0.92
0.31
,0.01
,0.01
,0.01
0.29
0.19
,0.05
,0.05
,0.01
,0.05
,0.05
,0.01
0.15
,0.05
,0.01
,0.05
0.06
0.53
,0.01
,0.01
0.16
,0.01
,0.05
,0.01
,0.01
,0.05
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
0.86
0.47
,0.01
0.60
0.96
0.05
0.20
0.35
0.18
0.47
0.34
0.81
0.22
0.37
0.74
0.97
0.65
0.60
0.13
0.75
0.60
0.42
0.11
0.55
,0.05
1Values are means with pooled SE. Means in a row without a common letter differ, P , 0.05.
2SC, Sildenafil citrate.
Viagra, amino acids, and fetal growth255
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