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Placenta 29 (2008) 539e548

Effect of Variable Long-Term Maternal Feed Allowance on the

Development of the Ovine Placenta and Fetus
S.P. Quigley a,b, D.O. Kleemann a, S.K. Walker a, P.A. Speck b, S.R. Rudiger b, G.S. Nattrass b,
M.J. DeBlasio c, J.A. Owens c,*
a
South Australian Research and Development Institute, Turretfield Research Centre, Rosedale, SA 5350, Australia
b
South Australian Research and Development Institute, Livestock Systems Alliance, Roseworthy Campus,
University of Adelaide, Roseworthy, SA 5371, Australia
c
Research Centre for Early Origins of Adult Disease, School of Paediatrics and Reproductive Health,
University of Adelaide, Adelaide, SA 5005, Australia
Accepted 27 February 2008

Abstract

Maternal feed allowance during pregnancy can affect the development of the ovine placenta and fetus. The impact of variations in feed
allowance prior to as well as throughout pregnancy has received less attention. Ewes were offered 0.6 (R), 1.2 (C) or 1.8 (AL) maintenance
requirements from 89 days before conception until day 133 of pregnancy. Ewes were euthanased on days 50, 92 and 133 of pregnancy. Ewe
live weight and body condition score, maternal and fetal metabolic and hormonal profiles, fetal body dimensions and organ weights, and the
number, weight and morphology of placentomes were measured. Maternal live weight and condition score were lower in R compared to AL
ewes at all stages of pregnancy (P < 0.05). Plasma glucose and albumin concentrations of R ewes were significantly reduced (P < 0.05) at
mid and late gestation, respectively. Placental components were generally unresponsive to long term variations in maternal feed allowance. How-
ever, placental weight was significantly (P < 0.05) correlated with fetal weight at days 50 (r ¼ 0.59) and 133 (r ¼ 0.69) of gestation. By late
gestation growth-retarded singleton fetuses from R ewes were 19% lighter (P < 0.05), with reduced abdominal (9%) and thoracic (10%) girths
(P<0.05) but of similar crown-rump length compared with fetuses from AL ewes. These differences were associated with significantly reduced
IGF-I concentrations in fetal plasma (P < 0.05). In conclusion, maternal, placental and fetal adaptations to long established planes of variable
maternal feed allowance were able to maintain fetal growth during early and mid-pregnancy while fetal growth restriction, associated with re-
duced fetal IGF-I levels, became apparent in late pregnancy.
Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Sheep; Nutrition; Fetal development

1. Introduction nutrition [2,3], maternal nutrient partitioning (e.g. adolescent

Perturbations of the pre-natal environment can affect the
development of the fetus and placenta, subsequently impacting
on post-natal viability and productivity. These latter aspects
have implications for the fields of agriculture and medicine.
The effects of intrauterine growth retardation (IUGR) via
artificial methods (e.g. carunclectomy) [1], maternal under-
* Corresponding author. Tel.: þ61 8 8524 9665; fax: þ61 8 8524 9088.
E-mail address: julie.owens@adelaide.edu.au (J.A. Owens).
ewe model) [4,5] and litter size (e.g. prolific ewe model) [6]
on ovine fetal development are widely reported. Severe restric-
tions of substrate supply may result in maternal, placental and
fetal adaptations to the altered pre-natal environment, which
allows survival of offspring, often at the expense of normal
fetal development. There is increasing evidence from epidemi-
ological and experimental studies that the programming of
fetal development may be particularly sensitive to maternal
nutrient status during the peri-conception period. Changes in
maternal live weight [7] and differences in ewe body condition
score [8] prior to mating have been related to altered

0143-4004/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.placenta.2008.02.014
540 S.P. Quigley et al. / Placenta 29 (2008) 539e548
utero-placental weights at days 53e56 and 65 of gestation,
respectively, in sheep. However, no studies appear to have in-
vestigated if these differences persist throughout pregnancy.
Perturbed fetal development may be apparent as altered
fetal/birth weight, length, girth and disproportionate tissue
growth. There is a tendency for substrates to be directed to
maintain essential tissues and organs, such as the brain, at
the expense of less vital ones. As a result, size at birth, muscle
development and growth [9,10], wool follicle formation [11],
adiposity [6,12] and gastrointestinal tract development [13]
may be altered in growth restricted fetuses and newborns.
Such perturbations in development may affect neonatal sur-
vival or influence post-natal productivity of livestock.
Adaptations of the fetus to restricted nutrient supply not
only alter post-natal performance but are also considered
likely precursors to adulthood disease in humans. These fetal
adaptations are often associated with fetal hypoglycaemia, hy-
poxaemia and modifications to the insulin-like growth factor
(IGF) and hypothalmo-pituitary-adrenal axes. A number of ep-
idemiology studies have implicated these fetal adaptations to
placental insufficiency with diseases later in life [14].
The following experiment examined the hypothesis that
long-term pre- and post-conception feed allowance of the
ewe induces adaptations in feto-placental and maternal charac-
teristics and affects the development of the ovine fetus. Some
of the mechanisms responsible for these adaptations were
investigated.
2. Materials and methods
The experimental protocol was reviewed and approved by the Primary In-
dustries and Resources South Australia, Animal Ethics Committee in accor-
dance with the Australian Code of Practice for the Care and Use of Animals
for Scientific Purposes [15].
2.1. Animals, feeding, mating and experimental design
Multiparous South Australian Merino ewes (n ¼ 119), greater than 3 years
of age at the commencement of the study, were ranked by live weight
(64.5  0.8 kg, mean  SEM) and randomly allocated to three feed allowance
treatment groups. The treatments were control (C, n ¼ 30), restricted (R,
50%C, n ¼ 60) and ad libitum (AL, 150%C, n ¼ 60), in which ewes were
fed at 120%, 60% and 180% of metabolisable energy (ME) requirements, re-
spectively. The maintenance ME requirements of a 64 kg non-pregnant ewe
were calculated from AFRC [16]. C ewes were offered 120% of maintenance
ME requirements in an attempt to maintain body conditions score throughout
pregnancy. AL ewes were fed such that there was greater than 10% feed re-
fusals each day, which was calculated to be an average daily intake of
180% of ME requirements. Ewes were fed a pelleted diet (90.7% dry matter
(DM), 9.5 MJ ME kg1 DM and 120 g kg1 crude protein), consisting of ce-
real and lucerne hays, barley, oats, lupins, oat bran, almond shells, limestone
and molasses (Johnsons Stockfeeds, Kapunda, SA, Australia). All of the die-
tary components were decreased or increased in equal proportions in the R and
AL treatments, respectively. The ewes were fed daily in their treatment groups
from 89 days prior to mating until the day of slaughter on either day 50 (early),
92 (mid) or 133 (late) of pregnancy. Ewe live weight and condition score [17]
were monitored at weekly intervals. After 75 days of treatment feeding, ewes
were treated with controlled intra-vaginal drug release devices (EAZI-
BREEDÔ CIDRÒ Sheep and Goat Device, Pharmica and Upjohn, Australia)
for twelve days. Vasectomized rams, fitted with harnesses and crayons, were
put with the ewes at CIDR removal. Crayon-marked ewes were inseminated
48 h later via laparoscopy (day 0) with fresh semen collected from a single
ram. Pregnancy and litter size was determined by ultrasonography (Echo Cam-
era SSD-500, 3.5 MHz probe; Aloka Co Ltd, Tokyo, Japan) on day 44 of ges-
tation. Non-pregnant ewes were subsequently removed from the experiment
while pregnant ewes within treatments were stratified on litter size and ranked
on live weight and randomly allocated to three sample cohorts based on ges-
tational age (days 50, 92 and 133 of gestation).
2.2. Euthanasia and fetal dissection
Ewes were penned as a group on the mornings of euthanasia, left undis-
turbed for 30 min and then bled via jugular venipuncture. They were subse-
quently slaughtered by captive-bolt pistol and exsanguination. The
reproductive tracts were immediately recovered by hysterotomy, blood col-
lected from the umbilical arteries and the fetuses euthanased with a sodium
pentobarbitone overdose. Blood samples were collected into Lithium Heparin
tubes and plasma samples were stored at 20  C until analysis.
Fetal weight, crown rump length, abdominal girth, thoracic girth, skull
width, skull length, litter size and sex were recorded. The liver, kidneys, heart,
brain and lungs were removed and weighed. The longissimus dorsi, semitendi-
nosus and supraspinatus muscles were removed from the left side of late ges-
tation fetuses and weighed. Absolute and relative weights were determined for
all tissues removed from the fetuses.
2.3. Placental measurements
The total number of placentomes, weight of individual placentomes, and
the number and weight of placentomes per fetus (in the case of multiple litters)
were recorded. Placental weight was defined as the aggregate weight of indi-
vidual placentomes. The morphology of individual placentomes was character-
ised according to their level of eversion [18] at day 92 and 133. Placentomes
were classified as type A (inverted), B, C or D (increasing levels of eversion,
respectively). The number of each placentome type was divided by the total
number of placentomes per ewe and the proportion of each type of placentome
determined per ewe.
2.4. Maternal and fetal metabolic and hormone profiles
Maternal and fetal plasma glucose (GLUC HK, Roche Diagnostic Sys-
tems, Germany), albumin (Albumin Plus BCG, Roche Diagnostic Systems)
and urea (UREA/BUN, Roche Diagnostic Systems) and maternal plasma
non-esterified fatty acids (NEFA C, Wako Pure Chemicals, Japan), cholesterol
(CHOD-PAP, Roche Diagnostic Systems) and triglyceride (TRIG, Roche Di-
agnostic Systems) concentrations were measured as indicators of nutritive sta-
tus on the COBAS MIRA automated analysis system (Roche Diagnostic
Systems). The intra-assay coefficients of variation of the glucose, albumin,
urea, NEFA, cholesterol and triglycerides assays were 1.8, 7.3, 6.2, 3.1, 2.9
and 4.2%, respectively.
Plasma concentrations of fetal IGF-I and IGF-II were determined on
a sub-population of fetuses (n ¼ 9 or 10 for R and AL fetuses at day 92
and 133 of gestation). IGF-I and IGF-II were separated from binding proteins
by size exclusion HPLC of plasma at pH 2.5 [19]. IGF-I and IGF-II concen-
trations were then determined in neutralised chromatography fractions using
a commercially available ELISA kit, according to the manufacturer’s instruc-
tions (Diagnostic System Laboratories, Inc., Texas, USA). The intra-assay co-
efficients of variation of the IGF-I and IGF_II assays were 3.2 and 8.8%,
respectively.
2.5. Statistical analysis
Analysis of variance procedures (GLM procedure in SAS) [20] were used
to interpret the significance of main effects and their first order interactions on
all variables. Data that were not normally distributed or exhibited unequal var-
iances were log transformed before analysis. Proportional placentome mor-
phology data were arcsin transformed before analysis to ensure stabilisation
of variance. Relationship coefficients (r) and associated probabilities (P) be-
tween maternal, placental and fetal parameters were established using Pearson
 S.P. Quigley et al. / Placenta 29 (2008) 539e548 541

product-moment correlations (CORR procedure in SAS) [21]. All correlations
were determined within each gestational stage, within litter size groupings and
within feed allowance treatments.
3. Results
3.1. Maternal live weight and condition score
Live weights and condition scores of R and AL ewes dif-
fered significantly from 50 days prior to insemination, at in-
semination and on allocated days of slaughter (P < 0.05,
Table 1). Ewe live weight increased significantly between
days 92 and 133 of pregnancy (P < 0.05) in all treatment
groups, even though feed allowances remained fixed at initial
treatment levels and condition scores of treatment groups con-
tinued to diverge. Nevertheless, AL ewes were 30e40%
heavier than their R counterparts at late gestation. Ewe live
weight at mating correlated positively with ewe condition
score at mating (P < 0.01, r ¼ 0.78) and ewe live weight
(P < 0.01, r ¼ 0.86) and condition score (P < 0.01, r ¼ 0.73)
on day of slaughter.
3.2. Maternal metabolic profile
Maternal plasma glucose concentration was lowest at day
133 of gestation (P < 0.05), while plasma urea and NEFA
concentrations were both greatest at this time (P < 0.05). Ma-
ternal plasma glucose concentration was reduced in R ewes
compared with AL ewes at each stage of gestation, although
this was only significant for twin bearing ewes at day 92
(P < 0.05, 4.6  0.6 and 6.6  0.5 mmol L1, R and AL, re-
spectively) and singleton bearing ewes at day 133 of gestation
(P < 0.05, 3.2  0.2 and 3.9  0.2 mmol L1, R and AL, re-
spectively). Similarly, maternal plasma albumin was signifi-
cantly reduced in singleton bearing R ewes compared with
singleton bearing AL ewes (P < 0.05) at each stage of gesta-
tion. Litter size had no effect on maternal plasma glucose, al-
bumin, urea, cholesterol and triglyceride concentration,
however NEFA concentration was elevated in R twin bearing
ewes compared with R singleton bearing ewes at day 92
(P < 0.01, 1.8  0.2 and 0.7  0.2 meq L1, respectively)
and day 133 of gestation (P < 0.01, 2.1  0.6 and
0.5  0.2 meq L1, respectively).

3.3. Placental characteristics

Table 1
Number, live weight and body condition score of ewes offered different feed Total placentome weight was significantly different at each
allowances prior to and throughout pregnancy
stage of gestation and greatest at day 92 (P < 0.05, data not
Stage of experiment Litter size Maternal feed allowance shown). The number of placentomes was similar at each stage
Restricted Control Ad libitum of pregnancy. Fetal to placentome weight ratio increased ap-
Number of ewes (n) proximately 10-fold with each successive stage of develop-
Days 89 and 50 59 30 60 ment examined (P < 0.01, data not shown). Maternal feed
Day 0 (insemination) 47 27 55 allowance had no effect on the total number of placentomes,
Day 44 (scanning) 23 18 40
total placentome weight, mean placentome weight or the fetal
Day 50 1 6 6 4
2 2 0 10 to placentome weight ratio in singleton or twin bearing ewes,
Day 92 1 4 4 7 at each stage of gestation (Table 2). The total placentome
2 4 2 6 weights from twin bearing pregnancies were heavier than sin-
Day 133 1 4 6 7 gleton bearing pregnancies at days 50, 92 and 133 of gestation
2 3 0 6
(P < 0.05, data not shown). There was no significant relation-
Ewe live weight (kg) ship between total placentome weight and ewe live weight at
Day 89 64.4  0.7 64.6  1.0 64.5  0.7 mating and slaughter at each stage of gestation. There was
Day 50 57.5  0.9a 63.0  1.3b 65.4  0.9b
Day 0 55.4  1.1a 65.5  1.5b 69.8  1.1c
a greater proportion of type A placentomes per ewe at day
Day 50 1 54.6  3.2a 64.4  3.2b 68.1  4.0b 50 of gestation than at days 92 and 133 (P < 0.01). At both
2 59.0  4.8a e 76.3  2.1b days 92 and 133 of gestation, maternal feed allowance had
Day 92 1 51.8  4.1a 70.2  4.1b 77.4  3.1b no effect on placentome morphology in singleton bearing
2 63.2  3.6a 78.4  5.1b 83.1  2.9b ewes. However, twin bearing R ewes had a greater proportion
Day 133 1 58.5  4.5a 77.3  3.7b 81.81  3.4b
2 65.8  4.4a e 91.2  3.1b
of everted placentomes than AL ewes at day 92 (type B, C and
D) and day 133 (type C and D) of gestation (P < 0.05). Type
Ewe body condition score
B, C and D placentomes were heavier than type A placen-
Day 89 3.3  0.1 3.2  0.1 3.3  0.1
Day 50 3.0  0.1a 3.1  0.1b 3.2  0.1b tomes from singleton and twin bearing ewes at both days 92
Day 0 2.9  0.1a 3.2  0.1b 3.5  0.1c and 133 of gestation (P < 0.05). While type D placentomes
Day 50 1 3.0  0.1a 3.3  0.1a 3.8  0.2b from twin bearing ewes were heavier than type D placentomes
2 3.0  0.2a e 3.8  0.1b from singleton bearing ewes at both days 92 and 133 of gesta-
Day 92 1 2.5  0.2a 3.5  0.2b 3.7  0.2c
tion (P < 0.05).
2 3.0  0.2a 3.5  0.2ab 3.8  0.1b
Day 133 1 2.4  0.2a 3.1  0.1b 3.8  0.1c
2 2.3  0.1a e 3.9  0.1b 3.4. Fetal body dimensions
Values are least-square means  standard error of the mean. Different alpha-
betical superscripts across the rows, indicate significant differences between Weight of singleton fetuses from AL feed allowance ewes
treatments (P < 0.05). was only significantly greater than those from R ewes at day
542 S.P. Quigley et al. / Placenta 29 (2008) 539e548

Table 2
Placental characteristics of singleton and twin bearing ewes offered different feed allowances prior to and throughout pregnancy
Parameter Litter size Maternal feed allowance
Restricted Control Ad libitum
Day 50 of pregnancy
Total placentome number 1 87.5  7.1 82.7  7.1 80.3  8.7
2 93.5  7.2 e 85.7  3.3
Total placentome weight (g) 1 124.6  12.3* 138.4  12.3 125.7  15.1**
2 293.7  54.6 e 235.0  24.4
Fetal:Placentome weight 1 0.1  0.02 0.1  0.02 0.1  0.02
2 0.1  0.03 e 0.2  0.02
Day 92 of pregnancy
Total placentome number 1 73.3  4.3 76.3  4.3 73.0  3.0**
2 98.5  6.4 98.5  9.0 89.5  5.2
Total placentome weight (g) 1 824.0  164.8 863.5  164.8 668.9  124.6**
2 1180.0  94.7 1029.6  133.9 998.8  77.3
Fetal:Placentome weight 1 0.9  0.1 1.0  0.1 1.1  0.1**
2 1.2  0.1 1.4  0.2 1.5  0.1
Day 133 of pregnancy
Total placentome number 1 72.5  9.3 76.3  7.6 69.6  7.0*
2 91.3  7.8 e 90.7  5.5
Total placentome weight (g) 1 411.8  58.4** 557.0  47.7 482.2  44.2
2 810.8  122.1 e 639.1  86.3
Fetal:Placentome weight 1 9.1  0.8 7.9  0.6 9.7  0.6
2 8.4  3.2 e 13.3  2.2
Values are least-square means  standard error of the mean. No significant differences between treatments, within each litter size, were present. Within columns,
differences between litter sizes, within a treatment, are indicated by *(P < 0.05) and **(P < 0.01).

133 (P < 0.05, Table 3). There was no significant difference in
fetal crown rump length between fetuses collected from AL
and R ewes at any stage of gestation. By day 133 of gestation
R singleton fetuses were significantly thinner (P < 0.05), with
smaller abdominal (319.3  11.3 and 349.9  8.6 mm) and
thoracic (331.7  10.5 and 367.4  7.7 mm) girths than AL
fetuses. Fetal weight was correlated positively with total pla-
centome weight at days 50 and 133 of gestation, but not at
day 92 (Fig. 1).
3.5. Fetal organ development
The restriction of maternal feed allowance reduced the ab-
solute (P < 0.05) and relative (P < 0.05) weight of the lungs
of singleton fetuses at day 50 of gestation (data not shown).
Restriction of maternal feed allowance increased the absolute
(46.3  1.7 and 41.6  1.4 g) and relative (70.7  2.0 and
58.3  1.6 g kg1 fetal weight) liver weights of R compared
with AL twin fetuses at day 92 (P < 0.05); maternal feed al-
lowance had no affect on organ development of singleton fe-
tuses at day 92. At day 133, absolute liver weights of R
singleton fetuses were reduced in response to restricted mater-
nal feed allowance (P < 0.05, Table 4), although relative liver
weights were similar between R and AL singleton fetuses. The
livers of R twin fetuses were proportionately larger than AL
twin fetuses (P < 0.05) at day 133. While absolute brain
weights of day 133 singleton fetuses were unaffected by ma-
ternal feed allowance, R fetuses had proportionately larger
brains than AL singleton fetuses (P < 0.05). The brain to liver
weight ratio was reduced in twin fetuses from feed restricted
ewes at both days 92 (P < 0.05, 0.3  0.02 and 0.4  0.02,
R and AL respectively) and 133 (P < 0.05, 0.5  0.03 and
0.6  0.02, R and AL respectively) of gestation. Fetal liver
and brain weights, as a proportion of fetal weight, correlated
positively (P < 0.01, r ¼ 0.70) and negatively (P < 0.01,
r ¼  0.57) with placental weight at day 133 of gestation,
respectively (Fig. 1), indicative of asymmetric growth
retardation.
3.6. Fetal muscle weights
Singleton fetuses had significantly (P < 0.01) heavier
longissimus (40.9  1.4 and 33.4  1.7 g) and supraspinatus
(10.7  0.5 and 8.5  0.6 g) muscles than twin fetuses. In
R singleton fetuses the weights of the longissimus, semitendi-
nosus and supraspinatus muscles were reduced by 19%,
23.5% and 21.3% respectively compared with AL singleton
fetuses. In R twin fetuses, the weights of the longissimus,
semitendinosus and supraspinatus muscles were reduced by
17.4%, 23.4% and 12.4% respectively, compared with AL
twin fetuses although this was only significant (P < 0.05)
for the longissimus muscle. The relative weight of the semite-
ndinosus, longissimus and supraspinatus muscles (g kg1 fe-
tal body weight) were unaffected by maternal feed allowance
or litter size.
3.7. Fetal metabolic profile
Fetal plasma glucose concentration decreased (P < 0.05)
and fetal plasma albumin concentration increased (P < 0.05)
 S.P. Quigley et al. / Placenta 29 (2008) 539e548 543

Table 3
Body dimensions of fetuses collected at days 50, 92 and 133 of pregnancy from ewes offered variable feed allowances prior to and throughout gestation
Parameter Litter size Maternal feed allowance
Restricted Control Ad libitum
Day 50 of pregnancy
Fetal weight (g) 1 16.0  0.7 17.3  0.7 18.0  1.0
2 18.7  1.0 e 18.1  0.5
Fetal crown rump length (mm) 1 97.2  2.9 101.9  2.9 104.5  3.6
2 103.3  3.0 e 105.5  1.3
Fetal abdominal girth (mm) 1 56.7  2.1 56.0  2.1 58.5  2.6
2 61.0  2.3 e 58.7  1.0
Fetal thoracic girth (mm) 1 54.3  1.2 55.8  1.2 55.3  1.4
2 57.5  1.6 e 58.9  0.7
Skull width (mm) 1 14.7  0.4 15.7  0.4 15.9  0.5
2 15.3  0.6 e 15.2  0.3
Skull length (mm) 1 24.5  0.3a 25.2  0.3b 26.4  0.3b
2 25.3  0.6 e 25.5  0.3
Day 92 of pregnancy
Fetal weight (g) 1 680.9  26.5 695.8  26.5 685.3  20.0
2 660.6  21.8 688.8  30.8 712.7  17.8
Fetal crown rump length (mm) 1 332.0  14.4 334.8  14.4 336.9  10.9
2 345.0  11.6 317.3  16.3 320.3  9.4
Fetal abdominal girth (mm) 1 202.0  6.0 188.5  6.0 198.7  4.6
2 194.9  5.0ab 182.3  7.1a 201.7  4.1b
Fetal thoracic girth (mm) 1 187.8  3.2 194.3  3.2 191.9  2.4
2 187.8  2.0a 185.8  2.8a 196.0  1.6b
Skull width (mm) 1 43.8  0.7 44.0  0.8 45.2  0.5
2 44.3  0.5 43.4  0.8 44.9  0.4
Skull length (mm) 1 78.4  1.1a 81.5  1.3ab 81.5  0.8b
2 80.71  .0 78.6  1.5 82.0  0.8
Day 133 of pregnancy
Fetal weight (g) 1 3573  287a 4333  234ab 4524  217b**
2 3386  126 e 3676  89
Fetal crown rump length (mm) 1 513  13a 553  1.0b 536  10ab
2 515  9 e 525  6
Fetal abdominal girth (mm) 1 319  11a 343  9ab 350  9b**
2 309  7 e 315  5
Fetal thoracic girth (mm) 1 326  11a 346  9ab 366  9b**
2 331  4 e 334  3
Skull width (mm) 1 62  1 63  1 64  1*
2 59  1 e 61  1
Skull length (mm) 1 118  2 123  2 122  2
2 116  2 e 119  1
Values are least-square means  standard error of the mean. Different alphabetical superscripts across the rows, indicate significant differences between treatments
(P < 0.05). Within columns, significant difference between litter sizes, within a treatment, is indicated by *(P < 0.05) and **(P < 0.01).

as gestation progressed. Fetal plasma glucose (P < 0.05) was
greater in twin fetuses from AL ewes than twin fetuses from
R ewes at day 92 of gestation (Table 5). However, maternal
feed allowance had no effect on albumin or urea concentra-
tions within each stage of gestation. Litter size had no effect
on plasma glucose and albumin concentrations; however
plasma urea concentration was significantly higher in single-
ton fetuses compared with twin fetuses from R ewes at day
92 of gestation. Fetal plasma glucose correlated positively
with fetal weight (P < 0.05, r ¼ 0.34) and placentome weight
(P < 0.05, r ¼ 0.36) at day 133 of gestation.
3.8. Fetal plasma IGF-I and IGF-II
Fetal plasma IGF-I concentration increased between day 92
and 133 of gestation (P < 0.01, Table 5). IGF-II concentration
was not significantly different between day 92 and day 133 of
gestation. Singleton fetuses from R and AL ewes had similar
plasma IGF-I concentrations at both days 92 and 133 of ges-
tation. Twin fetuses from AL ewes had higher plasma IGF-I
concentrations than twin fetuses from R ewes at both days
92 (P < 0.05) and 133 (P < 0.05) of gestation. Fetal plasma
IGF-II concentration was unaffected by maternal feed allow-
ance and litter size. There were no significant relationships be-
tween fetal plasma IGF-I and IGF-II and fetal weight,
placental weight, liver weight and plasma glucose at day 92
of gestation. However, by late gestation fetal weight, placental
weight and fetal plasma glucose concentration were all corre-
lated positively (P < 0.05) with fetal plasma IGF-I concentra-
tion (Fig. 2). No significant relationships existed between fetal
plasma IGF-II and fetal or placental parameters at day 133 of
gestation.
544 S.P. Quigley et al. / Placenta 29 (2008) 539e548

Fig. 1. Relationship between placental and fetal weights at day 50 (a), 92 (c) and 133 (e) of gestation and between placental weight and relative fetal liver weight
(b), relative fetal brain weight (d) and fetal liver:brain weight (f) at day 133 of gestation. Singleton (filled symbols) and twin (unfilled symbols) fetuses were col-
lected from ewes offered restricted (7), control (,) and ad libitum (B) feed allowances prior to and throughout gestation. Positive relationships existed between
placental and fetal weight at day 50 (r ¼ 0.44; P < 0.01) and day 133 (r ¼ 0.69; P < 0.001) and relative fetal liver weight at day 133 (r ¼ 0.70; P < 0.001). Neg-
ative relationships existed between placental weight and relative fetal brain weight (r ¼  0.57; P < 0.001) and fetal liver:brain weight (r ¼  0.79; P < 0.001) at
day 133.

4. Discussion of fetal growth. Restricting maternal feed allowance decreased

The present study is unique in the timing of onset and
duration of the maternal feed restriction imposed. It is the only
study in which fetal, placental and maternal characteristics
were quantified at three stages of gestation in ewes that were
already significantly divergent in live weight and body condi-
tion score from approximately 50 days prior to conception as
well as throughout gestation.
Differential maternal feed allowance coupled with variable
litter size, resulted in compensatory adaptations of the mother,
placenta and fetus, which enabled fetal survival at the expense
live weight and condition score of ewes which, together with
lowered maternal plasma glucose and albumin concentrations,
suggests energy and protein deficiencies and the mobilization
of fat reserves.
In the present study, placental weight and the number of
placentomes were generally unresponsive to variations in
long-term maternal feed allowance imposed 89 days prior to
insemination, in both singleton and twin pregnancies, at all
stages of pregnancy. The classical studies of Everitt [22] dem-
onstrated that feed intake during pregnancy had a profound in-
fluence on placental weights in sheep. However, recent
 S.P. Quigley et al. / Placenta 29 (2008) 539e548 545

Table 4
Organ weights of fetuses collected at day 133 of pregnancy from ewes offered variable feed allowances prior to and throughout gestation
Tissue Litter size Maternal feed allowance
Absolute weight (g) Relative weight (g kg1 fetal weight)
Restricted Control Ad libitum Restricted Control Ad libitum
a b b a b
Liver 1 100.7  11.6 143.4  9.5 131.8  8.8 * 27.9  1.7 33.3  1.4 28.8  1.3a*
2 95.3  4.9 e 90.0  3.5 28.2  0.8a e 24.4  0.6b
Heart 1 28.4  3.5 33.7  2.9 34.6  2.7* 8.0  0.5 7.8  0.4 7.6  0.4
2 28.8  1.7 e 28.7  1.2 8.5  0.4 e 7.8  0.3
Lungs 1 127.2  12.3a 161.1  10.1ab 164.4  9.3b* 36.2  3.4 37.3  2.8 36.9  2.6
2 135.5  7.7 e 131.2  5.5 40.2  2.2 e 35.8  1.6
Kidney 1 18.0  2.1 23.3  1.7 21.8  1.6* 5.1  0.4 5.4  0.4 4.8  0.3
2 18.5  0.7 e 17.4  0.5 5.5  0.3a e 4.8  0.2b
Brain 1 53.4  2.3 56.0  1.9 55.0  1.7 15.1  0.9a 13.0  0.7ab 12.3  0.7b*
2 50.3  1.6 e 53.2  1.1 14.9  0.5 e 14.5  0.3
Brain:Liver 1 0.6  0.05a 0.4  0.04b 0.4  0.04ab* e e e
2 0.5  0.03a e 0.6  0.02b e e e
Values are least-square means  standard error of the mean. Different alphabetical superscripts across the rows, indicate significant differences between treatments
(P < 0.05). Within columns, significant difference between litter sizes, within a treatment, are indicated by *(P < 0.05).

evidence suggests that the mechanisms by which maternal nu- between control and nutrient restricted (70% of ME require-
trient status regulates placental development and function are ments from 22 days post-conception) ewes at days 45 and 90
more complex than the simple supply of nutrients. For exam- of gestation but there was a tendency towards decreased pla-
ple, the timing, duration and severity of the intervention, the cental weights at day 135 of gestation in nutrient restricted
age of the ewe and the long-term management system imposed ewes [23]. In contrast, ewes that were nutrient restricted
may all influence how the placenta responds to nutrient supply (50% of ME requirements) between days 28 and 77 of gesta-
in sheep. No differences in placental weights were reported tion had increased placental weights at day 144 of gestation
[24]. An acute restriction, between days 85 and 90 of gesta-
tion, reduced placental weights at day 90 of gestation but
Table 5
Metabolites and insulin-like growth factors in the circulation of fetuses not at day 133 [25]. With respect to peri-conception nutrient
collected at days 92 and 133 of pregnancy from ewes offered variable feed status, lean ewes, 12 weeks prior to conception and maintained
allowances prior to and throughout gestation as such until slaughter, had similar placentome weights to fat
Parameter Litter size Maternal feed allowance and moderate body condition score ewes at day 65 of gestation
Restricted Control Ad libitum [8], while offering ewes 70% of ME requirements from
45 days prior to conception until day 53e56 of gestation
Day 92 of pregnancy
Glucose (mmol L1) 1 1.3  0.4 1.7  0.4 1.4  0.3 also had no influence on placental weight at that time [7]. Nu-
2 0.7  0.2a 0.8  0.3a 1.7  0.2b trient supply to ewes at different ages also has variable affects
Albumin (g L1) 1 19.0  0.9 18.0  1.0 20.2  0.6 on placental development, with over-nutrition of adolescent
2 20.1  1.7 20.5  2.4 21.5  1.4 [26] and adult [27] ewes resulting in decreased and unchanged
Urea (mmol L1) 1 5.4  0.3ab** 6.2  0.3b 5.3  0.2a
2 4.9  0.2 6.3  0.2 5.1  0.1
placental weights compared to control fed ewes of a similar
IGF-I (ng ml1) 1 13.2  0.7 e 12.8  0.7 age. Interestingly, under-nutrition of adolescent ewes resulted
2 11.9  0.6a e 14.1  0.5b in smaller fetuses at day 130 of gestation despite no differ-
IGF-II (ng ml1) 1 172.8  17.0 e 163.3  17.0 ences in placental mass at day 90 and 130 of gestation [28].
2 168.8  13.0 e 147.0  12.0 Variable placental development was also reported when ewes
Day 133 of pregnancy adapted to a harsh production system, with inadequate nutri-
Glucose (mmol L1) 1 0.6  0.2 0.6  0.2 0.6  0.2 tion, or a more sedentary lifestyle, with adequate nutrition,
2 0.4  0.1 0.5  0.1
e
were fed either 50% or 100% of ME requirements from
Albumin (g L1) 1 28.1  2.3 30.1  1.9 33.1  1.9
2 29.1  1.4 e 30.3  1.0 days 22 to 78 of gestation [29], suggesting that long-term en-
Urea (mmol L1) 1 5.5  0.8 5.5  0.6 7.1  0.6 vironmental or intergenerational influences may also deter-
2 5.8  0.7 e 6.7  0.5 mine the response of the placenta to variable nutrient supply.
IGF-I (ng ml1) 1 16.2  2.7 e 20.3  2.4 In the present study, fetal weight was correlated with pla-
2 11.5  0.6a e 18.0  0.8b cental weight in early and late gestation, consistent with other
IGF-II (ng ml1) 1 198.0  19.6 e 192.7  17.5
2 181.7  28.9 e 198.1  35.4 studies [7,30]. The lack of any relationship between placental
weight and fetal weight at day 92 of gestation may be attrib-
Values are least-square means  standard error of the mean. Different alpha-
betical superscripts across the rows, indicate significant differences between uted to the compensatory overgrowth of placentomes at mid
treatments (P < 0.05). Within columns, significant difference between litter gestation. It has been demonstrated that nutrient restriction in-
sizes, within a treatment, is indicated by **(P < 0.01). duces a conversion of type A to type C or D placentome
546 S.P. Quigley et al. / Placenta 29 (2008) 539e548

Fig. 2. Relationship between fetal plasma IGF1 and fetal (a) and placental (b) weights and glucose concentration (c) at day 133 of gestation. Singleton (filled
symbols) and twin (unfilled symbols) fetuses were collected from ewes offered restricted (7) or ad libitum (B) feed allowances prior to and throughout gestation.
There was a positive relationship between fetal plasma IGF1 concentration and fetal weight (r ¼ 0.74; P < 0.001), placental weight (r ¼ 0.48; P < 0.05) and fetal
plasma glucose concentration (r ¼ 0.59; P < 0.01).

morphology in sheep [23,29,31]. It has been suggested that
this conversion is associated with an increase in size and vas-
cularisation of placentomes [32], thereby increasing their ca-
pacity for nutrient exchange between the maternal and fetal
compartments. It is possible that the progression of placen-
tome morphology in the present study disrupted the relation-
ship between placental and fetal weights at day 92 of
gestation. Altered placentome morphology was not sufficient
to compensate for the low nutrient availability in R ewes at
day 133 of gestation in the present study however.
Fetal growth restriction was evident by day 133 of gestation
in ewes that were offered a restricted feed allowance. The rel-
ative absence of retarded fetal growth in response to restricted
maternal feed allowance at early and mid gestation suggests
that the adaptive mechanisms of the ewe and the placenta
were able to satisfactorily compensate at these early stages
of gestation, when the conceptus has much lower nutrient re-
quirements for growth compared to near-term. However, in
late gestation, when fetal growth accelerates rapidly relative
to placental size maternal and placental adaptations may
have been unable to maintain adequate nutrient supply. This
resulted in late gestation fetuses which were lighter and thin-
ner, had disproportionate tissue growth and decreased plasma
glucose and IGF-I concentrations, compared with normal
counterparts. Further, a brain sparing effect was evident in
these growth retarded fetuses, where growth of the brain
was maintained at the expense of less vital organs, such as
muscle, liver and lungs. This phenomenon has been com-
monly observed in the fetus subject to nutrient deprivation
in many previous studies and reflects physiological adapta-
tions that divert scarce resources to the brain at the expense
of other tissues [1]. The decreased muscle mass resulting
from maternal under-nutrition and increasing litter size evi-
dent in late gestation fetuses in the present study is of signif-
icance to animal production. Reductions in muscle mass in the
range of 12e28%, across a range of muscles, may have a sig-
nificant impact on post-natal muscle growth and yield of live-
stock [10]. While normal lung development is essential for the
survival of the neonate, aberrant skeletal muscle, liver and
lung development during gestation have been implicated in
the development of adult onset diseases, including type 2 di-
abetes and obstructive airway disease. Interestingly, lung
weight of day 50 fetuses was reduced by restriction of mater-
nal feeding but this was not reflected in twins. A possible ex-
planation is that maternal feed restriction may increase
placental tissue as an adaptive response, particularly in twins
[7] and through this mechanism reduce the effects of nutrient
restriction on organ growth.
 S.P. Quigley et al. / Placenta 29 (2008) 539e548
Restricted nutrient supply to the mother may have indi-
rectly reduced the supply of substrates available for growth
of the fetus and altered the production and circulating concen-
trations of IGF-I in the fetus. It was apparent by late gestation
that fetal plasma IGF-I was positively associated with fetal
weight, placental weight and fetal glucose concentration. Fur-
ther, circulating IGF-I concentrations were lower in fetuses
whose mothers received restricted nutriment, consistent with
circulating IGF-I regulating fetal growth in the last third of
pregnancy [33]. In support, circulating IGF-I is decreased
with maternal under-nutrition [34,35], carunclectomy [36]
and hypoxaemia [37] in the fetal sheep. Further, IGF-I knockout
mice have impaired growth [38], while maternal under-nutri-
tion [39] and carunclectomy [40] decrease the expression of
IGF-I mRNA expression in the liver and skeletal muscle of fetal
sheep, respectively. It is, therefore, possible that restricted nutri-
ent supply to the developing fetus reduced the expression of
IGF-I in tissues. In support, it has been demonstrated that pro-
vision of external glucose, in the presence of under-nutrition,
elevated fetal IGF-I levels [41] while IGF-I infusion increased
protein synthesis in fetal skeletal muscle when amino acid and
glucose concentrations were maintained at constant levels [42].
Therefore, it is possible that low plasma IGF-I concentrations in
the present study may have limited protein accretion in specific
tissues and in the fetuses as a whole.
Despite evidence that IGF-II is also involved in fetal devel-
opment, the present study revealed no such associations. This
finding is in contrast to other studies which have reported pos-
itive [36] and negative [43] associations between fetal weight
and IGF-II in fetal sheep. However, these associations
emerged in late gestation after the pre-partum cortisol surge,
which inhibits IGF-II production by fetal tissues [44] and
may be greater in the fetuses of feed allowance restricted ewes.
In conclusion, the impact of long established planes of vari-
able maternal nutrition in the sheep on feto-placental-maternal
growth and physiological responses was investigated in this
study. Restricted maternal feed allowance in this model re-
sulted in asymmetric IUGR by late gestation, compared to me-
dium or ad libitum maternal feed allowance. In addition, fetal
growth appeared to be regulated by placental weight, indepen-
dent of maternal nutritional status, during early and late gesta-
tion. Restricted fetal growth during late gestation was
characterised by reduced circulating IGF-I concentrations.
Overall, substantial reductions in maternal nutrient availability
resulted in fetal growth-restriction that only became evident
late in gestation.
Acknowledgements
The assistance of Karen Kind, Kathy Gatford and Melissa
Bradbury (Research Centre for Research Centre for Early Or-
igins of Adult Disease, University of Adelaide) and the Turret-
field Farm staff in fetal and placental collections is gratefully
acknowledged. Simon Humphrys (Primegro LTD) conducted
the IGF assays on fetal plasma samples. This work was sup-
ported by SARDI Livestock Systems.
547
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