BIOLOGY OF REPRODUCTION 70, 1–11 (2004)
Published online before print 17 September 2003.
DOI 10.1095/biolreprod.103.020982
Cloned Cattle Fetuses with the Same Nuclear Genetics Are More Variable Than
Contemporary Half-Siblings Resulting from Artificial Insemination and Exhibit Fetal
and Placental Growth Deregulation Even in the First Trimester1
Rita S.F. Lee,2,3 A. James Peterson,3 Martyn J. Donnison,3 Susan Ravelich,4 Anita M. Ledgard,3 Ning Li,3
Jan E. Oliver,3 Andria L. Miller,3 Fleur C. Tucker,3 Bernhard Breier,4 and David N. Wells3
Reproductive Technologies Group,3 AgResearch, Ruakura Research Centre, Hamilton 2001, New Zealand
The Liggins Institute,4 University of Auckland, Grafton, Auckland, New Zealand
ABSTRACT
12] and rabbit [13]; and in a carnivore, the cat [14]. To
date, the technology remains very inefficient and technically demanding. The biggest challenge facing the somatic
cell NT technology is the high rate of pregnancy failure as
well as fetal morbidity and mortality. Pregnancy failure occurs throughout the entire gestation, from embryo transfer
to parturition [15–19]. Cloned offspring tend to have increased birth weight and poorer postnatal survival and
health. However, those that do survive to maturity are fertile and produce normal offspring [15].
The predominant cause of pregnancy failure associated
with NT in cattle and sheep is believed to be either failure
of the placenta to form or abnormal placental development
and function. Abnormal placental development has also
been reported in the cloning of mice [12, 20, 21]. The failure of placental formation may account for most first-trimester losses in cattle, because fetal development is halted
in the absence of the placenta. The absence of vascularization of the chorioallantoic membranes was implicated as
a possible cause for the failure of placentation [16]. Abnormal placental development and function probably accounts for a high proportion of postimplantation fetal losses
and may contribute to inadequate mammary gland development in the surrogate recipients and the failure of signaling in preparation for parturition [1, 2, 19]. In cattle, the
major cause of fetal mortality is the acute, excessive accumulation of allantoic and, to a lesser extent, amniotic
fluid, which is collectively referred to as the hydrops syndrome or hydropsy. In the experience of this group, approximately 60% of the fetal losses between Day 120 and
full gestation may be attributed to or associated with this
syndrome.
The aim of the present study was to compare placental
and fetal development in NT cattle pregnancies with closely
matched control pregnancies generated either by artificial
insemination (AI) or after embryo transfer of in vitro-produced (IVP) embryos. Pregnancies were assessed at the
start of placentome formation (Day 50), when the placentomes were completely formed (Day 100), and during the
period when hydropsy frequently occurs (Day 150). Contributing factors, such as the genetic background of the fetuses, age of the surrogate recipients and their reproductive
history, and nutrition during the pregnancy, were controlled
in the experimental design. Use of the same sire allowed
comparison of fetal and placental development in the offspring from one sire, although it was not possible to control
for maternal gene contribution. The cell line chosen to generate NT embryos had been demonstrated previously to be
totipotent, with the production of 20 viable cloned calves.
However, previous pregnancies from NT embryos derived
The cloning of cattle by somatic cell nuclear transfer (NT) is
associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome),
and fetal overgrowth. Fetal and placental development was investigated at Day 50, during placentome formation; at Day 100,
when placentation was completed; and at Day 150, when the
hydrops syndrome frequently develops. The NT fetuses were
compared with contemporary half-siblings generated from in vitro-produced embryos or by artificial insemination (AI). Fetal
cotyledon formation and vascularization of the chorioallantoic
membranes was initiated normally in NT conceptuses, but fewer
cotyledons successfully formed placentomes. By Day 100, the
mean number of placentomes was significantly lower in surviving NT fetuses. Only those with normal placentome numbers
were represented in surviving NT pregnancies at Day 150. The
mean total caruncle tissue weight of the placentomes was significantly higher in the surviving NT groups at Days 100 and
150, irrespective of the placentome numbers, indicating that increased NT placental weight was caused by excessive uterine
tissue growth. By Day 100, NT fetuses exhibited growth deregulation, and those that survived to Day 150 were 17% heavier
than contemporary AI controls. Placentome, liver, and kidney
overgrowth accompanied the hydrops syndrome at Day 150. The
NT fetal overgrowth was not a consequence of in vitro embryo
culture and showed no correlation with placental overgrowth.
However, in vitro culture and incomplete reprogramming of the
donor genome are epigenetic effects that may override genetic
traits and contribute to the greater variability in placental and
fetal development in the NT group compared with AI half-siblings.
early development, embryo, placenta, pregnancy, uterus
INTRODUCTION
The successful production of viable offspring by cloning
with somatic cell nuclear transfer (NT) has been achieved
in ungulates, such as cattle [1–3], sheep [4], goat [5–7], pig
[8, 9], and mouflon [10]; in rodents, such as the mouse [11,
Supported by the Foundation for Research, Science and Technology, New
Zealand, grant C10X0018.
2
Correspondence: Rita S.F. Lee, Reproductive Technologies Group, AgResearch, Ruakura Research Centre, East St., Private Bag 3123, Hamilton
2001, New Zealand. FAX: 64 7 838 5628;
e-mail: rita.lee@agresearch.co.nz
1
Received: 7 July 2003.
First decision: 29 July 2003.
Accepted: 29 August 2003.
Q 2004 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
1
2
LEE ET AL.
from this cell line showed many of the problems associated
with clone pregnancies. The present experiment thus allowed us to assess mainly the effect of the NT technology
itself on fetal and placental development.
MATERIALS AND METHODS
All manipulations and treatments of animals involved in the present
study were conducted in accordance with the regulations of the New Zealand Animal Welfare Act of 1999. The generation of embryos for transfer
to recipients or AI of the heifers was carried out in two separate experiments, conducted approximately 2 mo apart, with equivalent numbers of
animals in each experiment. All dams or recipients were 2-yr-old heifers
of predominantly dairy or dairy cross-breeds. Oocytes were obtained from
ovaries of Friesian cows collected at the slaughterhouse, and the same
pool was used as a source of recipient cytoplasts for NT or was in vitro
fertilized to generate the IVP embryos. In vitro maturation of oocytes was
carried out as described previously [2]. To minimize the potential effect
of maternal breed on fetal growth and survival, the breed of the recipients
was kept as similar as possible for all three groups; however, it was not
possible to obtain enough animals from the same breed for these experiments. Only Friesian heifers were inseminated to obtain Friesian fetuses.
NT Embryos
The NT embryos were generated essentially as described previously
[2] using the same ovarian follicular cell line (EFC) from a Friesian cow
in that study. The cells used for NT in these studies had been passaged at
least nine times and previously cryopreserved. Cells were cultured in a 1:
1 mixture of Dulbecco modified Eagle medium and Ham F12 (Gibco, Life
Technologies, Gaithersburg, MA) supplemented with 10% (v/v) fetal calf
serum (FCS; Life Technologies) and 1 mM sodium pyruvate. Cells were
induced into a quiescent state by lowering the FCS concentration in the
medium to 0.5% and culturing for a further 9 days (experiment 1) or 11
days (experiment 2) before NT. After injection of the donor cells into the
perivitelline space, fusion was carried out at 22–24 h after the start of
maturation, and fused donor cell/cytoplast couplets were activated 27–28
h after the start of maturation. After activation, in vitro culture was carried
out as described by Wells et al. [22] in a biphasic AgResearch Synthetic
Oviduct Fluid medium (AgR SOF; AgResearch, Hamilton, New Zealand).
This medium was replaced on Day 4 of culture with fresh medium containing 10 mM 2,4-dinitrophenol as an uncoupler of oxidative phosphorylation [23]. The fatty acid-free bovine albumin (8 mg/ml) in the AgR
SOF was ABIVP (ICP Bio, Auckland, New Zealand). Forty-nine NT embryos judged to be of sufficiently good quality by a subjective grading
system were selected for transfer into recipients on Day 7 after fusion. Of
the heifers that received NT embryos, 20 of 25 in the first experiment and
11 of 24 in the second experiment were Friesian or Friesian cross-breeds.
The remainder were either Jersey cross-breeds or beef cattle.
IVP Embryos
In vitro-matured oocytes were fertilized with frozen-thawed spermatozoa in 50-ml drops under oil for 24 h as described previously [23]. The
semen used was from the same Friesian bull that sired the cow from which
the EFC cells were isolated. Zygotes were cultured as described for NT
embryos, and at Day 7 after fertilization, the embryos were graded, also
as described for the NT embryos. Of those receiving IVP embryos, 8 of
10 from the first experiment and six of nine from the second experiment
were Friesian or Friesian-cross. The remainder were beef breeds.
Artificial Insemination
Twenty-one Friesian heifers were synchronized for estrus using intravaginal controlled progesterone release devices (CIDR; Pharmacia, Ltd.,
Auckland, New Zealand) inserted for 12 days. On Day 8, all heifers were
injected with 1 ml of estrumate (Schering-Plough, Union, NJ), and the
devices were withdrawn after a further 4 days. The mean onset of estrus
was approximately 48 h later. Approximately 12 h after the onset of estrus,
the heifers were inseminated with frozen semen from the above-described
bull used for IVP.
Recipients of IVP or NT Embryos
Seventy healthy heifers were synchronized for estrus concurrent with
those heifers used for AI. Single IVP (n 5 19) or NT (n 5 49) embryos
were transferred nonsurgically into the uterine lumen ipsilateral to the
corpus luteum of each heifer on Day 7 after estrus was observed. The
transfer of single embryos avoided the complications of multiple pregnancies.
Pregnancy Monitoring and Morphometric Measurements
After embryo transfer or AI, all heifers were grazed on pasture together. Between Days 40 and 50 of gestation, all heifers were scanned by
transrectal ultrasonography using a Piemed 200 scanner with a linear 3.5to 5-MHz rectal probe (Philip-sweg, Maastricht, The Netherlands); pregnant animals were identified and subsequently scanned at monthly intervals until slaughter. Only the presence of a gestational sac was recorded
at the first scan. Subsequently, the presence of a fetal heartbeat was regarded as a sign of a viable pregnancy. A sample of pregnant animals
from each group was slaughtered at Days 50, 100, and 150 of gestation,
and the reproductive tracts were collected and transported back to the
laboratory within 1 h. Conceptuses were recovered from the uterus, and
morphometric measurements were carried out. The volumes of both allantoic and amniotic fluids were measured, and both fetal weights and crownrump (C-R) lengths were recorded. The fetal membranes with the cotyledons were weighed wet after all fluids were drained and the membranes
separated from the uterus and fetus. At Days 100 and 150, the heart, brain,
kidneys, and liver were weighed separately. The appearance of the fetus,
organs, uteroplacental units, and fetal membranes were noted. At Days
100 and 150, all caruncles of the uteroplacental units were cut from the
uterus after removal of the fetal cotyledons, combined, and weighed, and
the numbers were recorded.
Statistical Analyses
At each stage, the treatments were compared for each variable using
least significant differences calculated from analyses of variance. Certain
variables (fetal brain, heart, liver, and kidney weights) were also expressed
as a ratio to the fetal weight. An analysis of variance across stages was
done on log-transformed variables to check for treatment by stage interaction. Pairwise comparison of variances between treatment groups was
carried out with the F-test, assuming unequal variance. Differences were
considered to be significant at P # 0.05.
RESULTS
In Vitro Development of Postfusion NT Embryos
In the first experiment, 81% (105/129) of donor cell/
cytoplast couplets were fused successfully, and 104 reconstructed embryos were placed in culture, from which 45
blastocysts (43%) were judged subjectively to be of grades
1 and 2 and, thus, suitable for transfer to recipients. In the
second experiment, 83% (108/130) of donor cell/cytoplast
couplets were fused successfully. From the 105 of these
placed in culture, 45 developed to blastocysts (43%) of
grades 1 and 2. Thus, the development rate to grade 1 and
2 blastocysts for the NT embryos was similar in both experiments.
Pregnancy Rates
The pregnancy rate, as assessed by ultrasonography or
at slaughter with the recovery of a fetus, was similar between experiments 1 and 2 for all three treatment groups.
Therefore, the data were combined for simplicity of analyses.
Figure 1 shows the pregnancy rates of the three treatment groups at different stages of gestation. Pregnancy
rates after the first slaughter were calculated by taking into
account the number that were pregnant at the previous
slaughter and adding that to the remaining number that
were pregnant at the next ultrasound scanning or slaughter.
The data in Figure 1 did not take into account the health
and potential viability of the conceptus, only that a conceptus was detected at scanning or slaughter. The pregnancy rates were similar for all three groups at Day 50 (AI,
3
GROWTH DEREGULATION IN CLONED CATTLE FETUSES
going AI was 66.7% (14/21), which is consistent with the
reported mean conception rate of 64% for New Zealand
dairy herds after AI [24]. The conception rates for Friesian
heifers after embryo transfer was similar for NT (53.6%,
15/28) and IVP embryos (61.5%, 8/13). Conception rate
for IVP embryos in cross-bred heifers was 50% (3/6),
which is similar to the rate for Friesian heifers. The conception rate for cross-bred heifers after transfer of NT embryos was 81% (17/21), compared with 53.6% for Friesian
heifers; however, this difference was not significant (P 5
0.09, 2 3 2 exact test).
Morphometric Analysis
FIG. 1. Percentage of animals pregnant after AI (C) or embryo transfer
with single IVP (#) or NT (D) embryos. Pregnancy was determined either
by ultrasound scanning or with the recovery of a fetus at slaughter.
67%; IVP, 58%; NT, 65%). From then onward, NT pregnancies were continually lost, until only 40% of the recipients that received an NT embryo were still pregnant by
Day 150. No fetal losses were recorded with either the AI
or the IVP group after Day 50.
Maternal Breeds and Pregnancy Losses
To determine if maternal breed had any effect on embryonic survival, the conception rates were compared between the groups. At the first ultrasound scan between Days
40 and 50, the conception rate for Friesian heifers under-
The numbers of AI, IVP, and NT fetuses from which
data were obtained at Days 50, 100, and 150 are shown in
Table 1. Fetal fluid volumes at Days 100 and 150 sometimes were not recorded, because the tracts had ruptured
during slaughter and fluid from the amniotic and allantoic
compartments were mixed or, in some cases, partially lost.
During estimation of the mean fetal membrane weights,
those fetal membranes with large amounts of a gelatinous
substance were excluded from the analysis. The mean 6
SEM at each stage is presented in Table 1. The data are
also graphically represented (see Figs. 2 and 4–6) to display
the variability with stage and treatment, with the means for
each treatment being joined by a line to show how they
relate between the treatment groups.
C-R length and fetal weight. The C-R length of the fetus
is significantly correlated with gestation and is frequently
TABLE 1. Mean 6 SEM values for the morphometric measurements made at Days 50, 100 and 150.a
C-R length (cm)
Fetal weight (g)
Fetal membrane weight (g)
Total fluid volume (ml)
Allantoic fluid volume (ml)
Amniotic fluid volume (ml)
Total caruncle weight (g)
Caruncle numbers
Mean weight per caruncle (g)
Brain weight (g)
Heart weight (g)
Liver weight (g)
Kidney weight (g)
a
b
50
100
150
50
100
150
50
100
150
50
100
150
50
100
150
50
100
150
100
150
100
150
100
150
100
150
100
150
100
150
100
150
Significance of
comparisonsb
Treatment
Gestation
(days)
AI
3.97
18.78
39.99
4.43
283.4
2589
37.2
328
893
164
1655
6500
143
480
3866
20.7
1025
2477
120
802
103
109
1.17
7.37
7.43
37.2
2.55
22.6
19.8
113
1.98
19.7
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.10 (n 5 5)
0.43 (n 5 4)
0.33 (n 5 5)
0.24
2.4
149
2.1
36
117
13
196
444
11
213
542
1.8
38
315
11
28
15
10
0.15
0.92
0.51
3.3
0.08
1.53
0.9
9
0.05
0.9
IVP
3.94
18.79
38.93
4.02
302.8
2726
46.1
387
774
174
1609
5088
155
616
1981
18.1
744
2621
120
811
99
108
1.21
7.48
8.03
39.6
2.73
22.6
20.2
105
2.10
18.8
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.45
0.27
0.73
0.75
12.2
196
10.1
17
57
18
204
698
13
259
751
4.9
178
265
11
109
16
7
0.17
0.90
0.80
0.7
0.15
2.11
0.5
5
0.22
1.8
NT
(n 5 3)
(n 5 4)
(n 5 4)
(n 5 3)
(n 5 3)
3.83
18.59
38.36
4.45
320.6
3042
63.0
304
1360
213
1206
8033
194
235
6433
18.9
935
3985
214
1133
58
98
3.39
11.52
7.07
36.4
3.33
27.1
17.6
159
2.39
27.0
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.10
0.75
0.92
0.25
31.5
170
6.5
51
271
13
228
1800
13
146
1531
1.2
96
1021
44
86
9
10
0.67
1.52
0.26
1.5
0.44
2.11
1.2
10
0.42
2.8
(n 5 10)
(n 5 6)
(n 5 8)
(n 5 5)
(n 5 6)
(n 5 5)
(n 5 5)
(n 5 5)
(n 5 5)
(n 5 5)
NT:AI
NT:IVP
IVP:AI
—
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—
—
—
*
—
*
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—
—
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*
*
*
—
*
*
—
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*
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*
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—
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*
—
—
*
—
—
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*
*
*
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*
*
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*
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*
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—
—
—
Sample numbers for the variables at each stage of gestation and treatement are the same as that stated for C-R lengths, unless indicated otherwise.
—, Means are not significantly different; *, means are significantly different at P # 0.05.
4
LEE ET AL.
FIG. 2. Crown-rump (C-R) length, fetal
weight, and weight of combined fetal
membranes at Days 50, 100, and 150. The
means for each treatment (AI, IVP or NT)
are joined by a line.
used an indicator for the size of the fetal skeletal frame and
to estimate the age of the fetus. No significant difference
was found in the mean C-R length between treatments at
all stages of gestation (Fig. 2 and Table 1). By itself, the
C-R length did not indicate fetal overgrowth in either the
NT or IVP group compared with the AI group. No skeletal
abnormalities were observed.
The mean fetal weight was similar among all three
groups at Day 50 (Fig. 2 and Table 1). One NT fetus appeared to be slightly anemic, and the cotyledon-caruncle
attachment was poor, suggesting imminent pregnancy failure. One IVP fetus had both low fetal weight and C-R
length. An example of a Day 50 NT conceptus with exFIG. 3. A) Example of a Day 50 NT fetus
together with all its fetal membranes. The
chorion over the amniotic sac has been
peeled back to better show the fetus (amnion with fetus [A1F]). Note the absence
of fetal cotyledons at the tips of the chorioallantoic membranes (arrows). B) Closeup of the fetus and the membranes showing good vascularization of the cotyledons
(arrows). C) Head of a normal Day 100 AI
fetus. D) A Day 100 NT fetus with a shortened snout and thick, short neck. Bar 5 1
cm.
cellent vascular development is shown in Figure 3, A and
B. A positive linear correlation was found between the fetal
weight and (C-R length)3 at Day 50 in the AI (r 5 0.79, P
5 0.11) and IVP (r 5 0.99, P 5 0.09) groups, but the
correlation in the NT group was poorer (r 5 0.55, P 5
0.10).
At Day 100, the mean fetal weight (Table 1) in the NT
group was 320 6 32 g, compared with 303 6 12 g for the
IVP fetuses and 283 6 2 g for the AI group. Although the
means between the groups were not significantly different,
five of the six NT fetuses were more than 2 SD heavier
than the mean weight of AI fetuses, whereas only one in
four IVP fetuses was above this weight. Gross malforma-
GROWTH DEREGULATION IN CLONED CATTLE FETUSES
5
FIG. 4. Total fluid, allantoic fluid, and
amniotic fluid volumes at Days 50, 100,
and 150. The means for each treatment
(AI, IVP, or NT) are joined by a line.
tions were absent in all but one NT fetus, which had a
shortened snout and a thick neck in comparison with other
fetuses (Fig. 3, C and D). A strong, positive linear correlation was found between fetal weight and (C-R length)3 in
the AI (r 5 0.99, P 5 0.014) and NT (r 5 0.94, P 5 0.005)
groups but not in the IVP group (r 5 0.48, P 5 0.68). The
slope for this relationship was 7.5-fold higher in the NT
group compared with the AI group, indicating that weight
increase with C-R length was greater in the NT group.
By Day 150, the mean fetal weight had increased 9- to
10-fold in all groups (NT, 3042 6 170 g; IVP, 2726 6 196
g; AI, 2589 6 149 g) Although the mean fetal weight of
the NT group was only 17% and 11% higher than those of
the AI and IVP groups, respectively (not significantly different), five of the eight fetuses were greater than 2 SD
above the mean weight of the AI fetuses, whereas only one
in five and one in four were greater than this weight for
the AI and IVP groups, respectively. No gross fetal abnormalities were detected in any Day 150 fetus. At this stage
of gestation, the skin pigmentation was established, and it
was possible to tell that all fetuses were Friesian. No linear
correlation between the fetal weight and (C-R length)3 was
evident in any of the three groups at Day 150.
Fetal membrane weight. The combined wet weights of
the amniotic and chorioallantoic membranes containing the
fetal cotyledons are shown in Figure 2 and Table 1. These
fetal membranes are derived entirely from the extraembryonic tissues. At Day 50, all chorioallantoic membranes
were vascularized, and the fetal cotyledons were tenuously
attached to the uterine caruncles of the uterus. In all cases,
the vascularization and attachment of the cotyledonary
burrs to the caruncles were more advanced on the amniochorion and the allantochorion adjacent to the fetus than
they were toward the tips of the chorioallantoic sacs. The
number of visible cotyledonary burrs was similar in all
three groups, with an overall mean of 68 6 3. The mean
fetal membrane weight was significantly greater in NT conceptuses (63.0 6 6.5 g) compared with the AI group (37.2
6 2.1 g, P , 0.05) but not compared with the IVP group
(46.1 6 10.1 g). The highest membrane weight recorded
(101.5 g) was from the anemic NT fetus, in which attachment of the fetal cotyledons to the caruncles was poor.
The total membrane weight at Day 100 was highly var-
iable, but it was not significantly different between the
groups. In two cases in the NT group (one involving the
fetus shown in Fig. 3D), weights were not recorded because
of the extremely gelatinous nature; such gelatinous membranes were absent from either the AI or IVP group. All
fetal membranes were well vascularized and the fetal cotyledons firmly attached the caruncles. Two developmentally
retarded NT fetuses had correspondingly lower membrane
weights. Amniotic pustules, which were located on the inner surface of the amnion facing the fetus, were flat, whitish
plaques in the AI group, whereas in several cases in the
NT group, they were small, yellow spikes.
By Day 150, the mean fetal membrane weight was significantly higher (P , 0.05) in the NT group (1360 6 271
g) compared with either the AI (893 6 117 g) or IVP (774
6 57 g) group. This increased weight may be caused, in
part, by a greater total fetal fluid volume in several of the
NT pregnancies.
Fetal fluids. The total fetal fluid volume represented the
combined volumes of the allantoic and amniotic fluids. At
all three stages of gestation, no significant difference was
found in the mean allantoic, amniotic, or total fluid volume
between the treatment groups (Fig. 4 and Table 1). However, 3 of the 10 NT cases at Day 50 and three of the eight
cases at Day 150 had total fluid volumes greater than 2 SD
above the mean for the AI group at the respective stages
of gestation. The ratio of allantoic to amniotic fluid volume
varied with gestation. At Day 50, the allantoic fluid volume
was 9- to 10-fold higher than the amniotic fluid volume.
Increased total fluid volume in all three of the above-described Day 50 NT cases was caused by increased allantoic
fluid volume, which was greater than 2 SD above the mean
for the AI group.
At Day 100, allantoic fluid volumes were highly variable
in all three groups, ranging from 60 to 1300 ml (Fig. 5).
The volumes for two NT cases were not recorded, because
the tract had ruptured after slaughter in one case and the
allantoic sac was full of a jelly-like substance in the other.
Amniotic fluid volumes were less variable between individuals. One small NT fetus with only 36 placentomes had
low allantoic and amniotic fluid volumes, suggesting a loss
of ability to maintain appropriate fetal fluid volume.
At Day 150, three NT and one IVP allantoic and am-
6
LEE ET AL.
FIG. 5. Fetal brain, liver, heart, and kidney weights at Days 100 and 150 expressed as a percentage of the fetal
weight. The means for each treatment (AI,
IVP, or NT) are joined by a line. At Days
100 and 150, the means for the NT relative liver weight were significantly different from the AI and IVP groups (P ,
0.05).
niotic fluid volumes were not recorded, because the fetal
membranes had ruptured. Hydropsy was evident in two of
eight NT pregnancies and was likely in a third. All three
cases had total fluid volumes greater than 2 SD above the
AI mean, and two cases also had allantoic fluid volumes
that were 2 SD above the AI mean (20 and 12 L compared
with 6.6 6 0.4 L in the AI group). In the animal with 20
L of fluid, the amniotic fluid accounted for 8 L (AI, 2.4 6
0.3 L), an indication of polyhydramnios, which is far less
prevalent than hydrallantois in the hydrops syndrome associated with NT. Increased total fluid volume was associated with increased fetal and placental growth. In two of
the above-described cases, fetal weights and heart weights
were greater than 2 SD above the AI mean, and the kidney
and liver weights in all three cases were 2 SD above the
AI mean. All three cases had total caruncle weights greater
than 2 SD above the AI mean but normal caruncle numbers.
These three cases had the highest kidney and total caruncle
weights of any group at this gestation, but the fetuses were
not edematous.
Because the allantoic and amniotic fluids are contained
within the fetal membranes, we determined if a correlation
exists between the fetal membrane weight and the total fetal
fluid volume. A positive linear correlation was detected in
several instances, but no consistency was observed in this
relationship with either treatment or stage of gestation.
Thus, it was not always possible to attribute the increase in
fetal membrane weight to increased fluid accumulation.
Fetal organ development. The weights of the brain, liver,
heart, and kidney were compared among the three groups,
both with (Fig. 5, expressed as a percentage of fetal weight)
and without (Table 1) correction for the weight of the fetus.
No significant difference was observed in the mean brain
weight between groups at either Day 100 or 150. Mean
heart weights averaged over Days 100 and 150 were significantly higher (P , 0.05) in the NT group compared with
either control group; no difference was found between the
AI and IVP groups. However, when adjusted for gestation
and fetal weight, the difference between the NT group and
either control group was not significant.
No difference was observed in the mean liver weights
between the groups at Day 100 (Table 1). When corrected
for the fetal weight, the livers of NT fetuses were disproportionately lighter (P , 0.05) compared with either the AI
or IVP group. This trend was reversed by Day 150, when
the mean for the surviving NT group (159 6 10 g) was
significantly higher (P , 0.05) than that in either the AI
(113 6 9 g) or the IVP (105 6 5 g) group, even when
adjusted for fetal weight (P , 0.05). The mean NT liver
weight increased 9-fold between Days 100 and 150, compared with only 6.6- and 5-fold in the AI and IVP groups,
respectively, suggesting a ‘‘catch-up’’ growth, whereas the
fetal weight increase was similar in all three groups (9- to
10-fold). If this rate of growth was maintained for the rest
of gestation, the liver would be significantly enlarged by
the time these NT fetuses reached full term.
The mean kidney weights were not significantly different
between the groups at Day 100, with and without correction
for the fetal weight. At Day 150, kidneys from the NT
groups were significantly larger (P , 0.05) than those in
either control group (Table 1). However, when expressed as
a percentage of fetal weight (Fig. 5), the mean value for
the NT group (0.90% 6 0.06%) was still higher than that
for either the AI (0.77% 6 0.10%) or IVP (0.70% 6
0.14%) control group, although the difference was not significant.
No gross malformations were seen in the brain, liver,
heart, or kidneys at Day 100 or 150. However, livers from
one NT fetus at Day 100 and from another three NT fetuses
at Day 150 showed small, pale foci on the surface, giving
these livers a mottled appearance. The mottling may be
caused by fatty accumulation or hepatic congestion. In all
except one Day 150 case, mottling of the livers was associated with hepatic enlargement. An association between
liver mottling and cardiac enlargement was also seen in one
Day 100 and one Day 150 case. Three Day 150 NT kidneys
had dispersed clusters of fat cells on the capsules, and one
had a slightly misshapen kidney.
Placentome development. At the initiation of placentome formation at Day 50, we could detect no difference
between the three groups in the number of cotyledonary
burrs formed on the chorion. Five of the 10 NT conceptuses
had very red fetal cotyledons, suggestive of good vascularization (Fig. 3), compared with two from the five AI and
none from the IVP group (paler cotyledons).
The number of caruncles and their weights at Days 100
and 150 (Fig. 6) were used as indicators of placentome
numbers and weights, because the caruncles are the maternal tissues that, together with the fetal cotyledons, form the
placentomes. In both the AI and IVP groups, the numbers
of caruncles at Days 100 and 150 were very similar (Table
1), indicating that placentome numbers were fixed by Day
100. No difference was observed in the caruncle numbers
between the AI and IVP groups at either Day 100 or 150.
In contrast, the mean number of caruncles at Day 100 in
the surviving NT group (58 6 9) was significantly lower
(P , 0.05) than in either the AI (103 6 15) or IVP (99 6
16) group. Only 36 and 39 caruncles were recorded in two
GROWTH DEREGULATION IN CLONED CATTLE FETUSES
7
FIG. 6. Total caruncle weights (g), caruncle numbers, and average caruncle
weights (g) at Days 100 and 150. The
means for each treatment (AI, IVP, or NT)
are joined by a line.
NT pregnancies, and both these fetuses showed signs of
growth retardation accompanied by decreased fetal fluid
volume and membrane weight. Caruncle numbers in the
surviving NT group at Day 150 were similar to those in
the two control groups.
Despite lower caruncle numbers at Day 100, the mean
weight of all caruncles added together (Fig. 6 and Table 1)
was significantly higher (P , 0.05) in the NT pregnancies
(NT . AI 5 IVP). Together with the lower mean caruncle
numbers, this resulted in significantly increased (P , 0.05)
average caruncle weights in this group (NT . IVP 5 AI).
Figure 7 shows examples of uterine caruncles from an AI,
FIG. 7. A–D) Examples of Day 100 uteri
from an AI (A) with 66 caruncles, an IVP
(B) with 84 caruncles, an NT with only 36
caruncles (C), and an NT with 80 caruncles (D) showing the considerably larger
NT caruncles. The fetal membranes together with the fetal cotyledons have been removed. E) Sagittal cross-sections of a Day
150 NT and an IVP placentome showing
the increased thickening of the NT placentome. The white arrows indicate the fetal
side. Fetal villi growing into the caruncle
(‘‘streaks’’) are indicated by the black arrows. Bar 5 1 cm.
8
LEE ET AL.
TABLE 2. SD and significance of pairwise comparisons of within-group variances between treatment groups.
Significance of pairwise comparisonsa
SD
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
a
100 C-R length (cm)
100 fetal weight (g)
100 kidney weight (g)
100 heart weight (g)
150 liver weight (g)
150 kidney weight (g)
50 fetal membrane weight (g)
100 fetal membrane weight (g)
150 fetal membrane weight (g)
100 total caruncle weight (g)
100 ave. caruncle weight (g)
150 total caruncle weight (g)
100 amniotic fluid (ml)
150 amniotic fluid (L)
150 allantioc fluid (L)
150 total fluid (L)
AI
IVP
NT
NT:AI
NT:IVP
IVP:AI
0.85
4.9
0.10
0.16
19.6
2.05
4.7
70.1
262
21.1
0.30
63
76
0.70
1.21
0.99
0.54
24.4
0.44
0.30
28.2
8.03
17.6
33.2
114
21.0
0.34
217
357
0.46
1.30
1.40
1.83
77.1
1.04
1.08
50.4
11.5
20.6
124
767
108
1.34
243
215
2.28
3.42
5.09
NS
A
B
B
C
B
A
NS
C
C
C
C
NS
C
C
B
C
C
NS
C
NS
NS
NS
C
B
C
C
NS
NS
C
NS
C
NS
C
C
NS
NS
C
C
NS
NS
NS
NS
C
C
NS
NS
NS
NS, not significant; A, P # 0.001; B, P # 0.01; C, P # 0.05.
an IVP, and two NT pregnancies after removal of the fetal
membranes and associated cotyledons. The large, flat caruncles in Figure 7C were from a uterus in which only 36
were detected, whereas Figure 7D shows an NT example
with 80 caruncles and increased total and average caruncle
weights. A negative linear correlation was found between
the caruncle numbers and the average caruncle weight in
the AI (r 5 0.91, P 5 0.09) and IVP (r 5 0.81, P 5 0.19)
groups but not in the NT group (r 5 0.26, P 5 0.62).
Although the NT caruncle numbers were ‘‘normal’’ at
Day 150, the mean total caruncle weight was still significantly higher (NT . IVP 5 AI; P , 0.05), resulting in
significantly higher (P , 0.05) average caruncle weight
(NT . IVP 5 AI). A negative linear correlation between
the average caruncle weight and caruncle number was evident in both the AI (r 5 0.98, P 5 0.002) and NT (r 5
0.70, P 5 0.05) groups but not in the IVP group (r 5 0.11,
P 5 0.88). The larger of the NT placentomes assumed fistlike structures and were thicker in the sagittal cross-section
compared with AI or IVP placentomes (Fig. 7E), which
were more likely to be flat, discoid structures.
Individual variability. Plots of the morphometric data in
Figures 2 and 4–6 demonstrate considerably variability in
every parameter examined, particularly within the NT
group. We performed F-tests to compare the variances between the groups. Table 2 shows the SD for the variables
and the significance of the pairwise comparisons of variances between treatment groups. Despite all NT fetuses
having the same nuclear genetics of the cell line, a significantly greater variability was observed in the fetal weight
at Day 100 than in the contemporary AI (P , 0.001) or
IVP (P , 0.05) group. The IVP group was also more variable compared with the AI group (P , 0.05). In addition,
the NT kidney weights at Day 100 showed greater variability compared with the AI group (P , 0.05); however,
this may be caused, in part, by the greater variability in the
NT fetal weight. The Day 150 NT liver and kidney weights
were more variable compared with those in the AI, but not
with those in the IVP, group.
Greater variability in the NT group was most evident in
the components forming the uteroplacental tissues. Day 50
fetal membrane weights were significantly more variable in
the NT group compared with the AI group (P , 0.001) but
not with the IVP group (P 5 0.30). Membrane weights of
Day 50 IVP fetuses were also more variable compared with
the AI group (P , 0.05). At later stages of gestation, some
of the variability in fetal membrane weights may be caused,
in part, by greater variability in fetal fluid volumes (Table
2). By the time placentation was complete at Day 100, the
total caruncle and average caruncle weights in the NT
group were significantly more variable compared with either the AI (P , 0.05) or IVP (P , 0.05) group. Total
caruncle weights of both the NT (P , 0.05) and the IVP
(P , 0.05) groups were significantly more variable than the
weights of the AI group at Day 150. The analyses suggest
that greater variability occurs in fetal membrane and placental development in the NT group and, to a certain extent,
in the IVP group compared with the AI group.
DISCUSSION
A plethora of developmental abnormalities associated
with cloning by NT have been reported in several species
[16–20]. This considerable variation in abnormalities may
be caused by the different cell types and lines used, the
different methods of producing and culturing NT embryos,
and species differences in the mode of placentation and
gestational physiology. The in vitro culture of bovine embryos itself has been associated with abnormal phenotypes
[25] and the ‘‘large offspring syndrome’’ frequently associated with NT. In the present study, the number of confounding factors was minimized to allow comparison of NT
fetal development with contemporary half-siblings produced by AI or from embryos after in vitro fertilization.
The inclusion of IVP embryos allowed us to evaluate any
potential effect of embryo culture on subsequent in utero
development.
Failure of extraembryonic membrane development has
been postulated to be one of the potential causes of pregnancy failure before or around the time of placentome formation with cattle IVP and NT embryos. In our previous
experience, approximately 20%–25% of IVP conceptuses
were defective in allantoic development [26]. In the present
study, we did not see a single case of allantoic aplasia or
failure of allantoic vascularization at Day 50. It is unclear
whether this resulted from a change in the source of the
bovine albumin added to the embryo culture medium, from
a product obtained from Sigma (St. Louis, MO) to the current source [27], because these were the first embryos produced with this modified medium that had been transferred
GROWTH DEREGULATION IN CLONED CATTLE FETUSES
back to recipients and allowed to develop further. However,
we cannot exclude failure of allantoic development as one
of the causes of IVP and NT embryonic losses (30%–40%)
before Day 50. Clearly, vasculogenesis and the initiation of
fetal cotyledon development were unimpaired in the surviving Day 50 NT conceptuses in the present study.
Abnormal placental development and overgrowth are
hallmark pathologies associated with cloning by NT. The
significantly lower number of caruncles and, hence, placentomes in the NT group at Day 100 is consistent with the
report of Hill et al. [18]. Cotyledonary burr formation and
vascularization were unimpaired at Day 50, which suggests
that a high proportion of NT fetal cotyledons fail to interact
appropriately with the uterine caruncles to form placentomes. Some NT pregnancies in which no placentomes
were found by Day 70 have been encountered (unpublished
data). Placentome numbers in surviving Day 150 NT pregnancies were similar to those in the control groups, an indication that, perhaps, those with unusually low placentome
numbers were lost before Day 150 (3/5 and 0/6 were lost
in experiments 1 and 2, respectively).
Despite the decrease in caruncle numbers at Day 100,
total caruncle weight in the NT group was significantly
higher, suggesting growth overcompensation. The loss of
an inverse correlation between the average caruncle weight
and the caruncle number in the NT group suggests that
mechanisms normally regulating the total weight of placentomes are defective in NT pregnancies at Day 100. Even
when the caruncle numbers were normal in surviving Day
150 NT pregnancies, the total caruncle weights remained
significantly higher, suggesting a loss of placental growth
regulation even in apparently ‘‘normal’’ NT pregnancies.
The increased caruncle weights suggest that NT placental
overgrowth in cattle involved excessive uterine tissue
growth, perhaps in response to factors secreted by the fetal
cotyledons. Whereas the mouse placenta is made predominantly of conceptus-derived tissues, the maternal tissues in
the bovine placentome form an equivalent or greater part
of the structure. The ratio of maternal to fetal tissue weight
in the bovine placentome is 1–1.2 between Days 70 and
130 and 1.5–1.8 from Day 130 onward [28]. The larger,
thicker NT placentomes resembled those of later stages of
gestation in normal cattle pregnancy, indicating that NT
placentome growth was advanced for gestation. No correlation was found between total caruncle and fetal weight,
suggesting that placental overgrowth in NT is neither a
cause nor an indicator of fetal overgrowth nor a compensatory mechanism to overcome placental insufficiency in
fetal intrauterine growth retardation. Fetal and placental
overgrowth were closely associated in mouse cloning [20].
Whether this was caused, in part, by the smaller litter sizes
in clone pregnancies is unknown. In polytocous species,
such as the rat, mouse, rabbit, and guinea pig, the placental
and fetal weights are inversely related to the number of
fetuses in the litter.
Fetal overgrowth and the ‘‘large offspring syndrome’’ in
sheep and cattle have been associated with in vitro culture
of embryos [29–32]. Recently, an association between in
vitro fertilization in humans and the Beckwith-Wiedemann
syndrome (BWS), a human fetal overgrowth syndrome, has
been reported [33]. The IVP fetuses reported in the present
study were not significantly heavier than the AI fetuses at
all three stages of gestation. Therefore, in vitro culture is
unlikely to be a significant contributor to the NT fetal overgrowth in five of the six Day 100 fetuses and in five of the
eight Day 150 fetuses in the present study. However, in
9
vitro culture did have subtle effects on the development of
IVP fetuses, such as the greater variability in fetal weight
and C-R lengths, and it may contribute to the greater variability of NT fetal growth. Increased NT fetal weight at
Days 100 and 150 was not accompanied by greater C-R
length. Because most of the fetal weight gain normally occurs during the few weeks before parturition, a modest,
11%–17% gain in mean weight just past midgestation could
result in considerably heavier NT calves at birth. The mean
live birth weight of singleton NT calves produced from this
cell line was 40.4 6 1.3 kg (n 5 16), compared with the
mean live birth weight of 34.4 6 0.7 kg (n 5 74) for Friesian heifers born through AI on various farms around the
region (Dr. Susanne Meier, Dexcel, New Zealand; personal
communication), a weight increase of 17%.
The unusually high incidence of hydropsy and its associated fetal mortality in the NT cloning of cattle emphasizes
the need to understand the underlying mechanisms of hydropsy. Fetal fluid homeostasis in cattle is still not fully
understood. In the present study, perturbed fluid homeostasis was evident as early as Day 50, when the placentomes
were not yet fully formed. At Day 150, the hydrops syndrome was associated with fetal, placental, and organ overgrowth; whether this association holds true for NT fetuses
generated with other cell lines needs further investigation.
Organomegaly involving the liver, kidney, and heart and an
increased incidence of polyhydramnios are also seen with
BWS in humans and in mouse models of the syndrome
[34]. The degree of penetrance of the growth deregulation
phenotype in NT fetuses is reminiscent of the BWS phenotype: It is highly variable, and not all organs are affected
in the same way in every individual. The association between excessive fetal fluid accumulation and renal and placental growth deregulation may indicate impairment of renal and placental function. Although the placenta is the
major organ regulating the fetal environment, the fetal kidney also plays an important role in the regulation of fetal
arterial pressure, fluid and electrolyte homeostasis, acidbase balance, and hormone synthesis [35]. In ruminants,
fetal urine contributes to the allantoic and amniotic fluid.
Reports have appeared of kidney defects [36] and impaired
renal function in cloned offspring [15] as well as impaired
liver function in cloned mice at 3 and 14 mo of age [37].
The growth deregulation in the liver and kidney is of particular concern, because these two organs play critical roles
in the health of an animal.
An interesting observation in the present study was the
greater variability within the cohort of NT fetuses with the
same nuclear genetics than within the cohorts of AI and
IVP fetuses, given that sexual dimorphism in the growth of
AI and IVP fetuses is expected to increase the variability
within these two control groups. This suggests that nuclear
genetic background alone does not account entirely for the
phenotype. The cause of this variability is unknown, but
heterogeneity in epigenetic modifications in donor nuclei
and incomplete epigenetic reprogramming after NT could
affect the expression of key regulatory genes during embryonic development. This may lead to greater ‘‘noise’’ in
the expression of other genes further downstream, resulting
in greater variability. It is, however, impossible to know if
the somatic cells have acquired mutations either in vivo or
during culture before NT. Such mutations could conceivably result in phenotypic variability between cloned offspring and the animal from which the cells were isolated
or between individual cloned offspring.
The NT cloned animals also differ from monozygotic
10
LEE ET AL.
twins in that the mitochondrial genes are often different
between individuals. However, conflicting evidence exists
regarding whether the mitochondrial DNA is derived solely
from the recipient cytoplast, as in the case of cloned sheep
[38], or from both the cytoplast and donor somatic cell, as
is common in NT cattle [39]. Therefore, even though the
nuclear genome may be ‘‘identical,’’ NT animals from the
same cell line may have different mitochondrial genotypes
and, in cattle, may be mosaic for mitochondrial DNA subtypes. Because mitochondrial genes play a key role regulating the energy threshold and metabolic activity of all
cells, they may have a significant effect on offspring performance.
Loss of imprinting of IGF2 [40], paternal uniparental
disomy involving the cluster of imprinted genes containing
IGF2 [41], and mutations in the p57kip2 gene [42], also in
this cluster, have been implicated in BWS, but the underlying mechanism of growth deregulation in this syndrome
remains unknown. In cloned mouse fetuses, widespread
misexpression of genes, including those that are imprinted,
has been reported [43]. Many of these genes, including
those affected in BWS, are excellent candidates for future
investigations comparing gene expression profiles using the
material collected in the present study.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
ACKNOWLEDGMENTS
We thank Harold Henderson, David Duganzich, and Neil Cox for their
invaluable help with data analyses; Martin Berg and Jacqui Forsyth for
embryo transfer and AI of the heifers; Ljiljana Popovic and Don Saywell
for generating the IVP embryos; John Lange for animal husbandry; and
Alan Julian and staff of the former Animal Health Laboratories, Hamilton,
for the use of their pathology laboratory for the sample collection.
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