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ABSTRACT
In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell
donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell
nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single
AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two
sources of oocytes were compared, fusion rate was higher using in vivomatured oocytes as
recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitromatured
oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two
groups: recipients (n 24) receiving 25 embryos and recipients (n 26) receiving 30 embryos. Twelve recipients (46.2%) receiving 30 embryos were diagnosed to be pregnant, while
no pregnancies were established in recipients receiving 25 NT embryos. Also, to determine
the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer
on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of
embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n 17), versus day 5 (n 4) or day 6 (n 3) was significantly different. Of the 12 pregnant recipients,
nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%)
from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight
died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and
bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed
that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first
wild carnivores to be produced by nuclear transfer.
INTRODUCTION
1Audubon
Center for Research of Endangered Species, New Orleans, Louisiana 70131 USA.
of Animal Sciences, LSU Agricultural Center, Baton Rouge, Louisiana 70803 USA.
3School of Veterinary Medicine, University of California Davis, Davis, California 95616 USA.
4Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148 USA.
2Department
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Chemicals
Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated.
Oocyte maturation
For in vitro maturation, cumulusoocyte complexes (COC) were recovered from ovaries of domestic cats within 25 h after ovariohysterectomy. Selected COCs were cultured in modified
TCM-199 containing 1 IU/mL hCG, 0.5 IU/mL
eCG, 10 g/mL epidermal growth factor, and 3
mg/mL BSA (Fraction V, fatty acid free; Serological Proteins, Kankakee, IL) for 24 h in 5% CO2,
5% O2 and 90% N2 at 38C (Gmez et al., 2003).
To obtain in vivomatured oocytes, domestic
cats were treated with a total of 34 IU of porcinefollicle stimulating hormone (FSH, Sioux Biochemical, Sioux Center, IA) given in decreasing
daily doses (s.c.) for 4 days, followed by 3 IU of
porcine-luteinizing hormone (im; LH, Sioux Biochemical) on day 5. At 2426 h after LH injection,
oocytes were collected by laparoscopic aspiration
of mature ovarian follicles (Gmez et al., 2000).
In vivomatured oocytes were used for NT immediately after aspiration.
249
Nuclear transfer
The protocol for nuclear transfer has been described previously (Gmez et al., 2003). Briefly,
denuded metaphase II oocytes were incubated
for 15 min at 38C in Ca2-free and Mg2-free
modified Tyrodes salt solution supplemented
with 1% MEM nonessential amino acids, 3
mg/mL BSA, 30 mM NaHCO3, 0.36 mM pyruvate, 1 mM glutamine, 50 g/mL gentamicin
(ECM medium), 20 g/mL Hoechst 33342 and 20
g/mL cytochalasin B (CCB). After incubation,
oocytes were enucleated in ECM medium in
which supplemental NaHCO3 was reduced to 15
mM and to which 15 mM Hepes, 20 g/mL CCB
and 2 mg/mL sucrose were added. The first polar body and 10% of the underlying cytoplasm
were drawn into an enucleation pipette (20 m,
OD). Removal of the metaphase II plate was confirmed by brief exposure to epifluorescence microscopy.
A vial containing AWC fibroblast cells synchronized in G0/G1 phase by serum starvation
(Gmez et al., 2003) was thawed prior to being
used for NT, and a single AWC fibroblast cell was
introduced into the perivitelline space of each
enucleated oocyte. For fusion, each NT couplet,
in a solution of 0.3 M mannitol and 0.1 mM Mg2,
was placed between two stainless-steel electrodes
spaced 120 m apart (LF-101; Nepa Gene, Tokyo,
Japan) that were controlled by micromanipulators. Membrane fusion was induced by applying
a 3-sec AC pre-pulse of 20V, 1 MHz followed by
two 30-sec DC pulses of 240V/mm at intervals
of 0.5 sec. Following the fusion pulses, couplets
were washed and cultured in IVC-1 medium supplemented with 7.8 mM calcium lactate (IVC-1
Ca2) for 30 min., at which time fusion was evaluated visually by confirming the presence or absence of the donor cell in the perivitelline space.
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After 23 h of culture in IVC-1 Ca2, activation was induced by placing fused couplets between two electrodes in a fusion chamber containing 3 mL of a solution of 0.3 M mannitol, 0.1
mM Mg2, and 0.05 mM Ca2 and exposing them
to two 60-sec DC pulses of 120 V/mm. Then,
couplets were incubated in 30-L droplets of
IVC-1 medium supplemented with 10 g/mL cycloheximide and 5 g/mL CCB at 38C in 5% CO2
in air for 4 h.
Embryo culture
After activation, reconstructed embryos were
cultured in 500 L of IVC-1 medium in 5% CO2,
5% O2, and 90% N2 at 38C. Some couplets were
transferred to the oviducts of recipient females on
day 1. The remaining couplets were cultured until day 2 or 3, at which time, after uncleaved couplets were recorded and removed, embryos were
placed into 500 L of fresh IVC-1 medium containing 1% MEM essential amino acids (IVC-1
EAA). Then, on day 5, the number of morulae
was recorded and all embryos were moved into
Tyrodes solution containing 1% NEAA, 2% EAA,
10% FBS, and the same supplements as in IVC-1
medium (IVC-2 medium) and cultured until
transfer to a recipient.
Embryo transfer
Cloned embryos were transferred into the
oviducts or uteri of 50 gonadotropin-treated domestic short hair (DSH) recipients. Day 1 embryos were transferred to the oviducts of recipients, some of which had served as oocyte donors
on day 0 (n 2) and others of which served as
recipients only after induction of ovulation (n
14) using a lower dose of FSH (13 IU) and a
higher dose of LH (5 IU) than the oocyte donors
received. For oviductal embryo transfer, the abdominal cavity was visualized via a 5-mm endoscope inserted just above the umbilicus, and a 16
gauge 6.35 cm stainless steel trocar/cannula
was inserted transabdominally near the midline
5 cm below the umbilicus. Then, a 14.5-cm tom
cat catheter (3.5 French, Sherwood Medical, St.
Louis, MO) was passed through the 16-gauge
cannula and directed through the infundibulum
overlaying the ovary and into the upper ampullary portion of the oviduct. A 50-cm length of
sterile polyethylene tubing (PE 10, no. 427400,
Becton Dickinson, Sparks, MD) containing the
GMEZ ET AL.
Microsatellite analysis
To determine the clonal status of all kittens,
DNA was extracted using standard phenol/chloroform techniques from the fibroblast cells and
blood of the AWC donor, blood of the recipient
dam, umbilical cord blood of live kittens, or
spleen of dead kittens. Standard sodium hydroxide isolation was used to isolate DNA from cheek
swabs of live kittens. Feline derived microsatel-
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Statistical analyses
The Chi square test was used to test the differences between treatments. Statistical difference was set at the p 0.05 level.
RESULTS
Embryo production after NT
A total of 2,565 in vivo (n 1751) and in vitro
(n 814) matured domestic cat oocytes were mechanically enucleated and electrically fused to
frozen-thawed AWC fibroblasts. Fusion efficiency was higher when in vivomatured oocytes
(1696/1751; 96.8%) were used as recipient cytoplasts as compared with in vitromatured oocytes
(736/814, 90.4%, p 0.05). In contrast, cleavage
rate of embryos reconstructed with in vitromatured oocytes (435/511, 85.1%) was higher than
that of embryos reconstructed with in vivomatured oocytes (944/1194, 79.1%, p 0.05). Some
reconstructed couplets (n 727) were not included in the cleavage rate evaluation because
they were transferred to domestic cat recipients
on day 1 before cleavage occurred.
To determine the number of reconstructed embryos that should be transferred per recipient to
Of the 12 pregnant recipients, nine (75%) pregnancies developed to term (60 days). In the
TABLE 1.
EFFECTS
OF
IN
DOMESTIC CAT
AWC NT embryos
Average
per recipient
Embryos per recipient
25
30
Total
ET, day
Total, n
7
6
5
1
6
5
1
142
172
50
102
183
417
486
1552
Recipients
n SD
Total, n
9
9
2
4
5
9
12
50
15.7
19.1
25.0
25.5
36.6
46.3
40.5
5.5
4.4
0
0.5
4.1
12.1
10.5
Pregnant, n (%)
0
0
0
0
2
4
6
12
(0)
(0)
(0)
(0)
(40.0)
(44.4)
(50.0)
(24.0)
Fetuses, n
3
4
17
24
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GMEZ ET AL.
three (25%) other pregnancies, a total of five fetuses were present at day 2123, four of which
were reabsorbed between days 30 and 40, and one
which was spontaneously aborted on day 48 of
gestation (Table 2).
Six of the nine term pregnancies, consisting of
a total of eight fetuses, were not hormonally
treated during the last 710 days of pregnancy
(Table 2). Two of these recipients each had a single kitten that died in uteri within 24 h of delivery by Caesarean section on days 64 and 67 of
gestation. Histological analysis of the two kittens
showed no indications of infectious disease. No
inflammatory or other degenerative lesions were
recognizable in the tissue sections and all organs
appeared to be developing normally. One of the
two kittens had moderate haemorrhage within
the choroids plexus of the brain, possibly due to
hypoxia that occurred after placental separation.
The other four recipients delivered six kittens
by Caesarean section on days 6267 of pregnancy
after vaginal bleeding occurred. Of the six kittens,
Total
fetuses
days
2123
Fetuses
reabsorbed
days
3040
Delivery
day
ET
day
Embryos
transferred
n
1
2
3
4
5
6a
7a
5
1
1
6
5
1
6
41
42
56
39
35
59
42
1
2
2
1
1
4
2
1
2
1
48
64
67
62
62
8a
9a
5
1
43
34
1
2
62
67
10b
11b
5
1
39
40
1
5
69
65
Stillborn
1
1c
1c
1d,e
1c
1d
12b
Total
aPregnant
35
24
67
1c
7
Live 36 h
Healthy
1e
1
1f
1
1
1
1
1.g
1e
1f
7
Weight, g
90.4
108.7
60.5
95.4
102.2
77.0
107.0
50.0
98.3
92.5
73.0
86.9
71.9
111.0
105.2
88.7
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Placental analysis
A total of 11 placentae were analyzed. Macroscopic analyses showed that five placentae had
FIG. 1. African wildcat cloned kittens (A) Ditteaux born on 6th August 2003, at two months after birth. (B) Miles
and Otis born on 15th November 2003, at seven days after birth.
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GMEZ ET AL.
TABLE 3.
ANALYSIS
OF
AWC donor
Jazz
Feline
markers
Fibroblasts
Blood
Cheek
cells
Umbilical
cord
FCA075
FCA097
FCA201
FCA224
FCA229
FCA293
FCA305
FCA441
FCA018
FCA145
FCA651
FCA674
132/134
140/146
153/155
160/178
158/162
179/187
191/201
158/166
233/233
174/174
135/135
148/148
132/134
140/146
153/155
160/178
158/162
179/187
191/201
158/166
233/233
174/174
135/135
148/148
132/134
140/146
153/155
160/178
158/162
179/187
191/201
158/166
233/233
174/174
135/135
148/148
132/134
140/146
153/155
160/178
158/162
179/187
191/201
158/166
233/233
174/174
135/135
148/148
DISCUSSION
In the present study, we demonstrated that
AWC kittens can be produced after transfer of
embryos derived by fusion of adult somatic cells
from one species with enucleated oocytes of a
closely related species (domestic cat). We have
shown that, under our current laboratory conditions, transfer of at least 30 NT embryos per recipient was required for establishing a pregnancy. Also, the length of in vitro culture affected
the number of embryos that successfully underwent fetal development. The primary causes of
peri-natal mortality were placental separation,
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higher fusion rates with in vivomatured cytoplasts and higher cleavage rates with in vitromatured cytoplasts. In a previous study, we found
no differences in blastocyst development in vitro
when in vivomatured and in vitromatured cat
oocytes were used as recipient cytoplasts of AWC
cells (Gmez et al., 2003). A primary purpose of
the present study was to find out if AWC kittens
could be produced by transfer of NT embryos to
domestic cat recipients. Because we were unable
to produce NT pregnancies after transferring
17 embryos per recipient in a previous study
(Gmez et al., 2003), in the present study in order to transfer 25 embryos per recipient, for
most transfers it was necessary to combine reconstructed embryos derived from both in
vitromatured and in vivomatured domestic cat
cytoplasts. The one domestic cat recipient receiving NT embryos derived from in vivomatured
cytoplasts did establish pregnancy and produce
a live kitten. We do not know if the other NT kittens originated from embryos derived from in
vivomatured or in vitromatured cytoplasts. Certainly, the comparative developmental competence of in vitromatured versus in vivomatured
cat cytoplasts requires investigation.
Several developmental abnormalities occur in
pregnancies from somatic cell nuclear transferderived embryos, including a high rate of
abortion during early gestation and a number of
perinatal complications. One complication we encountered in some cats was the occurrence of
vaginal bleeding by day 6267 of pregnancy without overt signs of labor. Although the kittens
were delivered within the normal gestation period of domestic cats (6271 days; Herron, 1977)
and African Wildcats (5663 days; Nowell and
Jackson, 1996b), the kittens died shortly after delivery from respiratory failure, a condition usually associated with premature delivery.
In domestic cats, serum progesterone levels begin a slow decline by day 50 of pregnancy, progressing to a precipitous decrease during the last
week prior to parturition (Verhage et al., 1976).
Although we did not measure serum progesterone levels in pregnant recipients, it seems possible that, since the recipient domestic cats were
gestating AWC cloned fetuses, feto-placental endocrine signaling may have been inadequate. As
a result, progesterone levels may have shown an
abnormally early decrease, starting a cascade of
pre-parturient events that resulted in vaginal
bleeding. Consequently, with the last three preg-
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GMEZ ET AL.
hypoglycemia. This disorder involves the deregulation of several genes, including insulin-like
growth factor, type 2 (IGF2) and cyclin-dependent kinase inhibitor 1C (CDKN1C; Murrel et al.,
2004). The product of IGF2 is a major fetal growth
factor, and is associated with overgrowth in fetuses derived from sheep embryos cultured in
vitro before transfer to recipients (Young et al.,
2001). Recently, in humans, an association was
found between babies produced by in vitro technology and the incidence of B-WS (DeBaun et al.,
2003). In our study, a major abnormality found in
three cloned kittens was incomplete closure of
ventral body wall musculature with exteriorization of abdominal organs; however, pathology associated with overgrowth was not observed.
Likewise, knockout mice for CDKN1C have been
found to manifest an anterior wall defect but do
not exhibit fetal overgrowth or other features of
B-WS (Fitzpatrick et al., 2002). Whether body wall
pathology is caused by gene deregulation or is
merely a consequence of another wall anomaly is
not known.
The influence of possible mitochondrial heteroplasmy and/or a deleterious mtDNA effect on
neonatal death of the cloned AWC kittens cannot
be discounted. Indeed, in future studies the precise mitochondrial inheritance pattern in AWC
cloned kittens will be evaluated to determine its
influence on survival rate.
The effectiveness of our cloning procedure was
shown by the birth of healthy AWC kittens. These
births represent the first wild carnivores to be
produced by NT and further expand the growing list of species in which cloned offspring have
been produced. Our success may be attributable
to improvements in the embryo culture system
(Gmez et al., 2003), refinements made to the nuclear transfer procedure (Gmez et al., 2003), increasing the number of embryos transferred, reducing the culture interval before transfer and
modification of current protocols successfully
used in other species to manage pregnancy and
improve viability of offspring (Ptak et al., 2002).
While there are major limitations to current
cloning technologies, knowledge about nuclear
transfer procedures is advancing rapidly and
technology is improving. Researchers should be
accessible to the possibilities offered by nuclear
transfer as one of the assisted reproduction techniques potentially useful for the conservation of
endangered species, which in some cases, may offer the prospect of species continuation rather
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ACKNOWLEDGMENTS
We are grateful to Stella Sullivan for her excellent cat care and hand rearing some of the
cloned kittens. In addition, we thank Dr. Karen
Miller, Dr. Steve Miller, Barbara Vincent and the
Audubon Nature Institute veterinary staff for
their help with the surgery procedures; Jackie
Coulon for coordinating and collecting ovaries
from the veterinary clinics; and the staff and interns from the Species Survival Center for the 24
h per day cat observations towards the end of
each pregnancy.
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