rizos et al., 2002 ambiente in vitro vs in vivo

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MOLECULAR REPRODUCTION AND DEVELOPMENT 61:234–248 (2002)
Consequences of Bovine Oocyte Maturation,
Fertilization or Early Embryo Development
In Vitro Versus In Vivo: Implications for
Blastocyst Yield and Blastocyst Quality
DIMITRIOS RIZOS, FABIAN WARD, PAT DUFFY, MAURICE P. BOLAND, AND PATRICK LONERGAN*
Department of Animal Science and Production and Conway Institute for Biomedical and Biomolecular Research,
University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland
ABSTRACT The aim of this study is to
examine the effect of bovine oocyte maturation,
fertilization or culture in vivo or in vitro on the
proportion of oocytes reaching the blastocyst stage,
and on blastocyst quality as measured by survival
following vitrification. In Experiment 1, 4 groups of
oocytes were used: (1) immature oocytes from 2–
6 mm follicles; (2) immature oocytes from >6 mm
follicles; (3) immature oocytes recovered in vivo just
before the LH surge; and (4) in vivo matured oocytes.
Significantly more blastocysts developed from oocytes
matured in vivo than those recovered just before the LH
surge or than oocytes from 2–6 mm follicles. Results
from >6 mm follicles were intermediate. All blasto-
cysts had low survival following vitrification. In Experi-
ment 2, in vivo matured oocytes were either (1)
fertilized in vitro or (2) fertilized in vivo by artificial
insemination and the resulting presumptive zygotes
recovered on day 1. Both groups were then cultured
in vitro. In vivo fertilized oocytes had a significantly
higher blastocyst yield than those fertilized in vitro.
Blastocyst quality was similar between the groups.
Both groups had low survival following vitrification.
In Experiment 3a, presumptive zygotes produced
by in vitro maturation (IVM)/fertilization (IVF) were
cultured either in vitro in synthetic oviduct fluid, or in
vivo in the ewe oviduct. In Experiment 3b, in vivo
matured/in vivo fertilized zygotes were either surgically
recovered on day 1 and cultured in vitro in synthetic
oviduct fluid, or were nonsurgically recovered on day 7.
There was no difference in blastocyst yields between
groups of zygotes originating from the same source
(in vivo or in vitro fertilization) irrespective of whether
culture took place in vivo or in vitro. However, there
was a dramatic effect on blastocyst quality with those
blastocysts produced following in vivo culture surviving
vitrification at significantly higher rates than their
in vitro cultured counterparts. Collectively, these
results indicate that the intrinsic quality of the oocyte
is the main factor affecting blastocyst yields, while the
conditions of embryo culture have a crucial role in
determining blastocyst quality. Mol. Reprod. Dev.
61: 234–248, 2002. ß 2002 Wiley-Liss, Inc.
Key Words: oocyte development; in vitro fertiliza-
tion; ovum pick-up; embryo; gamete biology
INTRODUCTION
The development of bovine oocytes to the blastocyst
stage following maturation, fertilization and culture
in vitro is limited to about 30–40%. There is now a
growing amount of evidence to suggest that while
culture conditions can impact on the developmental
potential of the early embryo, the intrinsic quality of
the oocyte is the key factor determining the proportion
of oocytes developing to the blastocyst stage (Sirard and
Blondin, 1996). In support of this idea, oocytes derived
from large follicles are more competent than those
derived from small follicles (Pavlok et al., 1992;
Lonergan et al., 1994). Also, a greater proportion of
in vivo matured oocytes reach the blastocyst stage
compared to those produced in vitro (Greve et al., 1987;
Leibfried-Rutledge et al., 1987; Marquant-Le Guienne
et al., 1989; McCaffrey et al., 1991; Van Soom et al.,
1992).
While still enclosed within the follicle, especially the
one destined to become the dominant follicle, the oocyte
undergoes significant changes that play a key role in its
acquisition of developmental competence. A number of
ultrastructural and molecular changes occurring dur-
ing oocyte development have been linked to develop-
mental competence (Assey et al., 1994b; Hyttel et al.,
1997). Also, in vitro maturation (IVM) has been
associated with certain abnormalities in the oocyte
(Hyttel et al., 1986, 1988, 1989).
In addition to the proportion of oocytes developing to
the blastocyst stage, the quality of these embryos is
important. Despite extensive research in terms of
ß 2002 WILEY-LISS, INC.
DOI 10.1002/mrd.1153
Grant sponsor: EU Commission; Grant number: QLK3-CT-1999-
00104; Grant sponsor: Greek State Scholarships Foundation.
*Correspondence to: Patrick Lonergan, Ph.D., Department of Animal
Science & Production, University College Dublin, Lyons Research
Farm, Newcastle, County Dublin, Ireland.
E-mail: pat.lonergan@ucd.ie
Received 18 May 2001; Accepted 1 August 2001
increasing the yield of blastocysts from immature
oocytes, the quality of in vitro produced embryos, in
terms of survival following cryopreservation, has
continually lagged behind that achieved with in vivo
derived embryos. In vitro, produced bovine embryos
differ from those produced in vivo in many important
respects (Massip et al., 1995; Wright and Ellington,
1995) including darker cytoplasm, lower density (Pol-
lard and Leibo, 1994), more triglycerides and less lipids
from other classes (Abd El Razek et al., 2000), swollen
blastomeres (Van Soom et al., 1992), a more fragile zona
pellucida (Duby et al., 1997), a slower growth rate and
higher thermal sensitivity (Leibo and Loskutoff, 1993),
differences in intercellular communication (Boni et al.,
1999), and a higher incidence of chromosomal abnorm-
alities (Viuff et al., 1999; Slimane et al., 2000), all of
which may contribute to the higher sensitivity to
cryoinjury exhibited by such embryos.
It is unclear, which parts of the process of embryo
production are important in determining such para-
meters as blastocyst yields and blastocyst quality. The
aim of this study was to examine in detail the
sequential steps of bovine embryo production in vivo
and in vitro (i.e., maturation, fertilization, and early
embryo development) in an effort to understand, which
steps of the process are important in determining
oocyte/embryo developmental competence and embryo
quality, respectively. While some of these aspects have
been studied previously in isolation, this is the first
time, to our knowledge, that such studies have been
carried out in parallel.
MATERIALS AND METHODS
Oocyte Collection and IVM
Cumulus oocyte complexes (COCs) were obtained by
aspirating follicles from the ovaries of heifers, either at
slaughter or using ovum pick-up (OPU), depending on
the experiment. After four washes in PBS supplement-
ed with 36 mg/ml pyruvate, 50 mg/ml gentamycin,
and 0.5 mg/ml bovine serum albumin (BSA) (Sigma,
St. Louis, MO, catalogue number A-9647), groups of up
to 50 COCs were placed in 500 ml maturation medium
in four-well dishes (Nunc, Roskilde, Denmark), and
cultured for 24 hr at 398C under an atmosphere of 5%
CO2 in air with maximum humidity. The maturation
medium was TCM-199 supplemented with 10% (v/v)
fetal calf serum (FCS, Sigma, F-2442) and 10 ng/ml
epidermal growth factor (Sigma, E-4127).
Sperm Preparation and
In Vitro Fertilization (IVF)
For in vitro fertilization (IVF), COCs were washed
four times in PBS and then in fertilization medium
before being transferred in groups of up to 50 into four-
well dishes containing 250 ml of fertilization medium
(Tyrode’s medium with 25 mM bicarbonate, 22 mM Na-
lactate, 1mM Na-pyruvate, and 6 mg/ml fatty acid-free
BSA). In addition, 10 mg/ml heparin–sodium salt
(184 units/mg heparin, Calbiochem, San Diego, CA)
were added. Motile spermatozoa were obtained by
centrifugation of frozen-thawed semen (Dairygold A.I.
Station, Mallow, Ireland) on a discontinuous Percoll
(Pharmacia, Uppsala, Sweden)density gradient (2.5 ml
45% (v/v) Percoll over 2.5 ml 90% (v/v) Percoll) for 8 min
at 700g at room temperature. Viable spermatozoa,
collected at the bottom of the 90% fraction were washed
in HEPES-buffered Tyrode’s and pelleted by centrifu-
gation at 100g for 5 min. Spermatozoa were counted in
a hemocytometer and diluted in the appropriate volume
of fertilization medium to give a concentration of
2�106 spermatozoa/ml. A 250 ml aliquant of this
suspension was added to each fertilization well to
obtain a final concentration of 1�106 spermatozoa/ml.
Plates were incubated for 24 hr at 398C under an
atmosphere of 5% CO2 in air with maximum humidity.
Semen from the same bull was used for all experiments.
In Vitro Culture (IVC)
At approximately 20 hr post insemination (hpi), pre-
sumptive zygotes were denuded by gentle vortexing,
washed four times in PBS and twice in culture medium
(synthetic oviduct fluid, SOF) before being transferred
to 25 ml culture droplets (25 embryos/droplet). Fetal calf
serum (10%, v/v) was added 24 hr after placement in
culture. Cleavage was assessed 48 hpi and blastocyst
development was recorded on days 6–8 (day 0ˆday of
IVF).
In Vivo Production of Oocytes and Embryos
The protocol used for the in vivo production of oocytes
and embryos is described in Table 1. Beef cross heifers
were synchronized using a CIDR device (InterAg,
Hamilton, New Zealand) for 8 days. Three days before
CIDR removal, heifers received 2 ml (15 mg) of
prostaglandin F2a analogue (PG, Prosolvin, Intervet,
Dublin, Ireland). Heifers were checked for standing
estrus (ˆday 0). The dominant follicle was ablated by
transvaginal aspiration on day 8 of the estrous cycle.
TABLE 1. Protocol to Obtain In Vivo Oocytes/Embryos
Day Time (hr) Action
ÿ10 CIDR in
ÿ5 Prostaglandin
ÿ2 CIDR removal
0 Estrus
8 Dominant follicle ablation
10 FSH am & pm
11 FSH am & pm
12 FSH am & pm
0 Prostaglandin pm
13 FSH am & pm
14 40 Ovum pick up–oocytes just before LH
surge
40 Induction of LH surge with GnRH
48 AI for day 1 and day 7 in vivo embryos
15 60 AI for day 1 and day 7 in vivo embryos
60 Ovum pick up–in vivo matured
oocytes
16 Surgical embryo recovery of day 1 in
vivo zygotes
21 Nonsurgical embryo recovery of day 7
in vivo embryos
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 235
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Beginning on day 10, animals were superovulated with
a total of 180 mg FSH (Folltropin, Vetrepharm Canada,
Inc., London, Ontario, Canada) given as twice daily
injections over four days on a decreasing dose schedule.
Luteolysis was induced with 15 mg PG given on day 12.
To obtain oocytes just before the expected time of the
LH surge (i.e., before the resumption of meiosis), OPU
was performed 40 hr after PG (Callesen et al., 1986;
Greve et al., 1995; Vos et al., 1996). To obtain in vivo
matured oocytes, heifers received 0.25 mg GnRH (2.5 ml
Fertagyl, Intervet) at 40 hr after PG and OPU was
performed approximately 20 hr later. This treatment
has been shown to induce a preovulatory LH surge 2–
3 hr after injection (Bordignon et al., 1997; van de
Leemput et al., 1999). Only oocytes with an expanded
cumulus cell mass were used.
To produce day 1 (1- to 2-cell stage) and day 7
(blastocyst stage) embryos, heifers were inseminated
with frozen-thawed semen at 48 and 60 hr after PG
injection. The same semen batch as used in IVF was
used for artificial insemination. Day 1 embryos were
collected by mid-ventral laparotomy approximately
90 hr after the PG injection by flushing the oviducts
with PBS supplemented with 5% FCS. Day 7 embryos
were recovered by nonsurgical flushing 9 days after PG.
In Vivo Culture of IVM/IVF Presumptive
Zygotes in the Ewe Oviduct
Presumptive zygotes produced following IVM/IVF
were surgically transferred by mid ventral laparotomy
to the ligated ewe oviduct (approximately 100 per ovi-
duct) at 20 hpi. Embryos were recovered from the ewe 6
days later (i.e., day 7 post insemination) at surgery by
flushing the oviduct with 20 ml of PBS. On recovery, the
number of blastocysts was recorded and expressed as a
fraction of the total number of embryos recovered.
Embryos were then cultured overnight till day 8.
Blastocyst Vitrification
Blastocysts were vitrified using the open pulled straw
(OPS) method described by Vajta et al. (1998) in a final
solution containing 20% ethylene glycol and 20%
DMSO. Warmed blastocysts were cultured in 25 ml
droplets of M199‡ 10% FCS in the presence of a granu-
losa cell monolayer (Rizos et al., 2001), and examined at
24, 48, and 72 hr post-warming. Survival was defined as
re-expansion and maintenance of the blastocoel. The
hatching rate was also recorded and expressed as a
percentage of those embryos surviving at 72 hr post-
warming.
Experimental Design
The experimental design is illustrated in Figure 1.
The three experiments outlined below were concerned
with the effect of maturation, fertilization and culture,
respectively, either in vivo or in vitro, on the proportion
of oocytes developing to the blastocyst stage. Blasto-
cysts produced in these three experiments were used to
examine the effect of production method on blastocyst
quality as measured by survival following vitrification/
warming.
Experiment 1: Oocyte Maturation
In Vivo vs. In Vitro
The aim of Experiment 1 was to evaluate the im-
portance of the events surrounding oocyte maturation.
Four groups of oocytes were used: (1) immature oocytes
from 2–6 mm follicles from slaughterhouse ovaries,
nˆ388; (2) immature oocytes from > 6 mm follicles
from slaughterhouse ovaries, nˆ99; (3) immature
oocytes recovered by OPU just before the LH surge,
nˆ102; and (4) in vivo matured oocytes recovered by
OPU, nˆ 134. Following recovery, Groups 1–3 were
submitted to IVM for 24 hr as described above, while
Fig. 1. Experimental design. Bovine oocytes were matured either in vitro (IVM) or in vivo (VivoM),
fertilized either in vitro (IVF) or in vivo (VivoF) and cultured in vitro (IVC) or in vivo (VivoC).
236 D. RIZOS ET AL.
Group 4 oocytes were partially denuded by brief expo-
sure to 0.05% hyaluronidase and immediately insemi-
nated. The experimental design was such that oocytes
from all 4 groups were inseminated at the same time.
At approximately 20 hr post insemination presump-
tive zygotes were transferred to SOF droplets for
embryo culture. In order to study the kinetics of
cleavage, culture droplets were examined at exactly
24, 27, 30, 33, 36, 42, and 48 hpi. At each time-point, the
number of cleaved embryos was recorded. We have
previously shown that such repeated observations do
not impact on the overall blastocyst yield (Lonergan
et al., 1999a).
Experiment 2: Oocyte Fertilization
In Vivo vs. In Vitro
The aim of Experiment 2 was to assess the impor-
tance of fertilization in vitro or in vivo on subsequent
blastocyst development and quality. In vivo matured
oocytes were either (1) fertilized in vitro (nˆ 134) or (2)
fertilized in vivo by artificial insemination and the
resulting presumptive zygotes recovered on day
1 (nˆ 69). Both groups were then cultured in vitro. As
a control, a group of oocytes (nˆ 388) recovered from 2–
6 mm follicles from the ovaries of slaughtered heifers
were put through IVM/IVF/IVC in parallel.
Experiment 3: Embryo Culture
In Vivo vs. In Vitro
The aim of Experiment 3 was to assess the impact of
the culture in vitro or in vivo on the yield and quality of
blastocysts. In Experiment 3a, presumptive zygotes
produced by IVM/IVF were cultured either in vitro in
SOF (nˆ463), or in vivo in the ewe oviduct (nˆ775). In
Experiment 3b, in vivo matured/in vivo fertilized
zygotes were either surgically recovered on day 1 and
cultured in vitro in SOF (nˆ 69), or remained in vivo
and werenonsurgically recovered on day 7 (nˆ 98). As
a control, a group of zygotes (nˆ388) produced by IVM/
IVF were cultured in vitro in parallel.
In all three experiments, the overall cleavage rate
was recorded at 48 hpi and blastocyst development was
recorded on days 6, 7, and 8 pi. To assess blastocyst
quality, in each experiment, day 7 blastocysts from
each group were vitrified/warmed using the OPS
method as described above, and survival rate was
recorded every 24 hr up to 72 hr post-warming in
parallel with nonvitrified control embryos.
Statistical Analysis
Differences in cleavage and development of zygotes to
the blastocyst stage as well as data on the proportion of
blastocysts surviving cryopreservation were compared
using chi-square analysis. A P value less than 0.05 was
considered significant.
RESULTS
Experiment 1
The average follicle size (mm) from which oocytes
were recovered at OPU was 11.4�1.8 (nˆ 92) for
oocytes recovered just before the LH surge and
13.5�0.5 (nˆ87) for in vivo matured oocytes. There
was no difference in oocyte cleavage rate following IVF
or the yield of blastocysts by day 6, irrespective of
the origin of the oocyte (Table 2). However, by day 7
and day 8, significantly more blastocysts developed
from oocytes matured in vivo (58.2%) than those re-
covered just before the LH surge (39.2%) or those from
2–6 mm follicles (38.9%). Oocytes from large follicles
(>6 mm) resulted in an intermediate blastocyst yields
(46.5%).
The kinetics of early cleavage is shown in Figure 2.
With regard to the three groups of oocytes matured
in vitro (2–6 mm, > 6 mm, LH), the kinetics of cleavage
are consistent with the subsequent blastocyst develop-
ment; ranking the groups based on the proportion of
oocytes cleaved by 30 hpi mirrored their ranking based
on overall blastocyst yields (see Table 2). In contrast,
while oocytes matured in vivo had the highest blas-
tocyst yields of all groups, they exhibited an apparently
slower kinetics of cleavage.
In terms of blastocyst quality (Fig. 3), there was no
difference in the survival or hatching rate of control
(nonvitrified) blastocysts amongst the four groups.
TABLE 2. Effect of Bovine Oocyte Origin on In Vitro Embryo Development (Experiment 1)
Oocyte origin N
Cleaved
n (%)
Blastocyst yield
Day 6 Day 7 Day8
n (%) n (%) n (%)
In vitro 2–6 mm 388 319 (82.2) 65 (16.8) 123a (31.7) 151 (38.9)
In vitro > 6 mm 99 87 (87.9) 23 (23.2) 38a,b (38.4) 46a,b (46.5)
Before LH surge 102 90 (88.2) 17 (16.7) 36a (35.3) 40a (39.2)
In vivo matured 134 117 (87.3) 30 (22.4) 65b (48.5) 78b (58.2)
P value NS NS 0.05 0.01
a,bValues in the same column with different superscripts differ significantly.
N, total number of oocytes.
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 237
In contrast, survival following vitrification was rela-
tively low in all groups ranging from <40% at 24 hr
post-warming to <20% in all groups by 72 hr post-
warming (see Figure 3 for statistical comparisons).
There were no differences in hatching rate either in
control or vitrified blastocysts.
Experiment 2
The results of Experiment 2 are shown in Figure 4.
There was no difference in cleavage rate when in vivo
matured oocytes were fertilized in vivo (92.8%) or
in vitro (87.3%). However, in vivo fertilization resulted
in a significantly higher cleavage rate (P<0.05) than
the control in vitro-matured group. In vivo fertilized
oocytes had a significantly higher blastocyst yields
(P<0.01) than both in vitro fertilized groups on days 6,
7, and 8 (73.9 vs. 58.2% and 39.2% on day 8, for in vivo
fertilized, in vivo matured/in vitro fertilized and in vitro
matured/in vitro fertilized oocytes, respectively). In
addition, in vivo matured/in vitro fertilized oocytes
yielded significantly more blastocysts (P< 0.001) than
in vitro matured oocytes on day 7 and day 8.
Similar to the results of Experiment 1, there was no
difference in the survival of control (nonvitrified)
blastocysts (Fig. 5). In addition, survival and hatching
rate following vitrification was not different between
the groups, with survival ranging from < 40% at 24 hr
to < 20% at 72 hr post-warming.
Experiment 3
The results of Experiment 3a are presented in
Table 3. The recovery rate of embryos from the ligated
ewe oviduct was 64.3%. The cleavage rate of in vitro
cultured zygotes was 82.5%; a figure for the cleavage
rate of in vivo cultured zygotes was not obtainable
due to the degeneration of nondeveloping embryos in
the oviduct. However, there was no difference in the
proportion of oocytes developing to the blastocyst stage
by day 7 or 8 between the two culture systems (34.1 vs.
34.5%).
In contrast, there was a marked difference in the
quality of the blastocysts produced in the two culture
systems. While there was no difference in the survival
and hatching rates of control (nonvitrified) blastocysts
(Fig. 6), following vitrification and warming, signifi-
cantly more blastocysts (P< 0.001) from the ewe
oviduct survived at all time points and hatched than
their in vitro counterparts (88.0 vs. 5.6% survival,
respectively at 72 hr).
The results of culturing in vivo matured/in vivo
fertilized zygotes either in vivo or in vitro (Experiment
3b) are presented in Table 4. The cleavage rate and
Fig. 2. Kinetics of first cleavage following in vitro fertilization of bovine oocytes from 2–6 mm follicles
(nˆ289), > 6 mm follicles (nˆ47), oocytes recovered just prior to the expected time of the LH surge
(nˆ63) and oocytes matured in vivo (nˆ 82).
238 D. RIZOS ET AL.
Fig. 3. Effect of oocyte origin on bovine blastocyst quality assessed
in terms of survival following vitrification. Blastocysts were derived
from oocytes from 2–6 mm follicles (control: nˆ 34, vitrified: nˆ57),
>6 mm follicles (control: nˆ 5, vitrified: nˆ 25), oocytes recovered just
prior to the expected time of the LH surge (control: nˆ5, vitrified:
nˆ 28) and oocytes matured in vivo (control: nˆ 20, vitrified: nˆ 40).
Different superscripts indicate significant differences (P< 0.05)
between treatments at a given time point.
Fig. 4. Effect of fertilization in vivo or in vitro on the cleavage rate
at 48 hr post insemination and the blastocyst yield on days 6, 7, and 8.
In vivo matured bovine oocytes were either fertilized in vitro (nˆ 134)
or in vivo by artificial insemination (nˆ 69). Both groups were
cultured in vitro in parallel with a control group of IVM/IVF/IVC
oocytes (nˆ388). Different superscripts indicate significant differ-
ences (P< 0.05) between treatments.
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 239
blastocyst yields of in vivo produced zygotes was
unaffected by the site of culture (in vitro vs. cow
oviduct). In addition, both in vivo groups resulted in
significantly higher cleavage (P<0.05) and blastocyst
yields (P<0.001) at all time points than the in vitro
control.
In terms of blastocyst quality, as in Experiment 3a, it
was clear that culture system had a dramatic effect on
survival following vitrification. There was no difference
in the survival of control (nonvitrified) blastocysts at 24
or 48 hr. At 72 hr, significantly more in vivo produced
blastocysts survived compared to those derived from
IVM/IVF/IVC (Fig. 7). In vitro culture, irrespective of
origin of the zygote, resulted in significantly lower
survival and hatching (P< 0.001) following vitrification
than culture in vivo (69.6 vs. 0% and 1.8% survival
at 72 hr, for in vivo matured /fertilized /cultured, in vivo
matured /fertilized /in vitro culture and in vitro
matured /fertilized /cultured embryos, respectively).
Figure 8 presents a summary of all the data from the
three experiments relating to oocyte quality measured
in terms of blastocyst development, and blastocyst
quality measured in terms of survival following
vitrification. The farther along the developmental axis
from oocyte to blastocystthat the oocyte/embryo was
removed from the in vivo environment and placed
in vitro, the greater the development. In terms of
quality, irrespective of the method of production of the
zygotes, culture in vivo resulted in blastocysts of
significantly higher quality.
The morphology of bovine embryos produced in vitro
and in vivo is shown in Figure 9. In vitro produced
embryos tended to have a darker overall appearance,
larger blastomeres at the early cleavage stages coupled
Fig. 5. Effect of fertilization in vivo or in vitro on bovine blastocyst
quality assessed in terms of survival following vitrification. Blas-
tocysts were derived from in vivo matured oocytes, which were either
fertilized in vitro (control: nˆ20, vitrified: nˆ 40) or in vivo (control:
nˆ 21, vitrified: nˆ26) or from IVM/IVF/IVC oocytes (control: nˆ 34,
vitrified: nˆ 57). Different superscripts indicate significant differ-
ences (P< 0.05) between treatments at a given time point.
TABLE 3. Effect of Culture of IVM/IVF Zygotes In Vitro in Synthetic Oviduct Fluid
or In Vivo in the Ewe Oviduct on Embryo Development (Experiment 3a)
Culture
system
Zygotes
transferred
n
Embryos
recovered
n (%)
Cleaved
n (%)
Blastocyst yield
Day 7
n (%)
Day 8
n (%)
In vitro 463 — 382 (82.5) 127 (27.4) 158 (34.1)
In vivo 775 498 (64.3) — 121 (24.3) 172 (34.5)
P value NS NS
240 D. RIZOS ET AL.
with a much reduced perivitelline space and more
defined compaction of the morula prior to blastulation.
A representative sample of blastocysts produced by
IVM/IVF/IVC, following culture in the ewe oviduct or
produced totally in vivo is illustrated in Figure 10.
Consistent with the data described above relating to
blastocyst quality as assessed by survival following
vitrification, IVM/IVF zygotes cultured in vivo resulted
in blastocysts with a similar morphology to those
produced totally in vivo, with IVM/IVF/IVC blastocysts
being considerably darker.
DISCUSSION
Oocyte Maturation
The results clearly demonstrate that oocytes matured
in vivo are more competent than those matured in vitro.
This in agreement with several previous studies (Greve
et al., 1987; Leibfried-Rutledge et al., 1987; Marquant-
Le Guienne et al., 1989; Bordignon et al., 1997; van de
Leemput et al., 1999). In the study of Bordignon et al.
(1997), blastocyst development of oocytes with ex-
panded cumulus fertilized immediately after recovery
was 60% in GnRH-treated animals (using similar
timings to the present study, i.e., OPU 60 hr post PG).
Our results are entirely consistent with these findings.
However, in many of these studies the sources of in vivo
and in vitro matured oocytes were different, coming
from preovulatory and 2–8-mm follicles, respectively.
To take account of this, we used immature oocytes
recovered just before the expected time of the LH surge
in vivo (i.e., just prior to resumption of meiosis) as a
control as well as oocytes from 2–6 mm follicles and
>6 mm follicles as it has previously been demonstrated
that oocytes from these follicle sizes differ in terms
of developmental competence (Pavlok et al., 1992;
Lonergan et al., 1994). Consistent with these observa-
tions, we observed that oocytes from follicles >6 mm
resulted in significantly more blastocysts than those
from 2–6 mm follicles.
One unexpected result was the relatively low blas-
tocyst yields from oocytes recovered just before the LH
surge (39.2%), which was not significantly higher
than our routine laboratory control using oocytes from
2–6 mm follicles from slaughterhouse ovaries (38.9%).
It is well known that superovulation can result in
abnormal endocrine profiles in the animal leading to
abnormal preovulatory LH release (Callesen et al.,
1986, 1987) and this is often associated with impaired
oocyte maturation and ultimately reduced embryo
quality (Callesen et al., 1986; Goff et al., 1986). It
could also be argued that oocytes recovered from
GnRH-treated heifers had been exposed to a more
synchronous signal for meiotic resumption, resulting in
the superior blastocyst yields observed in this study.
With regard to the kinetics of first cleavage, we have
previously demonstrated a clear relationship between
the time of first cleavage post-insemination in vitro and
Fig. 6. Effect of culture of IVM/IVF zygotes in vitro (control: nˆ20, vitrified: nˆ 54) or in vivo in the
ewe oviduct (control: nˆ 9, vitrified: nˆ50) on blastocyst quality assessed in terms of survival following
vitrification. Different superscripts indicate significant differences (P< 0.001) between treatments at a
given time point.
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 241
developmental competence with those oocytes cleaving
earliest after IVF being more likely to reach the
blastocyst stage than their later-cleaving counterparts
(Dinnyes et al., 1999; Lonergan et al., 1999a). In
Experiment 1, we observed a similar relationship
amongst the groups that were matured in vitro (2–
6 mm, >6 mm, LH); ranking of the groups based on
the proportion of oocytes cleaving by 30 hpi mirrored
their ranking based on overall blastocyst yields. In
contrast, however, the in vivo matured oocytes, despite
being inseminated at the same time as the other three
groups, had an apparently slower kinetics of cleavage,
but nonetheless resulted in the highest blastocyst
yields. This apparent contradiction is difficult to
explain.
Further, evidence for the fact that the intrinsic
quality of the oocyte determines the blastocyst yields
comes from the results of Experiment 3, where in vitro
matured/fertilized oocytes resulted in a blastocyst
yields of approximately 35% irrespective of whether
they were cultured in vitro or in vivo, while >70% of in
vivo matured /fertilized oocytes reached that stage
following culture either in vitro or in vivo (Table 4).
Differences have been reported between in vivo- and
in vitro-matured oocytes, which may explain the
observed differences in developmental competence.
TABLE 4. Effect of Culture of In Vivo Matured/Fertilized Bovine Zygotes In Vitro or
In Vivo on Embryo Development (Experiment 3b)
Treatment N
Cleaved
n (%)
Blastocyst yield
Day 6
n (%)
Day 7
n (%)
Day 8
n (%)
Control 388 319a (82.2) 65a (16.8) 123a (31.7) 151a (38.9)
In vivo matured/
fertilized
69 64b (92.8) 34b (49.3) 49b (71.0) 51b (73.9)
In vivo matured/
fertilized/cultured
98 90b (91.8) — 73a (74.5) 80b (81.6)
P value 0.05 0.001 0.001 0.001
a,bValues in the same column with different superscripts differ significantly.
N, total number of zygotes; control, oocytes from 2–6 mm follicles from slaughterhouse ovaries
used in IVM/IVF/IVC.
Fig. 7. Effect of culture of in vivo matured/fertilized zygotes in vitro
(control: nˆ 21, vitrified: nˆ 26) or in vivo (control: nˆ 22, vitrified:
nˆ 46) on blastocyst quality assessed in terms of survival following
vitrification. A group of blastocysts derived from IVM/IVF/IVC were
treated in the same way (control: nˆ34, vitrified: nˆ 57). Different
superscripts indicate significant differences (P<0.01) between treat-
ments at a given time point.
242 D. RIZOS ET AL.
Cumulus expansion is usually more extensive following
maturation in vivo (Suzuki et al., 1996). There is a high
degree of homogeneity amongst oocytes matured in vivo
at the ultrastructural level; this contrasts with the
ultrastructural heterogeneity exhibited by in vitro
matured oocytes, even when a uniform population of
the latter is selected prior to IVM (de Loos et al., 1992).
IVM has been shown to result in deviations ranging
from asynchrony between nuclear, cytoplasmic, and
cumulus maturation to outright degeneration.
Assey et al. (1994a) reported that bovine oocytes
aspirated from dominant follicles before the LH surge
display alterations in their nuclear and cytoplasmic
morphology which according to the authors, are a
prerequisitefor the acquisition of full developmental
competence. This would indicate that not only final
oocyte maturation (i.e., the processes occurring
between LH surge and ovulation) is significant, but
also the period preceding the LH surge may be
important for the establishment of developmental
competence.
Oocyte Fertilization
The results of Experiment 2 in which a higher
proportion of in vivo matured oocytes developed to
blastocysts following fertilization in vivo compared
with fertilization in vitro suggests that the events
around the time of fertilization might be important in
determining the developmental competence of the
oocyte. However, whether or not fertilization in vivo
per se was solely responsible for the observed increase
in blastocyst yields is questionable. It should be noted
that the in vivo fertilized oocytes were ovulated oocytes;
this is in contrast to the in vivo matured/in vitro
fertilized group, in which oocytes were recovered from
preovulatory follicles just prior to the expected time of
ovulation. In unstimulated cattle, ovulation occurs
approximately 24 hr after the LH peak, while following
superovulation, ovulations occur from 24–33 hr after
the peak (Callesen et al., 1986). This would suggest that
a proportion of the oocytes removed from preovulatory
follicles may not have completed maturation and this
may have contributed to a lower blastocyst yields.
To address this question, we attempted the fertiliza-
tion of in vitro matured bovine oocytes in the sheep
oviduct using GIFT, involving the transfer of matured
oocytes and sperm to the oviduct simultaneously, or the
transfer of matured oocytes to the oviduct of a ewe
previously inseminated with bovine sperm (results not
shown). Irrespective of the method used only a very low
Fig. 8. Summary graph showing effect of oocyte origin on the yield
and quality of bovine blastocysts. Blastocysts were derived from
oocytes from (1) slaughterhouse ovaries from 2–6 mm or (2) >6 mm
follicles, (3) recovered by ovum pick up just prior to the expected time
of the LH surge, or (4) following maturation in vivo, (5) matured and
fertilized in vitro and cultured in the ewe oviduct, (6) matured/
fertilized in vivo and cultured in vitro, or (7) matured/fertilized/
cultured in vivo. Arrows indicate that the further along the
developmental axis the oocyte/embryo is removed from the in vivo
environment the greater the blastocyst development. Culture in vivo,
irrespective of the origin of the oocyte results in blastocysts of superior
quality to the culture in vitro.
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 243
proportion of oocytes were fertilized and none devel-
oped to blastocysts. Other authors have similarly
attempted the in vivo fertilization of in vitro matured
bovine oocytes in the inseminated rabbit (Sreenan,
1970; Hunter et al., 1972; Trounson et al., 1977), sheep
(Sreenan, 1970), and cow oviduct (Trounson et al.,
1977; Newcomb et al., 1978; Myers et al., 1992) with
limited success, although Newcomb et al. (1978) did
report the birth of twin calves following one such
Fig. 9. Morphology of bovine embryos produced in vivo (A–E) or
in vitro (F–J). Images are representative of matured oocytes (A and
F), 2-cell embryos (B and G), 8-cell embryos (C and H), morulae (D and
I), and blastocysts (E and J).
Fig. 10. Morphology of bovine blastocysts produced by (A) IVM/
IVF/IVC, (B) in vivo culture in the sheep oviduct of IVM/IVF zygotes,
and (C) superovulation and nonsurgical embryo recovery on day 7.
244 D. RIZOS ET AL.
attempt. It would seem that such an approach is
fraught with technical difficulties which only cloud
the issue.
Embryo Culture
In vivo, the oviduct is the site of fertilization and
early embryo development. The oviductal environment
can support embryonic growth up to the blastocyst
stage across a wide range of species following trans-
species transfer. Ligated rabbit oviducts have been
used extensively for the development of embryos from
many species including sheep (Averill et al., 1955;
Lawson et al., 1972), cattle (Sreenan et al., 1968;
Boland, 1984; Ectors et al., 1993), pigs (Polge et al.,
1972), and horses (Allen et al., 1976). Sheep oviducts
have been shown to support the growth of cow
(Eyestone et al., 1987; Galli and Lazzari, 1996;
Gutierrez-Adan et al., 1996; Enright et al., 2000; Rizos
et al., 2001), and pig (Prather et al., 1991) embryos. The
mouse oviduct has also been used to support the
development of zygotes from the cow (Krisher et al.,
1989; Sharif et al., 1991), and hamster (Minami et al.,
1988).
In the present study, the blastocyst yields from IVM/
IVF oocytes was unaffected irrespective of whether
culture took place in vitro or in vivo in the ewe oviduct
(Experiment 3a). This is consistent with a previous
report from our group (Enright et al., 2000). Jimenez
et al. (2001) observed that culture in the ligated sheep
oviduct resulted in similar blastocyst yields to culture
in vitro (26 vs. 27%). However, if ligation was not
carried out and the embryos were allowed to go into the
uterus, development was significantly reduced (Jime-
nez et al., 2001). This observation is diffcult to reconcile
with the results of Rexroad and Powell (1999) and
Talbot et al. (2000), who demonstrated that the uterus
of the ewe supports normal development of bovine
blastocysts after transfer at day 7 and recovery at day
14, demonstrating that bovine embryos can undergo
continued development in the reproductive tract of
ewes, when transferred either as 4-cell embryos or as
expanded or hatched blastocysts.
In the corollary to this experiment, we compared
development of in vivo matured/fertilized zygotes
following culture either in vitro or in vivo in the heifer
oviduct. Consistent with the observations described
above, there was no difference in blastocyst yields
between the two culture systems. The combined results
of these two experiments clearly demonstrate that the
proportion of oocytes developing to the blastocyst stage
is not determined by the culture system, but rather by
the origin of the oocytes. In addition, it is clear that
oocytes from a common source will result in similar
blastocyst development even when culture takes place
in different environments.
While culture of IVM/IVF zygotes in the ewe oviduct
did not affect blastocyst yields, the oviduct environment
of the intermediate recipient clearly improved the
overall quality of IVM/IVF blastocysts, as measured
by survival following cryopreservation. Similarly, the
results of Experiment 3b demonstrate that the in vitro
culture of zygotes derived from oocytes of high devel-
opmental potential (in vivo matured/fertilized) is
sufficient to result in blastocysts of low cryotolerance,
similar to those resulting from IVM/IVF/IVC. Consis-
tent with these and previous results from our group
(Enright et al., 2000), Pugh et al. (2001) observed that
culture of bovine embryos in the sheep oviduct
improved the frozen but not the fresh embryo survival
following transfer. Tervit et al. (1994) found that, while
culture in the oviduct did not affect the proportion of
sheep embryos judged to be of freezeable quality, the
percentage of embryos surviving post thaw was higher
for IVM/IVF embryos after culture in the oviduct than
culture in SOF. Holm et al. (1994) demonstrated that
IVC of IVM/IVF derived ovine zygotes reduced embryo
viability by 15–25% compared with in vivo culture.
Galli and Lazzari (1996) reported that there were no
differences between culture in the ewe oviduct or
culture in vitro in terms of blastocyst formation at
day 8. However, in agreement with our results, they
observed major differences in quality with embryos
cultured in the ewe oviduct and those collected from
superovulated donors being superior in terms of
sensitivity to freezing/thawing.
Differences have been described at the ultrastruc-tural level which may in part explain the variation in
cryotolerance observed amongst groups of embryos. In
a recent study by Fair et al. (2001), it was demonstrated
that blastocysts derived from the in vivo culture in the
ewe oviduct of IVM/IVF zygotes displayed only minor
morphological differences from those produced comple-
tely in vivo by superovulation. In contrast, in vitro
produced blastocysts exhibited a range of character-
istics associated with reduced cryotolerance such as a
wider perivitelline space, vacuoles in the trophblast
cells, a sparse population of microvilli, a greatly
reduced network of intercellular connections and a
large increase in lipid content. Similar observations
have been reported by other authors (Abe et al., 1999;
Crosier et al., 2000).
What is it that controls the development of a
blastocyst of high quality? Differences in gene expres-
sion exist between in vitro and in vivo embryos, which
may explain the differences in quality observed.
Wrenzycki et al. (1996) demonstrated that expression
of the connexin 43 gene at the blastocyst stage differs
between bovine embryos produced in vitro and those
produced in vivo. This gene is involved in the formation
of a protein that gives rise to gap junctions between
cells. Poor gap junction formation is associated with
poor cell compaction and is a common occurrence in IVP
embryos. Also, accelerated development in vitro due to
serum addition (Carolan et al., 1995; Lonergan et al.,
1999b) may affect gene regulation and transcription,
resulting in well-documented developmental abnorm-
alities such as foetal oversize in the bovine (Young et al.,
1998).
It has also been demonstrated that the gene expres-
sion in the developing embryo can be influenced by the
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 245
culture environment in vitro (Wrenzycki et al., 1999;
Natale et al., 2000). Knijn et al. (2001) compared gene
expression in blastocysts derived from in vivo- or
in vitro-matured bovine oocytes. No differences were
observed in relative abundance for four genes studied,
suggesting that maturation is not the major step in the
IVP process affecting expression of these genes in the
embryo. This would be consistent with the findings of
Wrenzycki et al. (1999) and those of the present study,
reinforcing the point that the culture system is the
major determinant of blastocyst quality, irrespective of
the origin of the oocyte.
The culture environment can also have a significant
effect on embryo metabolism, which may have implica-
tions for embryo quality. Embryos generated in a
completely defined medium have lower rates of glyco-
lysis than those in serum (Krisher et al., 1999).
Khurana and Niemann (2000) examined energy meta-
bolism in in vivo- and in vitro-derived bovine embryos.
In general, the pattern was similar; however, IVP
embryos exhibited a 2-fold higher rate of anaerobic
glycolysis and produced more lactate. Culture for
72 hr of in vivo produced blastocysts resulted in
lactate production similar to that of in vitro produced
blastocysts.
CONCLUSION
In conclusion, events before ovulation determine the
ultimate fate of the oocyte but events occurring
between the zygote and blastocyst stages determine
the blastocyst quality. As pointed out by Hendriksen
et al. (2000), the observation that oocytes with identical
developmental conditions up to the LH surge (i.e.,
initiation of meiotic resumption) differ in their ability to
reach the blastocyst stage depending on whether they
undergo maturation in vivo or in vitro highlights the
fact that current IVM methods can still be improved.
One route towards improving embryo yields may be the
prematuration of the oocyte prior to maturation. While
some recent results are encouraging in that oocytes can
be reversibly maintained at the germinal vesicle stage
without affecting subsequent blastocyst yields (Lone-
rgan et al., 1997; Kubelka et al., 2000; Lonergan et al.,
2000; Mermillod et al., 2000; Motlik et al., 2000), it has
not yet been demonstrated that such an approach
can improve the developmental ability of an oocyte.
In addition, there is little if any evidence to demon-
strate that such blocked oocytes can result in the birth
of normal offspring. A more feasible approach in the
short term at least will probably be to modify the
conditions of in vitro culture to ensure that those
blastocysts that do develop are of the highest possible
quality.
ACKNOWLEDGMENTS
D. Rizos was supported by a grant from the Greek
State Scholarships Foundation. We thank M. Wade for
excellent technical assistance, and T. Harte, and the
staff at Lyons for help in maintaining the animals.
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