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ABSTRACT The aim of this study is to Key Words: oocyte development; in vitro fertiliza-
examine the effect of bovine oocyte maturation, tion; ovum pick-up; embryo; gamete biology
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 INTRODUCTION
following vitrification. In Experiment 1, 4 groups of The development of bovine oocytes to the blastocyst
oocytes were used: (1) immature oocytes from 2± stage following maturation, fertilization and culture
6 mm follicles; (2) immature oocytes from > 6 mm in vitro is limited to about 30±40%. There is now a
follicles; (3) immature oocytes recovered in vivo just growing amount of evidence to suggest that while
before the LH surge; and (4) in vivo matured oocytes. culture conditions can impact on the developmental
Significantly more blastocysts developed from oocytes potential of the early embryo, the intrinsic quality of
matured in vivo than those recovered just before the LH the oocyte is the key factor determining the proportion
surge or than oocytes from 2±6 mm follicles. Results of oocytes developing to the blastocyst stage (Sirard and
from > 6 mm follicles were intermediate. All blasto- Blondin, 1996). In support of this idea, oocytes derived
cysts had low survival following vitrification. In Experi- from large follicles are more competent than those
ment 2, in vivo matured oocytes were either (1) derived from small follicles (Pavlok et al., 1992;
fertilized in vitro or (2) fertilized in vivo by artificial Lonergan et al., 1994). Also, a greater proportion of
insemination and the resulting presumptive zygotes in vivo matured oocytes reach the blastocyst stage
recovered on day 1. Both groups were then cultured compared to those produced in vitro (Greve et al., 1987;
in vitro. In vivo fertilized oocytes had a significantly Leibfried-Rutledge et al., 1987; Marquant-Le Guienne
higher blastocyst yield than those fertilized in vitro. et al., 1989; McCaffrey et al., 1991; Van Soom et al.,
Blastocyst quality was similar between the groups. 1992).
Both groups had low survival following vitrification. While still enclosed within the follicle, especially the
In Experiment 3a, presumptive zygotes produced one destined to become the dominant follicle, the oocyte
by in vitro maturation (IVM)/fertilization (IVF) were undergoes signi®cant changes that play a key role in its
cultured either in vitro in synthetic oviduct fluid, or in acquisition of developmental competence. A number of
vivo in the ewe oviduct. In Experiment 3b, in vivo ultrastructural and molecular changes occurring dur-
matured/in vivo fertilized zygotes were either surgically ing oocyte development have been linked to develop-
recovered on day 1 and cultured in vitro in synthetic mental competence (Assey et al., 1994b; Hyttel et al.,
oviduct fluid, or were nonsurgically recovered on day 7. 1997). Also, in vitro maturation (IVM) has been
There was no difference in blastocyst yields between associated with certain abnormalities in the oocyte
groups of zygotes originating from the same source (Hyttel et al., 1986, 1988, 1989).
(in vivo or in vitro fertilization) irrespective of whether In addition to the proportion of oocytes developing to
culture took place in vivo or in vitro. However, there the blastocyst stage, the quality of these embryos is
was a dramatic effect on blastocyst quality with those important. Despite extensive research in terms of
blastocysts produced following in vivo culture surviving
vitrification at significantly higher rates than their
in vitro cultured counterparts. Collectively, these Grant sponsor: EU Commission; Grant number: QLK3-CT-1999-
00104; Grant sponsor: Greek State Scholarships Foundation.
results indicate that the intrinsic quality of the oocyte
*Correspondence to: Patrick Lonergan, Ph.D., Department of Animal
is the main factor affecting blastocyst yields, while the Science & Production, University College Dublin, Lyons Research
conditions of embryo culture have a crucial role in Farm, Newcastle, County Dublin, Ireland.
determining blastocyst quality. Mol. Reprod. Dev. E-mail: pat.lonergan@ucd.ie
61: 234±248, 2002. ß 2002 Wiley-Liss, Inc. Received 18 May 2001; Accepted 1 August 2001
increasing the yield of blastocysts from immature centrifugation of frozen-thawed semen (Dairygold A.I.
oocytes, the quality of in vitro produced embryos, in Station, Mallow, Ireland) on a discontinuous Percoll
terms of survival following cryopreservation, has (Pharmacia, Uppsala, Sweden) density gradient (2.5 ml
continually lagged behind that achieved with in vivo 45% (v/v) Percoll over 2.5 ml 90% (v/v) Percoll) for 8 min
derived embryos. In vitro, produced bovine embryos at 700g at room temperature. Viable spermatozoa,
differ from those produced in vivo in many important collected at the bottom of the 90% fraction were washed
respects (Massip et al., 1995; Wright and Ellington, in HEPES-buffered Tyrode's and pelleted by centrifu-
1995) including darker cytoplasm, lower density (Pol- gation at 100g for 5 min. Spermatozoa were counted in
lard and Leibo, 1994), more triglycerides and less lipids a hemocytometer and diluted in the appropriate volume
from other classes (Abd El Razek et al., 2000), swollen of fertilization medium to give a concentration of
blastomeres (Van Soom et al., 1992), a more fragile zona 2 106 spermatozoa/ml. A 250 ml aliquant of this
pellucida (Duby et al., 1997), a slower growth rate and suspension was added to each fertilization well to
higher thermal sensitivity (Leibo and Loskutoff, 1993), obtain a ®nal concentration of 1 106 spermatozoa/ml.
differences in intercellular communication (Boni et al., Plates were incubated for 24 hr at 398C under an
1999), and a higher incidence of chromosomal abnorm- atmosphere of 5% CO2 in air with maximum humidity.
alities (Viuff et al., 1999; Slimane et al., 2000), all of Semen from the same bull was used for all experiments.
which may contribute to the higher sensitivity to
cryoinjury exhibited by such embryos. In Vitro Culture (IVC)
It is unclear, which parts of the process of embryo At approximately 20 hr post insemination (hpi), pre-
production are important in determining such para- sumptive zygotes were denuded by gentle vortexing,
meters as blastocyst yields and blastocyst quality. The washed four times in PBS and twice in culture medium
aim of this study was to examine in detail the (synthetic oviduct ¯uid, SOF) before being transferred
sequential steps of bovine embryo production in vivo to 25 ml culture droplets (25 embryos/droplet). Fetal calf
and in vitro (i.e., maturation, fertilization, and early serum (10%, v/v) was added 24 hr after placement in
embryo development) in an effort to understand, which culture. Cleavage was assessed 48 hpi and blastocyst
steps of the process are important in determining development was recorded on days 6±8 (day 0 day of
oocyte/embryo developmental competence and embryo IVF).
quality, respectively. While some of these aspects have
been studied previously in isolation, this is the ®rst In Vivo Production of Oocytes and Embryos
time, to our knowledge, that such studies have been The protocol used for the in vivo production of oocytes
carried out in parallel. and embryos is described in Table 1. Beef cross heifers
were synchronized using a CIDR device (InterAg,
MATERIALS AND METHODS Hamilton, New Zealand) for 8 days. Three days before
Oocyte Collection and IVM CIDR removal, heifers received 2 ml (15 mg) of
Cumulus oocyte complexes (COCs) were obtained by prostaglandin F2a analogue (PG, Prosolvin, Intervet,
aspirating follicles from the ovaries of heifers, either at Dublin, Ireland). Heifers were checked for standing
slaughter or using ovum pick-up (OPU), depending on estrus ( day 0). The dominant follicle was ablated by
the experiment. After four washes in PBS supplement- transvaginal aspiration on day 8 of the estrous cycle.
ed with 36 mg/ml pyruvate, 50 mg/ml gentamycin,
and 0.5 mg/ml bovine serum albumin (BSA) (Sigma, TABLE 1. Protocol to Obtain In Vivo Oocytes/Embryos
St. Louis, MO, catalogue number A-9647), groups of up Day Time (hr) Action
to 50 COCs were placed in 500 ml maturation medium
in four-well dishes (Nunc, Roskilde, Denmark), and ÿ10 CIDR in
cultured for 24 hr at 398C under an atmosphere of 5% ÿ5 Prostaglandin
ÿ2 CIDR removal
CO2 in air with maximum humidity. The maturation 0 Estrus
medium was TCM-199 supplemented with 10% (v/v) 8 Dominant follicle ablation
fetal calf serum (FCS, Sigma, F-2442) and 10 ng/ml 10 FSH am & pm
epidermal growth factor (Sigma, E-4127). 11 FSH am & pm
12 FSH am & pm
Sperm Preparation and 0 Prostaglandin pm
In Vitro Fertilization (IVF) 13 FSH am & pm
14 40 Ovum pick up±oocytes just before LH
For in vitro fertilization (IVF), COCs were washed surge
four times in PBS and then in fertilization medium 40 Induction of LH surge with GnRH
before being transferred in groups of up to 50 into four- 48 AI for day 1 and day 7 in vivo embryos
15 60 AI for day 1 and day 7 in vivo embryos
well dishes containing 250 ml of fertilization medium 60 Ovum pick up±in vivo matured
(Tyrode's medium with 25 mM bicarbonate, 22 mM Na- oocytes
lactate, 1mM Na-pyruvate, and 6 mg/ml fatty acid-free 16 Surgical embryo recovery of day 1 in
BSA). In addition, 10 mg/ml heparin±sodium salt vivo zygotes
(184 units/mg heparin, Calbiochem, San Diego, CA) 21 Nonsurgical embryo recovery of day 7
in vivo embryos
were added. Motile spermatozoa were obtained by
236 D. RIZOS ET AL.
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).
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 237
Group 4 oocytes were partially denuded by brief expo- recorded on days 6, 7, and 8 pi. To assess blastocyst
sure to 0.05% hyaluronidase and immediately insemi- quality, in each experiment, day 7 blastocysts from
nated. The experimental design was such that oocytes each group were vitri®ed/warmed using the OPS
from all 4 groups were inseminated at the same time. method as described above, and survival rate was
At approximately 20 hr post insemination presump- recorded every 24 hr up to 72 hr post-warming in
tive zygotes were transferred to SOF droplets for parallel with nonvitri®ed control embryos.
embryo culture. In order to study the kinetics of
cleavage, culture droplets were examined at exactly Statistical Analysis
24, 27, 30, 33, 36, 42, and 48 hpi. At each time-point, the Differences in cleavage and development of zygotes to
number of cleaved embryos was recorded. We have the blastocyst stage as well as data on the proportion of
previously shown that such repeated observations do blastocysts surviving cryopreservation were compared
not impact on the overall blastocyst yield (Lonergan using chi-square analysis. A P value less than 0.05 was
et al., 1999a). considered signi®cant.
Blastocyst yield
Fig. 2. Kinetics of ®rst cleavage following in vitro fertilization of bovine oocytes from 2±6 mm follicles
(n 289), > 6 mm follicles (n 47), oocytes recovered just prior to the expected time of the LH surge
(n 63) and oocytes matured in vivo (n 82).
In contrast, survival following vitri®cation was rela- the groups, with survival ranging from < 40% at 24 hr
tively low in all groups ranging from < 40% at 24 hr to < 20% at 72 hr post-warming.
post-warming to < 20% in all groups by 72 hr post-
warming (see Figure 3 for statistical comparisons). Experiment 3
There were no differences in hatching rate either in The results of Experiment 3a are presented in
control or vitri®ed blastocysts. Table 3. The recovery rate of embryos from the ligated
ewe oviduct was 64.3%. The cleavage rate of in vitro
Experiment 2 cultured zygotes was 82.5%; a ®gure for the cleavage
The results of Experiment 2 are shown in Figure 4. rate of in vivo cultured zygotes was not obtainable
There was no difference in cleavage rate when in vivo due to the degeneration of nondeveloping embryos in
matured oocytes were fertilized in vivo (92.8%) or the oviduct. However, there was no difference in the
in vitro (87.3%). However, in vivo fertilization resulted proportion of oocytes developing to the blastocyst stage
in a signi®cantly higher cleavage rate (P < 0.05) than by day 7 or 8 between the two culture systems (34.1 vs.
the control in vitro-matured group. In vivo fertilized 34.5%).
oocytes had a signi®cantly higher blastocyst yields In contrast, there was a marked difference in the
(P < 0.01) than both in vitro fertilized groups on days 6, quality of the blastocysts produced in the two culture
7, and 8 (73.9 vs. 58.2% and 39.2% on day 8, for in vivo systems. While there was no difference in the survival
fertilized, in vivo matured/in vitro fertilized and in vitro and hatching rates of control (nonvitri®ed) blastocysts
matured/in vitro fertilized oocytes, respectively). In (Fig. 6), following vitri®cation and warming, signi®-
addition, in vivo matured/in vitro fertilized oocytes cantly more blastocysts (P < 0.001) from the ewe
yielded signi®cantly more blastocysts (P < 0.001) than oviduct survived at all time points and hatched than
in vitro matured oocytes on day 7 and day 8. their in vitro counterparts (88.0 vs. 5.6% survival,
Similar to the results of Experiment 1, there was no respectively at 72 hr).
difference in the survival of control (nonvitri®ed) The results of culturing in vivo matured/in vivo
blastocysts (Fig. 5). In addition, survival and hatching fertilized zygotes either in vivo or in vitro (Experiment
rate following vitri®cation was not different between 3b) are presented in Table 4. The cleavage rate and
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 239
Fig. 3. Effect of oocyte origin on bovine blastocyst quality assessed prior to the expected time of the LH surge (control: n 5, vitri®ed:
in terms of survival following vitri®cation. Blastocysts were derived n 28) and oocytes matured in vivo (control: n 20, vitri®ed: n 40).
from oocytes from 2±6 mm follicles (control: n 34, vitri®ed: n 57), Different superscripts indicate signi®cant differences (P < 0.05)
>6 mm follicles (control: n 5, vitri®ed: n 25), oocytes recovered just between treatments at a given time point.
Fig. 4. Effect of fertilization in vivo or in vitro on the cleavage rate cultured in vitro in parallel with a control group of IVM/IVF/IVC
at 48 hr post insemination and the blastocyst yield on days 6, 7, and 8. oocytes (n 388). Different superscripts indicate signi®cant differ-
In vivo matured bovine oocytes were either fertilized in vitro (n 134) ences (P < 0.05) between treatments.
or in vivo by arti®cial insemination (n 69). Both groups were
240 D. RIZOS ET AL.
Fig. 5. Effect of fertilization in vivo or in vitro on bovine blastocyst n 21, vitri®ed: n 26) or from IVM/IVF/IVC oocytes (control: n 34,
quality assessed in terms of survival following vitri®cation. Blas- vitri®ed: n 57). Different superscripts indicate signi®cant differ-
tocysts were derived from in vivo matured oocytes, which were either ences (P < 0.05) between treatments at a given time point.
fertilized in vitro (control: n 20, vitri®ed: n 40) or in vivo (control:
blastocyst yields of in vivo produced zygotes was matured /fertilized /in vitro culture and in vitro
unaffected by the site of culture (in vitro vs. cow matured /fertilized /cultured embryos, respectively).
oviduct). In addition, both in vivo groups resulted in Figure 8 presents a summary of all the data from the
signi®cantly higher cleavage (P < 0.05) and blastocyst three experiments relating to oocyte quality measured
yields (P < 0.001) at all time points than the in vitro in terms of blastocyst development, and blastocyst
control. quality measured in terms of survival following
In terms of blastocyst quality, as in Experiment 3a, it vitri®cation. The farther along the developmental axis
was clear that culture system had a dramatic effect on from oocyte to blastocyst that the oocyte/embryo was
survival following vitri®cation. There was no difference removed from the in vivo environment and placed
in the survival of control (nonvitri®ed) blastocysts at 24 in vitro, the greater the development. In terms of
or 48 hr. At 72 hr, signi®cantly more in vivo produced quality, irrespective of the method of production of the
blastocysts survived compared to those derived from zygotes, culture in vivo resulted in blastocysts of
IVM/IVF/IVC (Fig. 7). In vitro culture, irrespective of signi®cantly higher quality.
origin of the zygote, resulted in signi®cantly lower The morphology of bovine embryos produced in vitro
survival and hatching (P < 0.001) following vitri®cation and in vivo is shown in Figure 9. In vitro produced
than culture in vivo (69.6 vs. 0% and 1.8% survival embryos tended to have a darker overall appearance,
at 72 hr, for in vivo matured /fertilized /cultured, in vivo larger blastomeres at the early cleavage stages coupled
Blastocyst yield
Zygotes Embryos
Culture transferred recovered Cleaved Day 7 Day 8
system n n (%) n (%) n (%) n (%)
P value NS NS
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 241
Fig. 6. Effect of culture of IVM/IVF zygotes in vitro (control: n 20, vitri®ed: n 54) or in vivo in the
ewe oviduct (control: n 9, vitri®ed: n 50) on blastocyst quality assessed in terms of survival following
vitri®cation. Different superscripts indicate signi®cant differences (P < 0.001) between treatments at a
given time point.
with a much reduced perivitelline space and more To take account of this, we used immature oocytes
de®ned compaction of the morula prior to blastulation. recovered just before the expected time of the LH surge
A representative sample of blastocysts produced by in vivo (i.e., just prior to resumption of meiosis) as a
IVM/IVF/IVC, following culture in the ewe oviduct or control as well as oocytes from 2±6 mm follicles and
produced totally in vivo is illustrated in Figure 10. > 6 mm follicles as it has previously been demonstrated
Consistent with the data described above relating to that oocytes from these follicle sizes differ in terms
blastocyst quality as assessed by survival following of developmental competence (Pavlok et al., 1992;
vitri®cation, IVM/IVF zygotes cultured in vivo resulted Lonergan et al., 1994). Consistent with these observa-
in blastocysts with a similar morphology to those tions, we observed that oocytes from follicles > 6 mm
produced totally in vivo, with IVM/IVF/IVC blastocysts resulted in signi®cantly more blastocysts than those
being considerably darker. from 2±6 mm follicles.
One unexpected result was the relatively low blas-
tocyst yields from oocytes recovered just before the LH
DISCUSSION
surge (39.2%), which was not signi®cantly higher
Oocyte Maturation than our routine laboratory control using oocytes from
The results clearly demonstrate that oocytes matured 2±6 mm follicles from slaughterhouse ovaries (38.9%).
in vivo are more competent than those matured in vitro. It is well known that superovulation can result in
This in agreement with several previous studies (Greve abnormal endocrine pro®les in the animal leading to
et al., 1987; Leibfried-Rutledge et al., 1987; Marquant- abnormal preovulatory LH release (Callesen et al.,
Le Guienne et al., 1989; Bordignon et al., 1997; van de 1986, 1987) and this is often associated with impaired
Leemput et al., 1999). In the study of Bordignon et al. oocyte maturation and ultimately reduced embryo
(1997), blastocyst development of oocytes with ex- quality (Callesen et al., 1986; Goff et al., 1986). It
panded cumulus fertilized immediately after recovery could also be argued that oocytes recovered from
was 60% in GnRH-treated animals (using similar GnRH-treated heifers had been exposed to a more
timings to the present study, i.e., OPU 60 hr post PG). synchronous signal for meiotic resumption, resulting in
Our results are entirely consistent with these ®ndings. the superior blastocyst yields observed in this study.
However, in many of these studies the sources of in vivo With regard to the kinetics of ®rst cleavage, we have
and in vitro matured oocytes were different, coming previously demonstrated a clear relationship between
from preovulatory and 2±8-mm follicles, respectively. the time of ®rst cleavage post-insemination in vitro and
242 D. RIZOS ET AL.
Blastocyst yield
Control 388 319a (82.2) 65a (16.8) 123a (31.7) 151a (38.9)
In vivo matured/ 69 64b (92.8) 34b (49.3) 49b (71.0) 51b (73.9)
fertilized
In vivo matured/ 98 90b (91.8) Ð 73a (74.5) 80b (81.6)
fertilized/cultured
developmental competence with those oocytes cleaving yields. This apparent contradiction is dif®cult to
earliest after IVF being more likely to reach the explain.
blastocyst stage than their later-cleaving counterparts Further, evidence for the fact that the intrinsic
(Dinnyes et al., 1999; Lonergan et al., 1999a). In quality of the oocyte determines the blastocyst yields
Experiment 1, we observed a similar relationship comes from the results of Experiment 3, where in vitro
amongst the groups that were matured in vitro (2± matured/fertilized oocytes resulted in a blastocyst
6 mm, > 6 mm, LH); ranking of the groups based on yields of approximately 35% irrespective of whether
the proportion of oocytes cleaving by 30 hpi mirrored they were cultured in vitro or in vivo, while > 70% of in
their ranking based on overall blastocyst yields. In vivo matured /fertilized oocytes reached that stage
contrast, however, the in vivo matured oocytes, despite following culture either in vitro or in vivo (Table 4).
being inseminated at the same time as the other three Differences have been reported between in vivo- and
groups, had an apparently slower kinetics of cleavage, in vitro-matured oocytes, which may explain the
but nonetheless resulted in the highest blastocyst observed differences in developmental competence.
Fig. 7. Effect of culture of in vivo matured/fertilized zygotes in vitro treated in the same way (control: n 34, vitri®ed: n 57). Different
(control: n 21, vitri®ed: n 26) or in vivo (control: n 22, vitri®ed: superscripts indicate signi®cant differences (P < 0.01) between treat-
n 46) on blastocyst quality assessed in terms of survival following ments at a given time point.
vitri®cation. A group of blastocysts derived from IVM/IVF/IVC were
BOVINE OOCYTE AND EMBRYO DEVELOPMENT 243
Fig. 8. Summary graph showing effect of oocyte origin on the yield fertilized in vivo and cultured in vitro, or (7) matured/fertilized/
and quality of bovine blastocysts. Blastocysts were derived from cultured in vivo. Arrows indicate that the further along the
oocytes from (1) slaughterhouse ovaries from 2±6 mm or (2) >6 mm developmental axis the oocyte/embryo is removed from the in vivo
follicles, (3) recovered by ovum pick up just prior to the expected time environment the greater the blastocyst development. Culture in vivo,
of the LH surge, or (4) following maturation in vivo, (5) matured and irrespective of the origin of the oocyte results in blastocysts of superior
fertilized in vitro and cultured in the ewe oviduct, (6) matured/ quality to the culture in vitro.
Cumulus expansion is usually more extensive following blastocysts following fertilization in vivo compared
maturation in vivo (Suzuki et al., 1996). There is a high with fertilization in vitro suggests that the events
degree of homogeneity amongst oocytes matured in vivo around the time of fertilization might be important in
at the ultrastructural level; this contrasts with the determining the developmental competence of the
ultrastructural heterogeneity exhibited by in vitro oocyte. However, whether or not fertilization in vivo
matured oocytes, even when a uniform population of per se was solely responsible for the observed increase
the latter is selected prior to IVM (de Loos et al., 1992). in blastocyst yields is questionable. It should be noted
IVM has been shown to result in deviations ranging that the in vivo fertilized oocytes were ovulated oocytes;
from asynchrony between nuclear, cytoplasmic, and this is in contrast to the in vivo matured/in vitro
cumulus maturation to outright degeneration. fertilized group, in which oocytes were recovered from
Assey et al. (1994a) reported that bovine oocytes preovulatory follicles just prior to the expected time of
aspirated from dominant follicles before the LH surge ovulation. In unstimulated cattle, ovulation occurs
display alterations in their nuclear and cytoplasmic approximately 24 hr after the LH peak, while following
morphology which according to the authors, are a superovulation, ovulations occur from 24±33 hr after
prerequisite for the acquisition of full developmental the peak (Callesen et al., 1986). This would suggest that
competence. This would indicate that not only ®nal a proportion of the oocytes removed from preovulatory
oocyte maturation (i.e., the processes occurring follicles may not have completed maturation and this
between LH surge and ovulation) is signi®cant, but may have contributed to a lower blastocyst yields.
also the period preceding the LH surge may be To address this question, we attempted the fertiliza-
important for the establishment of developmental tion of in vitro matured bovine oocytes in the sheep
competence. oviduct using GIFT, involving the transfer of matured
oocytes and sperm to the oviduct simultaneously, or the
Oocyte Fertilization transfer of matured oocytes to the oviduct of a ewe
The results of Experiment 2 in which a higher previously inseminated with bovine sperm (results not
proportion of in vivo matured oocytes developed to shown). Irrespective of the method used only a very low
244 D. RIZOS ET AL.
attempt. It would seem that such an approach is results of Experiment 3b demonstrate that the in vitro
fraught with technical dif®culties which only cloud culture of zygotes derived from oocytes of high devel-
the issue. opmental potential (in vivo matured/fertilized) is
suf®cient to result in blastocysts of low cryotolerance,
Embryo Culture similar to those resulting from IVM/IVF/IVC. Consis-
In vivo, the oviduct is the site of fertilization and tent with these and previous results from our group
early embryo development. The oviductal environment (Enright et al., 2000), Pugh et al. (2001) observed that
can support embryonic growth up to the blastocyst culture of bovine embryos in the sheep oviduct
stage across a wide range of species following trans- improved the frozen but not the fresh embryo survival
species transfer. Ligated rabbit oviducts have been following transfer. Tervit et al. (1994) found that, while
used extensively for the development of embryos from culture in the oviduct did not affect the proportion of
many species including sheep (Averill et al., 1955; sheep embryos judged to be of freezeable quality, the
Lawson et al., 1972), cattle (Sreenan et al., 1968; percentage of embryos surviving post thaw was higher
Boland, 1984; Ectors et al., 1993), pigs (Polge et al., for IVM/IVF embryos after culture in the oviduct than
1972), and horses (Allen et al., 1976). Sheep oviducts culture in SOF. Holm et al. (1994) demonstrated that
have been shown to support the growth of cow IVC of IVM/IVF derived ovine zygotes reduced embryo
(Eyestone et al., 1987; Galli and Lazzari, 1996; viability by 15±25% compared with in vivo culture.
Gutierrez-Adan et al., 1996; Enright et al., 2000; Rizos Galli and Lazzari (1996) reported that there were no
et al., 2001), and pig (Prather et al., 1991) embryos. The differences between culture in the ewe oviduct or
mouse oviduct has also been used to support the culture in vitro in terms of blastocyst formation at
development of zygotes from the cow (Krisher et al., day 8. However, in agreement with our results, they
1989; Sharif et al., 1991), and hamster (Minami et al., observed major differences in quality with embryos
1988). cultured in the ewe oviduct and those collected from
In the present study, the blastocyst yields from IVM/ superovulated donors being superior in terms of
IVF oocytes was unaffected irrespective of whether sensitivity to freezing/thawing.
culture took place in vitro or in vivo in the ewe oviduct Differences have been described at the ultrastruc-
(Experiment 3a). This is consistent with a previous tural level which may in part explain the variation in
report from our group (Enright et al., 2000). Jimenez cryotolerance observed amongst groups of embryos. In
et al. (2001) observed that culture in the ligated sheep a recent study by Fair et al. (2001), it was demonstrated
oviduct resulted in similar blastocyst yields to culture that blastocysts derived from the in vivo culture in the
in vitro (26 vs. 27%). However, if ligation was not ewe oviduct of IVM/IVF zygotes displayed only minor
carried out and the embryos were allowed to go into the morphological differences from those produced comple-
uterus, development was signi®cantly reduced (Jime- tely in vivo by superovulation. In contrast, in vitro
nez et al., 2001). This observation is diffcult to reconcile produced blastocysts exhibited a range of character-
with the results of Rexroad and Powell (1999) and istics associated with reduced cryotolerance such as a
Talbot et al. (2000), who demonstrated that the uterus wider perivitelline space, vacuoles in the trophblast
of the ewe supports normal development of bovine cells, a sparse population of microvilli, a greatly
blastocysts after transfer at day 7 and recovery at day reduced network of intercellular connections and a
14, demonstrating that bovine embryos can undergo large increase in lipid content. Similar observations
continued development in the reproductive tract of have been reported by other authors (Abe et al., 1999;
ewes, when transferred either as 4-cell embryos or as Crosier et al., 2000).
expanded or hatched blastocysts. What is it that controls the development of a
In the corollary to this experiment, we compared blastocyst of high quality? Differences in gene expres-
development of in vivo matured/fertilized zygotes sion exist between in vitro and in vivo embryos, which
following culture either in vitro or in vivo in the heifer may explain the differences in quality observed.
oviduct. Consistent with the observations described Wrenzycki et al. (1996) demonstrated that expression
above, there was no difference in blastocyst yields of the connexin 43 gene at the blastocyst stage differs
between the two culture systems. The combined results between bovine embryos produced in vitro and those
of these two experiments clearly demonstrate that the produced in vivo. This gene is involved in the formation
proportion of oocytes developing to the blastocyst stage of a protein that gives rise to gap junctions between
is not determined by the culture system, but rather by cells. Poor gap junction formation is associated with
the origin of the oocytes. In addition, it is clear that poor cell compaction and is a common occurrence in IVP
oocytes from a common source will result in similar embryos. Also, accelerated development in vitro due to
blastocyst development even when culture takes place serum addition (Carolan et al., 1995; Lonergan et al.,
in different environments. 1999b) may affect gene regulation and transcription,
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