Theriogenology 65 (2006) 126136

www.journals.elsevierhealth.com/periodicals/the

Contribution of the oocyte to embryo quality

Marc-Andre Sirard *, Francois Richard, Patrick Blondin,
Claude Robert
Centre de Recherche en Biologie de la Reproduction, Department of Animal Sciences,
Laval University, Pav. Comtois, Sainte-Foy, Que., Canada G1K 7P4

Abstract
The ability of a bovine embryo to develop to the blastocyst stage, to implant and to generate a healthy
offspring is not a simple process. To clarify the importance of the contribution of the oocyte to the
embryo quality, it is important to define more precisely the different types of competence expressed by
oocytes. The ability to resume meiosis, to cleave upon fertilization to develop into a blastocyst, to induce
pregnancy and to generate an healthy offspring are all separate events and succeeding in the first events
does not ensure the success of subsequent ones. Furthermore, these events are associated with the three
types of maturation processes observed in the oocyte: meiotic, cytoplasmic and molecular. These
abilities vary also upon the type of follicle the oocytes is removed from. Larger or slow-growing follicles
have been shown to foster better eggs than small or actively growing follicles. Hormonal stimulation can
also affect oocyte competence with the nature of the effect (positive or negative) depending on timing
and dose. This complex situation requires better definition of the contribution of each factor affecting the
oocyte competence and the resulting embryo quality.
# 2005 Elsevier Inc. All rights reserved.
Keywords: Oocyte competence; Follicle; RNA; Differentiation

1. Introduction
The literature concerning bovine embryo quality has become very abundant. The
influence of the oocyte quality on the developmental potential of the embryo has been
recognized in the cow more clearly that in any other species. Because multiple factors are
involved and a vast amount of data have been published, it may seem difficult to have a
* Corresponding author. Tel.: +1 418 656 2131x7359; fax: +1 418 656 3766.
E-mail address: marc-andre.sirard@crbr.ulaval.ca (M.-A. Sirard).
0093-691X/$ see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2005.09.020

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clear idea of the concept of competence and the factors acting upon it. Therefore, it is
timely to clarify the concepts of oocyte competence, oocyte maturation and follicular
differentiation with regards to resulting embryo quality.

2. Oocyte competence
The following is a comprehensive enumeration and a short description of the five levels
of oocyte competence. They represent key steps that characterize developmental
competence:
(1)
(2)
(3)
(4)
(5)

Ability
Ability
Ability
Ability
Ability

to
to
to
to
to

resume meiosis.
cleave following fertilization.
develop to the blastocyst stage.
induce a pregnancy and bring it to term.
develop to term in good health.

2.1. Ability to resume meiosis

Even if the ability to resume meiosis is probably the easiest to measure, it underlies a
spatio-temporal synchrony of cell cycle molecules. When mammalian oocytes, including
those from the cow, are removed from their follicles, they have the ability to spontaneously
resume meiosis [1]. No stimulating agents are required. Meiotic resumption can be
visualised under the microscope by the first polar body extrusion or with specific dyes to
stain the metaphase. This resumption of meiosis is believed to be a consequence of the
absence of a follicular inhibitor still unidentified in domestic animals [2,3]. In the cow, the
oocyte acquires the ability to form a metaphase plate when reaching its full size in the
growing follicle just before antrum formation [4]. In some species, the capacity to reach
metaphase I is acquired before the capacity to reach metaphase II [5]. In cows, the two
seems to be acquired at the same time, but the low abundance of cell cycle activator can
result in an metaphase I arrest [6], indicating that the two capacities require distinct
molecules.
2.2. Ability to cleave following fertilization
The capacity to cleave is almost automatic and is an intrinsic potential within the fully
grown oocytes of large mammals because it can occur in absence of fertilization by a
simple activation stimulus (electrical current, ethanol) [7]. When cleavage does not occur,
it is not clear if it is the consequence of a dysfunctional sperm that failed to activate the
oocyte or the oocyte itself did not have the ability to undergo the first cell division.
The assessment of hundreds of presumptive zygotes that did not cleave 36 h postinsemination indicates that most of them were not fertilized and only a small proportion
had an incomplete decondensed sperm or asynchronized pronuclei (Sirard, personal
observation). This observation indicates that a negligible subpopulation of full-size oocytes
obtained from slaughterhouse ovaries are incompetent to trigger the downstream processes

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following fertilization and cleave. This situation is different than in the mouse where the
ability to activate/cleave following fertilization is acquired later than the capacity to
resume meiosis [8].
2.3. Ability to develop to the blastocyst stage
This is the most controversial and yet key marker of oocyte competence commonly used
by most laboratories. It is obvious that a fertilized oocyte must reach the blastocyst stage
within 69 days in the proper culture conditions to have a significant chance of inducing a
pregnancy and producing an offspring. The ability to sustain the first week of embryonic
development is clearly influenced by the follicular status from which the oocyte is obtained
(see below) indicating that this developmental potential is inherent within certain oocytes.
Since most early embryos that do not reach the blastocyst stage are blocked at or close to
the maternal to zygotic transition (MZT)-stage, which occurs at the eight-cell stage in
cattle [9], one could speculate that incompetent oocytes fail to appropriately activate the
embryonic genome.
The early developmental program embedded in the oocyte through the accumulation of
proteins and RNA is likely to be responsible for the proper execution of the embryonic
genome activation. The use of transcription inhibitors such as a-amanitin during the first
few days following fertilization results in normal cleavage until the four- to eight-cell stage
[9]. Thus, transcriptional activation of the new embryonic genome is required for
development beyond MZT [9]. Such activation may well depend on the activation or
translation of some maternal transcription factors already stored in competent oocytes that
make this activation possible [10].
Since blastocysts can be classified based on their morphology, it is clear that their
quality varies depending on criteria such as number of cells, trophectoderm to inner-cell
mass ratio, blastocoele expansion, overall appearance, etc. (IETS Manual). Their ability to
survive cryopreservation and induce a pregnancy is also affected by their apparent
morphology as well as by their origin (in vitro or in vivo). From these visual assessments,
one can approximately predict pregnancy rates supporting the perception that good
blastocysts can result in pregnancy. Therefore the competence to achieve the blastocyst
stage is more an assessment of the normalcy of the oocyte than anything else. Even in
cloning experiments, most often the blastocyst rate obtained is lower or closer to the IVF
controls but never above, indicating the intrinsic limitation, of a proportion of immature
oocytes obtained from slaughterhouse ovaries [11]. Incidentally, none of the attempts to
improve culture conditions for bovine embryos has produced consistent blastocyst
production rates above 3040%. Only modifications of superovulatory treatments to
control follicular growth have had a significant effect on increasing these rates [12]. This
suggests that developmental competence is mainly acquired during oocyte growth within
the follicle by an unknown mechanism [13].
2.4. Ability to induce a pregnancy
As mentioned above, all blastocysts are not equal and do not always result in a
pregnancy once transferred in suitable recipients. Part of this failure can be attributed to the

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recipient. However, blastocysts that originate from oocytes matured in vitro result in
lower rates of gestation compared to their in vivo counterparts [14]. If we exclude the
influence of genetics (some oocyte donors are better than others) or sperm selection
(some bulls also achieve higher blastocyst rate and quality than others [15]), the quality
of blastocysts resulting from in vivo or in vitro matured oocytes collected from small
growing follicles and submitted to identical fertilization and embryo culture procedures
should be comparable. Therefore what could explain the higher rate of gestation failure
[14] associated with the in vitro procedure? It is reasonable to think that the ability to go
to term is influenced by events occurring before the blastocyst stage and could be
explained by either faulty culture conditions and/or by the incomplete oocyte
programming before aspiration from its follicle. Up to now, most of the negative
consequences of culture have been associated with the culture media post-fertilization,
but what about in vivo conditions in the follicle before the oocyte is removed as well as
culture conditions during maturation?
Could we be using oocytes that have not completed their imprinting by collecting
them from small growing follicles? There are only a few studies with in vivo matured
oocytes in cows [1618] and they were not designed to explain if the deleterious effect
observed following in vitro maturation comes from incomplete oocyte programming or
inadequate culture conditions. Recent studies indicate that induction of follicular
differentiation by manipulation of the ovarian stimulation protocol, namely FSH
starvation or coasting, can result in the recovery of germinal-vesicle stage (immature)
oocytes where most are capable of developing to the blastocyst stage following
completion of in vitro procedures. Moreover, the embryonic developmental rates
obtained from this procedure equal or even surpass the blastocyst rates obtained with
in vivo matured oocytes submitted to in vitro fertilization and culture although the two
were not compared in the same experiment [12]. Overall, the comparison between the
in vivo matured and the in vitro matured oocyte recovered at different times before the
LH surge support a progressive influence of the follicular differentiation on oocyte
competence.
2.5. Ability to develop to term in good health
One of the most surprising discoveries from the use of in vitro production of
embryos in cattle is the effect of the procedure on the health of the offspring. The most
studied of these effects, the Large Calf Syndrome (LCS), does not systematically occur
after vitro culture and varies in frequency according to the different laboratories [19].
Its presence is a warning that culture conditions at the early embryonic stage may have
direct impacts much later in life. The principal cause could be related to the oocyte
maturation period, possibly due to an incomplete acquisition of developmental
competence at onset of maturation as mentioned above, or as a consequence of
suboptimal culture conditions. Therefore the follicular environment could have an
influence not only on oocyte quality and female fertility but on the offsprings health as
well. It is known that the uterine environment can affect fetal development and impact
the offsprings health but the ovarian influence is becoming an additional source of
epigenetic influence that must be explored [20].

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3. Oocyte maturation
The three levels of oocyte maturation are described here to improve clarity and to allow
dissection of the association of follicular to intra-oocytes events. These levels are:
(1) Meiotic maturation
(2) Cytoplasmic maturation
(3) Molecular maturation
3.1. Meiotic maturation
As described above for meiotic competence, meiotic maturation is the cascade of
nuclear events that is induced either by the LH surge or by the removal of the oocyte
from its follicular environment. These events are programmed to occur in the oocyte
upon the removal of a still unidentified inhibitory substance. Once allowed to proceed,
maturation promoting factor (MPF), a protein complex composed of cyclin B1 and
P32cdc2, is synthesized and/or activated depending on the species and thereafter, the cell
cycle machinery becomes activated and the oocyte undergoes the first metaphase and
extrusion of the first polar body before arresting after the formation of the second
metaphase under the influence of the cytostatic factor (CSF). The timing of meiotic
maturation is quite precise and defined [21] allowing it to be used as a reference for other
intra-oocyte events.
3.2. Cytoplasmic maturation
Cytoplasmic maturation is not as clearly defined as the meiotic process since it
encompasses events both visible and invisible to the microscope. The early description of
cytoplasmic maturation is based on ultrastructural observations during the few days before
the LH surge when the oocytes awaits the ovulation signal. The first evidence of
cytoplasmic competence occurs when the oocyte stop its preparation phase (RNA and
protein synthesis) by modifying the transcription and translation machinery (nucleolus
condensation and ribosome depletion) [4,22,23]. A second series of changes occur close to
the LH surge which result in a re-distribution of organelles such as the mitochondria and
the cortical granules along with the changes occurring with the cell progression to
metaphase [24]. The second aspect of maturation that is often included as part of the
cytoplasmic maturation process is the accumulation of specific molecules, largely
unidentified, which prepare the oocyte for post-fertilization events. This component has
been referred to before as oocyte capacitation [18] but for the purpose of this explanation it
will be referred to as molecular maturation.
3.3. Molecular maturation
Molecular maturation is the least defined of the three types of oocyte maturation. As
indicated above, most of fully grown oocytes undergo normal meiotic and cytoplasmic
maturation although only a subset of them will develop to the blastocyst stage. The

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difference between a developmentally capable oocyte and an incompetent one can be

related to the differentiation state of the follicle of origin and these differences are not
always visible in the oocyte at the ultrastructural level. Currently, the most popular
hypothesis is that specific mRNA and possibly some proteins are produced and added to the
oocytes stockpile in the last few days before ovulation. These unique capacitators
would give the ovary a last word on the outcome of ovulation by altering the developmental
ability of the gamete produced. Logically, there are several good evolutionary reasons for
such a mechanism that are beyond the scope of this chapter. What comes out of all these
observations is that special instructions, coming from a specific follicular environment and
accumulated in the oocyte, are essential to promote the proper molecular cascades for
embryonic genome activation and the development to the blastocyst stage. Since no clear
discrepancies associated with the first two types of oocyte maturation have been identified
to be responsible for developmental competence, it is believed that molecular maturation
represents the closest association with the intrinsic capacity of an oocyte to reach the
blastocyst stage and probably beyond.

4. Follicular influence on oocyte competence

To dissect the influence of the follicular environment on the oocytes acquisition of
developmental competence, it is important to distinguish a number of relevant follicular
phases as follows:
(1) The preantral phase
(2) The growing phase
a. FSH dependent
b. FSH independent
(3) The early atretic phase
(4) The late atretic phase
(5) The dominant phase
(6) The plateau phase
(7) The preovulatory phase
(8) The post LH phase
4.1. The preantral phase
As described before [4], oocytes from preantral follicles cannot complete meiosis
because of incomplete meiotic, cytoplasmic and molecular maturation.
4.2. The growing phase
4.2.1. FSH dependent
This category includes the cohort of small antral follicles that can respond to FSH either
in a normal estrous cycle leading to a single dominant follicle or in an ovarian stimulation
scheme which leads to ovulation in a multi-dominant paradigm. These follicles contain

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oocytes with two to four layers of cumulus cells and very little, if any, signs of atresia.
Because these follicles are actively growing, it is very difficult to aspirate them from living
animals using ultrasound or laparoscopy and when removed in the growing phase (under
FSH stimulation) they display very limited competence [25,26]. The oocytes from these
follicles are partially competent only if maintained in the follicles post-mortem at body
temperature [27].
4.2.2. FSH independent
Once follicles reach a diameter of 8.5 mm in non-stimulated animals, they acquire LH
receptor in the granulosa layers and become less dependent of the FSH support [28].
Normally, in cows, only the follicle corresponding to the dominant follicle reaches this
status during each follicular wave. It is possible to induce the production of several of these
follicles with exogenous FSH support and, surprisingly, when oocytes are collected in the
active growing phase, they also display low developmental competence even if their size
often exceeds 79 mm [13].
4.3. The early atretic phase
The subordinate follicles and the dominant follicle in its demise contain oocytes of
relatively high developmental potential as measure by the blastocyst rate [29]. This
situation is potentially explained by similarities between maturing dominant follicle and
follicles in their early phases of atresia which might send similar maturation promoting
signals to the oocyte [27]. The increased competence level displayed by oocytes collected
from early atretic follicles and the reduced competence level of oocytes collected from late
atretic follicles can be induced artificially by prolonging folliculogenesis with external
FSH support followed by FSH withdrawal for 2448 h to obtain early atretic follicles or for
more than 72 h to collect oocytes from late atretic follicles [27].
4.4. The late atretic phase
The follicles entering the late atretic phase contain oocytes with a disrupted cumulus
layer which can be easily classified and result in a poor development rate to the blastocyst
stage [25]. A surprising observation is that atretic follicles above 5 mm (large subordinates
that were striving for dominance) often contain oocytes with partially expanded outer
layers of cumulus (as if the oocyte was trying to mature) and consequently display the
cumulus morphology change normally occurring after the LH surge.
4.5. The dominant phase
The dominant follicle is sustaining a fast growing rate for a few days and then reaches a
slower growth rate correlated with a higher estradiol output indicative of further follicular
differentiation [28]. Once these changes occur, the developmental potential of the oocyte
rapidly increases [18,29]. If this dominant follicle then faces a high progesterone level from
a persistent corpus luteum, it will not ovulate and the next follicular wave will emerge. The
potential of the oocyte within the non-ovulating dominant follicle is probably maintained a

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few days but very few studies have analysed these oocytes in non-stimulated conditions.
The fact that prostaglandin injection can lead to ovulation of these follicles and subsequent
pregnancy following insemination provides additional evidence that these follicles
contains good eggs [30].
4.6. The plateau phase
This phase is naturally occurring between the establishment of dominance and the
resultant reduction of growth and the preovulatory period where progesterone
concentrations decrease sharply and LH pulsatility increases [31]. This phase can be
induced artificially by withdrawing FSH for 48 h after three consecutive days of
stimulation [12] and result in oocytes with a high competence to reach the blastocyst
stage.
4.7. The preovulatory phase
This phase is coincident with low progesterone concentrations and can be obtained
without stimulation on days 1920 of the oestrous cycle or with prostaglandin injections
any day after wave emergence or with FSH stimulation and prostaglandin. The oocytes
obtained in these conditions have a competence level close to that of oocytes that mature in
vivo [18].
4.8. The post LH phase
Oocytes collected at different times after the LH surge posses a high degree of
competence to reach the blastocyst stage although the variability across animals reflects the
delicate equilibrium between the normal ovulation and the stimulated multiple ovulation
obtained with FSH. Because pregnancy rates are quite high (>85%) in non-stimulated and
inseminated heifers, it is assumed that most, if not all, of the oocytes produced in the
normal non-stimulated conditions are fully competent. By contrast, it is well known that a
significant proportion of eggs obtained after ovarian stimulation are not retrieved at the
blastocyst stage on day 7 post-insemination, supporting a variable level of competence
within an oocyte pool from different follicles. Such a result would suggest that, upon
stimulation, the ovary produces some late-growing follicles that ovulate oocytes of low
competence. Since follicles acquire the ability to ovulate with the appearance of LH
receptors in granulosa cells at relatively small diameter [28], follicles still growing under
the influence of FSH may be rushed to ovulation without the appropriate differentiation
stage so important to oocyte quality.

5. The effect of ovarian stimulation on oocyte competence

As indicated in previous sections, further evidence of follicular influence on oocyte
potential can be obtained by hormonal stimulation. Developmental competence is on
hold during growth and this is seen in follicles from all size. To activate that competence,

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a process of follicular differentiation must be induced either through dominance or early

atresia. Therefore to maximize the assisted reproductive success, it is important to better
understand the different physiological steps that trigger the passage from follicular growth
to differentiation and ideally to reproduce this normal pattern using the right combination
of gonadotropins. For example, it could be physiologically logical to increase the LH
support and decrease the FSH support as stimulation progresses to better reflect the in vivo
processes and promote the final slow follicular growth accompanied with high
differentiation that results in better eggs.
By using molecular approaches to define the changes occurring in these follicles, it will
become possible to understand the differentiation processes that lead to the oocytes
increased developmental capacity. Preliminary analysis using subtractive molecular
approaches has revealed a number of good candidates both from the follicle side [32] and
the oocyte itself [33]. These early analyses are pointing to the early luteinization events as
indicators of the type of differentiation associated with increased competence.
The signalling from the follicle to the oocyte is certainly more complex than only one
given factor at one given threshold. The analysis of the competence of oocyte from
different follicles also indicates that it is not an all or nothing event. When oocytes originate
from differentiated follicles, they have a higher ability to produce a morphologically
healthy blastocyst which is normally associated with higher pregnancy rates. Although we
have not found in the literature a report that show directly the comparison of blastocyst
from IVP following ovarian stimulation versus in vivo, the fact that the ovarian stimulation
results in more blastocyst of good quality is supportive of an higher quality. The molecular
dissection of all aspects of both follicular and oocyte differentiation is the only way to
understand the complexity of these processes and to bring significant improvements to the
assisted reproduction procedures in such animals as the cow.
To illustrate the window of the acquisition of competence within the follicle, a figure
may be helpful (Fig. 1). During each follicular waves a number of follicles enter the rapid
growth phase and as they exit from it, the oocyte inside them acquires part or all the

Fig. 1. Schematic illustration of the growth of follicles in waves during the estrous cycle in cows. The grey-shaded
areas represent windows in which the competence of the oocyte is likely to increase significantly. The darker
follicles are the more differentiated ones and the stippled follicles are atretic.

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135

information required to proceed to the blastocyst stage once fertilized. In regular cycle,
only the second or third wave will result in ovulation of an oocyte with the maximum
potential. The distinction between the growing follicles and the receding ones or the ones
reaching the plateau phase is probably resulting in difference of competence according to
the size of the follicle at inflexion.
It must be added that competence is temporary and maybe lost with more advanced
stages of atresia. It is not known if the competence factors disappears or other components
comes into play to prevent the development of an oocyte from a follicle in a more advance
stages of degeneration.

6. Conclusion
It is clear that the follicle can profoundly influence the quality of the oocyte obtained at
ovulation and, as a result, the quality of embryo obtained. There is also growing evidence
that the ovary resists ovarian stimulation by decreasing the quality of the oocytes it
produces. This brings back in perspective the importance of moderating the hormonal
stimulation protocols based on physiological considerations to optimize the yield of high
quality, transferable embryos in animals as well as in humans.

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