Journal of Animal and Feed Sciences, 15, 2006, 525–542

Pig embryo production by in vitro maturation

and fertilization of ovarian oocytes. A review

L. Kątska-Książkiewicz1

Rzeszów University, Branch Campus of the Faculty of Biotechnology,

Department of Bioengineering
36-100 Kolbuszowa, Poland

(Received 27 March 2006; revised version 31 July 2006; accepted 6 November 2006)

ABSTRACT

In addition to their major role in food production, pigs have become an increasingly important
species in biomedical applications involving the production of pharmaceutical products and as
donors of organs for xenotransplantation. They are also used as a model for studies of human
diseases. In pigs, as in other mammals, immature oocytes released from ovarian follicles resume
meiosis and complete maturation in culture. Although several systems have been established to
generate embryos in vitro, the quality of embryos produced in vitro is inferior to those produced in
vivo. This review focuses on recent achievements in the development and identification of defined
conditions for the in vitro production of porcine embryos. It also discusses the effects of oocyte-
donor age, size of follicles used for oocyte recovery, synchronization of meiosis before IVM,
supplementations of media for IVM, IVF and embryo culture, oxygen tension during culture and the
ways for overcoming polyspermy.

KEY WORDS: pig, oocyte, IVM, IVF, embryo culture

INTRODUCTION

The development and identification of defined in vitro conditions for oocyte

maturation and fertilization is required by biotechnological researches since most
of the new reproductive technologies rely on these basic techniques. In vitro
embryo production (IVP) in pigs will contribute to reproductive biotechnology
including cloning and transgenesis and will generate multiple opportunities for the
4
Corresponding author: e-mail: [email protected]
526 PIG EMBRYO IN VITRO PRODUCTION

use of these modified animals as bioreactors of pharmaceutical products, donors of

organs for xenotransplantation, donors of embryonic stem cell lines or as a model
for studies of human diseases. Furthermore, the development of efficient in vitro
techniques would allow the production of large numbers of mature oocytes and
embryos in shorter time and with lower costs than from those produced in vivo.
The in vitro developmental competence of pig IVM/IVF oocytes was first
reported by Matiolli et al. (1989). Then, piglets were born in 1993 from IVP
porcine embryos transferred to donors at 2 to 4-cell stage (Yoshida et al., 1993).
Since then, several laboratories have developed and improved porcine IVP
systems. However, in spite of the progress, the quality of in vitro mature oocytes
and in vitro produced embryos is inferior to those produced in vivo. The major
problems include improper oocyte maturation, both nucleus and cytoplasmic,
high polyspermy and unsatisfactory quality of in vitro developed blastocysts.
This review will focus on recent advances in the identification and development
of defined in vitro conditions for porcine IVP technology.

OOCYTE IN VITRO MATURATION

Many of the events that prepare the female gamete for fertilization and make
it capable of supporting the initiation and continuation of embryonic development
take place during oocyte maturation. Maturation of the oocyte is connected
with achievement of the competence to undergo three aspects of maturation,
i.e. nuclear, cytoplasmic and genomic (Opiela and Kątska-Książkiewicz, 2004,
2005). Generally, looking into the basic aspects of oocyte meiotic maturation,
fully-grown follicular oocytes of most mammals are arrested at G2 phase of the
first meiosis (germinal vesicle stage - GV), and resume meiosis in vivo in response
to specific signals, such as the preovulatory peak in LH, or in vitro after being
released from the follicular environment and cultured in vitro for 20 to 24 h. This
resumption is characterized by germinal vesicle breakdown (GVBD), chromosome
condensation, and spindle formation. Oocytes then proceed toward metaphase I,
anaphase I, telophase I, and without any chromosome decondensation, they enter
meiosis II. Oocytes, reaching the metaphase stage of the second meiotic division
(MII), accompanied by extrusion of the first polar body, are then arrested again.
Resumption of meiosis in pig oocytes (Sun and Nagai, 2003) is controlled by
a complex cascade of phosphorylation and dephosphorylation events that lead
to activation of the M-phase promoting factor (MPF). The MPF induces M-
phase in eukaryotic cells, including oocytes. The MPF is a composite formed
by a catalytic subunit (p34cdc2), and a regulatory subunit (cyclin B) and this
complex displays a serine/threonine kinase activity. During S and G2 phases,
KĄTSKA-KSIĄŻKIEWICZ L. 527

association of these two subunits leads to the formation of pre-MPF, the inactive
form of MPF, which is converted into the active complex by phosphorylation and
dephosphorylation events. In the natural oestrous cycle, nuclear, cytoplasmic
and genomic maturation are three series of events that occur simultaneously
during oocyte maturation. Therefore, a nuclear matured oocyte normally means
that it achieves full developmental potential if fertilized. However, in oocyte
maturation in vitro these series of maturation events may be dissociated, resulting
in the loss of developmental potential due to impaired cytoplasmic maturation
and/or asynchrony of nuclear and cytoplasmic maturation (Opiela and Kątska-
Książkiewicz, 2004, 2005). Despite the fact that more than 20 years have been
dedicated to optimizing oocyte maturation in vitro in a number of species, in
vitro matured oocytes still have an overall developmental competence that is
far from normal and the kind of developmental abnormalities that are most
frequently recorded, such as the large offspring syndrome in ruminants, point to
aberrant epigenetic changes as the probable underlying cause (Niemann et al.,
2002).
In the pig, meiotic competence of oocytes is reached in ovarian follicles
with a diameter of 3 mm or more (Marchal et al., 2002). Oocytes from larger
follicles usually are more competent than those from smaller ones (Liu et al.,
2002; Marchal et al., 2002), and those from sows develop better than those
from prepubertal gilts (Marchal et al., 2001; Ikeda and Takahashi, 2003;
Sherrer et al., 2004). The heterogeneity of oocytes from different sources
leads to asynchronous meiotic progression during IVM, especially because pig
oocytes need a longer culture period (42 to 48 h) than those of other species.
Reducing nuclear morphological variation, i.e. meiotic synchronization, before
maturation by preincubation without gonadotropins (Funahashi et al., 1997a)
or with dibutyryl cAMP (Funahashi et al., 1997b; Somfai et al., 2003) appears
to enhance pig oocyte development. Butyrolactone I and roscovitine, which are
specific inhibitors of Cdc2 (a universal G2/M-phase regulator) have been found
to arrest meiosis in vitro (Motlik et al., 1998; Wu et al., 2002; Le Beux et al.,
2003). These substances reversibly block meiosis resumption and may be used
to synchronize subsequent nuclear maturation (Hirao et al., 2003; Le Beux et al.,
2003). However, there is little evidence to suggest any significant improvement
in oocyte developmental competence and no proof of full-term development
in any species has been reported (Fair, 2003; Lonergan et al., 2003). Protein
synthesis is essential for meiotic resumption of oocytes in vitro in the pig, as
in other mammals (Figure 1). It has been shown (Le Beux et al., 2003; Ye et
al., 2005) that cycloheximide (CHX), a nonspecific protein synthesis inhibitor,
can reversibly block meiotic resumption in porcine oocytes, improving their
developmental competence.
528 PIG EMBRYO IN VITRO PRODUCTION

Figure 1. An immature pig oocyte surrounded by compact, dense cumulus cell layers, suitable for
in vitro maturation (x100)

Recent IVM methods allow achievement in pig, as in other mammalian species,

80 to 90% of oocytes in MII stage (Figure 2). Similarly as in other mammals, a
large percentage of in vitro matured pig oocytes do not reach cytoplasmic maturity.
In vitro conditions may cause several disruptions. These include movement of
mitochondria to the inner cytoplasm (Sun et al., 2001; Torner et al., 2004), protein
synthesis (Ellederova et al., 2006), transport of signals, ions (especially calcium)
and other substances (Petr et al., 2000; Sun et al., 2001; Sun and Nagai, 2003). As
a consequence of these disruptions incomplete maturation may occur due to the
deficiency in some factors needed for full cytoplasmic maturation. To synchronize
meiotic resumption, IVM conditions have been improved in recent years, through
the realization of the importance of the redox state and glutathione content in
relation to cysteine in the maturation medium (Nagai, 2001).
For pig oocyte maturation several culture media have been successfully
used, such as North Carolina State University (NCSU) containing taurine and

Figure 2. In vitro matured pig oocyte at metaphase II stage. The chromosomes arranged in metaphase
plate and first polar body are indicated (x1000)
KĄTSKA-KSIĄŻKIEWICZ L. 529

hypotaurine - NCSU-23 (Yoneda et al., 2004) or sorbitol - NCSU-37 (Kikuchi

et al., 2002; Yoshioka et al., 2003; Karja et al., 2004; Suzuki et al., 2004).
Some research groups use Tissue Culture Medium 199 - TCM 199 (Qian et
al., 2003; Booth et al., 2005; Skrzyszowska et al., 2005) or modified Krebs-
Ringer medium TALP (Yoshida et al., 1993). Maturation medium is usually
supplemented with small quantities of additional substances, that have appeared
to be beneficial for oocyte maturation and subsequent embryo development.
Supplementation of culture medium with cysteine, cysteamine, cycloheximide,
glutamine, hormones: FSH, HCG, eCG, leptin, epidermal growth factor,
β-mercaptoethanol, follicular fluid, serum or serum albumin improve cytoplasmic
maturation as indicated by the higher developmental competence (Grupen et al.,
1995; Singh et al., 1997; Abeydeera et al., 1998; Bing et al., 2001; Jeong and
Yang, 2001; Marchal et al., 2001; Yoshioka et al., 2003; Craig et al., 2004; Ye et
al., 2005).
The process of cumulus cell expansion that occurs both in vivo and in vitro
is one of the major indicators of oocyte maturation. It has been shown that
supplementation of maturation medium with porcine follicular fluid increases
cumulus expansion and cytoplasmic maturation in in vitro matured pig oocytes
(Yoshida et al., 1993; Rath et al., 1995; Funahashi et al., 1997a). Indeed,
supplementation of IVM medium with follicular fluid has been applied by several
authors (Yoshida et al., 1993; Marchal et al., 2001; Kikuchi et al., 2002; Qian et
al., 2003; Yoshioka et al., 2003; Suzuki et al., 2004; Skrzyszowska et al., 2005).
Oxygen tension during IVM is also an important factor in cytoplasmic maturity
for in vitro development to blastocysts (Kikuchi et al., 2002; Park et al., 2005).
When cumulus-oocyte complexes were matured in vitro in the medium NCSU-37
under 5% O2 or 20% O2, fertilized under 5% O2, and subsequently cultured under
5% O2, there were no significant differences in blastocyst rates (Kikuchi et al.,
2002). However, the quality of blastocysts, as evaluated by total cell number, was
better after IVM under 5% O2 than under 20% O2. Contrary to this observation,
the investigations of Macháty et al. (1998) have indicated that embryos developed
from oocytes that matured in medium NCSU-23, under 20% O2 showed a higher
number of nuclei than those developed under 5% O2. Recent investigations of Park
et al. (2005) have shown that high oxygen tension (20%) during IVM significantly
improved blastocyst formation (23 vs 13%) after IVF than low oxygen (5%) but
did not improve the rates of nuclear maturation, sperm penetration, monospermic
fertilization, pronuclear formation, cleavage and blastocyst cell number. On the
basis of these investigations we can conclude that oxygen content during IVM
should be correlated with type of culture medium used.
Nuclear maturation of oocytes is easily assessed morphologically by the
presence of the first polar body, but there is a lack of non-invasive methods to
530 PIG EMBRYO IN VITRO PRODUCTION

evaluate cytoplasmic maturation. Until now, the only reliable method for assessing
the competence of an oocyte is its post-fertilization development.

IN VITRO FERTILIZATION

Fertilization is the cascade of the cellular mechanisms that pass the genome
from one generation to the next and initiate development of a new organism.
A typical mammalian egg freshly ovulated or in vitro matured is enclosed by
two layers: an outer layer of corona cells and inner, extracellular matrix, the
zona pellucida. To reach the egg plasma membrane, sperm must penetrate both
layers in steps requiring sperm motility, sperm surface enzymes, and sperm
secreted enzymes. Binding to the oocyte zona pellucida induces the sperm cell
to undergo the acrosomal reaction in which the outer acrosomal membrane fuses
with the overlying plasma membrane (Primakoff and Myles, 2002). Sperm bind
transiently to the oocyte zona pellucida and the plasma membrane and then fuse.
This exocytotic event results in the release of hydrolytic enzymes, principally
the trypsin like acrosin, and in the exposure of new membrane domains, both of
which are essential for the fertilization process. Signaling in the sperm is induced
by sperm adhesion to the zona pellucida, and signaling in the oocyte by gamete
fusion (Primakoff and Myles, 2002).
However, mammalian spermatozoa are unable to fertilize the oocyte
immediately after ejaculation. They require a period of incubation in the female
reproductive tract or in the appropriate in vitro conditions in order to acquire the
capacity to fertilize. During this time, the spermatozoa undergo a poorly defined
process of maturation known as capacitation (Breitbart, 2003). Sperm capacitation
includes a cascade of biochemical changes that must occur before spermatozoa
can effectively interact with an oocyte. This involves activation of adenylyl
cyclase, protein tyrosine phosphorylation and actin polymerization, cholesterol
efflux, increases in calcium ions and changes in sperm motility (Breitbart,
2003). It had been generally accepted that spermatozoa are translationally and
transcriptionally silent; however, the latest investigations of Gur and Breitbart
(2006) have demonstrated, for the first time, incorporation of labeled amino acids
into polypeptides during sperm capacitation. These authors also demonstrated that
protein translation in sperm involves mitochondrial but not cytoplasmic ribosomes
and that inhibition of protein translation significantly reduced sperm motility,
capacitation and in vitro fertilization rate. The importance of the mitochondrion
for pig fertilization has also been shown by El Shourbagy et al. (2006). Their
data suggest that mitochondrial number is important for fertilization outcome and
embryonic development. Furthermore, a mitochondrial pre-fertilization threshold
KĄTSKA-KSIĄŻKIEWICZ L. 531

may ensure that, as mitochondria are diluted out during post-fertilization cleavage,
there are sufficient copies of mtDNA per blastomere to allow transmission of
mtDNA to each cell of the embryo after the initiation of mtDNA replication during
the early postimplantation stages (El Shourbagy et al., 2006).
Following sperm penetration into the cytoplasm of a mature oocyte, the highly
condensed chromatin of the sperm nucleus first decondenses and the protamines
are replaced by histones. After a short period of chromatin recondensation a final
phase of decondensation and the formation of the interphase male pronucleus,
surrounded by a new organized pronuclear membrane, occurs. During this sperm
chromatin structure reorganization, and particularly during protamine-histone
replacement, profound DNA epigenetic modifications take place. At the same
time the maternal genome is also modified and prepared for integration with the
paternal genome (Gioia et al., 2005).
During pig in vitro fertilization, sperm penetration begins at 3 h post-insemination
(Ding et al., 1992). Sperm penetration quickly induces the resumption of meiosis
and cortical reaction that blocks polyspermy. By 5 h, decondensing sperm head and
anaphase II plate are observed in half of the oocytes, and by 8 h, both female and
male pronuclei are formed (Ding et al., 1992). In appropriate in vitro conditions
60 to 70% of in vitro mature pig oocytes may be penetrated by spermatozoa;
however, the rate of normally fertilized oocytes, i.e. monospermic fertilization is
rather low while exceptionally high incidence of polyspermic fertilization has been
observed. Polyspermy is considered to be one of the persistent and most difficult
problems to overcome in pig IVF. Incidence of polyspermy in porcine eggs in vivo
can reach 30 to 40%, and polyspermy rate in the in vitro fertilized eggs can be
as high as 65% (Xia et al., 2001). The reasons for the occurrence of polyspermy
in pig oocytes are not clear, and our knowledge about the exact mechanisms for
preventing polyspermy in this species is relatively poor. Low developmental
rates of in vitro produced porcine embryos may, therefore, be caused not only
by inadequate culture conditions but by a high incidence of polyspermy, a lethal
condition in mammals (Hunter and Nicol, 1988). Although polyspermic porcine
embryos can develop to blastocysts, they have fewer numbers of inner cell mass
cells compared with monospermic embryos (Han et al., 1999). Polyspermic
penetration in vitro is caused by a delayed zona reaction and/or the simultaneous
penetration by a number of spermatozoa with a reacted acrosome (Wang et al.,
1999; Funahashi and Nagai, 2001; Yoshioka et al., 2003).
To reduce the incidence of polyspermic penetration several systems of sperm
capacitation have been developed. It has been shown that methylxanthines, such
as caffeine and theophylline, can enhance the ability of sperm to penetrate in
vitro matured porcine oocytes stimulating and maintaining sperm motility by
acting as phosphodiesterase inhibitors, presumably by elevating cAMP levels
532 PIG EMBRYO IN VITRO PRODUCTION

(Yoshioka et al., 2003; Funahashi and Romar, 2004). The replacement of caffeine
with adenosine in a porcine IVF system increased the incidence of monospermic
penetration (Funahashi and Nagai, 2001; Yoshioka et al., 2003). Addition of
cysteine to the maturation medium, the precursor of glutathione, can increase
intracellular glutathione levels resulting in an enhancement of male pronucleus
formation and the percentage of zygotes developing to blastocysts (Yoshioka et
al., 2003). It has also been reported that follicular fluid may decrease the incidence
of polyspermic penetration of porcine oocytes (Abeydeera, 2002). Furthermore,
incubation of spermatozoa with oviductal epithelial cells during capacitation prior
to IVF and fertilization may reduce polyspermy by 40 to 50% (Nagai and Moor,
1990). Supplementation of culture medium with bovine serum albumin (BSA)
accelerated the ability of porcine spermatozoa to penetrate in vitro mature oocytes
(Suzuki et al., 1994). Serum albumin has numerous properties that can play a
role in capacitation, including removal or alteration of sperm membrane surface
components and alteration of cholesterol:phospholipid ratios in sperm membranes
(Suzuki et al., 1994). According to Suzuki et al. (1994) replacement of BSA with
polyvinyl alcohol decreases the number of polyspermic oocytes and the number
of spermatozoa per penetrated oocyte. Also anti-hyaluronidase oligosaccharide
derived from chondroitin sulphate A effectively reduces the incidence of
polyspermy during IVF of porcine oocytes promoting the normal fertilization
process as has recently been shown by Tatemoto et al. (2005). However, according
to Rath et al. (2005) the only way to avoid polyspermic penetration is to reduce
the number of spermatozoa per oocyte used for IVF. The amount of spermatozoa
depends on the treatment of the sperm and has to be set for each individual boar
(Rath et al., 2005). Recent investigations of Koo et al. (2005) have also shown that
the spermatozoa concentration during in vitro fertilization may be important for
developmental competence and quality of pig embryos.
All three steps of in vitro embryo production, i.e. maturation, fertilization and
embryo culture are closely mutually dependent. The capability of reprogramming
the male chromatin after fertilization is dependent on the quality of oocyte
maturation (Gioia et al., 2005). In fact, as has been shown by Gioia et al. (2005),
while in about 80% of in vivo matured and in vitro fertilized pig oocytes the male
pronucleus underwent a process of active demethylation and showed a condition
of histone H4 hyperacetylation, only 40% of IVM/IVF zygotes displayed a
similar pronucleus remodeling asymmetry. However, oocytes that carried out the
first part of maturation in vivo (up to GVBD) and then completed the process in
vitro, displayed the same pronucleus asymmetry as oocytes matured entirely in
vivo (Gioia et al., 2005). Therefore, in addition to the improvement of IVM/IVF
procedures, improvement of the in vitro embryo culture procedure seems to be the
more important factor needed to obtain viable blastocysts.
KĄTSKA-KSIĄŻKIEWICZ L. 533

EMBRYO CULTURE

The development of in vitro produced porcine embryos from the two-cell to

the hatched blastocyst stage (Figure 3a-f) requires adequate culture conditions.

Figure 3. Pig embryos at: a. 2-cell stage; b. 4-cell stage; c. 8-cell stage; d. morula; e. the late
blastocyst; f. hatching blastocyst

In spite of intensive efforts of several investigators, there are very few satisfactory
protocols for embryo culture in vitro. The main problem in pig embryo culture is
relatively low quality in terms of total cell number compared to in vivo embryos
of the same chronological age (Macháty et al., 1998). For example, the total cell
number of blastocysts was reported to range from 30 to 38 on day 6 after IVF,
while the mean number of cells in 6-day-old expanded blastocysts developed in
vivo was twice as high, i.e. 74 cells (Abeydeera and Day, 1997; Wang et al., 1997).
Furthermore, blastocyst development is a poor indicator of embryo viability. The
most valid criterion of embryo viability is in vivo development to term following
embryo transfer to a synchronized recipient.
534 PIG EMBRYO IN VITRO PRODUCTION

In many studies oriented on in vitro culture of pig embryos, several modifications

of the culture conditions were used. These modifications included different media
(Wang et al., 1997; Macháty et al., 1998; Gajda and Smorąg, 2004; Im et al.,
2004), differentiated volumes of culture medium, medium covered with mineral
oil or without covering (Yoshioka et al., 2002, 2003; Gil et al., 2003; Im et al.,
2004, 2005), group embryo culture with differentiated distances between embryos
(Stokes et al., 2005) and supplementation of medium with different ingredients
(Ka et al., 1997; Swain et al., 2002; Karja et al., 2004; Booth et al., 2005; Craig et
al., 2005; Lee et al., 2005).
For pig embryo culture several media have been successfully used: North
Carolina State University containing taurine and hypotaurine - NCSU 23 (Macháty
et al., 1998; Lee et al., 2003; Gajda and Smorąg, 2004; Im et al., 2004; Yoneda
et al., 2004; Skrzyszowska et al., 2005) or NCSU containing sorbitol - NCSU
47 (Kikuchi et al., 2002; Gajda and Smorąg, 2004; Karja et al., 2004), Carles
Rosenkrans 2 - CR2 (Bettauser et al., 2000), potassium simplex optimized medium
- KSOM (Macháty et al., 1998), modified Calot, Ziomek, Bavister-CZB (Gajda
and Smorąg, 2004), Beltsville embryo culture medium - BECM 3 (Onishi et al.,
2000; Im et al., 2004), porcine zygote medium - PZM 4 (Yoshioka et al., 2003) or
modified Whitten’s medium (Onishi et al., 2000).
The comparative study of Gajda and Smorąg (2004) on in vitro development of
in vivo produced 2 to 4-cell pig embryos cultured in different media (NCSU-23,
NCSU-37 and CZB) has revealed that the highest percentage of embryos reached
the blastocyst stage in NCSU-23 (89.2%) followed by NCSU-37 (78.9%) and CZB
(67.8%) medium. The average total number of cells in the blastocyst developed in
NCSU-23, NCSU-37 and CZB media was 139.5±32.8, 71.9±36.6 and 58.3±8.6,
respectively (Gajda and Smorąg, 2004).
The developmental competence of porcine embryos can be markedly affected
by protein supplementation (Ka et al., 1997). Preimplantation embryos can resume
and produce amino acids in a manner dependent upon the stage of development
that may be predictive of subsequent viability. The investigations of Booth et al.
(2005) have shown that the net rates of depletion and uptake of amino acids by pig
embryos vary between amino acids, the day of embryo development and the type
of embryos present at a given stage of development.
The in vitro development of porcine zygotes to the blastocyst stage may be
facilitated by culture in groups, suggesting a role for autocrine/paracrine factors.
Recent reports of Stokes et al. (2005) on culture of in vitro produced and in vivo
derived porcine embryos have suggested a role for, as yet unknown diffusible
paracrine/autocrine factors released by early porcine embryos, in promoting the
growth of neighbouring embryos in vitro. The development of individual zygotes
to the blastocyst stage was optimal when they were cultured at a distance of
KĄTSKA-KSIĄŻKIEWICZ L. 535

between 81 and 160 μm. As the distance between the embryos was increased,
blastocyst rates declined significantly. Blastocyst volume and cell number (both
inner cell mass and trophectoderm) were also increased when the distance apart
was between 81 and 160 μm. Culturing embryos in groups at different stages
of development suggested that group culture confers a greater advantage to
development after the activation of the genome. Group culture of in vivo derived
embryos showed a weak distance effect. This advantage was observed to a lesser
extent by in vivo derived zygotes which are likely to have been better conditioned
for development in vitro by being conceived in the female reproductive tract.
Recently Lee et al. (2005) have pointed to a favourable effect of supplementing
pig oocyte and embryo culture medium with insulin and metformin. When added
during the entire IVM and IVC, insulin increased the developmental potential
of porcine oocytes and embryos, and metformin enhanced the action of insulin.
The effects of insulin and metformin were associated with oocyte GSH content
and tyrosine kinase activity. Insulin significantly increased oocyte GSH content
and metformin significantly enhanced the action of insulin on GSH content and
tyrosine kinase activity compared to insulin alone. Recent studies have also
suggested that leptin plays an important role in embryo development (Craig et
al., 2005). As has already been mentioned, leptin increases oocyte maturation in
vitro, and inclusion of leptin in both IVM and embryo culture medium further
increased blastocyst development compared to when leptin was included in the
embryo culture alone. These results have suggested that lepin has a synergistic
role on both oocyte maturation and preimplantation embryo development (Craig
et al., 2005).
In addition, the oxygen concentration has been shown to be a major factor
causing a difference in the developmental rates of porcine IVM/IVF embryos
between in vivo and in vitro environments. The oxygen concentration in the
mammalian oviduct and uterus is about 5% (Fisher and Bavister, 1993), whereas
in vitro cultured embryos are usually maintained under 5% CO2 and 95% air,
i.e. 20% O2. Reduction of the O2 concentration from 20 to 5% has been shown
to enhance embryonic development in humans (Dumoulin et al., 1995; Catt and
Henman, 2000), cattle (Liu and Foote, 1995; Lim et al., 1999), sheep (Thompson et
al., 1990) and mice (Goto et al., 1992; Dumoulin et al., 1995).The developmental
rate to the blastocyst stage of IVM/IVF porcine embryos cultured under 5% O2
was significantly higher than that of embryos cultured under 20% O2 (Yoneda et
al., 2004). On the other hand, there was no difference in the developmental rate to
the blastocyst stage between in vivo fertilized oocytes cultured under 5% O2 and
20% O2 (Yoneda et al., 2004).
The metabolism of porcine embryos produced both in vivo and in vitro is
different from that of other species, as they metabolize glucose throughout
536 PIG EMBRYO IN VITRO PRODUCTION

preimplantation development (Swain et al., 2002; Karja et al., 2004). Some

reports showed that glucose inhibits embryo development before compaction or
before the blastocyst stage in mice (Gardner and Leese, 1988) and cattle (Kim
et al., 1993). Investigations of Swain et al. (2002) showed that pig embryos use
glucose via glycolysis in significant amounts at all stages examined, regardless of
embryo origin. In vitro derived embryos have significantly increased glycolytic
activity after the eight cell stage, whereas in vivo derived embryos have increased
glycolysis at the blastocyst stage. In vivo derived embryos have higher rates of
glycolysis compared with in vitro derived embryos. Glucose usage through the
Krebs cycle for in vitro- and in vivo derived embryos increased significantly at
the blastocyst stage. Pig embryos produced in vitro used constant amounts of
glutamine throughout development, whereas in vivo derived embryos increased
glutamine usage after the eight-cell stage. Pyruvate use was minimal at all stages
examined for both in vitro- and in vivo derived pig embryos, showing significant
increases at the blastocyst stage. Krebs cycle metabolism of pyruvate, glutamine
and glucose by in vivo derived embryos was higher than that by in vitro derived
embryos (Swain et al., 2002). Therefore it can be concluded that in vitro culture
conditions produce pig embryos with altered metabolic activity, which may
compromise embryo viability (Swain et al., 2002). It has been shown by Karja
et al. (2004) that the replacement of pyruvate and lactate with glucose at 58 h of
culture significantly enhanced the rate of blastocyst production. Successful culture
conditions allowing the expanded blastocysts to be obtained on Day 6 of embryo
culture with the mean cell number of 80 cells were reported by Kikuchi (2004).
Embryo culture was carried out for the first 2 days in the conditioned medium
obtained from the culture of porcine oviductal epithelial cells, then medium was
supplemented with glucose (Kikuchi, 2004). Moreover, some of these blastocysts
possessed the ability to develop to term. When Day 5 expanded blastocysts (50
blastocysts per recipient) were transferred to an oestrus-synchronized recipient
they farrowed 8 normal piglets, and out of Day 6 expanded blastocysts transferred
to two recipients a total of 11 piglets were obtained (Kikuchi, 2004).
In spite of obtaining satisfactory results of pig embryo development in the
conditioned medium (Kikuchi, 2004), there are intensive efforts aimed to develop
chemically defined porcine culture medium (Yoshioka et al., 2002, 2003; Cui
et al., 2004; Kishida et al., 2004; Booth et al., 2005). Because a chemically
defined medium eliminates undefined factors present in biological materials
such as follicular fluid, serum or serum albumin and/or co-culture with somatic
cells, application of a chemically defined medium to IVP of embryos has certain
advantages. For instance, the use of defined media facilitates the analysis of the
physical action of substances on the development of preimplantation embryos,
improves reliability of formulations, yields a higher reproducibility of results, and
KĄTSKA-KSIĄŻKIEWICZ L. 537

ensures biosafety of culture media by elimination of protein preparations, which

may be contaminated with pathogens (Bavister, 1995). However, up to now the
complete chemically defined systems for porcine IVP have not been developed. For
example, although there was no significant difference in cleavage rates between
newborn calf serum and polyvinyl alcohol supplemented media containing both
cysteine and epidermal growth factor, the rate of blastocyst development was
significantly lower in the defined medium than in the serum containing medium
(Kishida et al., 2004). In spite of these lower results, it may be expected that in the
near future chemically defined media for oocyte and embryo culture will be used
in practical applications of pig IVP.
In conclusion, application of advanced embryo production technologies
is essential for the progression of animal breeding and for the use of animals
in the production of pharmaceuticals and for xenotransplantation purposes.
However, the associated in vitro techniques have inherent problems, which from
a mechanistic viewpoint, are not well understood. For widespread use of IVP
technology it is essential to remove all the side effects observed following culture.
The greatest concern is related to the production loss as a consequence of lowered
embryo quality, high rates of conceptus loss and to the deviations in pregnancy,
parturition and the development of offspring. It should be emphasized however
that many normal offspring have been born using IVP and that the method may be
successfully used, albeit with low efficiency and with concerns regarding the well
being of the offspring.

ACKNOWLEDGEMENTS

The figures presented in the article were done by my colleagues: Dr. Maria
Skrzyszowska (Figure 1) and Dr. Barbara Gajda (Figures 3a-f) from the National
Research Institute of Animal Production in Balice (Poland) and Figure 2 was
done by Dr. Hannelore Alm from the Research Institute for the Biology of Farm
Animals, Dummerstorf/Rostock (Germany).
538 PIG EMBRYO IN VITRO PRODUCTION

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